Stability and sensory analysis of walnut polypeptide liquid: response surface optimization

ABSTRACT

Bioactive peptides are small molecular peptides with some physiologically active functions. These peptides have been shown to possess antibacterial, antioxidant, and blood pressure lowering activities. Although walnuts are rich in high-quality plant proteins, the extracted walnut dregs have low utilization by the walnut industry chain. Therefore, the aim of the study was to investigate the nutritional value and antioxidant activity of walnut dregs in order to increase the added value of walnut by-products. Using the response surface methodology, the optimum process parameters of purification and stability for walnut polypeptide liquid were determined as follows: pH 4.5, 8 column/hours (h) column speed and 3:2 ratio of anion to cation. The scavenging reaction rates for hydroxyl radicals and superoxide anion by 3, 5, 10 u ultrafiltration membrane fractionation screening were found to be 73.3% and 64.4%, 66.1% and 58.9%, 59.2%, and 51.6%, respectively. After comparison, walnut polypeptide liquid showed some degree of antioxidant capacity. The stability of walnut polypeptide liquid was optimized by the response surface. Under the homogeneous pressure of 35 Mpa, the use of a certain amount of stabilizers improved the stability of walnut polypeptide liquid. In conclusion, with the addition of additives, the optimum values of parameters for walnut polypeptide liquid obtained using response surface methodology were determined as follows: the ratio of solid to liquid was 15%; the amount of protein sugar was 0.2%; the amount of citric acid was 0.25%; and the addition of walnut powder flavor was 0.15%. Also, the study has provided a theoretical basis for the waste utilization of walnut by-products, and partial support for the intensive processing of the walnut industry chain.

KEYWORDS:

• Walnut
• polypeptides
• stability
• response surface
• sensory evaluation

Introduction

Polypeptides (or peptides) are composed of different kinds of amino acids, which are combined to form peptide chains. These peptide chains are then folded to form protein molecules. Studies have reported that polypeptides have good physical properties and are easily absorbed and utilized by organisms.^[1,2] One of these peptides is bioactive peptides, which are small molecular peptides with physiologically active functions. These peptides have antibacterial, antioxidant, and blood pressure lowering activities.^[3] Unlike proteins, peptides are more easily digested and absorbed by human body.^[4] There are many ways to prepare polypeptides. For example, natural antioxidant peptides can be extracted from plants.^[5] Also, polypeptides can be produced by the chemical hydrolysis and enzymatic hydrolysis of proteins. Currently, several studies have isolated many different types of peptides from plant proteins such as rice, peas, and soybeans^[6,7], which have provided a theoretical basis for further development of peptide products.

Walnuts (Juglans regia L.) are widely cultivated worldwide.^[8–10] Studies have reported that walnuts contain 14–18% protein.^[11,12] Its fat content is as high as 70%. The extracted walnut dregs also contain about 30% protein, and contain higher levels of glutamic acid and arginine acid. It has a digestive rate of more than 85% in the human body.

As a high-quality protein and high-quality antioxidant, its utilization rate in China is still very low. The extracted walnut dregs are often used as animal feed or directly landfilled, which cause a waste of high-quality resources and pollution to the ecological environment. Therefore, the aim of the study was to investigate the nutritional value and antioxidant activity of walnut dregs in order to increase the added value of walnut by-products. Also, this study would provide a theoretical basis for the industry to develop and produce new functional products using walnut by-products.

Materials and methods

Materials and equipment

Materials used were as follows: degreased walnut dregs (were obtained after walnut oil extraction) (Xinjiang Zhongya Food Co., Ltd., Xinjiang, China); alkaline protease (enzyme activity 800,000 U/g) and papain (enzyme activity 800,000 U/g) (Nanning Pangbo Biological Co., Ltd., Nanning, China); 732-type cation and D301-type anion exchange resin (Hebei Langfang Shengquan Chemical Co., Ltd., Hebei, China); Gly-Gly-Tyr-Arg (Sigma Company, USA); ultrafiltration membrane (Bestech Instrument Technology (Beijing) Co., Ltd., Beijing, China); carboxymethylcellulose sodium (CMC-Na), xanthan gum (SG), and sucrose fatty acid ester (SE) were all food-grade and supplied by Henan Zhengzhou Longxin Biotechnology Co., Ltd., Henan, China; white sugar, protein sugar, citric acid, and walnut powder flavor were all food-grade and supplied by Beijing North Xiaguang Food Additive Co., Ltd., Beijing, China.

Equipments used were as follows: AL204-1C analytical balance (Shanghai Mettler Toledo Instrument Co., Ltd., Shanghai, China); DHG-9140A electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China); TU-1810 UV-Vis spectrophotometer (Beijing Pu Analysis General Instrument Co., Ltd., Beijing, China); magnetic DDS-307 conductivity meter (Shanghai Jingke Co., Ltd., Shanghai, China); Ultrafiltration device (Millipore Company, USA); 85-2A thermostatic magnetic stirrer (Jiangsu Changzhou Jintan District Huanyu Scientific Instrument Factory, Jiangsu, China); TDL-50A low speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory, Shanghai, China); Q/L704-2000 ultra high pressure homogenizer (Hebei Langfang General Machinery Co., Ltd., Hebei, China); BL-75A vertical pressure steam sterilizer (Shanghai East Asia Pressure Vessel Manufacturing Co., Ltd., Shanghai, China).

Experimental methods

Preparation of samples: Preparation of walnut dreg protein was as follows: degreased walnut dregs were dried followed by coarse pulverization through 80 mesh sieve. After that, the suspension (stock-to-liquid ratio 1:10) was adjusted to pH 9.0–9.5 and centrifuged at 4800 rpm for 25 min (min). The supernatant was adjusted to pH 4.0–4.5 followed by centrifugation at 4800 rpm for 25 min. The precipitate was washed to neutral pH and freeze-dried.^[13]

Walnut dreg chime proteolysis was performed as follows: walnut dreg protein was pulverized into coarse powder which was further ground and passed through 80 mesh sieve. The suspension (concentration 6–8%) was adjusted to pH 7.0. After that, the suspension underwent microwave pretreatment (microwave power 500 W, microwave time 5 min, microwave temperature 50°C) followed by enzymatic hydrolysis (alkaline protease to papain ratio = 2:1; substrate concentration 6–8%; pH 7.5; temperature 50°C) for 3 h. After that, the suspension was heated at 90°C to inactivate the enzyme for 10 min. Then, the suspension was centrifuged at 5000 rpm for 20 min and the supernatant was obtained. Operation points were performed as follows: after microwave enzymatic treatment of walnut dreg protein, the enzymatically hydrolyzed antioxidant polypeptide obtained by enzymatic hydrolysis was used as a sample for desalination and ultrafiltration.

Walnut polypeptide liquid content

Production of standard curve

The modified method by Lu et al.^[14] and trichloroacetic acid preparation were used to prepare the following standard solutions: 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL standard solution of different concentrations and sequentially weighed 6.0 mL of different concentrations of standard solution. After adding 4.0 mL of biuret reagent, the solution was mixed and allowed to stand for 15 min. The solution was then centrifuged at 4800 r/min for 15 min. The absorbance (OD) of the obtained supernatant was measured at 540 nm. The ordinate was calibrated as the absorbance value. The abscissa was calibrated as the concentration of the peptide, and a standard curve was made.

Methods for determining peptide content

After accurately weighing 3 mL walnut dreg polypeptide liquid, 10% 3 mL trichloroacetic acid was added to the liquid. The mixture was allowed to stand for 15 min and centrifuge at 4800 rpm for 15 min. The supernatant (6.0 mL) was added with 4.0 mL biuret reagent. The supernatant was then allowed to stand for 15 min followed by centrifugation at 4800 rpm for 15 min. The absorbance (OD) of supernatant was measured at 540 nm. The polypeptide in the walnut polypeptide liquid was calculated according to the tetrapeptide standard curve drawn.

Single factor experiments to optimize desalination rate

This study employed an anion-cation mixed bed desalination method. The pH of the polypeptide solution was selected at 3.5, 4.5, 5.5, 6.5, and 7.5 under the conditions of an anion-cation exchange resin ratio of 3:2 and a column speed of 8 times column/h. Under the conditions of pH 4.5 of the polypeptide liquid and 8 times column/h of the column, the ratio of different anion-cation exchange resins was selected as follows: 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2. Under the condition that the pH of the polypeptide liquid was 4.5 and the ratio of the anion-cation exchange resin was 3:2, different column speeds were selected as follows: 2, 5, 8, 10, and 15 times column/h. From these three aspects, the antioxidant activity and desalination rate of walnut polypeptide liquid were compared and analyzed. The desalination rate and peptide content were used as indicators to determine the optimum pH of the peptide liquid, the ratio of anion-cation exchange resin and the flow rate of the column. Equation 1(1) $\mathrm{D}\mathrm{e}\mathrm{s}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}=\frac{{\mathrm{K}}_0-{\mathrm{K}}_1}{{\mathrm{K}}_0}\mathrm{x}100$(1) shows the calculation of desalination rate:

(1) $\mathrm{D}\mathrm{e}\mathrm{s}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}=\frac{{\mathrm{K}}_0-{\mathrm{K}}_1}{{\mathrm{K}}_0}\mathrm{x}100$(1)

Where: K₀ is the conductivity before treatment; K₁ is the conductivity after treatment.

Ultrafiltration separation process of walnut polypeptide liquid

Supplementary Figure 1 shows a schematic view of a membrane device, mainly composed of a pressure device and a membrane filtration device. Using the 3, 5, and 10 KDa in the ultrafiltration membrane and a certain pressure-assisted membrane filtration, walnut polypeptide liquid was separated by ultrafiltration. The collected filtered different filtrates were used to determine their antioxidant capacity.

Single factor experiments for walnut polypeptide liquid stability

Emulsification stability was analyzed by selecting CMC-Na, SG, SE and homogenization pressure as independent variables.

Optimization stability of walnut polypeptide liquid

In the single factor test results, combined with the Box–Benhnken test design, CMC-Na (X₁), SG (X₂), SE (X₃), and homogenization pressure (X₄) were used as independent variables to emulsify the stability of walnut polypeptide solution (Y). The response surface optimization analysis for the dependent variable is shown in Table 1.

Table 1. Levels of test factors for the optimization stability of walnut polypeptide liquid

Level	Four-factor level table
	X1 -CMC-Na (%)	X2-SG (%)	X3-SE (%)	X4-Homogeneous pressure (MPa)
−1	0.01	0.02	0.02	30
0	0.02	0.03	0.03	35
1	0.03	0.04	0.04	40

Single factor experiments of walnut polypeptide liquid preparation

During the preparation process of walnut polypeptide liquid, the effect of the ratio of solid-to-liquid, protein sugar, citric acid, walnut powder flavor were determined.^[15–17] The ratio of solid-to-liquid is 5%, 10%, 15%, 20% and 25%, respectively. The amount of protein sugar added was 0.05%, 0.1%, 0.2%, 0.3%, and 0.4%, respectively (the ratio of white sugar to protein sugar was 3:1 and the white sugar was added according to the amount of protein sugar added). The amount of citric acid added was 0.15%, 0.20%, 0.25%, 0.30%, and 0.35%, respectively. The amount of walnut powder flavor added was 0, 0.10%, 0.15%, 0.20%, and 0.25%, respectively.

Optimization of response surface methodology for sensory experiments

The sensory evaluation was graded by 10 taste sensitive participants who had worked in research related to agricultural products. The indicators of the black wheat nutritional powder samples were graded according to the sensory evaluation criteria and the scores were recorded. Based on the single factor tests, according to the Box–Benhnken center combination, the ratio of solid-to-liquid (X₁), protein sugar (X₂), citric acid (X₃), and walnut powder flavor (X₄) were selected to prepare the peptide liquid (Y) (Table 2).

Table 2. Test factor and level table for response surface methodology. Based on the Box–Benhnken Center combination, the ratio of material to liquid (X₁), protein sugar (X₂), citric acid (X₃) and walnut powder flavor (X₄) were selected, the sensory test was optimized using the formulated polypeptide solution (Y) as the response value

Level	Four-factor level table
	X1-Material liquid ratio (%)	X2-Protein sugar (%)	X3-Citric acid (%)	X4-9115 Walnut powder flavor (%)
−1	10	0.1	0.20	0.10
0	15	0.2	0.25	0.15
1	20	0.3	0.30	0.20

Walnut polypeptide liquid sensory evaluation criteria

According to the Chinese National Standards^[18,19], the color, top aroma, sweetness, and mouthfeel of walnut polypeptide liquid were evaluated. The scoring standards are shown in Table 3.

Table 3. Walnut peptide liquid taste rating standards according to the Chinese National standards

Category	First grade	Secondary grade	Third grade
Color (20)	Uniform color, white or milky white (16–20)	Rendered white gray (10–15)	Color shades are different, or have other colors (1–9)
Head fragrance (20)	With aroma of walnut oil, no other odor (16–20)	Lighter or heavier walnut flavor, no other odor (10–15)	There are other odors (1–9)
Sweetness (30)	Moderate sweetness, no other bad taste (20–30)	Sweet or sweet taste, no other bad taste (10–19)	A little sweet, with other flavors (1–9)
Taste (30)	Good taste, no bitter taste (20–30)	Slightly bitter and astringent, unpleasant (10–19)	Poor taste, bitter taste (1–9)

Walnut polypeptide liquid index detection

According to the Chinese National Standards^[20], the peptide content of the walnut polypeptide liquid, coliform bacteria, pathogenic bacteria (Salmonella, Shigella and Staphylococcus aureus), and other indicators were tested.

Statistical analysis

Data were compared and analyzed using ANOVA (SPSS 18.0, SPSS Inc, Chicago, IL, USA). Graphic drawing was performed using the Center Combine Box-Behnken of Design Expert 8.0.6 and Excel 2003 software. All experiments were conducted in triplicate.

Results and discussion

Standard curve

A tetrapeptide standard curve was drawn to determine the amount of peptide in the walnut polypeptide liquid. Supplementary Figure 2 shows the standard curve. The linear relationship between mass concentration and absorbance showed a good state in the concentration range of 0.2–1.2 mg/mL. The equation was: y = 0.0612x-0.0207; R2 = 0.9994. The content of the polypeptide is calculated by the calibration and equation.

Analysis of single factor test results of desalination rate

Effect of changing the ph value of peptide liquid on desalination

The effect of pH change on the desalination effect is shown in Supplementary Figure 3. Under the condition that the ratio of anion-cation exchange resins was 3:2 and the column speed was 5 times column/h, the pH changes at different points were selected: 3.5, 4.5, 5.5, 6.5, and 7.5. Therefore, the desalination rate and the peptide content index of the polypeptide solution are different under different pH changes. Within a certain range of indicators, the desalting rate and the peptide content decreased with the increase of pH value (Supplementary Figure 3). This may be due to an increase in the pH of the polypeptide liquid, which caused a relatively high degree of ionization in the liquid, resulting in a corresponding increase in the loss of the polypeptide. When the pH value was 4.5 and the isoelectric point of the walnut dreg protein was between 4.0 and 4.5, the corresponding large amount of protein containing macromolecular salts would settle. Therefore, the desalination rate and peptide content of the polypeptide liquid were the highest, 91.26% and 88.69%, respectively, which were 1.49 and 1.45 times of the lowest value. Therefore, pH 4.5 was selected because of its optimum effect.

The effect of the ratio of anion-cation exchange resin on desalination

Supplementary Figure 4 shows the effect of the change in the ratio of anion and cation exchange on the desalination. At pH 4.5 and 5 column/h column speed, different ratios of anion and cation changes were selected: 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2. Within a certain range of indicators, the desalination rate and the peptide content did not change much with the adjustment ratio (Supplementary Figure 4). Improper ratio of anion to cation directly affected the pH of the polypeptide liquid. When the ion content in the system was high, the desalination became insufficient, thereby reducing the desalination rate and correspondingly reducing the content of the polypeptide. When the ratio of anion-cation exchange resin was 3:2, the desalination rate and peptide content of the polypeptide liquid had the highest values, 82.56% and 74.92%, respectively, which were 1.37 and 1.22 times of the lowest values. Therefore, the anion-cation exchange ratio was determined to be 3:2, which gave a better effect.

Effect of column speed on desalination

Supplementary Figure 5 shows the effect of column speed on the desalination. At pH 4.5 and the anion to cation ratio was 3:2, different column speeds were selected: 2, 5, 8, 10, and 15 columns/h. Within a certain range of indicators, as the speed of the column increased, the lack of contact between the exchange resin and the polypeptide solution resulted in a decrease in the desalination rate and the polypeptide loss rate (Supplementary Figure 5). With the decreasing column speed, the desalination rate and the polypeptide loss rate increased, and the resin adsorbed a large amount of polypeptide. When the column speed was 8, 10, and 15 times column/h, the desalination rates of the polypeptide liquid were 83.7%, 84.3%, and 85.9%, respectively, showing that the difference was not significant; the peptide content of the polypeptide liquid was 60.2%, 54.8%, and 53.7%, respectively, and the highest value was 1.12 times of the lowest value. Therefore, the speed of the column of the walnut polypeptide solution was determined to be 8 times column/h.

Analysis of polypeptide ultrafiltration separation

The enzymatic preparation of walnut dreg polypeptide was generally carried out by hydrolyzing protein molecules into macromolecular peptides or smaller dipeptides. Therefore, the relatively large molecular peptides obtained were converted into relatively active small peptides by ultrafiltration. Most of the walnut polypeptides have a molecular weight below 3 KDa. Therefore, the ultrafiltration membranes of 3, 5, and 10 KDa were selected for ultrafiltration of the polypeptide liquid.

Table 4 shows the antioxidant capacity of walnut polypeptide liquid. The molecular weight was increasing continuously, but the antioxidant capacity of the polypeptide liquid was rather small, which showed that the antioxidant capacity of the small molecule peptide was relatively stronger. In comparison with the stock solution, the highest clearance rate was 1.65 and 1.75 times of the stock solution. Therefore, the use of ultrafiltration technology has certain significance for purifying walnut polypeptides and retaining their high activity.

Table 4. Antioxidant capacity of walnut polypeptide liquid

Number	Molecular weight cut off	OH (%)	O2^−(%)
1	3 u	73.3	64.4
2	5 u	66.1	58.9
3	10 u	59.2	51.6
4	Liquid	44.4	36.8

Analysis of single factor test results of peptide stability

Effect of CMC-Na, SG, and SE addition on emulsion stability

Supplementary Figure 6 shows the effect of CMC-Na, SG, and SE addition of on the emulsion stability. At a pressure of 30 MPa, different addition amounts of CMC-Na, SG, and SE were selected. In the range of 0.01%-0.05%, when the addition amount of CMC-Na, SG, and SE was gradually increased, the stability also showed a certain increase. When the addition amounts were 0.02%, 0.03%, and 0.03%, the stability tended to be a gentle state, and even if the addition amount was increased, there was no large fluctuation. Therefore, 0.02%, 0.03%, and 0.03% were determined as the optimum addition amount of CMC-Na, SG, and SE in the polypeptide liquid, and the emulsion stability was 71.4%, 90.6%, and 81.2%, respectively.

Effect of changing homogenization pressure on emulsion stability

Supplementary Figure 7 shows the effect of homogenization pressure change on emulsion stability. The dosages of CMC-Na, SG and SE were 0.02%, 0.03%, and 0.03%, respectively, and the different pressures of 25, 30, 35, 40, and 45 Mpa were selected to homogenize the polypeptide solution. In the range of pressure 25–45 Mpa, the homogenization pressure showed a certain increasing trend, and the emulsion stability of the corresponding peptide liquid showed a mild increase. When the homogenization pressure was at 35 Mpa, there was no significant change in the emulsion stability, and the growth rate was basically stable. Under the premise of saving consumables and achieving stable stability, the homogenization pressure was selected to be 35 Mpa with emulsion stability of 97.46%.

Response surface optimization of polypeptide stability

From the single factor data, 29 sets of experimental data were optimized in combination with the Box-Behnken response surface method. The results are shown in Table 5. In the experiment, the addition amount of CMC-Na (X₁), SG (X₂), SE (X₃) and the homogenization pressure (X₄) were used as the corresponding variables, and the response value was the emulsion stability (Y). A linear regression analysis of variance was performed to obtain a response model:

Table 5. Test sample data and results using Box-Behnken design for walnut polypeptide liquid

Number	X1-CMC-Na (%)	X2-SG (%)	X3-SE (%)	X4-Homogeneous pressure (Mpa)	Y-Emulsification stability (%)
1	0.03	0.03	0.04	35	69.9
2	0.02	0.03	0.04	30	87.6
3	0.02	0.04	0.04	35	70.0
4	0.02	0.02	0.04	35	91.3
5	0.03	0.02	0.03	35	78.7
6	0.02	0.02	0.03	40	70.1
7	0.02	0.03	0.04	40	71.3
8	0.01	0.03	0.03	40	60.6
9	0.02	0.03	0.02	40	49.7
10	0.02	0.03	0.03	35	88.3
11	0.01	0.04	0.03	35	86.5
12	0.02	0.03	0.03	35	89.7
13	0.02	0.04	0.03	40	43.9
14	0.01	0.02	0.03	35	71.7
15	0.01	0.03	0.04	35	79.8
16	0.03	0.03	0.03	40	95.2
17	0.02	0.03	0.03	35	93.4
18	0.02	0.02	0.03	30	73.0
19	0.02	0.03	0.03	35	81.2
20	0.01	0.03	0.03	30	78.3
21	0.03	0.03	0.03	30	57.5
22	0.02	0.04	0.02	35	43.6
23	0.01	0.03	0.02	35	78.8
24	0.02	0.03	0.03	35	97.2
25	0.02	0.03	0.02	30	78.4
26	0.02	0.04	0.03	30	39.5
27	0.02	0.02	0.02	35	78.7
28	0.03	0.03	0.02	35	28.2
29	0.03	0.04	0.03	35	34.1

$\begin{array}{rl}& Y=89.95-{13.13}_{\ast }{X}_1-{7.88}_{\ast }{X}_2+{8.32}_{\ast }{X}_3-{2.04}_{\ast }{X}_4-{14.91}_{\ast }{X}_1{X}_2+{4.01}_{\ast }{X}_1{X}_3+{2.11}_{\ast }{X}_1{X}_4\\ & +{9.95}_{\ast }{X}_2{X}_3+{14.13}_{\ast }{X}_2{X}_4+{2.97}_{\ast }{X}_3{X}_4-{13.98}_{\ast }{X_1}^2-{10.31}_{\ast }{X_2}^2-{9.16}_{\ast }{X_3}^2-{12.30}_{\ast }{X_4}^2\end{array}$Supplementary Table 1 shows the variance and significance analysis of the stability of the walnut polypeptide solution. The regression linear equation had a significant difference at the F_0.01 level; R² = 0.78, indicating that the linearity was fitting and could accurately reflect the authenticity of the experiment. Among them, the quadratic term X₃² and the first term X₂, X₄, X₁*X₃, and X₂*X₃ behave as significant differences. The primary term X₃, X₁*X2, and X₁*X₄ showed highly significant differences. Combined with software analysis, when CMC-Na, SG, and SE were at 0.02%, 0.03%, and 0.03%, and the homogenization pressure was 35 MPa, the emulsion stability of walnut polypeptide liquid reached the optimal value of 96.8%. The response surface optimization results are shown in Supplementary Figure 8.

Analysis of sensory results using single factor test

Effect of solid to liquid ratio on sensory results

As shown in Supplementary Figure 9, when the amount of protein sugar added was 0.2%, the citric acid was 0.25%, the walnut powder flavor was 0.15% and the ratio of solid to liquid was 15%, the sensory score had the highest value (87 points). However, as the ratio of solid to liquid increased, the moisture content gradually increased, the amount of soluble solids gradually decreased, and the liquid became sparse. Therefore, a decrease in the sensory score was reported.

Effect of the addition amount of protein sugar, citric acid, and walnut powder on the sensory results

As shown in Supplementary Figure 10, when the ratio of solid to liquid was 15% and the amount of protein sugar was 0.2%, the sensory score was optimal (70 points). With the gradual addition of protein sugar, the sweetness showed a corresponding increase trend, and the increase of sweetness directly affected the taste, which caused the sensory score to decrease continuously. When the ratio of solid to liquid was 15% and the dosage of citric acid was 0.25%, the sensory score was the highest (85 points). In the initial stage, with the gradual addition of citric acid, the acidity value of walnut polypeptide liquid increased accordingly. Appropriate adjustment of the amount of citric acid added, to some extent, would adjust the ratio of sugar to acid of the emulsion, thereby providing more mouthfeel. When the ratio of solid to liquid was 15% and the amount of walnut powder flavor was 0.15%, the sensory score was optimal (89 points). However, with the gradual addition of walnut powder flavor, the top notes were getting heavier and the fragrance deviates from the aroma of the original walnut polypeptide liquid, which made the sensory score decreased continuously.

Response surface optimization of sensory experiments

The experiment was carried out according to the Box-Behnken response surface, and the results are shown in Supplementary Table 2. Using the Design Expert software, the solid-to-liquid ratio (X₁), protein sugar (X₂), citric acid (X₃), walnut powder flavor (X₄) as the response variable, and the sensory score as the response value (Y) for regression analysis. The model was presented as follows:$\begin{array}{rl}& Y=81.80-0.33\ast X_1-0.42\ast X_2-0.50\ast X_3+0.083\ast X_4-16.25\ast X_1\ast X_2-11.75\ast X_1\ast X_3\\ & -10.00\ast X_1\ast X_4-12.75\ast X_2\ast X_3-0.75\ast X_2\ast X_4-0.50\ast X_3\ast X_4-17.90\ast {X_1}^2\\ & -14.53\ast {X_2}^2-1.90\ast {X_3}^2-5.78\ast {X_4}^2\end{array}$

It can be seen from Supplementary Table 3 that R² = 0.9059, which showed that the experimental simulation results were better. The quadratic terms X₁² and X₂² were extremely significant, and the differences between X₁*X₂, X₁*X₃, X₁*X₄, X₂*X₃, and X₃² were highly significant, so the optimal conditions could be obtained: The ratio of solid to liquid was 15%; the amount of protein added was 0.2%; the amount of citric acid added was 0.25%; and the amount of walnut powder was 0.15%. The sensory score was 89 points, and the response surface results are shown in Supplementary Figure 11.

Walnut polypeptide liquid index detection

The walnut polypeptide liquid was sterilized at 121°C and 0.2 Mpa for 10 min. The results of the test after sterilization are shown in Supplementary Table 4. Various indicators of walnut polypeptide liquid met the Chinese national standards.

Conclusion

It can be seen from the single factor test that walnut polypeptide liquid was purified by the anion-cation mixed bed method. The pH of the peptide solution, the ratio of the anion-cation exchange resin and the speed of the column were directly affected by the desalination rate and peptide content. Although the oxidation resistance was somewhat reduced by this method, the desalination rate was greatly improved. Filtration of the polypeptide using a 3 KDa ultrafiltration membrane was an ideal method, in part because the antioxidant capacity of the polypeptide changes with molecular weight.

The optimum parameters determined by single factor experiments were as follows: pH 4.5, anion to cation ratio of 3:2, and column speed of 8 times column/h. The clearance rate of hydroxyl radicals and superoxide anion in walnut polypeptide liquid was 73.3% and 64.6%, which were 1.65 and 1.75 times of the stock solution, respectively, indicating that the walnut peptide liquid also had some degree of antioxidant capacity. Combined with the response surface, when CMC-Na, SG, and SE and homogenization pressure were 0.02%, 0.03%, 0.03%, and 35 MPa, respectively, the optimal stability of walnut polypeptide solution was 96.8%.

In conclusion, the formula of walnut polypeptide liquid obtained by single factor test in this study was as follows: the ratio of solid to liquid was 15%, the amount of protein sugar added was 0.2%, the amount of citric acid added was 0.25%, and the amount of walnut powder flavor was 0.15%. The sensory evaluation was 89 points. Sterilization treatment was carried out for 10 min at 121°C and 0.2 Mpa. Different detection indices of walnut polypeptide liquid are in line with the Chinese National Standards. Therefore, the functional walnut polypeptide liquid beverage in this study has provided important theoretical data for its commercial production by the industry.

Acknowledgments

The authors would like to thank the Major Science and Technology Project of Xinjiang Uygur Autonomous Region, China (2016A01001-2), Major Science and Technology Project of Xinjiang Uygur Autonomous Region, China (2017A01001-4) and Key Technology Research and Development Program of Xinjiang Uygur Autonomous Region, China (2017B01003-3) for funding this project. All authors declare no conflict of interest.

Additional information

Funding

This work was supported by the Major Science and Technology Project of Xinjiang Uygur Autonomous Region, China [2016A01001-2], Major Science and Technology Project of Xinjiang Uygur Autonomous Region, China [2017A01001-4] and Key Technology Research and Development Program of Xinjiang Uygur Autonomous Region, China [2017B01003-3].