# Study on Extraction of Peanut Protein and Oil Bodies by Aqueous Enzymatic Extraction and Characterization of Protein

Authors: Chen Liu; Lihua Hao; Fusheng Chen; Chenxian Yang
Journal: Journal of Chemistry
 (2020)

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## Abstract

Cell wall degrading enzymes break down the cell wall by degrading the main cell wall components and destroying structure of the cell wall without influencing the protein. Effects of various enzymes (Viscozyme® L, cellulase, hemicellulase, and pectinase) on the molecular weight distribution of peanut protein and yield of peanut protein and oil bodies during an aqueous enzymatic extraction process were investigated in this study. The molecular weight distribution of peanut protein was not changed, and Viscozyme® L was selected to assist peanut protein and oil bodies extraction by the aqueous extraction process. The aqueous enzymatic extraction process was optimized by a signal factor experiment and response surface methodology, and the optimal condition was enzyme hydrolysis temperature of 52°C, solid-liquid ratio of 1 : 4, enzyme concentration of 1.35%, and enzyme hydrolysis time of 90 min. A peanut protein yield of 78.60% and oil bodies yield of 48.44% were achieved under the optimal condition. Compared with commercial peanut protein powder (CPPP), the solubility and foaming properties of peanut protein powder obtained by aqueous enzymatic extraction (AEEPPP) were a little lower. However, the functional properties of foam stability, emulsifying activity, emulsifying stability, water holding capacity, and oil holding capacity of AEEPPP were better than that of CPPP.


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## Body

## 1. Introduction

Aqueous enzymatic extraction process is a promising method to facilitate the recovery of oils and proteins from oilseeds with the assistance of enzymes. Notably, in the aqueous enzymatic extraction system, water is used as the extraction solvent, which has many advantages compared with conventional extraction [1]. As an innovative technology for extracting peanut protein and oil, aqueous enzymatic extraction has the advantages of having no organic solvent use, low energy consumption, and green and environmental protection and has mild reaction conditions and product safety [2]. Oil extraction by aqueous enzymatic extraction requires a low degree of refining and fewer antioxidant treatments, and protein can be recycled at the same time [3–5]. The principle of aqueous enzymatic extraction is to destroy the peanut cell structure by mechanical disruption; then, internal macromolecular complexes (lipoprotein, lipopolysaccharide, and cell wall polysaccharides) are hydrolyzed by enzymes to facilitate the release of oil and protein. The oil and protein can be separated according to the density differences and the affinity differences to each component of oil-water [6, 7].Protease, pectinase, cellulase, and hemicellulase are widely used in aqueous enzymatic extraction of peanut protein and oil. Protease can substantially improve the yield of oil and protein by hydrolyzing peanut protein into smaller molecules [8, 9]. Thus, protein processing properties (solubility, emulsification, and foamability) will reduce because of hydrolysis of protein and formation of a stubborn emulsion mass in the process of aqueous enzymatic extraction of oil peanut and protein [10–12]. The enzymes (cellulase, hemicellulase, and pectinase) break down the cell wall by degrading the main wall components and destroying the structure of cell wall without influencing the protein. Bisht et al. [13] showed that a single cellulase, hemicellulase, pectinase, or their complex enzyme could effectively increase the oil yield at an appropriate concentration. Especially, with the combination of cellulase and pectinase (14.22% increase), Szydłowska-Czerniak et al. [14] found that the yield of rapeseed oil extracted by pectinase and cellulase were higher than the traditional method, and the effect of pectinase was better than cellulase. Viscozyme® L, a compound cell wall degradation enzyme, promotes the release of protein and oil bodies by degrading cellulose, hemicellulose, and pectin, which retains the original functional properties of protein [15, 16].Effects of various cell wall degrading enzymes (Viscozyme® L, cellulase, hemicellulase, and pectinase) on the molecular weight distribution of peanut protein and the yield of peanut protein and oil bodies were studied as part of this paper. Viscozyme® L was used to extract peanut protein and oil bodies, with the extraction conditions optimized by a signal factor experiment and response surface methodology, subsequently. Peanut protein obtained by aqueous enzymatic extraction was unhydrolyzed, and its functional properties were analyzed by comparison with CPPP.


## 2. Materials and Methods

### 2.1. Materials

Peanut samples were purchased from a local market (Zhengzhou, China) and stored at 4°C until used. The composition (g/100 g dry matter) of peanuts is 46.84% oil, 24.44% protein, 4.65% crude fiber, 4.63% water, and 2.35% ash. CPPP was purchased from Henan Liangjian Inc. (Henan, China). Viscozyme® L, with the nominal activity of 5086 U/mL (main ingredients: cellulase, hemicellulase, and arabinase), and cellulase, with the nominal activity of 1835 U/mL, were purchased from Novozymes (Novo, China). Hemicellulase, with the nominal activity of 2500 U/g, and pectinase, with the nominal activity of 500 U/g, were purchased from Sigma Chemical Company (St. Louis, USA). All the other reagents used were of analytical grade.


### 2.2. Peanut Microstructure

Central cotyledons of peanut seeds were cut into 1-2 mm sections transversely by a razor blade, fixed in 2.5% (v/v) glutaraldehyde sodium phosphate buffer (0.1 M, pH 7.2) at 4°C for 12 h, and then were washed with the same sodium phosphate butter three times. Samples were postfixed in 1% (w/v) osmium tetroxide 2 h and washed with sodium phosphate buffer three times, 30 min each time; subsequently, they were dehydrated in acetone. Dehydrated samples were infiltrated progressively and embedded in Spurr resin. Embedded samples were cut into ultrathin sections (50–70 nm thick) with an ultramicrotome (UC5; Leica, Wetzlar, Germany). Sections were mounted on copper grids, stained with 2% uranyl acetate and Reynold’s lead citrate, and then examined with a transmission electron microscope (TEM) (H-7650; Hitachi Chiyoda-ku, Tokyo, Japan), operated at an accelerating voltage of 80 kV [17]. Images were recorded with a 4K CCD camera (832 ORIUS; Gatan, Pleasanton, CA, USA). Three repeats were performed for peanut seeds, and more than 10 ultrathin sections per block were examined. Statistics of the diameter of protein bodies and oil bodies were conducted on the TEM (6000×) with the Nano Measurer 1.2; ImageJ statistics for the number of protein bodies and oil bodies were conducted in the area of 1000 µm2. The results were expressed as mean ± standard error.


### 2.3. Preparation of Peanut Protein and Oil Bodies

Peanut protein and oil bodies were extracted using the method of grading extraction for peanut protein [18] and aqueous enzymatic extraction for oil bodies of maize germ [19], respectively, with some modifications (Figure 1). The skinless peanut seeds were ground by a high-speed universal grinder (FW-100; Beijing Ever Bright Medical Treatment Inc., Beijing, China). Twenty grams of peanut powder were dispersed in deionized water with 1 : 5 (wt/vol) solid-liquid ratio. The enzymolysis of the mixture was conducted in a digital water-bathing constant temperature vibrator (THZ-82; Jintan Huafeng Instrument Inc., Changzhou, China) for 2 h at 50°C. Following, the enzyme was deactivated by placing the samples in a boiling water bath for 5 min. The cooled solution was transferred into a centrifuge tube and was centrifuged (DZ267-32C6; Anting Scientific Instrument Factory, Shanghai, China) at 5000 ×g for 20 min. The floating layer (oil bodies) and the lower precipitation (peanut protein) were separated. The floating layer (oil bodies) was dried at 50°C for over 10 h in a vacuum-drying oven (DZF-2B; Beijing Ever Bright Medical Treatment Inc., Beijing, China), and the lower precipitation was freeze-dried (LGJ-25; Beijing Sihuan Scientific Instrument Inc., Beijing, China) for 24 h and then weighed.Figure 1
Process diagram of enzymatic extraction of peanut protein and oil bodies.The peanut protein content was measured with an Automatic Kjeldahl Apparatus (K1100; Jinan Haineng Instrument Inc., Shandong, China), with yield of peanut protein and oil bodies calculated using the following formulas:(1)yield of peanut protein=protein content of precipitateprotein of content peanut×100%,yield of oil bodies=dry weight  of oil bodiesweight of peanut×100%.


### 2.4. SDS-PAGE

Molecular weight of peanut protein extracted by various enzymes was determined by SDS-PAGE according to the method of Du et al. [20]. For each sample, 0.1 g dried peanut protein, extracted by different enzymes, was dispersed in 20 mL 0.01 mol/L phosphate buffer (pH 7.2). Ten microliter diluent samples were taken for electrophoresis with 5% stacking gel and 12% resolving gel; the gels were scanned on an Amersham Imager 600 gel image system.


### 2.5. Optical Microscopy of Oil Bodies

Dried peanut oil bodies (0.1 g), extracted by various enzymes, were dispersed in 1 mL phosphate buffer (pH 7.0; 0.01 mol/L). The diluent of the oil bodies (1-2 drops) was dripped on a glass slide and covered by a coverslip to observe the morphology of oil bodies by Optical Microscope (E100; Nikon Inc., Tokyo, Japan).


### 2.6. Optimization of the Aqueous Enzymatic Extraction Process

Peanut protein and oil bodies were extracted using Viscozyme® L according to the aqueous enzymatic extraction process described in Section2.3. The enzyme hydrolysis temperature (35–70°C), solid-liquid ratio (1 : 2–1 : 7), enzyme concentration (0.25–2.0%), and enzyme hydrolysis time (20–160 min) were varied independently, keeping other parameters unchanged, as to obtain the optimum conditions.


### 2.7. Response Surface Methodology (RSM)

According to the results of the single factor experiment, the Central Composite Design of Design Expert 8.05b software was used to design a response surface optimization experiment. Enzyme hydrolysis temperature (A), solid-liquid ratio (B), enzyme concentration (C), and enzyme hydrolysis time (D) worked as influencing factors, while the yield of peanut protein (Y1) and the yield of oil bodies (Y2) worked as response values. Encoding of factor levels is shown in Table 1.Table 1
Levels of response surface experiments.
Factors
Code
Levels
−2
−1
0
1
2
Enzyme hydrolysis temperature (°C)
A
40
45
50
55
60
Solid-liquid ratio (g/mL)
B
1 : 2
1 : 3
1 : 4
1 : 5
1 : 6
Enzyme concentration (%)
C
0.75
1.00
1.25
1.50
1.75
Enzyme hydrolysis time (min)
D
40
60
80
100
120


### 2.8. Determination of the Functional Properties of Peanut Protein Powder

#### 2.8.1. Solubility

Five grams of peanut protein powder were dissolved in 10 mL, 0.02 mol/L phosphate buffer solution (pH 7.0), and stirred with an electromagnetic stirrer at 25°C. Suspended liquid was centrifuged at 5000 ×g for 20 min and then 2 mL supernatant was extracted to determine the content of protein by a Kjeldahl Nitrogen Determiner [21]:(2)solubility=protein content in supernateprotein content of peanut×100%.


#### 2.8.2. Foamability and Foam Stability

Determination of peanut protein powder foamability was based on the method of Yu et al. [22]. Two grams of peanut protein powder were dissolved in 100 mL of 0.01 mol/L phosphate buffer solution (pH 7.0). Twenty milliliters of prepared protein solution were homogenized at 10,000 ×g for 1 min. The homogenized protein solution was quickly transferred to a 100 mL measuring cylinder for the determination of foaming property and foam stability. The foaming property equaled the increased volume percentage of protein suspension after homogenization; the foam stability was the percentage of reserved foam after standing for 30 min:(3)foaming  property=V020×100%,foam stabilty=VtV0×100%,where V0: the foam volume read when the protein solution was homogenized and rapidly transferred to a 100 mL measuring cylinder and Vt: the foam volume retained after 30 min.


#### 2.8.3. Emulsifying Activity (EAI) and Emulsifying Stability (ESI)

Determination of emulsification of peanut protein powder was based on the method by Qiu et al. [23] with some modification. Peanut protein powder (0.1 g) was dissolved in 100 mL, 0.01 mol/L phosphate buffer solution (pH 7.0) to prepare 0.1% sample solution, a 21 mL solution sample, combined with 7 mL oil were homogenized at 10,000 ×g for 1 min at ambient temperature. A 50 μL solution was taken from the bottom of sample solution during homogenizing at 0 min and 10 min and was then dispersed in 5 mL 0.1% SDS to determine the absorbance value at 500 nm ultraviolet-visible spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan). Emulsifying activity (EAI) and emulsifying stability (ESI) were calculated as follows:(4)EAImL/g=2×2.303×A0×dilution ratioC×∅×104,where A0: the absorbance value at 0 min, C: protein concentration in aqueous solution before forming emulsion, and Ø: volume fraction of oil in emulsion. (5)ESImin=A0A0−A10×t,where A0: the absorbance value at 0 min, t: the time interval of measuring the absorbance was 10 min, and A10: the absorbance value at 10 min.


#### 2.8.4. Water Holding Capacity and Oil Holding Capacity

Determination of emulsification of peanut protein powder was based on the method of He et al. [24]. One gram of peanut protein powder combined with 20 mL distilled water or peanut oil were placed in a predried centrifuge tube, stirred evenly, and stood for 30 min. The reduction in volume of supernatant liquid was given by water absorption or oil absorption of the sample after centrifuging at 3000 ×g for 20 min.


### 2.9. Statistical Analysis

All measurements were repeated at least three times using duplicate samples, and the results were given as means ± standard deviations. The data were statistically analyzed using the software of Design Expert 8.05b, Origin 8.5, and SPSS 19.0. Significance of differences was defined atp<0.05.


## 2.1. Materials

Peanut samples were purchased from a local market (Zhengzhou, China) and stored at 4°C until used. The composition (g/100 g dry matter) of peanuts is 46.84% oil, 24.44% protein, 4.65% crude fiber, 4.63% water, and 2.35% ash. CPPP was purchased from Henan Liangjian Inc. (Henan, China). Viscozyme® L, with the nominal activity of 5086 U/mL (main ingredients: cellulase, hemicellulase, and arabinase), and cellulase, with the nominal activity of 1835 U/mL, were purchased from Novozymes (Novo, China). Hemicellulase, with the nominal activity of 2500 U/g, and pectinase, with the nominal activity of 500 U/g, were purchased from Sigma Chemical Company (St. Louis, USA). All the other reagents used were of analytical grade.


## 2.2. Peanut Microstructure

Central cotyledons of peanut seeds were cut into 1-2 mm sections transversely by a razor blade, fixed in 2.5% (v/v) glutaraldehyde sodium phosphate buffer (0.1 M, pH 7.2) at 4°C for 12 h, and then were washed with the same sodium phosphate butter three times. Samples were postfixed in 1% (w/v) osmium tetroxide 2 h and washed with sodium phosphate buffer three times, 30 min each time; subsequently, they were dehydrated in acetone. Dehydrated samples were infiltrated progressively and embedded in Spurr resin. Embedded samples were cut into ultrathin sections (50–70 nm thick) with an ultramicrotome (UC5; Leica, Wetzlar, Germany). Sections were mounted on copper grids, stained with 2% uranyl acetate and Reynold’s lead citrate, and then examined with a transmission electron microscope (TEM) (H-7650; Hitachi Chiyoda-ku, Tokyo, Japan), operated at an accelerating voltage of 80 kV [17]. Images were recorded with a 4K CCD camera (832 ORIUS; Gatan, Pleasanton, CA, USA). Three repeats were performed for peanut seeds, and more than 10 ultrathin sections per block were examined. Statistics of the diameter of protein bodies and oil bodies were conducted on the TEM (6000×) with the Nano Measurer 1.2; ImageJ statistics for the number of protein bodies and oil bodies were conducted in the area of 1000 µm2. The results were expressed as mean ± standard error.


## 2.3. Preparation of Peanut Protein and Oil Bodies

Peanut protein and oil bodies were extracted using the method of grading extraction for peanut protein [18] and aqueous enzymatic extraction for oil bodies of maize germ [19], respectively, with some modifications (Figure 1). The skinless peanut seeds were ground by a high-speed universal grinder (FW-100; Beijing Ever Bright Medical Treatment Inc., Beijing, China). Twenty grams of peanut powder were dispersed in deionized water with 1 : 5 (wt/vol) solid-liquid ratio. The enzymolysis of the mixture was conducted in a digital water-bathing constant temperature vibrator (THZ-82; Jintan Huafeng Instrument Inc., Changzhou, China) for 2 h at 50°C. Following, the enzyme was deactivated by placing the samples in a boiling water bath for 5 min. The cooled solution was transferred into a centrifuge tube and was centrifuged (DZ267-32C6; Anting Scientific Instrument Factory, Shanghai, China) at 5000 ×g for 20 min. The floating layer (oil bodies) and the lower precipitation (peanut protein) were separated. The floating layer (oil bodies) was dried at 50°C for over 10 h in a vacuum-drying oven (DZF-2B; Beijing Ever Bright Medical Treatment Inc., Beijing, China), and the lower precipitation was freeze-dried (LGJ-25; Beijing Sihuan Scientific Instrument Inc., Beijing, China) for 24 h and then weighed.Figure 1
Process diagram of enzymatic extraction of peanut protein and oil bodies.The peanut protein content was measured with an Automatic Kjeldahl Apparatus (K1100; Jinan Haineng Instrument Inc., Shandong, China), with yield of peanut protein and oil bodies calculated using the following formulas:(1)yield of peanut protein=protein content of precipitateprotein of content peanut×100%,yield of oil bodies=dry weight  of oil bodiesweight of peanut×100%.


## 2.4. SDS-PAGE

Molecular weight of peanut protein extracted by various enzymes was determined by SDS-PAGE according to the method of Du et al. [20]. For each sample, 0.1 g dried peanut protein, extracted by different enzymes, was dispersed in 20 mL 0.01 mol/L phosphate buffer (pH 7.2). Ten microliter diluent samples were taken for electrophoresis with 5% stacking gel and 12% resolving gel; the gels were scanned on an Amersham Imager 600 gel image system.


## 2.5. Optical Microscopy of Oil Bodies

Dried peanut oil bodies (0.1 g), extracted by various enzymes, were dispersed in 1 mL phosphate buffer (pH 7.0; 0.01 mol/L). The diluent of the oil bodies (1-2 drops) was dripped on a glass slide and covered by a coverslip to observe the morphology of oil bodies by Optical Microscope (E100; Nikon Inc., Tokyo, Japan).


## 2.6. Optimization of the Aqueous Enzymatic Extraction Process

Peanut protein and oil bodies were extracted using Viscozyme® L according to the aqueous enzymatic extraction process described in Section2.3. The enzyme hydrolysis temperature (35–70°C), solid-liquid ratio (1 : 2–1 : 7), enzyme concentration (0.25–2.0%), and enzyme hydrolysis time (20–160 min) were varied independently, keeping other parameters unchanged, as to obtain the optimum conditions.


## 2.7. Response Surface Methodology (RSM)

According to the results of the single factor experiment, the Central Composite Design of Design Expert 8.05b software was used to design a response surface optimization experiment. Enzyme hydrolysis temperature (A), solid-liquid ratio (B), enzyme concentration (C), and enzyme hydrolysis time (D) worked as influencing factors, while the yield of peanut protein (Y1) and the yield of oil bodies (Y2) worked as response values. Encoding of factor levels is shown in Table 1.Table 1
Levels of response surface experiments.
Factors
Code
Levels
−2
−1
0
1
2
Enzyme hydrolysis temperature (°C)
A
40
45
50
55
60
Solid-liquid ratio (g/mL)
B
1 : 2
1 : 3
1 : 4
1 : 5
1 : 6
Enzyme concentration (%)
C
0.75
1.00
1.25
1.50
1.75
Enzyme hydrolysis time (min)
D
40
60
80
100
120


## 2.8. Determination of the Functional Properties of Peanut Protein Powder

### 2.8.1. Solubility

Five grams of peanut protein powder were dissolved in 10 mL, 0.02 mol/L phosphate buffer solution (pH 7.0), and stirred with an electromagnetic stirrer at 25°C. Suspended liquid was centrifuged at 5000 ×g for 20 min and then 2 mL supernatant was extracted to determine the content of protein by a Kjeldahl Nitrogen Determiner [21]:(2)solubility=protein content in supernateprotein content of peanut×100%.


### 2.8.2. Foamability and Foam Stability

Determination of peanut protein powder foamability was based on the method of Yu et al. [22]. Two grams of peanut protein powder were dissolved in 100 mL of 0.01 mol/L phosphate buffer solution (pH 7.0). Twenty milliliters of prepared protein solution were homogenized at 10,000 ×g for 1 min. The homogenized protein solution was quickly transferred to a 100 mL measuring cylinder for the determination of foaming property and foam stability. The foaming property equaled the increased volume percentage of protein suspension after homogenization; the foam stability was the percentage of reserved foam after standing for 30 min:(3)foaming  property=V020×100%,foam stabilty=VtV0×100%,where V0: the foam volume read when the protein solution was homogenized and rapidly transferred to a 100 mL measuring cylinder and Vt: the foam volume retained after 30 min.


### 2.8.3. Emulsifying Activity (EAI) and Emulsifying Stability (ESI)

Determination of emulsification of peanut protein powder was based on the method by Qiu et al. [23] with some modification. Peanut protein powder (0.1 g) was dissolved in 100 mL, 0.01 mol/L phosphate buffer solution (pH 7.0) to prepare 0.1% sample solution, a 21 mL solution sample, combined with 7 mL oil were homogenized at 10,000 ×g for 1 min at ambient temperature. A 50 μL solution was taken from the bottom of sample solution during homogenizing at 0 min and 10 min and was then dispersed in 5 mL 0.1% SDS to determine the absorbance value at 500 nm ultraviolet-visible spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan). Emulsifying activity (EAI) and emulsifying stability (ESI) were calculated as follows:(4)EAImL/g=2×2.303×A0×dilution ratioC×∅×104,where A0: the absorbance value at 0 min, C: protein concentration in aqueous solution before forming emulsion, and Ø: volume fraction of oil in emulsion. (5)ESImin=A0A0−A10×t,where A0: the absorbance value at 0 min, t: the time interval of measuring the absorbance was 10 min, and A10: the absorbance value at 10 min.


### 2.8.4. Water Holding Capacity and Oil Holding Capacity

Determination of emulsification of peanut protein powder was based on the method of He et al. [24]. One gram of peanut protein powder combined with 20 mL distilled water or peanut oil were placed in a predried centrifuge tube, stirred evenly, and stood for 30 min. The reduction in volume of supernatant liquid was given by water absorption or oil absorption of the sample after centrifuging at 3000 ×g for 20 min.


## 2.8.1. Solubility

Five grams of peanut protein powder were dissolved in 10 mL, 0.02 mol/L phosphate buffer solution (pH 7.0), and stirred with an electromagnetic stirrer at 25°C. Suspended liquid was centrifuged at 5000 ×g for 20 min and then 2 mL supernatant was extracted to determine the content of protein by a Kjeldahl Nitrogen Determiner [21]:(2)solubility=protein content in supernateprotein content of peanut×100%.


## 2.8.2. Foamability and Foam Stability

Determination of peanut protein powder foamability was based on the method of Yu et al. [22]. Two grams of peanut protein powder were dissolved in 100 mL of 0.01 mol/L phosphate buffer solution (pH 7.0). Twenty milliliters of prepared protein solution were homogenized at 10,000 ×g for 1 min. The homogenized protein solution was quickly transferred to a 100 mL measuring cylinder for the determination of foaming property and foam stability. The foaming property equaled the increased volume percentage of protein suspension after homogenization; the foam stability was the percentage of reserved foam after standing for 30 min:(3)foaming  property=V020×100%,foam stabilty=VtV0×100%,where V0: the foam volume read when the protein solution was homogenized and rapidly transferred to a 100 mL measuring cylinder and Vt: the foam volume retained after 30 min.


## 2.8.3. Emulsifying Activity (EAI) and Emulsifying Stability (ESI)

Determination of emulsification of peanut protein powder was based on the method by Qiu et al. [23] with some modification. Peanut protein powder (0.1 g) was dissolved in 100 mL, 0.01 mol/L phosphate buffer solution (pH 7.0) to prepare 0.1% sample solution, a 21 mL solution sample, combined with 7 mL oil were homogenized at 10,000 ×g for 1 min at ambient temperature. A 50 μL solution was taken from the bottom of sample solution during homogenizing at 0 min and 10 min and was then dispersed in 5 mL 0.1% SDS to determine the absorbance value at 500 nm ultraviolet-visible spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan). Emulsifying activity (EAI) and emulsifying stability (ESI) were calculated as follows:(4)EAImL/g=2×2.303×A0×dilution ratioC×∅×104,where A0: the absorbance value at 0 min, C: protein concentration in aqueous solution before forming emulsion, and Ø: volume fraction of oil in emulsion. (5)ESImin=A0A0−A10×t,where A0: the absorbance value at 0 min, t: the time interval of measuring the absorbance was 10 min, and A10: the absorbance value at 10 min.


## 2.8.4. Water Holding Capacity and Oil Holding Capacity

Determination of emulsification of peanut protein powder was based on the method of He et al. [24]. One gram of peanut protein powder combined with 20 mL distilled water or peanut oil were placed in a predried centrifuge tube, stirred evenly, and stood for 30 min. The reduction in volume of supernatant liquid was given by water absorption or oil absorption of the sample after centrifuging at 3000 ×g for 20 min.


## 2.9. Statistical Analysis

All measurements were repeated at least three times using duplicate samples, and the results were given as means ± standard deviations. The data were statistically analyzed using the software of Design Expert 8.05b, Origin 8.5, and SPSS 19.0. Significance of differences was defined atp<0.05.


## 3. Results and Discussion

### 3.1. Morphology of Protein Bodies and Oil Bodies in Cotyledon Cells of Peanut

Oil bodies are small spheres composed of neutral lipids, phospholipids wrapping neutral lipids, and proteins embedded in the phospholipid layer, which wrap the protein bodies in cytoplasm [25]. The transmission electron micrograph of peanut is shown in Figure 2. The shape of peanut protein bodies and oil bodies are spherical and oval, with few irregular shapes. The oil bodies were distributed in the cells, and the protein bodies were wrapped tightly by oil bodies.Figure 2
Transmission electron micrograph of peanut (PB: protein body; OB: oil body).The diameter of oil bodies varies from 0.5 to 2.5μm for different oil plants, and the diameter of protein bodies is larger than that of oil bodies [26]. In this study, the average diameter of protein bodies was 3.34 ± 0.79 μm, and the number of protein bodies was 16 ± 2. The average diameter of oil bodies was 1.43 ± 0.18 μm, and the number of oil bodies was 147 ± 12. The particle size of protein bodies was obviously larger than oil bodies, but the number of protein bodies was less than that of oil bodies in the peanut cell.


### 3.2. Effects of Enzymes on Peanut Protein and Oil Bodies

The main components of plant cell walls are cellulose, hemicellulose, and pectin. Viscozyme® L, cellulase, hemicellulase, and pectinase were used to facilitate the degradation of the peanut cell wall to extract more peanut protein and oil bodies. These enzymes had no effect on protein; thus, protein was unhydrolyzed.


#### 3.2.1. SDS-PAGE

SDS-PAGE of peanut protein extracted by various enzymes and CPPP is shown in Figure3, and the molecular weight distribution was consistent. Krishna et al. [27] and Shokraii et al. [28] analyzed protein subunits of different peanut varieties, and their results were similar to those in this study. The results here indicate that the molecular weight distribution of peanut protein was not changed by Viscozyme® L, cellulase, hemicellulase, or pectinase.Figure 3
SDS-PAGE of peanut protein extracted by various enzymes: (1) standard protein; (2) Viscozyme® L; (3) cellulase; (4) hemicellulase; (5) pectinase; (6) CPPP.


#### 3.2.2. Microscopy of Oil Bodies

Microscope images of oil body suspensions extracted by Viscozyme® L, cellulase, hemicellulase, and pectinase are shown in Figure4. The size of some oil bodies in Figures 4(a) and 4(b) was obviously bigger than the peanut oil bodies in its original state believed to be because oil bodies collided into each other and merged into big oil bodies during mechanical crashing and enzymatic hydrolysis. With the enzymatic hydrolysis, the size of oil bodies increased, and the distribution was not uniform, which was beneficial to the yield of oil bodies. Figures 4(c) and 4(d) show peanut oil bodies were complete, regular, and distributed uniformly, illustrating the effects of hemicellulase and pectinase on release of oil bodies were smaller than with Viscozyme® L and cellulase.Figure 4
Microscopy of oil bodies extracted by various enzymes: (a) Viscozyme® L; (b) cellulase; (c) hemicellulase; (d) pectinase. All photos are at the same magnification. Bar represents 10μm.
(a)
(b)
(c)
(d)


#### 3.2.3. Effect of Enzymes on Yield of Peanut Proteins and Oil Bodies

The yield of peanut protein and oil bodies extracted by various enzymes is shown in Table2. As a control experiment, the yield of peanut protein and oil bodies extraction without any enzyme was 73.3% ± 0.3 and 29.1% ± 0.7, respectively. As shown in Table 2, the yield of peanut protein and oil bodies extracted by various enzymes was higher than control experiment significantly (p<0.05). The highest yield of oil bodies was 41.1% ± 0.9 extracted by Viscozyme® L, which had significant differences (p<0.05) with cellulase and hemicellulase. The yield of peanut protein was lowest extracted by Viscozyme® L, but there were no significant differences with cellulase and pectinase. In conclusion, Viscozyme® L was selected as the enzyme to extract peanut protein and oil bodies.Table 2
Effects of various enzymes on yield of peanut protein and oil bodies.
Serial number
Type of enzyme
Yield of peanut protein (%)
Yield of oil bodies (%)
1
—
73.3 ± 0.3c
29.1 ± 0.7d
2
Viscozyme® L
81.5 ± 0.8b
41.1 ± 0.9a
3
Cellulase
83.5 ± 0.9ab
37.7 ± 0.5bc
4
Hemicellulose
85.5 ± 0.7a
35.4 ± 1.1c
5
Pectinase
83.2 ± 0.9b
39.8 ± 1.4ab
Note. Different letters mean significant difference (p<0.05).


### 3.3. Effects of Operation Parameters on the Yield of Peanut Protein and Oil Bodies

Temperature is a key factor affecting enzyme activity, and the yield of peanut protein and oil bodies effected by different temperatures is shown in Figure5(a). Peanut protein yield first increased and then decreased and reached a maximum (78.86%) at 55°C. The yield of oil bodies followed the same trend as protein, but it reached its maximum (38.86%) at 50°C. The extraction rate of peanut protein reaches a maximum (88.69%) at 1 : 2 solid-liquid ratio and however decreased and achieved a balance with the increase in solid-liquid ratio (Figure 5(b)). The reason could be that water-soluble whey protein and salt-soluble arachin and conarachin gradually dissolved with the increase in solid-liquid ratio; then, arachin and conarachin were nondegradable because their change in the spatial structure trended to be stable [29, 30]. The yield of oil bodies increased first and then decreased, reaching a maximum (42.37%) at solid-liquid ratio 1 : 4. The increase in aqueous solution could promote the decomposition of peanut cell wall structure, but continuous increase in the solid-liquid ratio decreased the yield of oil bodies for the decrease in enzyme concentration. The yield of peanut protein and oil bodies had a trend of increasing first and then decreasing and both reached maximum (68.45% and 39.28%, respectively) at 1.25% (Figure 5(c)). The damage degree of peanut cell wall is increased with the increase in enzyme concentration protein and oil bodies in the cell that are fully released. The yield of peanut protein and oil bodies tend to be stable when concentration of enzyme increases to a certain degree. The yield of peanut protein reached maximum (79.91%) at 80 min, and the yield of oil bodies increased between 20 and 80 min and then stabilized after this (Figure 5(d)). At a certain time, enzymes were in full contact with the substrate; thus, the reaction rate was fast, and enzymatic hydrolysis was sufficient. When the reaction time increased, the concentration of the substrate and reaction rate decreased; thus, the yield of peanut protein and oil bodies trend to be stable.Figure 5
Effect of different enzyme hydrolysis temperature, solid-liquid ratio, enzyme concentration, and enzyme hydrolysis time on peanut protein and oil bodies yield.
(a)
(b)
(c)
(d)


### 3.4. Result of Response Surface Optimization Test

Response surface test designed by the Central Composite Design of Design Expert 8.05b is shown in Table3. The results of the variance analysis on the yield of peanut protein and oil bodies are shown in Tables 4 and 5, respectively. Regression analysis was carried out on the results, and the polynomial regression model of response value of the yield of peanut protein and oil bodies is given in equations (6) and (7), respectively:(6)Y1=80.61+3.96A−12.91B+3.56C+2.26D−2.50AB+0.81AC−0.63AD+2.91BC+0.91AD+0.77CD−4.64A2−2.74B2−2.20C2−3.56D2,(7)Y2=47.09−0.60A+3.66B+1.72C+0.99D+0.45AB−0.03AC−0.51AD+0.057CD+0.38BD−0.18CD−0.57A2−1.85B2−2.91C2−0.27D2.Table 3
Program and experimental results of response surface methodology.
Serial number
A (°C)
B (g/mL)
C (%)
D (min)
Y
1 (%)
Y
2 (%)
1
60
1 : 4
1.25
80
70.52
45.22
2
50
1 : 2
1.25
80
91.49
33.69
3
50
1 : 6
1.25
80
52.10
44.76
4
50
1 : 4
1.25
80
80.81
47.56
5
55
1 : 5
1.00
100
52.09
44.96
6
45
1 : 5
1.00
60
40.83
43.01
7
55
1 : 3
1.00
100
83.57
34.06
8
40
1 : 4
1.25
80
57.92
43.50
9
55
1 : 3
1.00
60
89.43
35.05
10
45
1 : 5
1.00
100
48.53
46.76
11
50
1 : 4
1.25
80
82.67
47.12
12
55
1 : 5
1.00
60
44.17
42.58
13
45
1 : 3
1.50
60
66.65
38.77
14
50
1 : 4
1.25
80
80.97
48.27
15
45
1 : 5
1.50
100
56.51
48.40
16
55
1 : 3
1.50
60
87.23
38.43
17
50
1 : 4
1.25
40
60.35
42.99
18
55
1 : 5
1.50
60
59.36
45.00
19
50
1 : 4
1.25
120
76.72
48.14
20
45
1 : 3
1.00
60
78.33
36.39
21
50
1 : 4
1.25
80
78.62
48.06
22
50
1 : 4
1.75
80
84.82
39.62
23
55
1 : 3
1.50
100
90.58
36.67
24
50
1 : 4
0.75
80
63.10
30.41
25
55
1 : 5
1.50
100
59.65
48.05
26
50
1 : 4
1.25
80
84.40
45.50
27
45
1 : 3
1.50
100
77.78
42.11
28
50
1 : 4
1.25
80
76.19
46.00
29
45
1 : 5
1.50
60
54.42
47.79
30
45
1 : 3
1.00
100
73.12
39.45Table 4
Experiment data for test validation.
Parameter
Extraction rate of peanut protein (%)
Yield of oil bodies (%)
Mean
78.60
48.44
Predicted values
80.47
47.73
Relative error
2.32
1.49Table 5
Functional properties of peanut protein powder.
Sample
Solubility (%)
Emulsifying activity (mL/g)
Emulsifying stability (min)
Foaming property (%)
Foam stability (%)
Water holding capacity (mL/g)
Oil holding capacity (mL/g)
AEEPPP
5.8 ± 0.7
22.5 ± 0.9
54.2 ± 1.0
20.5 ± 0.4
51.2 ± 1.2
7.4 ± 0.4
18.5 ± 0.7
CPPP
6.4 ± 0.5
20.5 ± 0.8
22.9 ± 0.5
21.0 ± 0.3
42.9 ± 0.9
6.2 ± 0.2
16.7 ± 0.5The results of the effect of factors A, B, C, and D on the extraction rate of peanut protein indicated that the order of positive influence for each factor was solid-liquid ratio > enzyme hydrolysis temperature > concentration of enzyme > enzyme hydrolysis time. Linear terms (A, B, and C), quadratic terms (A2, B2, C2, and D2), and the interaction term (BC) of the response surface equation for peanut protein had a significant effect (p<0.05) on the extraction rate of peanut protein. This indicated that the effect of the experimental factors on the response value was not a linear relationship, but a quadratic relationship, and the change of response value (extraction rate of peanut protein) was very complex.The order of positive influence for each factor on the yield of oil bodies was solid-liquid ratio > concentration of enzyme > enzyme hydrolysis time > enzyme hydrolysis temperature. Linear terms (B, C, and D) and quadratic terms (B2 and C2) of response surface equation for peanut protein had a significant effect (p<0.05) on the extraction rate of peanut protein. This indicated that the effect of experimental factors on response values was also a quadratic relationship.The polynomial regression models of response value of the yield of peanut protein and oil bodies were significant (p<0.05), while testing the lose effectiveness of fit for the models was not significant (p<0.05). Analysis of variance indicated that the model was consistent with the data.The nonsignificant terms were removed after the analysis of variance of the response surface equation for the yield of peanut protein and oil bodies, giving the final regression model. The polynomial regression model of the yield of peanut protein and oil bodies is given in equations (8) and (9), respectively:(8)Y1=80.61+3.96A−12.91B+3.56C+2.26D+2.91BC−4.64A2−2.74B2−2.20C2−3.56D2,(9)Y2=46.25+3.66B+1.72C+0.99D−1.75B2−2.80C2.Results for analysis of variance of the modified model for the extraction rate peanut protein and yield of oil bodies showed thatR2 and adjusted R2 were close to 1, which indicated the modified model could predict the extraction rate of peanut protein and the yield of oil bodies, well [31, 32].


### 3.5. Effects of Interaction on the Yield of Peanut Protein

The interaction term (BC) of the response surface equation for peanut protein had a significant effect on its yield (p=0.042); the interaction between the solid-liquid ratio and enzyme concentration on the yield of peanut protein is shown in Figure 6.Figure 6
Effect of different solid-liquid ratios and enzyme concentrations on the yield of peanut protein.Figure6 shows that the yield of peanut protein continued to increase with the increase in the solid-liquid ratio and the enzyme concentration, simultaneously. For a certain peanut raw material, there are optimal dissolution and precipitation ratio and enzyme dosage for aqueous enzymatic extraction. Water can promote the dissolution and precipitation of peanut protein but however dilutes the enzyme concentration in excess, which hinders the interaction between the enzyme and cell walls.


### 3.6. Verification Test of Response Surface Optimization

Optimizing for the yield of peanut protein was conducted solely, and the predicted value reached 92.67% at enzyme hydrolysis temperature 52.5°C, solid-liquid ratio 1 : 2.6, enzyme concentration 1.44%, and enzyme hydrolysis time 93.1 min. The yield of peanut protein in verification tests was 89.87%, indicating that the regression model of the yield of protein had good predictability. The yield of oil bodies was optimized solely, and the predicted value reached 48.66% at enzyme hydrolysis temperature 57.7°C, solid-liquid ratio 1 : 5.2, enzyme concentration 1.30%, and enzyme hydrolysis time 87 min. The yield of oil bodies in the verification test reached 49.14%, indicating that the regression model of the yield of oil bodies had good predictability.Considering that the yield of peanut protein and oil bodies cannot reach their maximum simultaneously, technological parameters were optimized by giving priority to oil bodies. The predicted values for the yield of protein and oil bodies were 80.47% and 47.73%, respectively, at the optimal condition of enzyme hydrolysis temperature 52.1°C, solid-liquid ratio 1 : 4, enzyme concentration 1.35%, and enzyme hydrolysis time 96.7 min. Considering the technical parameters and the feasibility of the operation, a verification test was conducted with the conditions of enzyme hydrolysis temperature 52°C, solid-liquid ratio 1 : 4, enzyme concentration 1.35%, and enzyme hydrolysis time 90 min and was repeated ten times to verify the reliability of the models prediction results. Results are shown in Table4, and the relative errors between measured and predicted values of the yield of peanut protein and oil bodies were 2.32% and 1.49%, respectively, which were smaller than their standard deviation. The results indicated that the measured values agreed with the predicted values well, and the model had a good degree of fitting [33].


### 3.7. Functional Properties of Peanut Protein Powder

Viscozyme® L promotes the release of proteins and oil bodies by reducing the binding of cellulose and other components to protein. Viscozyme® L has no effect on the molecular weight distribution and structure of protein, retaining the original functional properties of the protein well [34, 35]. As shown in Table 5, the solubility and foaming property of AEEPPP were lower than CPPP slightly because Viscozyme® L lightly acted on the cell wall of the peanut, not changing the molecular weight of peanut protein, thus having poor solubility. Overall, the foam stability, emulsifying activity, and emulsifying stability of AEEPPP were better than CPPP.The water holding capacity and oil holding capacity refer to the maximum amount of water or oil that can be combined with a certain amount of protein after full absorption of water or oil and centrifugation [36, 37]. The holding capacities of water and oil of AEEPPP were both higher than CPPP, indicating that the former has greater advantages in food processing.


## 3.1. Morphology of Protein Bodies and Oil Bodies in Cotyledon Cells of Peanut

Oil bodies are small spheres composed of neutral lipids, phospholipids wrapping neutral lipids, and proteins embedded in the phospholipid layer, which wrap the protein bodies in cytoplasm [25]. The transmission electron micrograph of peanut is shown in Figure 2. The shape of peanut protein bodies and oil bodies are spherical and oval, with few irregular shapes. The oil bodies were distributed in the cells, and the protein bodies were wrapped tightly by oil bodies.Figure 2
Transmission electron micrograph of peanut (PB: protein body; OB: oil body).The diameter of oil bodies varies from 0.5 to 2.5μm for different oil plants, and the diameter of protein bodies is larger than that of oil bodies [26]. In this study, the average diameter of protein bodies was 3.34 ± 0.79 μm, and the number of protein bodies was 16 ± 2. The average diameter of oil bodies was 1.43 ± 0.18 μm, and the number of oil bodies was 147 ± 12. The particle size of protein bodies was obviously larger than oil bodies, but the number of protein bodies was less than that of oil bodies in the peanut cell.


## 3.2. Effects of Enzymes on Peanut Protein and Oil Bodies

The main components of plant cell walls are cellulose, hemicellulose, and pectin. Viscozyme® L, cellulase, hemicellulase, and pectinase were used to facilitate the degradation of the peanut cell wall to extract more peanut protein and oil bodies. These enzymes had no effect on protein; thus, protein was unhydrolyzed.


### 3.2.1. SDS-PAGE

SDS-PAGE of peanut protein extracted by various enzymes and CPPP is shown in Figure3, and the molecular weight distribution was consistent. Krishna et al. [27] and Shokraii et al. [28] analyzed protein subunits of different peanut varieties, and their results were similar to those in this study. The results here indicate that the molecular weight distribution of peanut protein was not changed by Viscozyme® L, cellulase, hemicellulase, or pectinase.Figure 3
SDS-PAGE of peanut protein extracted by various enzymes: (1) standard protein; (2) Viscozyme® L; (3) cellulase; (4) hemicellulase; (5) pectinase; (6) CPPP.


### 3.2.2. Microscopy of Oil Bodies

Microscope images of oil body suspensions extracted by Viscozyme® L, cellulase, hemicellulase, and pectinase are shown in Figure4. The size of some oil bodies in Figures 4(a) and 4(b) was obviously bigger than the peanut oil bodies in its original state believed to be because oil bodies collided into each other and merged into big oil bodies during mechanical crashing and enzymatic hydrolysis. With the enzymatic hydrolysis, the size of oil bodies increased, and the distribution was not uniform, which was beneficial to the yield of oil bodies. Figures 4(c) and 4(d) show peanut oil bodies were complete, regular, and distributed uniformly, illustrating the effects of hemicellulase and pectinase on release of oil bodies were smaller than with Viscozyme® L and cellulase.Figure 4
Microscopy of oil bodies extracted by various enzymes: (a) Viscozyme® L; (b) cellulase; (c) hemicellulase; (d) pectinase. All photos are at the same magnification. Bar represents 10μm.
(a)
(b)
(c)
(d)


### 3.2.3. Effect of Enzymes on Yield of Peanut Proteins and Oil Bodies

The yield of peanut protein and oil bodies extracted by various enzymes is shown in Table2. As a control experiment, the yield of peanut protein and oil bodies extraction without any enzyme was 73.3% ± 0.3 and 29.1% ± 0.7, respectively. As shown in Table 2, the yield of peanut protein and oil bodies extracted by various enzymes was higher than control experiment significantly (p<0.05). The highest yield of oil bodies was 41.1% ± 0.9 extracted by Viscozyme® L, which had significant differences (p<0.05) with cellulase and hemicellulase. The yield of peanut protein was lowest extracted by Viscozyme® L, but there were no significant differences with cellulase and pectinase. In conclusion, Viscozyme® L was selected as the enzyme to extract peanut protein and oil bodies.Table 2
Effects of various enzymes on yield of peanut protein and oil bodies.
Serial number
Type of enzyme
Yield of peanut protein (%)
Yield of oil bodies (%)
1
—
73.3 ± 0.3c
29.1 ± 0.7d
2
Viscozyme® L
81.5 ± 0.8b
41.1 ± 0.9a
3
Cellulase
83.5 ± 0.9ab
37.7 ± 0.5bc
4
Hemicellulose
85.5 ± 0.7a
35.4 ± 1.1c
5
Pectinase
83.2 ± 0.9b
39.8 ± 1.4ab
Note. Different letters mean significant difference (p<0.05).


## 3.2.1. SDS-PAGE

SDS-PAGE of peanut protein extracted by various enzymes and CPPP is shown in Figure3, and the molecular weight distribution was consistent. Krishna et al. [27] and Shokraii et al. [28] analyzed protein subunits of different peanut varieties, and their results were similar to those in this study. The results here indicate that the molecular weight distribution of peanut protein was not changed by Viscozyme® L, cellulase, hemicellulase, or pectinase.Figure 3
SDS-PAGE of peanut protein extracted by various enzymes: (1) standard protein; (2) Viscozyme® L; (3) cellulase; (4) hemicellulase; (5) pectinase; (6) CPPP.


## 3.2.2. Microscopy of Oil Bodies

Microscope images of oil body suspensions extracted by Viscozyme® L, cellulase, hemicellulase, and pectinase are shown in Figure4. The size of some oil bodies in Figures 4(a) and 4(b) was obviously bigger than the peanut oil bodies in its original state believed to be because oil bodies collided into each other and merged into big oil bodies during mechanical crashing and enzymatic hydrolysis. With the enzymatic hydrolysis, the size of oil bodies increased, and the distribution was not uniform, which was beneficial to the yield of oil bodies. Figures 4(c) and 4(d) show peanut oil bodies were complete, regular, and distributed uniformly, illustrating the effects of hemicellulase and pectinase on release of oil bodies were smaller than with Viscozyme® L and cellulase.Figure 4
Microscopy of oil bodies extracted by various enzymes: (a) Viscozyme® L; (b) cellulase; (c) hemicellulase; (d) pectinase. All photos are at the same magnification. Bar represents 10μm.
(a)
(b)
(c)
(d)


## 3.2.3. Effect of Enzymes on Yield of Peanut Proteins and Oil Bodies

The yield of peanut protein and oil bodies extracted by various enzymes is shown in Table2. As a control experiment, the yield of peanut protein and oil bodies extraction without any enzyme was 73.3% ± 0.3 and 29.1% ± 0.7, respectively. As shown in Table 2, the yield of peanut protein and oil bodies extracted by various enzymes was higher than control experiment significantly (p<0.05). The highest yield of oil bodies was 41.1% ± 0.9 extracted by Viscozyme® L, which had significant differences (p<0.05) with cellulase and hemicellulase. The yield of peanut protein was lowest extracted by Viscozyme® L, but there were no significant differences with cellulase and pectinase. In conclusion, Viscozyme® L was selected as the enzyme to extract peanut protein and oil bodies.Table 2
Effects of various enzymes on yield of peanut protein and oil bodies.
Serial number
Type of enzyme
Yield of peanut protein (%)
Yield of oil bodies (%)
1
—
73.3 ± 0.3c
29.1 ± 0.7d
2
Viscozyme® L
81.5 ± 0.8b
41.1 ± 0.9a
3
Cellulase
83.5 ± 0.9ab
37.7 ± 0.5bc
4
Hemicellulose
85.5 ± 0.7a
35.4 ± 1.1c
5
Pectinase
83.2 ± 0.9b
39.8 ± 1.4ab
Note. Different letters mean significant difference (p<0.05).


## 3.3. Effects of Operation Parameters on the Yield of Peanut Protein and Oil Bodies

Temperature is a key factor affecting enzyme activity, and the yield of peanut protein and oil bodies effected by different temperatures is shown in Figure5(a). Peanut protein yield first increased and then decreased and reached a maximum (78.86%) at 55°C. The yield of oil bodies followed the same trend as protein, but it reached its maximum (38.86%) at 50°C. The extraction rate of peanut protein reaches a maximum (88.69%) at 1 : 2 solid-liquid ratio and however decreased and achieved a balance with the increase in solid-liquid ratio (Figure 5(b)). The reason could be that water-soluble whey protein and salt-soluble arachin and conarachin gradually dissolved with the increase in solid-liquid ratio; then, arachin and conarachin were nondegradable because their change in the spatial structure trended to be stable [29, 30]. The yield of oil bodies increased first and then decreased, reaching a maximum (42.37%) at solid-liquid ratio 1 : 4. The increase in aqueous solution could promote the decomposition of peanut cell wall structure, but continuous increase in the solid-liquid ratio decreased the yield of oil bodies for the decrease in enzyme concentration. The yield of peanut protein and oil bodies had a trend of increasing first and then decreasing and both reached maximum (68.45% and 39.28%, respectively) at 1.25% (Figure 5(c)). The damage degree of peanut cell wall is increased with the increase in enzyme concentration protein and oil bodies in the cell that are fully released. The yield of peanut protein and oil bodies tend to be stable when concentration of enzyme increases to a certain degree. The yield of peanut protein reached maximum (79.91%) at 80 min, and the yield of oil bodies increased between 20 and 80 min and then stabilized after this (Figure 5(d)). At a certain time, enzymes were in full contact with the substrate; thus, the reaction rate was fast, and enzymatic hydrolysis was sufficient. When the reaction time increased, the concentration of the substrate and reaction rate decreased; thus, the yield of peanut protein and oil bodies trend to be stable.Figure 5
Effect of different enzyme hydrolysis temperature, solid-liquid ratio, enzyme concentration, and enzyme hydrolysis time on peanut protein and oil bodies yield.
(a)
(b)
(c)
(d)


## 3.4. Result of Response Surface Optimization Test

Response surface test designed by the Central Composite Design of Design Expert 8.05b is shown in Table3. The results of the variance analysis on the yield of peanut protein and oil bodies are shown in Tables 4 and 5, respectively. Regression analysis was carried out on the results, and the polynomial regression model of response value of the yield of peanut protein and oil bodies is given in equations (6) and (7), respectively:(6)Y1=80.61+3.96A−12.91B+3.56C+2.26D−2.50AB+0.81AC−0.63AD+2.91BC+0.91AD+0.77CD−4.64A2−2.74B2−2.20C2−3.56D2,(7)Y2=47.09−0.60A+3.66B+1.72C+0.99D+0.45AB−0.03AC−0.51AD+0.057CD+0.38BD−0.18CD−0.57A2−1.85B2−2.91C2−0.27D2.Table 3
Program and experimental results of response surface methodology.
Serial number
A (°C)
B (g/mL)
C (%)
D (min)
Y
1 (%)
Y
2 (%)
1
60
1 : 4
1.25
80
70.52
45.22
2
50
1 : 2
1.25
80
91.49
33.69
3
50
1 : 6
1.25
80
52.10
44.76
4
50
1 : 4
1.25
80
80.81
47.56
5
55
1 : 5
1.00
100
52.09
44.96
6
45
1 : 5
1.00
60
40.83
43.01
7
55
1 : 3
1.00
100
83.57
34.06
8
40
1 : 4
1.25
80
57.92
43.50
9
55
1 : 3
1.00
60
89.43
35.05
10
45
1 : 5
1.00
100
48.53
46.76
11
50
1 : 4
1.25
80
82.67
47.12
12
55
1 : 5
1.00
60
44.17
42.58
13
45
1 : 3
1.50
60
66.65
38.77
14
50
1 : 4
1.25
80
80.97
48.27
15
45
1 : 5
1.50
100
56.51
48.40
16
55
1 : 3
1.50
60
87.23
38.43
17
50
1 : 4
1.25
40
60.35
42.99
18
55
1 : 5
1.50
60
59.36
45.00
19
50
1 : 4
1.25
120
76.72
48.14
20
45
1 : 3
1.00
60
78.33
36.39
21
50
1 : 4
1.25
80
78.62
48.06
22
50
1 : 4
1.75
80
84.82
39.62
23
55
1 : 3
1.50
100
90.58
36.67
24
50
1 : 4
0.75
80
63.10
30.41
25
55
1 : 5
1.50
100
59.65
48.05
26
50
1 : 4
1.25
80
84.40
45.50
27
45
1 : 3
1.50
100
77.78
42.11
28
50
1 : 4
1.25
80
76.19
46.00
29
45
1 : 5
1.50
60
54.42
47.79
30
45
1 : 3
1.00
100
73.12
39.45Table 4
Experiment data for test validation.
Parameter
Extraction rate of peanut protein (%)
Yield of oil bodies (%)
Mean
78.60
48.44
Predicted values
80.47
47.73
Relative error
2.32
1.49Table 5
Functional properties of peanut protein powder.
Sample
Solubility (%)
Emulsifying activity (mL/g)
Emulsifying stability (min)
Foaming property (%)
Foam stability (%)
Water holding capacity (mL/g)
Oil holding capacity (mL/g)
AEEPPP
5.8 ± 0.7
22.5 ± 0.9
54.2 ± 1.0
20.5 ± 0.4
51.2 ± 1.2
7.4 ± 0.4
18.5 ± 0.7
CPPP
6.4 ± 0.5
20.5 ± 0.8
22.9 ± 0.5
21.0 ± 0.3
42.9 ± 0.9
6.2 ± 0.2
16.7 ± 0.5The results of the effect of factors A, B, C, and D on the extraction rate of peanut protein indicated that the order of positive influence for each factor was solid-liquid ratio > enzyme hydrolysis temperature > concentration of enzyme > enzyme hydrolysis time. Linear terms (A, B, and C), quadratic terms (A2, B2, C2, and D2), and the interaction term (BC) of the response surface equation for peanut protein had a significant effect (p<0.05) on the extraction rate of peanut protein. This indicated that the effect of the experimental factors on the response value was not a linear relationship, but a quadratic relationship, and the change of response value (extraction rate of peanut protein) was very complex.The order of positive influence for each factor on the yield of oil bodies was solid-liquid ratio > concentration of enzyme > enzyme hydrolysis time > enzyme hydrolysis temperature. Linear terms (B, C, and D) and quadratic terms (B2 and C2) of response surface equation for peanut protein had a significant effect (p<0.05) on the extraction rate of peanut protein. This indicated that the effect of experimental factors on response values was also a quadratic relationship.The polynomial regression models of response value of the yield of peanut protein and oil bodies were significant (p<0.05), while testing the lose effectiveness of fit for the models was not significant (p<0.05). Analysis of variance indicated that the model was consistent with the data.The nonsignificant terms were removed after the analysis of variance of the response surface equation for the yield of peanut protein and oil bodies, giving the final regression model. The polynomial regression model of the yield of peanut protein and oil bodies is given in equations (8) and (9), respectively:(8)Y1=80.61+3.96A−12.91B+3.56C+2.26D+2.91BC−4.64A2−2.74B2−2.20C2−3.56D2,(9)Y2=46.25+3.66B+1.72C+0.99D−1.75B2−2.80C2.Results for analysis of variance of the modified model for the extraction rate peanut protein and yield of oil bodies showed thatR2 and adjusted R2 were close to 1, which indicated the modified model could predict the extraction rate of peanut protein and the yield of oil bodies, well [31, 32].


## 3.5. Effects of Interaction on the Yield of Peanut Protein

The interaction term (BC) of the response surface equation for peanut protein had a significant effect on its yield (p=0.042); the interaction between the solid-liquid ratio and enzyme concentration on the yield of peanut protein is shown in Figure 6.Figure 6
Effect of different solid-liquid ratios and enzyme concentrations on the yield of peanut protein.Figure6 shows that the yield of peanut protein continued to increase with the increase in the solid-liquid ratio and the enzyme concentration, simultaneously. For a certain peanut raw material, there are optimal dissolution and precipitation ratio and enzyme dosage for aqueous enzymatic extraction. Water can promote the dissolution and precipitation of peanut protein but however dilutes the enzyme concentration in excess, which hinders the interaction between the enzyme and cell walls.


## 3.6. Verification Test of Response Surface Optimization

Optimizing for the yield of peanut protein was conducted solely, and the predicted value reached 92.67% at enzyme hydrolysis temperature 52.5°C, solid-liquid ratio 1 : 2.6, enzyme concentration 1.44%, and enzyme hydrolysis time 93.1 min. The yield of peanut protein in verification tests was 89.87%, indicating that the regression model of the yield of protein had good predictability. The yield of oil bodies was optimized solely, and the predicted value reached 48.66% at enzyme hydrolysis temperature 57.7°C, solid-liquid ratio 1 : 5.2, enzyme concentration 1.30%, and enzyme hydrolysis time 87 min. The yield of oil bodies in the verification test reached 49.14%, indicating that the regression model of the yield of oil bodies had good predictability.Considering that the yield of peanut protein and oil bodies cannot reach their maximum simultaneously, technological parameters were optimized by giving priority to oil bodies. The predicted values for the yield of protein and oil bodies were 80.47% and 47.73%, respectively, at the optimal condition of enzyme hydrolysis temperature 52.1°C, solid-liquid ratio 1 : 4, enzyme concentration 1.35%, and enzyme hydrolysis time 96.7 min. Considering the technical parameters and the feasibility of the operation, a verification test was conducted with the conditions of enzyme hydrolysis temperature 52°C, solid-liquid ratio 1 : 4, enzyme concentration 1.35%, and enzyme hydrolysis time 90 min and was repeated ten times to verify the reliability of the models prediction results. Results are shown in Table4, and the relative errors between measured and predicted values of the yield of peanut protein and oil bodies were 2.32% and 1.49%, respectively, which were smaller than their standard deviation. The results indicated that the measured values agreed with the predicted values well, and the model had a good degree of fitting [33].


## 3.7. Functional Properties of Peanut Protein Powder

Viscozyme® L promotes the release of proteins and oil bodies by reducing the binding of cellulose and other components to protein. Viscozyme® L has no effect on the molecular weight distribution and structure of protein, retaining the original functional properties of the protein well [34, 35]. As shown in Table 5, the solubility and foaming property of AEEPPP were lower than CPPP slightly because Viscozyme® L lightly acted on the cell wall of the peanut, not changing the molecular weight of peanut protein, thus having poor solubility. Overall, the foam stability, emulsifying activity, and emulsifying stability of AEEPPP were better than CPPP.The water holding capacity and oil holding capacity refer to the maximum amount of water or oil that can be combined with a certain amount of protein after full absorption of water or oil and centrifugation [36, 37]. The holding capacities of water and oil of AEEPPP were both higher than CPPP, indicating that the former has greater advantages in food processing.


## 4. Conclusion

This study investigated the effect of various enzymes (Viscozyme® L, cellulase, hemicellulase, and pectinase) on the molecular weight distribution of peanut protein, microscopy of oil bodies, and yield of peanut protein and oil bodies during the aqueous enzymatic extraction process. The molecular weight distribution of peanut protein was not changed by Viscozyme® L, cellulase, hemicellulase, or pectinase by comparing SDS-PAGE of peanut protein extracted by various enzymes with CPPP. The size of oil bodies extracted by Viscozyme® L and cellulase increased, and the distribution was not uniform, which was beneficial to the yield of oil bodies. However, oil bodies extracted by hemicellulase and pectinase were complete, regular, and distributed uniformly, illustrating the effects of hemicellulase and pectinase on release of oil bodies were smaller than with Viscozyme® L and cellulase. Viscozyme® L was selected as the enzyme for the single factor and response surface testing, and the yield of peanut protein and oil bodies reached 78.60% and 48.44%, respectively, at the optimal conditions of enzyme hydrolysis temperature 52°C, solid-liquid ratio 1 : 4, enzyme concentration 1.35%, and enzyme hydrolysis time 90 min. Study of the enzyme screening, process optimization, and effect of technological parameters on the yield of protein and oil bodies showed significance for aqueous enzymatic extraction, a possible realization for industrial production. The protein obtained was unhydrolyzed because Viscozyme® L has no effect on protein, and its functional properties were analyzed by comparison with CPPP. The solubility and foaming property of AEEPPP were lower than CPPP, but the foam stability, emulsifying activity, emulsifying stability, water holding capacity, and oil holding capacity of AEEPPP were better than CPPP, which provides a theoretical basis for the industrial production and application of AEEPPP.



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*Source: 5148967-2020-01-27.xml*