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Abstract ［Objectives］ This study was conducted to investigate the drying methods, functional and structure properties of dietary fiber (DF) from peanut shells.
［Methods］ Peanut shells were used as a raw material to prepare peanut shell dietary fiber (DF) by hot air drying (HA) and vacuum freeze drying (VF), respectively, and their functional and structural characteristics were compared in detail.
［Results］ The solubility, water holding capacity, oil holding capacity and swelling capacity of HA-DF and VF-DF were 2.15 %, 7.63 g/g, 7.73 g/g, 10.35 ml/g and 3.85 %, 14.98 g/g, 15.25 g/g, 15.85 ml/g, respectively. The total phenol contents were 2.623 and 5.173 mg GAE/g, respectively. The IC 50 values of· OH, O-2·and DPPH free radicals were 4.16 and 4.09 mg/ml, 7.90 and 3.32 mg/ml, and 3.19 and 3.09 mg/ml, respectively. The molecular weight of VF-DF was smaller, and it had narrow molecular weight distribution and denser particles. Electron microscopy showed that VF-DF had a porous network like honeycomb and swelled structure.
［Conclusions］ This study can provide a theoretical basis for the functional modification and comprehensive utilization of peanut shell dietary fiber.
Key words Peanut shell; Dietary fiber; Drying method; Functional property; Structural property
Received: February 28, 2020Accepted: April 15, 2020
Supported by Tangshan Science Project (19150204E).
Lei WANG (1982-), male, P. R. China, associate researcher, devoted to research about functional ingredients and food additives.
*Corresponding author. E-mail: 416167083@qq.com; caohuihui0526@126.com.
Dietary fiber is a variety of plant materials other than starch polysaccharides, mainly coming from the cell walls of animals and plants, including cellulose, lignin, wax, chitin, pectin, β-glucan, inulin and oligosaccharides, etc. It can be divided into insoluble dietary fiber and water-soluble dietary fiber［1-3］.
Dietary fiber refers to polysaccharides and lignin that cannot be broken down by human digestive enzymes. It has the function of absorbing water in the digestive system, thus increasing the volume of food in the intestine and stomach and increasing satiety. It can also promote gastrointestinal motility and relieve constipation. Meanwhile, dietary fiber can also absorb harmful substances in the intestine, facilitating excretion of harmful substances, and can improve intestinal flora, providing energy and nutrition for the proliferation of probiotics［4-8］. Dietary fiber is indispensable for a healthy diet. Fiber plays an important role in maintaining the health of the digestive system. The intake of sufficient fiber can also prevent cardiovascular disease, cancer, diabetes and other diseases, clean the digestive wall and enhance digestive function, dilute and accelerate the removal of carcinogens and toxic substances from food, and protect the vulnerable digestive tract and prevent colon cancer. Dietary fiber can slow down the digestion rate and excrete cholesterol the fastest, and thus can control blood sugar and cholesterol in the blood at the optimal level ［9-15］. China is the worlds largest peanut production and export country, and its planting area and annual output are among the highest in the world. The peanut industry produces a lot of by-products every year, which are potential dietary fiber resources. If these resources are not fully utilized, they will cause serious waste of resources and pollution to the environment. Therefore, processing these by-products of the peanut industry into natural dietary fiber can not only increase the added value of the peanut industry, but also bring huge social and economic benefits［16］. Vacuum freeze-drying technology is a drying method that reduces the temperature of a material below the eutectic point to freeze the moisture into ice, and provides sublimation heat at low pressure to directly sublimate it, thereby removing moisture from the material. In this study, the effects of different drying methods on the function and structural characteristics of dietary fiber were investigated, providing a theoretical basis for the development of the application value of peanut shell dietary fiber obtained by vacuum freeze-drying.
Material and Methods
Experimental materials
Peanut shells were provided by Tangshan Runze Cereals, Oils and Foods Co., Ltd.
The dietary fiber products obtained through processing of peanut shells were HA-DF and VF-DF. HA-DF: Starch and protein in the peanut shells were removed with temperature tolerant α-amylase and neutral protease, respectively. The treated peanut shells were then extracted with water and centrifuged, and the supernatant was subjected to ethanol precipitation and centrifugation. The two precipitates were merged, dried with hot air at 60 ℃ , crushed and sieved through 80 mesh sieve, giving a white powder. VF-DF: Starch and protein in the peanut shells were removed with temperature tolerant α-amylase and neutral protease, respectively. The treated peanut shells were then extracted with water and centrifuged, and the supernatant was subjected to ethanol precipitation and centrifugation. The two precipitates were merged, pre-frozen in a -20 ℃ refrigerator for 12 h, and then freeze-dried in vacuum at -50 ℃ and 100 Pa. The dried materials was crushed and sieved with an 80 mesh sieve, giving a white powder.
Experimental methods
Determination of water solubility (WS)
Water solubility determination: About 1 g of sample was weighed in a centrifuge tube, added with distilled water at a ratio of 1∶10, and mixed evenly. The mixture was stood at room temperature for 1 h and centrifuged under the condition of 3 000 r/min for 10 min. The supernatant and residue were collected, and dried and weighed separately. WS (%)=(Weight of supernatant after drying/Weight of sample powder)×100.
Determination of water holding capacity (WHC)
Water holding capacity refers to the amount of water bound by a certain amount of sample without external forces (except gravity and atmospheric pressure). For its determination, a 1.0 g of sample was accurately weighed into a 50 ml centrifuge tube, added with 20 ml of deionized water, and mixed well. The mixture was stood at 4 ℃ for 24 h, then centrifuged at 4 200 r/min for 15 min, and weighed.
WHC (g/g)=(Wet weight of sample after being saturated with water-Weight of sample powder)/Weight of sample powder
Oil holding capacity (OHC)
Oil holding capacity refers to the amount of olive oil that a certain amount of sample can bind. For its determination, a 1.0 g of sample was accurately weighed into a 50 ml centrifuge tube, added with 10 ml of olive oil, and mixed well. The mixture was stood at 4 ℃ for 1 h, then centrifuged at 4 200 r/min for 15 min, and weighed.
OHC (g/g)=(Wet weight of sample saturated with oil-Weight of sample powder)/Weight of sample powder
Determination of swelling capacity (SC)
Swelling capacity refers to the difference between the volume occupied by a certain amount of sample immersed in excess water after reaching equilibrium and its actual volume. For its determination, a 0.2 g of sample was accurately weighed in a graduated test tube, and its volume was recorded. The, 5.0 ml of distilled water was added into the test tube, followed by mixing well and standing at 4 ℃ for 18 h, and the volume of the sample after absorbing water was recorded.
SC (ml/g)=(Sample volume after swelling-Volume of sample powder)/Volume of sample powder
Determination of·OH scavenging rate
Referring to the Fenton reaction system model, in the reaction system (salicylic acid-ethanol solution 9 mmol/L, Fe2+ 9 mmol/L, H2O2 8.8 mmol/L), if substances with the ability to remove·OH are added, they will compete with salicylic acid for·OH, thereby reducing the amount of colored substances produced. According to the method with fixed reaction time, different concentrations of DF were added to the same volume of the reaction system, and the absorbance after adding the different concentrations of DF was measured at a wavelength of 510 nm, with distilled water as a blank control. The determined values were substituted into the scavenging rate calculation formula to calculate the ability of different concentrations of DF to scavenge·OH radicals. ·OH scavenging rate (%)=(1- A1-A2A3 )×100
Wherein A 1 is salicylic acid-ethanol 0.5 ml+DF 1.0 ml+Fe2+ 0.5 ml+H2O2 5.0 ml; A 2 is salicylic acid-ethanol 0.5 ml+DF 1.0 ml+distilled water 0.5 ml+H2O2 5.0 ml; and A 3 is salicylic acid-ethanol 0.5 ml+distilled water 1.0 ml+Fe2+ 0.5 ml+H2O2 5.0 ml.
Determination of O-2·scavenging rate
O-2·is generated by the pyrogallol auto-oxidation method. Specifically, 4.0 ml of 0.05 mol/L Tris-HCl buffer solution (pH 8.2) was heated in a 25 ℃ water bath for 20 min. Then, 1 ml of the test liquid and 1 ml of 25 mmol/L pyrogallol solution were added, followed by mixing. The obtained reaction liquid was heated in a water bath at 25 ℃ for 5 min to allow reaction, which was terminated by adding 100 μl of 8% HCl solution. The absorbance (A) was measured at a wavelength of 320 nm. A blank test was carried out with 1 ml of distilled water instead of the test liquid.
O-2·scavenging rate (%)=(1- A1-A2A3 )×100
Wherein A 1 is Tris-HCl 4 ml+DF 1.0 ml+pyrogallol 2 ml; A 2 is Tris-HCl 4 ml+DF 1.0 ml+distilled water 2 ml; and A 3 is Tris-HCl 4 ml+distilled water 1.0 ml+pyrogallol 2 ml.
Determination DPPH radical scavenging rate
DPPH free radicals are stable in 95% ethanol solution. And the solution has an absorption peak at a wavelength of 517 nm and is purple. When a free radical scavenger is present, the lone pair electrons of DPPH are paired, the color becomes lighter, the absorption at the maximum absorption wavelength becomes smaller, and the color change is in a stoichiometric relationship with the number of paired electrons, which can be used to evaluate the free radical scavenging situation. Specifically, 4 ml of the test liquid was added to 2 ml of 0.2 mmol/L DPPH free radical solution, and the absorbance ( Ai ) was measured at 517 nm after reacting in a water bath at 25 ℃ for 20 min. A blank test was carried out with 1 ml of distilled water instead of the test liquid.
DPPH scavenging rate (%)=(1- A1-A2A3 )×100
Wherein A 1 is DF 4 ml+DPPH free radical 2.0 ml; A 2 is DF 4 ml+95% ethanol 2.0 ml; and A 3 is distilled water 4 ml+DPPH free radical 2.0 ml.
Determination of total phenol content
Preparation of sample solution: At first, 1 g of sample powder was added into an Erlenmeyer flask, and added with 70% ethanol solution at a ratio of 1∶30 ( w/v ). The mixture was extracted with shaking in a constant temperature water-bath shaker (800 r/min) at 30 ℃ for 24 h. After the extraction, centrifugation was performed at 3 000 r/min for 10 min, and the obtained supernatant was diluted to 50 ml with the original solvent. The resulting solution was added into a brown bottle, which was placed in the refrigerator at -20 ℃ for later testing. A 0.5 ml of sample at a certain concentration was added with 2.5 ml of 0.2 N Folin-Ciocalteu reagent, shaken for 30 s and allowed to react for 5 min; the solution was then added with 2 ml of 7.5% ( w/v ) Na2CO3 solution, shaken for 20 s and allowed to react for 2 h; and the absorbance was measured at 760 nm, and a blank control using deionized water instead of the sample was also tested. The total phenol content was expressed in gallic acid equivalent (GAE/g, dry basis). Dietary fiber molecular weight analysis
High-performance gel permeation chromatography (GPC) was used to determine the molecular weight of the sample under following test conditions: instrument: DAWN EOS laser light scattering instrument of American wyatt company, experimental temperature: (35.0±0.1) ℃, solvent: 0.1 mol/L NaNO3 solution, laser wavelength: 690.0 nm, solvent flow rate: 0.5 ml/min, gel column: Shodex 806HQ analytical column, and injection volume: 300 μl.
Determination method: A DF sample was dissolved in deionized water to a concentration of 5 mg/ml. Into a 50 ml Erlenmeyer flask, 40 mg of the sample solution and 10 ml of mobile phase were added in order. After shaking, the solution was stood at room temperature for 8 h. LC determination was performed with an injection volume of 300 μl, and the chromatogram was recorded. The weight-average molecular weight was calculated using professional software.
Electron microscopic observation of dietary fiber
HA-DF and VF-DF were pulverized and sieved with a 0.5 mm sieve. Gold plating was performed by ion sputtering method. The prepared samples were analyzed and observed by scanning electron microscope, obtaining corresponding scanning electron microscope photos.
Data processing
Design-Expert 7.0 was adopted for experiment design and data processing.
Agricultural Biotechnology2020
Results and Analysis
Effects of different drying methods on water solubility, water holding capacity, oil holding capacity and swelling capacity of DF
It can be seen from Table 1 that the physical and chemical properties of peanut shell DF were improved after vacuum freeze-drying. The water solubility, water holding capacity, oil holding capacity and swelling capacity of HA-DF were 2.15%, 7.63 g/g, 7.73 g/g and 10.35 ml/g, respectively; and the values of VF-DF increased to 3.85%, 14.98 g/g, 15.25 g/g and 15.85 ml/g, respectively. The reason is that traditional drying can cause the material to shrink and damage the cells, while the structure of a sample will not be destroyed during the freeze-drying process, because the solid component is supported by the solid ice in its place and when the ice sublimates, it will leave pores in the remaining dry material, so that the product can keep its biological and chemical structure and the integrity of activity［17］.
Effects of different drying methods on total phenol content (TPC) and free radical scavenging effect of DF Modern medicine proves that free radicals produced by lipid oxidation play an important role in the initiation and promotion of cancer tumor formation. The free radicals produced by the body in the process of metabolism include superoxide anion free radicals, hydroxyl free radicals, and hydroperoxy free radicals. Among them, hydroxyl free radicals are the most dangerous free radicals, and polyphenols and polysaccharides in dietary fiber have the ability to scavenge superoxide anion free radicals and hydroxyl free radicals, and have proved to be unique in the treatment of cardiovascular diseases and Alzheimers disease. The changes of total phenol content in DF after different drying methods are shown in Fig. 1. After vacuum freeze-drying, total phenol content was increased to a certain extent. The total phenol contents in HA-DF and VF-DF were 2.623 and 5.173 mg GAE/g, respectively. The total phenol content can indirectly reflect the value of antioxidant capacity.
As can be seen from Fig. 2, within a concentration range from 2.0 to 12.0 mg/ml, the·OH scavenging rate had a linear relationship with the DF concentration, and the linear equations for HA-DF and VF-DF were y=10.029x+8.266 7 (R2=0.999 6) and y=12.689x+7.84 (R2=0.999 1) . According to the linear equations, the IC 50 (the mass concentration of DF when scavenging 50% of free radicals) values were 4.16 and 3.32 mg/ml, respectively.
As can be seen from Fig. 3, within a concentration range from 2.0 to 12.0 mg/ml, the O-2·scavenging rate was linearly related to the DF concentration. The linear equations for HA-DF and VF-DF were y=10.206x+8.28 (R2=0.999 1) and y=12.729x +9.433 3 (R2=0.999 7) , respectively. The IC 50 (mass concentration of DF when scavenging 50% of free radicals) values were 4.09 and 3.19 mg/ml, respectively, according to the linear equations.
As can be seen from Fig. 4, within a concentration range from 2.0 to 12.0 mg/ml, the DPPH scavenging rate was linearly related to the DF concentration. The linear equations for HA-DF and VF-DF were y=5.048 6x+10.113 (R2=0.999 3) and y=13.037x+9.686 7 (R2=0.999 2) , respectively. The IC 50 (mass concentration of DF when scavenging 50% of free radicals) values were 7.90 and 3.09 mg/ml, respectively, according to the linear equations.
Molecular weight analysis of dietary fiber
Weight-average molecular weight (MW): The molecular weight of all synthetic polymer compounds and the molecular weight of most natural polymer compounds are heterogeneous, they are mixtures of homologues with different molecular weights, and the average molecular weight calculated by molecular weight is the weight-average molecular weight, the value of which is equal to the sum of the products of the molecular weight of various molecule and their corresponding weight fractions. The number-average molecular weight (Mn) represents one of the average molecular weights of polymers, which is the sum of the products of the fractions of molecules with different molecular weights and their corresponding molecular weights. It can be seen from Table 2 that the weight-average molecular weight of HA-DF was 5.89×105 g/mol, which was greater than that of VF-DF (with a weight-average molecular weight of 2.43×105 g/mol), indicating that HA-DF has a more complete molecular chain. After vacuum freeze-drying, the polysaccharide molecular chain is cut to reduce the molecular weight.
Pd=Mw/Mn is called the polydispersity coefficient and is used to characterize the degree of dispersion. The larger the Pd, the more dispersed the molecular weights. When Pd=1, it means that the molecular weight of the polycondensation product is monodisperse (the same) (Pd=1.03-1.05 means that the molecular weight is approximately monodisperse); when Pd is about 2, the polycondensation product is a free radical product; when Pd=3-5, it means that the polycondensation product is branched; and when Pd=25-30, it means that the polycondensation product is PE. In this study, for HA-DF, Pd=3.23>3, which indicated that the HA-DF was branched. During the polymerization process of polymers, due to the chain transfer of free radical polymerization, as the monomers containing more than three functional groups in the polycondensation process participate in the polycondensation reaction, plus the presence of radiation cross-linking and chemical cross-linking reactions, branch structures extend on linear molecular chains. The branch structures of polymer chains can be generally divided into long branch structures and short branch structures. The length of a long branch chain can be equivalent to that of the main chain, and the length of a short branch chain is similar to that of the longer side chain［18］. However, the Pd of VF-DF was close to 1, indicating that after vacuum freeze-drying, the long branch chain part of the dietary fiber was cut short and the molecular weight distribution was uniform.
Electron microscopic observation of dietary fiber
Scanning electron microscope (SEM) is a new type of electron optical instrument that has developed rapidly in the past three decades. It is characterized by three-dimensional and realistic images showing the shape and size of particles, and has strong adaptability to samples. It is suitable for intuitive research on dietary fiber particles, and can take photos of its representative particle morphology.
Fig. 5 shows the scanning electron microscopy results of the two dietary fibers with a scanning multiple of 3 000 times. It can be seen from the figures that the particles of dietary fiber prepared by the two different drying methods were tiny, and the dietary fiber had slightly wrinkled surface and a loose and flaky structure. However, the VF-DF particles were finer, and the fiber had a clear honeycomb surface structure, evenly distributed. The microstructure of DF is closely related to its functional characteristics, so it is speculated that the VF-DF has good adsorption capacity of cholesterol, sodium cholate and nitrite［19］. Conclusions
Peanut shell dietary fiber HA-DF and VF-DF obtained by the two drying methods had water solubility of 2.15% and 3.85%, water holding capacity of 7.63 and 14.98 g/g, oil holding capacity of 7.73 and 15.25 g/g, and swelling capacity of 10.35 and 15.85 ml/g, respectively. The vacuum freeze-drying treatment improved the water solubility, water holding capacity, oil holding capacity and swelling capacity of peanut shell DF.
The total phenol contents of peanut shell dietary fiber HA-DF and VF-DF obtained by the two drying methods were 2.623 and 5.173 mg GAE/g, respectively, and the IC 50 values of the ·OH, O-2 ·and DPPH scavenging capacity were 4.16 and 3.32 mg/ml, 4.09 and 3.19 mg/ml, and 7.90 and 3.09 mg/ml, respectively. After vacuum freeze-drying treatment, the antioxidant activity of dietary fiber was improved.
The weight-average molecular weights of HA-DF and VF-DF were 5.89×105 and 2.43×105 g/mol, respectively, indicating that HA-DF had a more complete molecular chain, and after vacuum freeze-drying, the polysaccharide molecular chain was cut short, thereby reducing the molecular weight. The polydispersity coefficient of VF-DF was 1.07, and the molecular weight distribution tended to be concentrated.
Observed by scanning electron microscope, the particles of HA-DF and VF-DF were tiny and had slightly wrinkled surface and loose and clear flaky structure, which had pores for adsorbing other substances, but the VF-DF particles were denser and had smaller voids, and the surface had a clear honeycomb structure, evenly distributed.
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Editor: Yingzhi GUANGProofreader: Xinxiu ZHU
［Methods］ Peanut shells were used as a raw material to prepare peanut shell dietary fiber (DF) by hot air drying (HA) and vacuum freeze drying (VF), respectively, and their functional and structural characteristics were compared in detail.
［Results］ The solubility, water holding capacity, oil holding capacity and swelling capacity of HA-DF and VF-DF were 2.15 %, 7.63 g/g, 7.73 g/g, 10.35 ml/g and 3.85 %, 14.98 g/g, 15.25 g/g, 15.85 ml/g, respectively. The total phenol contents were 2.623 and 5.173 mg GAE/g, respectively. The IC 50 values of· OH, O-2·and DPPH free radicals were 4.16 and 4.09 mg/ml, 7.90 and 3.32 mg/ml, and 3.19 and 3.09 mg/ml, respectively. The molecular weight of VF-DF was smaller, and it had narrow molecular weight distribution and denser particles. Electron microscopy showed that VF-DF had a porous network like honeycomb and swelled structure.
［Conclusions］ This study can provide a theoretical basis for the functional modification and comprehensive utilization of peanut shell dietary fiber.
Key words Peanut shell; Dietary fiber; Drying method; Functional property; Structural property
Received: February 28, 2020Accepted: April 15, 2020
Supported by Tangshan Science Project (19150204E).
Lei WANG (1982-), male, P. R. China, associate researcher, devoted to research about functional ingredients and food additives.
*Corresponding author. E-mail: 416167083@qq.com; caohuihui0526@126.com.
Dietary fiber is a variety of plant materials other than starch polysaccharides, mainly coming from the cell walls of animals and plants, including cellulose, lignin, wax, chitin, pectin, β-glucan, inulin and oligosaccharides, etc. It can be divided into insoluble dietary fiber and water-soluble dietary fiber［1-3］.
Dietary fiber refers to polysaccharides and lignin that cannot be broken down by human digestive enzymes. It has the function of absorbing water in the digestive system, thus increasing the volume of food in the intestine and stomach and increasing satiety. It can also promote gastrointestinal motility and relieve constipation. Meanwhile, dietary fiber can also absorb harmful substances in the intestine, facilitating excretion of harmful substances, and can improve intestinal flora, providing energy and nutrition for the proliferation of probiotics［4-8］. Dietary fiber is indispensable for a healthy diet. Fiber plays an important role in maintaining the health of the digestive system. The intake of sufficient fiber can also prevent cardiovascular disease, cancer, diabetes and other diseases, clean the digestive wall and enhance digestive function, dilute and accelerate the removal of carcinogens and toxic substances from food, and protect the vulnerable digestive tract and prevent colon cancer. Dietary fiber can slow down the digestion rate and excrete cholesterol the fastest, and thus can control blood sugar and cholesterol in the blood at the optimal level ［9-15］. China is the worlds largest peanut production and export country, and its planting area and annual output are among the highest in the world. The peanut industry produces a lot of by-products every year, which are potential dietary fiber resources. If these resources are not fully utilized, they will cause serious waste of resources and pollution to the environment. Therefore, processing these by-products of the peanut industry into natural dietary fiber can not only increase the added value of the peanut industry, but also bring huge social and economic benefits［16］. Vacuum freeze-drying technology is a drying method that reduces the temperature of a material below the eutectic point to freeze the moisture into ice, and provides sublimation heat at low pressure to directly sublimate it, thereby removing moisture from the material. In this study, the effects of different drying methods on the function and structural characteristics of dietary fiber were investigated, providing a theoretical basis for the development of the application value of peanut shell dietary fiber obtained by vacuum freeze-drying.
Material and Methods
Experimental materials
Peanut shells were provided by Tangshan Runze Cereals, Oils and Foods Co., Ltd.
The dietary fiber products obtained through processing of peanut shells were HA-DF and VF-DF. HA-DF: Starch and protein in the peanut shells were removed with temperature tolerant α-amylase and neutral protease, respectively. The treated peanut shells were then extracted with water and centrifuged, and the supernatant was subjected to ethanol precipitation and centrifugation. The two precipitates were merged, dried with hot air at 60 ℃ , crushed and sieved through 80 mesh sieve, giving a white powder. VF-DF: Starch and protein in the peanut shells were removed with temperature tolerant α-amylase and neutral protease, respectively. The treated peanut shells were then extracted with water and centrifuged, and the supernatant was subjected to ethanol precipitation and centrifugation. The two precipitates were merged, pre-frozen in a -20 ℃ refrigerator for 12 h, and then freeze-dried in vacuum at -50 ℃ and 100 Pa. The dried materials was crushed and sieved with an 80 mesh sieve, giving a white powder.
Experimental methods
Determination of water solubility (WS)
Water solubility determination: About 1 g of sample was weighed in a centrifuge tube, added with distilled water at a ratio of 1∶10, and mixed evenly. The mixture was stood at room temperature for 1 h and centrifuged under the condition of 3 000 r/min for 10 min. The supernatant and residue were collected, and dried and weighed separately. WS (%)=(Weight of supernatant after drying/Weight of sample powder)×100.
Determination of water holding capacity (WHC)
Water holding capacity refers to the amount of water bound by a certain amount of sample without external forces (except gravity and atmospheric pressure). For its determination, a 1.0 g of sample was accurately weighed into a 50 ml centrifuge tube, added with 20 ml of deionized water, and mixed well. The mixture was stood at 4 ℃ for 24 h, then centrifuged at 4 200 r/min for 15 min, and weighed.
WHC (g/g)=(Wet weight of sample after being saturated with water-Weight of sample powder)/Weight of sample powder
Oil holding capacity (OHC)
Oil holding capacity refers to the amount of olive oil that a certain amount of sample can bind. For its determination, a 1.0 g of sample was accurately weighed into a 50 ml centrifuge tube, added with 10 ml of olive oil, and mixed well. The mixture was stood at 4 ℃ for 1 h, then centrifuged at 4 200 r/min for 15 min, and weighed.
OHC (g/g)=(Wet weight of sample saturated with oil-Weight of sample powder)/Weight of sample powder
Determination of swelling capacity (SC)
Swelling capacity refers to the difference between the volume occupied by a certain amount of sample immersed in excess water after reaching equilibrium and its actual volume. For its determination, a 0.2 g of sample was accurately weighed in a graduated test tube, and its volume was recorded. The, 5.0 ml of distilled water was added into the test tube, followed by mixing well and standing at 4 ℃ for 18 h, and the volume of the sample after absorbing water was recorded.
SC (ml/g)=(Sample volume after swelling-Volume of sample powder)/Volume of sample powder
Determination of·OH scavenging rate
Referring to the Fenton reaction system model, in the reaction system (salicylic acid-ethanol solution 9 mmol/L, Fe2+ 9 mmol/L, H2O2 8.8 mmol/L), if substances with the ability to remove·OH are added, they will compete with salicylic acid for·OH, thereby reducing the amount of colored substances produced. According to the method with fixed reaction time, different concentrations of DF were added to the same volume of the reaction system, and the absorbance after adding the different concentrations of DF was measured at a wavelength of 510 nm, with distilled water as a blank control. The determined values were substituted into the scavenging rate calculation formula to calculate the ability of different concentrations of DF to scavenge·OH radicals. ·OH scavenging rate (%)=(1- A1-A2A3 )×100
Wherein A 1 is salicylic acid-ethanol 0.5 ml+DF 1.0 ml+Fe2+ 0.5 ml+H2O2 5.0 ml; A 2 is salicylic acid-ethanol 0.5 ml+DF 1.0 ml+distilled water 0.5 ml+H2O2 5.0 ml; and A 3 is salicylic acid-ethanol 0.5 ml+distilled water 1.0 ml+Fe2+ 0.5 ml+H2O2 5.0 ml.
Determination of O-2·scavenging rate
O-2·is generated by the pyrogallol auto-oxidation method. Specifically, 4.0 ml of 0.05 mol/L Tris-HCl buffer solution (pH 8.2) was heated in a 25 ℃ water bath for 20 min. Then, 1 ml of the test liquid and 1 ml of 25 mmol/L pyrogallol solution were added, followed by mixing. The obtained reaction liquid was heated in a water bath at 25 ℃ for 5 min to allow reaction, which was terminated by adding 100 μl of 8% HCl solution. The absorbance (A) was measured at a wavelength of 320 nm. A blank test was carried out with 1 ml of distilled water instead of the test liquid.
O-2·scavenging rate (%)=(1- A1-A2A3 )×100
Wherein A 1 is Tris-HCl 4 ml+DF 1.0 ml+pyrogallol 2 ml; A 2 is Tris-HCl 4 ml+DF 1.0 ml+distilled water 2 ml; and A 3 is Tris-HCl 4 ml+distilled water 1.0 ml+pyrogallol 2 ml.
Determination DPPH radical scavenging rate
DPPH free radicals are stable in 95% ethanol solution. And the solution has an absorption peak at a wavelength of 517 nm and is purple. When a free radical scavenger is present, the lone pair electrons of DPPH are paired, the color becomes lighter, the absorption at the maximum absorption wavelength becomes smaller, and the color change is in a stoichiometric relationship with the number of paired electrons, which can be used to evaluate the free radical scavenging situation. Specifically, 4 ml of the test liquid was added to 2 ml of 0.2 mmol/L DPPH free radical solution, and the absorbance ( Ai ) was measured at 517 nm after reacting in a water bath at 25 ℃ for 20 min. A blank test was carried out with 1 ml of distilled water instead of the test liquid.
DPPH scavenging rate (%)=(1- A1-A2A3 )×100
Wherein A 1 is DF 4 ml+DPPH free radical 2.0 ml; A 2 is DF 4 ml+95% ethanol 2.0 ml; and A 3 is distilled water 4 ml+DPPH free radical 2.0 ml.
Determination of total phenol content
Preparation of sample solution: At first, 1 g of sample powder was added into an Erlenmeyer flask, and added with 70% ethanol solution at a ratio of 1∶30 ( w/v ). The mixture was extracted with shaking in a constant temperature water-bath shaker (800 r/min) at 30 ℃ for 24 h. After the extraction, centrifugation was performed at 3 000 r/min for 10 min, and the obtained supernatant was diluted to 50 ml with the original solvent. The resulting solution was added into a brown bottle, which was placed in the refrigerator at -20 ℃ for later testing. A 0.5 ml of sample at a certain concentration was added with 2.5 ml of 0.2 N Folin-Ciocalteu reagent, shaken for 30 s and allowed to react for 5 min; the solution was then added with 2 ml of 7.5% ( w/v ) Na2CO3 solution, shaken for 20 s and allowed to react for 2 h; and the absorbance was measured at 760 nm, and a blank control using deionized water instead of the sample was also tested. The total phenol content was expressed in gallic acid equivalent (GAE/g, dry basis). Dietary fiber molecular weight analysis
High-performance gel permeation chromatography (GPC) was used to determine the molecular weight of the sample under following test conditions: instrument: DAWN EOS laser light scattering instrument of American wyatt company, experimental temperature: (35.0±0.1) ℃, solvent: 0.1 mol/L NaNO3 solution, laser wavelength: 690.0 nm, solvent flow rate: 0.5 ml/min, gel column: Shodex 806HQ analytical column, and injection volume: 300 μl.
Determination method: A DF sample was dissolved in deionized water to a concentration of 5 mg/ml. Into a 50 ml Erlenmeyer flask, 40 mg of the sample solution and 10 ml of mobile phase were added in order. After shaking, the solution was stood at room temperature for 8 h. LC determination was performed with an injection volume of 300 μl, and the chromatogram was recorded. The weight-average molecular weight was calculated using professional software.
Electron microscopic observation of dietary fiber
HA-DF and VF-DF were pulverized and sieved with a 0.5 mm sieve. Gold plating was performed by ion sputtering method. The prepared samples were analyzed and observed by scanning electron microscope, obtaining corresponding scanning electron microscope photos.
Data processing
Design-Expert 7.0 was adopted for experiment design and data processing.
Agricultural Biotechnology2020
Results and Analysis
Effects of different drying methods on water solubility, water holding capacity, oil holding capacity and swelling capacity of DF
It can be seen from Table 1 that the physical and chemical properties of peanut shell DF were improved after vacuum freeze-drying. The water solubility, water holding capacity, oil holding capacity and swelling capacity of HA-DF were 2.15%, 7.63 g/g, 7.73 g/g and 10.35 ml/g, respectively; and the values of VF-DF increased to 3.85%, 14.98 g/g, 15.25 g/g and 15.85 ml/g, respectively. The reason is that traditional drying can cause the material to shrink and damage the cells, while the structure of a sample will not be destroyed during the freeze-drying process, because the solid component is supported by the solid ice in its place and when the ice sublimates, it will leave pores in the remaining dry material, so that the product can keep its biological and chemical structure and the integrity of activity［17］.
Effects of different drying methods on total phenol content (TPC) and free radical scavenging effect of DF Modern medicine proves that free radicals produced by lipid oxidation play an important role in the initiation and promotion of cancer tumor formation. The free radicals produced by the body in the process of metabolism include superoxide anion free radicals, hydroxyl free radicals, and hydroperoxy free radicals. Among them, hydroxyl free radicals are the most dangerous free radicals, and polyphenols and polysaccharides in dietary fiber have the ability to scavenge superoxide anion free radicals and hydroxyl free radicals, and have proved to be unique in the treatment of cardiovascular diseases and Alzheimers disease. The changes of total phenol content in DF after different drying methods are shown in Fig. 1. After vacuum freeze-drying, total phenol content was increased to a certain extent. The total phenol contents in HA-DF and VF-DF were 2.623 and 5.173 mg GAE/g, respectively. The total phenol content can indirectly reflect the value of antioxidant capacity.
As can be seen from Fig. 2, within a concentration range from 2.0 to 12.0 mg/ml, the·OH scavenging rate had a linear relationship with the DF concentration, and the linear equations for HA-DF and VF-DF were y=10.029x+8.266 7 (R2=0.999 6) and y=12.689x+7.84 (R2=0.999 1) . According to the linear equations, the IC 50 (the mass concentration of DF when scavenging 50% of free radicals) values were 4.16 and 3.32 mg/ml, respectively.
As can be seen from Fig. 3, within a concentration range from 2.0 to 12.0 mg/ml, the O-2·scavenging rate was linearly related to the DF concentration. The linear equations for HA-DF and VF-DF were y=10.206x+8.28 (R2=0.999 1) and y=12.729x +9.433 3 (R2=0.999 7) , respectively. The IC 50 (mass concentration of DF when scavenging 50% of free radicals) values were 4.09 and 3.19 mg/ml, respectively, according to the linear equations.
As can be seen from Fig. 4, within a concentration range from 2.0 to 12.0 mg/ml, the DPPH scavenging rate was linearly related to the DF concentration. The linear equations for HA-DF and VF-DF were y=5.048 6x+10.113 (R2=0.999 3) and y=13.037x+9.686 7 (R2=0.999 2) , respectively. The IC 50 (mass concentration of DF when scavenging 50% of free radicals) values were 7.90 and 3.09 mg/ml, respectively, according to the linear equations.
Molecular weight analysis of dietary fiber
Weight-average molecular weight (MW): The molecular weight of all synthetic polymer compounds and the molecular weight of most natural polymer compounds are heterogeneous, they are mixtures of homologues with different molecular weights, and the average molecular weight calculated by molecular weight is the weight-average molecular weight, the value of which is equal to the sum of the products of the molecular weight of various molecule and their corresponding weight fractions. The number-average molecular weight (Mn) represents one of the average molecular weights of polymers, which is the sum of the products of the fractions of molecules with different molecular weights and their corresponding molecular weights. It can be seen from Table 2 that the weight-average molecular weight of HA-DF was 5.89×105 g/mol, which was greater than that of VF-DF (with a weight-average molecular weight of 2.43×105 g/mol), indicating that HA-DF has a more complete molecular chain. After vacuum freeze-drying, the polysaccharide molecular chain is cut to reduce the molecular weight.
Pd=Mw/Mn is called the polydispersity coefficient and is used to characterize the degree of dispersion. The larger the Pd, the more dispersed the molecular weights. When Pd=1, it means that the molecular weight of the polycondensation product is monodisperse (the same) (Pd=1.03-1.05 means that the molecular weight is approximately monodisperse); when Pd is about 2, the polycondensation product is a free radical product; when Pd=3-5, it means that the polycondensation product is branched; and when Pd=25-30, it means that the polycondensation product is PE. In this study, for HA-DF, Pd=3.23>3, which indicated that the HA-DF was branched. During the polymerization process of polymers, due to the chain transfer of free radical polymerization, as the monomers containing more than three functional groups in the polycondensation process participate in the polycondensation reaction, plus the presence of radiation cross-linking and chemical cross-linking reactions, branch structures extend on linear molecular chains. The branch structures of polymer chains can be generally divided into long branch structures and short branch structures. The length of a long branch chain can be equivalent to that of the main chain, and the length of a short branch chain is similar to that of the longer side chain［18］. However, the Pd of VF-DF was close to 1, indicating that after vacuum freeze-drying, the long branch chain part of the dietary fiber was cut short and the molecular weight distribution was uniform.
Electron microscopic observation of dietary fiber
Scanning electron microscope (SEM) is a new type of electron optical instrument that has developed rapidly in the past three decades. It is characterized by three-dimensional and realistic images showing the shape and size of particles, and has strong adaptability to samples. It is suitable for intuitive research on dietary fiber particles, and can take photos of its representative particle morphology.
Fig. 5 shows the scanning electron microscopy results of the two dietary fibers with a scanning multiple of 3 000 times. It can be seen from the figures that the particles of dietary fiber prepared by the two different drying methods were tiny, and the dietary fiber had slightly wrinkled surface and a loose and flaky structure. However, the VF-DF particles were finer, and the fiber had a clear honeycomb surface structure, evenly distributed. The microstructure of DF is closely related to its functional characteristics, so it is speculated that the VF-DF has good adsorption capacity of cholesterol, sodium cholate and nitrite［19］. Conclusions
Peanut shell dietary fiber HA-DF and VF-DF obtained by the two drying methods had water solubility of 2.15% and 3.85%, water holding capacity of 7.63 and 14.98 g/g, oil holding capacity of 7.73 and 15.25 g/g, and swelling capacity of 10.35 and 15.85 ml/g, respectively. The vacuum freeze-drying treatment improved the water solubility, water holding capacity, oil holding capacity and swelling capacity of peanut shell DF.
The total phenol contents of peanut shell dietary fiber HA-DF and VF-DF obtained by the two drying methods were 2.623 and 5.173 mg GAE/g, respectively, and the IC 50 values of the ·OH, O-2 ·and DPPH scavenging capacity were 4.16 and 3.32 mg/ml, 4.09 and 3.19 mg/ml, and 7.90 and 3.09 mg/ml, respectively. After vacuum freeze-drying treatment, the antioxidant activity of dietary fiber was improved.
The weight-average molecular weights of HA-DF and VF-DF were 5.89×105 and 2.43×105 g/mol, respectively, indicating that HA-DF had a more complete molecular chain, and after vacuum freeze-drying, the polysaccharide molecular chain was cut short, thereby reducing the molecular weight. The polydispersity coefficient of VF-DF was 1.07, and the molecular weight distribution tended to be concentrated.
Observed by scanning electron microscope, the particles of HA-DF and VF-DF were tiny and had slightly wrinkled surface and loose and clear flaky structure, which had pores for adsorbing other substances, but the VF-DF particles were denser and had smaller voids, and the surface had a clear honeycomb structure, evenly distributed.
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