CN115669795A - Method for preparing quercetin-containing emulsion gel by squeezing pretreated soybean protein - Google Patents
Method for preparing quercetin-containing emulsion gel by squeezing pretreated soybean protein Download PDFInfo
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- CN115669795A CN115669795A CN202211418199.0A CN202211418199A CN115669795A CN 115669795 A CN115669795 A CN 115669795A CN 202211418199 A CN202211418199 A CN 202211418199A CN 115669795 A CN115669795 A CN 115669795A
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- soybean protein
- quercetin
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- emulsion gel
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 title claims abstract description 236
- 108010073771 Soybean Proteins Proteins 0.000 title claims abstract description 164
- 235000019710 soybean protein Nutrition 0.000 title claims abstract description 137
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 118
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- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 118
- 229960001285 quercetin Drugs 0.000 title claims abstract description 118
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- 238000000034 method Methods 0.000 title claims abstract description 72
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- 238000001125 extrusion Methods 0.000 claims description 38
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
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- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims description 16
- 229940114124 ferulic acid Drugs 0.000 claims description 16
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims description 16
- 235000001785 ferulic acid Nutrition 0.000 claims description 16
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- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 3
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
A method for preparing quercetin-containing emulsion gel by squeezing pretreated soybean protein belongs to the technical field of soybean protein processing. The invention provides a method for preparing an emulsion gel containing quercetin by extruding pretreated soybean protein, in order to carry quercetin and improve the antioxidant activity of quercetin, and specifically relates to a method for preparing an emulsion gel containing quercetin by taking defatted natural soybean protein as a raw material, covalently combining polyphenol with compound modified natural soybean protein by an extrusion-alkali method, and preparing the emulsion gel containing quercetin by using the compound modified soybean protein. The method further improves the emulsifying property and the gelling property of the soybean protein, so that the prepared emulsion gel has more excellent carrying property and lower cost, and is suitable for industrial production. The emulsion gel prepared by the invention has better encapsulation effect of carrying quercetin, higher bioavailability and stronger effect of protecting the antioxidant activity of quercetin.
Description
Technical Field
The invention belongs to the technical field of soybean protein processing, and particularly relates to a method for preparing quercetin-containing emulsion gel by extruding pretreated soybean protein.
Background
At present, a plurality of carriers for carrying quercetin are emulsion, particles, fibers, micelles, gels, liposomes and the like. After the carriers carry the quercetin, the problems of low water solubility, poor chemical stability, low bioavailability and the like of the quercetin are solved to a certain extent. However, the protective effects on the antioxidant activity of quercetin are not the same when these carriers carry quercetin. In consideration of the problem of food safety, these carriers are prepared by using food-based biomacromolecules as raw materials. However, natural food biomacromolecules have some defects in their own properties, and these proteins are often modified in order to prepare carriers with superior performance. Currently, a single modification method is often used to prepare protein emulsion gels, such as physical modification alone (extrusion alone, ultrasound alone, etc.), and chemical modification alone (alkaline polyphenol covalent bonding alone, phosphorylation alone, succinylation alone, etc.). No method for modifying protein by combining various modification methods and using the modified protein to prepare emulsion gel so as to improve the antioxidant activity of quercetin is found.
Disclosure of Invention
In order to carry quercetin and improve the antioxidant activity of quercetin, the invention provides a method for preparing an emulsion gel containing quercetin by extruding pretreated soybean protein, which comprises the following steps:
(1) Alkaline polyphenol covalent binding modified soy protein: dissolving the soybean protein freeze-dried powder in phosphate buffer solution, stirring for 2h at room temperature to prepare soybean protein solution with the concentration of 2g/100mL, and hydrating at 4 ℃ overnight; adjusting the pH value of the soybean protein solution to 9.0, adding ferulic acid to ensure that the concentration of the ferulic acid in the soybean protein solution is 150 mu mol/g protein, and magnetically stirring for 12h at room temperature; adjusting the pH of the mixed solution to 7.0, then filling the mixed solution into a dialysis bag, dialyzing the mixed solution for 24 hours by using phosphate buffer solution, and obtaining modified soybean protein by freeze drying;
(2) The extrusion treatment further modifies the soy protein: adding distilled water into the modified soybean protein obtained in the step (1), adjusting the water content of the modified soybean protein to 30% -70%, standing for 24h to balance water, performing extrusion treatment, drying the extruded sample in a drying oven at 45 ℃ to constant weight, grinding into powder, and preparing to obtain the extrusion-alkali polyphenol covalent bonding composite modified soybean protein;
(3) Dissolving the composite modified soybean protein obtained in the step (2) in a phosphate buffer solution, stirring for 2 hours at room temperature, and placing at 4 ℃ for hydration overnight to obtain a composite modified soybean protein solution; dissolving 20mg quercetin in 3mL ethanol solution with volume fraction of 25% at 65 deg.C, adding 10mL soybean oil, heating at 65 deg.C for 30min, and removing ethanol to obtain soybean oil containing quercetin; taking 10mL of the compound modified soybean protein solution, adding 1.76mL of soybean oil containing quercetin, stirring in a dark place, and homogenizing to obtain a crude emulsion; placing the crude emulsion in an ultrasonic cell pulverizer, treating for 15min under 400W water bath condition at 25 deg.C, centrifuging at low speed to remove air bubbles, adding gluconolactone to adjust pH to 4.5, magnetically stirring, and cold preserving at 4 deg.C for 12 hr to obtain emulsion gel.
Further limiting, the preparation method of the soybean protein freeze-dried powder in the step (1) comprises the steps of mixing defatted soybean powder and ultrapure water according to a material-liquid ratio of 1g.
Further, the phosphate buffer solution in the step (1) and the step (3) has the concentration of 0.02mol/L and the pH of 7.0.
Further defined, the pH is adjusted in step (1) by using 2mol/L NaOH solution and 2mol/L HCl solution.
Further defined, the cut-off molecular weight of the dialysis bag in the step (1) is 8000-14000Da.
Further defined, the dialysis of step (1) is performed with phosphate buffer every 6h to ensure removal of free phenolics.
Further defined, the step (2) is to adjust the moisture content of the modified soy protein to 40%.
Further, the conditions of the extrusion treatment in the step (2) are that the rotation speed of a screw of a main machine is 300r/min, the rotation speed of a screw of a feeding area is 200r/min, and the extrusion temperatures from the first area to the fifth area are respectively 175 ℃,140 ℃,120 ℃,100 ℃ and 60 ℃.
Further limiting, the feed-liquid ratio of the complex modified soybean protein in the step (3) to the phosphate buffer solution is 1g.
Further limiting, the stirring in the dark place in the step (3) is magnetic stirring in the dark place for 2 hours.
Further defined, the homogenization conditions in step (3) are 10000rpm and 2.5min.
Further, the low-speed centrifugation in the step (3) is performed under the condition of 1000rpm and stirring for 1min.
Further defined, the final concentration of gluconolactone in step (3) is 15mg/mL.
Further, the magnetic stirring time in the step (3) is 30s, and the reaction is carried out for 2h at 40 ℃ after the magnetic stirring.
The invention also provides the emulsion gel for protecting the antioxidant activity of the quercetin, which is obtained by the preparation method.
The invention has the beneficial effects that:
the invention takes natural soybean protein, ultrasonic modified soybean protein, extrusion treated modified soybean protein, free radical method polyphenol covalent bonding modified soybean protein, alkali method polyphenol covalent bonding modified soybean protein and extrusion-alkali method polyphenol covalent bonding composite modified soybean protein as gel matrix to prepare the soybean protein emulsion gel for carrying quercetin. The method for preparing the quercetin-containing emulsion gel from the modified soy protein is characterized in that the influence of different modification methods on the in-vitro digestion rule of the quercetin emulsion gel is investigated by researching the basic properties of the natural soy protein and the modified soy protein quercetin emulsion and the emulsion gel, the protection effect of a soy protein emulsion gel carrying system on the antioxidant activity of quercetin is discussed, the optimal soy protein modification method is determined, and the method for preparing the quercetin-containing emulsion gel from the modified soy protein is further obtained. The invention specifically takes defatted natural soybean protein as a raw material, utilizes an extrusion-alkali method polyphenol to covalently combine with compound modified natural soybean protein, and adopts the compound modified soybean protein to prepare the quercetin-containing emulsion gel.
The invention provides an excellent carrier for hydrophobic nutrient substance quercetin, and simultaneously provides a theoretical basis for carrying other hydrophobic substances, thereby having wide reference significance. Compared with the prior art, the method provided by the invention adopts the soy protein as the main raw material, so that the potential safety hazard caused by using the traditional synthetic gel matrix is eliminated; meanwhile, the emulsifying performance and the gel performance of the soybean protein are further improved by adopting various modes of physical modification, polyphenol modification and physical-polyphenol composite modification, so that the prepared emulsion gel has more excellent carrying performance and lower cost, and is suitable for industrial production. Compared with the soybean protein emulsion gel prepared from natural soybean protein, the soybean protein emulsion gel prepared from the modified soybean protein (particularly the soybean protein emulsion gel prepared from the compound modified soybean protein) has better encapsulation effect for carrying quercetin, higher bioavailability and stronger effect on the antioxidant activity protection effect of quercetin.
Drawings
Fig. 1 is a flow chart of preparation of soy protein lyophilized powder.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to specific embodiments and the accompanying drawings. The experimental procedures used in the examples which follow are conventional unless otherwise specified, and the materials, reagents, methods and apparatus used are conventional in the art and are commercially available to those skilled in the art without specific reference to any other materials, reagents, methods and apparatus.
Preparation of natural soybean protein:
the freeze-dried soybean protein powder used in the invention is natural soybean protein, the preparation flow chart is shown in figure 1, the freeze-dried soybean protein powder is prepared by using defatted soybean meal (the defatted soybean meal is ground into powder and then sieved by a 50-mesh sieve) as a raw material, and the preparation method specifically comprises the following steps:
mixing defatted soybean powder and ultrapure water according to a material-liquid ratio of 1g to 10mL, adjusting the pH value to 8.5, stirring in a water bath for 2 hours, centrifuging to obtain a supernatant, adjusting the pH value to 4.5, standing for 2 hours, centrifuging to obtain a precipitate, dissolving by using the ultrapure water, adjusting the pH value to 7.0 to obtain soybean protein, and freeze-drying to obtain the soybean freeze-dried powder.
The invention adopts different protein modification methods to obtain modified soybean protein, then utilizes natural soybean protein and the modified soybean protein obtained by different modification methods to prepare the emulsion gel containing quercetin, and finally determines the optimal modified soybean protein by measuring the properties of the emulsion gel.
The invention relates to a soybean protein modification method which comprises the following steps:
1. modification of natural soybean protein by ultrasonic treatment
Dissolving the soybean protein freeze-dried powder in ultrapure water (10%, w/v), magnetically stirring for 2h at room temperature, and then putting the mixture into a refrigerator at 4 ℃ for hydration overnight. Placing the soybean protein dispersion liquid in a flat-bottom conical flask, and carrying out ultrasonic treatment by using a JY92-2D ultrasonic cell crusher to prepare the ultrasonic modified soybean protein.
Optimizing ultrasonic conditions: setting the temperature of ultrasonic waves to be 55 ℃, setting the time to be 30min, setting the power to be 100W, 200W, 300W, 400W and 500W respectively, and selecting the optimal ultrasonic power; setting the ultrasonic power at 400W, the temperature at 55 ℃ and the time at 10min,20min, 30min, 40min and 50min respectively, and selecting the optimal ultrasonic time; the optimum ultrasonic temperature was selected by setting the ultrasonic power at 400W, the time at 30min, and the temperatures at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C, respectively.
The optimal ultrasonic treatment conditions are determined by the texture characteristics and water holding capacity of the prepared emulsion gel: 30mL of soybean protein solution (10%, W/v, pH 7.0) was poured into a 50mL three-necked flask, and the ultrasonically modified soybean protein was prepared under conditions of an ultrasonic power of 400W, an ultrasonic temperature of 55 ℃ and an ultrasonic time of 30 min.
2. Extrusion treatment for modifying natural soybean protein
Weighing certain mass of soybean protein freeze-dried powder, adding distilled water to adjust the water content to be 30%,40%,50%,60% and 70% (mass fraction, calculated on a protein dry basis), standing for 24 hours to balance the water content, and setting extrusion conditions, wherein the rotation speed of a main machine screw is 300r/min, the rotation speed of a feeding zone screw is 200r/min, and the extrusion temperatures from a first zone to a fifth zone are 175 ℃,140 ℃,120 ℃,100 ℃ and 60 ℃. And drying the extruded sample in a drying oven at 45 ℃ to constant weight, grinding the dried sample into powder, and preparing the extruded modified soybean protein.
The water content of the soybean protein freeze-dried powder after adding distilled water is optimized, and the optimal extrusion treatment conditions are determined through the texture characteristics and the water retention of the prepared emulsion gel: adding distilled water to the soybean protein to adjust the water content to 40 percent (mass fraction, calculated on a protein dry basis), standing for 24 hours to balance the water content, and setting extrusion conditions, wherein the rotating speed of a main machine screw is 300r/min, the rotating speed of a feeding area screw is 200r/min, and the extrusion temperature is 175 ℃,140 ℃,120 ℃,100 ℃ and 60 ℃ respectively from a first area to a fifth area. And drying the extruded sample in a drying oven at 45 ℃ to constant weight, grinding the dried sample into powder, and preparing the extruded modified soybean protein.
The invention respectively obtains the ultrasonic modified soybean protein and the extrusion modified soybean protein by utilizing the optimal parameters of the ultrasonic and extrusion modification methods, respectively prepares the emulsion gel according to the preparation method of the emulsion gel containing quercetin, and then compares the quality-structure characteristics and the water binding capacity of the obtained different emulsion gels.
The comparison result shows that the texture characteristics of the ultrasonic modified soy protein quercetin emulsion gel are that the hardness is 109.25 +/-3.87 g, the elasticity is 0.871 +/-0.003 mm, the cohesiveness is 0.487 +/-0.005, the chewiness is 52.03 +/-1.34, and the water retention is 84.20 +/-0.44%; the quality structure characteristic of the extrusion modified soy protein quercetin emulsion gel is that the hardness is 112.88 plus or minus 3.25g, the elasticity is 0.865 plus or minus 0.011mm, the cohesiveness is 0.492 plus or minus 0.009, the chewiness is 54.31 plus or minus 1.49, and the water retention is 84.90 plus or minus 0.25%. From the above comparison results, it is known that the modified soybean protein prepared by the extrusion modification method is excellent in the texture properties and water retention of the quercetin-containing emulsion gel, and therefore, the extrusion modification is determined as the optimum physical modification method.
3. Modification of natural soybean protein by polyphenol free radical method covalent bonding
Dissolving the soybean protein freeze-dried powder in phosphate buffer solution (PBS solution, 0.02mol/L, pH 7.0), wherein the concentration of the soybean protein is 2 percent (w/v), magnetically stirring for 2h at room temperature, and then placing the mixture in a refrigerator at 4 ℃ for overnight hydration; taking 0.1% (v/v) free radical system (5 mmol/LH) 2 O 2 0.1mol/L ascorbic acid, 0.1mol/LFeCl 3 ) Adding into the soybean protein solution, and stirring at room temperature for 2h to oxidize the soybean protein; adding ferulic acid to make the final concentration of soybean protein 2% (w/v), stirring at room temperature for 24h; and after the reaction is finished, adding 6-hydroxy-2, 5,7, 8-tetramethyl chromane-2-carboxylic acid (Trolox, 1 mmol/L) to terminate the oxidation reaction, putting the solution after the reaction into a 10kDa dialysis bag, dialyzing for 24h (changing the phosphate buffer solution every 6 h) by using a phosphate buffer solution (0.02 mol/L, pH 7.0), and freeze-drying for 24h at (-80 ℃ for 24 h) to obtain the free radical method polyphenol covalent binding modified soybean protein.
Optimizing the addition amount of the ferulic acid, changing the addition amount of the ferulic acid to ensure that the final concentrations of the ferulic acid are respectively 10 mu mol/g protein, 50 mu mol/g protein, 150 mu mol/g protein, 250 mu mol/g protein and 500 mu mol/g protein, and determining the optimal addition amount of the ferulic acid to be 150 mu mol/g protein through the texture characteristics and the water retention property of the prepared emulsion gel after optimization.
4. Modification of natural soybean protein by polyphenol alkali covalent bonding
Dissolving the soybean protein freeze-dried powder in phosphate buffer solution (PBS solution, 0.02mol/L, pH 7.0), wherein the concentration of the soybean protein is 2 percent (w/v), magnetically stirring for 2h at room temperature, and then placing the mixture in a refrigerator at 4 ℃ for overnight hydration; adjusting the soy protein solution to pH 9.0 with 2mol/L NaOH solution, adding ferulic acid to a final concentration of 2% (w/v) of soy protein, stirring the mixture at room temperature for 12h to ensure adequate reaction of FA with SPI, during which time pH 9.0 is maintained with NaOH solution; after the reaction is finished, adjusting the pH value to 7.0 by using 2mol/L HCl solution, putting the solution after the reaction into a dialysis bag with 10kDa, dialyzing for 24h (changing the phosphate buffer solution every 6 h) by using phosphate buffer solution (0.02 mol/L, pH 7.0), and freeze-drying (-80 ℃ for 24 h) to prepare the alkali polyphenol covalent binding modified soybean protein.
Optimizing the addition amount of the ferulic acid, changing the addition amount of the ferulic acid to ensure that the final concentrations of the ferulic acid are respectively 10 mu mol/g protein, 50 mu mol/g protein, 150 mu mol/g protein, 250 mu mol/g protein and 500 mu mol/g protein, and determining the optimal addition amount of the ferulic acid to be 150 mu mol/g protein through the texture characteristics and the water retention property of the prepared emulsion gel after optimization.
According to the invention, the polyphenol covalent bonding modified soybean protein by the free radical method and the polyphenol covalent bonding modified soybean protein by the alkaline method are respectively obtained by utilizing the optimal parameters of the polyphenol free radical covalent bonding and the polyphenol covalent bonding by the alkaline method, the emulsion gel is respectively prepared according to the following preparation method of the emulsion gel containing quercetin, and then the quality structure characteristics and the water retention of the obtained different emulsion gels are compared, and the result shows that the quality structure characteristics of the polyphenol covalent bonding modified soybean protein quercetin emulsion gel by the free radical method are that the hardness is 155.98 +/-4.83 g, the elasticity is 0.904 +/-0.009 mm, the cohesiveness is 0.532 +/-0.013, the chewiness is 83.18 +/-3.84, and the water retention is 86.13 +/-0.45%; the texture characteristics of the alkaline polyphenol covalent binding modified soy protein quercetin emulsion gel are that the hardness is 214.99 +/-4.98 g, the elasticity is 0.913 +/-0.013 mm, the cohesiveness is 0.543 +/-0.002, the chewiness is 118.28 +/-4.37, and the water retention is 90.26 +/-0.25%. The comparison results show that the quality structure property and the water retention performance of the quercetin-containing emulsion gel prepared from the soybean protein modified by the alkaline polyphenol covalent bonding prepared by the polyphenol alkaline covalent bonding modification method are better, so that the polyphenol alkaline covalent bonding modification method is determined to be the optimal chemical modification method.
5. Extrusion-alkali polyphenol covalent bonding composite modification method for modifying natural soybean protein
The invention combines the obtained optimal physical modification method with the optimal chemical modification method to obtain an extrusion-alkali method polyphenol covalent bonding composite modification method, which comprises the following steps:
dissolving the soybean protein freeze-dried powder in phosphate buffer solution (0.02 mol/L, pH 7.0), stirring at room temperature for 2h to prepare a soybean protein solution with the concentration of 2% (w/v), and putting the soybean protein solution into a refrigerator at 4 ℃ for hydration overnight; adjusting the pH of the soybean protein solution to 9.0 with 2mol/L NaOH solution, adding ferulic acid to make the concentration of ferulic acid in the soybean protein solution be 150 μmol/g protein, magnetically stirring the mixture at room temperature for 12h to ensure that FA and SPI react sufficiently, and maintaining the pH of the solution at 9.0 during the period; after the reaction is finished, adjusting the pH of the mixed solution to 7.0 by using 2mol/L HCl, then filling the reacted solution into a dialysis bag (with the molecular weight of 10000 Da), and dialyzing for 24h (changing water every 6 h) by using a phosphate buffer solution (0.02 mol/L, pH 7.0) at room temperature to ensure that free phenolic substances are removed; and finally, freeze-drying the mixture at-80 ℃ for 24h to prepare the alkaline polyphenol covalent binding modified soybean protein.
Adding distilled water into modified soybean protein covalently bound with alkaline polyphenol to adjust water content, standing for 24h to balance water content, and setting extrusion conditions. And drying the extruded sample in a drying oven at 45 ℃ to constant weight, grinding the sample into powder, and preparing the extrusion-alkali covalent bonding composite modified soybean protein.
Optimizing the extrusion conditions: the water content is respectively set to 30%,40%,50%,60% and 70% (mass fraction, based on dry protein basis), the mixture is placed for 24 hours to balance the water content, and the extrusion conditions are set as follows: the rotation speed of a screw of the main machine is 300r/min, the rotation speed of a screw of the feeding area is 200r/min, and the extrusion temperatures from the first area to the fifth area are respectively 175 ℃,140 ℃,120 ℃,100 ℃ and 60 ℃. The optimum moisture content is selected.
The extrusion treatment of the alkaline polyphenol covalent binding modified soybean protein determines the optimal conditions through the texture characteristics and the water retention of the prepared emulsion gel: adding distilled water into the modified soybean protein with covalent bonding of alkaline polyphenol to adjust the water content to 40% (mass fraction, calculated on a protein dry basis), standing for 24h to balance the water content, and setting the extrusion conditions, wherein the rotation speed of a main machine screw is 300r/min, the rotation speed of a feeding zone screw is 200r/min, and the extrusion temperatures from the first zone to the fifth zone are respectively 175 ℃,140 ℃,120 ℃,100 ℃ and 60 ℃. And drying the extruded sample in a drying oven at 45 ℃ to constant weight, grinding the sample into powder, and preparing the extrusion-alkali covalent bonding composite modified soybean protein.
The preparation method of the quercetin-containing emulsion gel comprises the following steps:
respectively dissolving natural soybean protein or modified soybean protein (ultrasonic modification, extrusion modification, free radical method polyphenol covalent bonding modification, alkaline method polyphenol covalent bonding modification, extrusion-alkaline method polyphenol covalent bonding composite modification) obtained by the different methods in phosphate buffer solution (0.01 mol/L, pH 7.0), magnetically stirring for 2h at room temperature, and placing in a refrigerator at 4 ℃ for hydration overnight to obtain soybean protein/modified soybean protein solution; dissolving 20mg quercetin in 65 deg.C 3mL ethanol solution (6.6 mg/mL) with volume fraction of 25%, adding 10mL soybean oil into the ethanol solution containing quercetin, heating at 65 deg.C for 30min, and evaporating to remove ethanol to obtain soybean oil containing quercetin; taking 10mL of soybean protein/modified soybean protein solution, adding 1.76mL of soybean oil containing quercetin, stirring at room temperature in a dark place for 2h by magnetic force, and homogenizing by using a high-speed homogenizer (10000 r/min,2.5 min) to obtain a crude emulsion; placing the crude emulsion in an ultrasonic cell pulverizer, performing ultrasonic treatment (400W, 15min) with a titanium probe with a diameter of 0.636cm, and maintaining the ultrasonic temperature at about 25 deg.C by ice-water bath to obtain O/W type soybean protein/modified soybean protein emulsion loaded with quercetin; and (3) carrying out low-speed centrifugation (1000 r/min,1 min) on the quercetin-loaded emulsion to remove air bubbles in the emulsion. Adding a certain amount of glucono-delta-lactone (GDL) powder into the emulsion to a final concentration of 15mg/mL, so that the pH value of the emulsion is gradually reduced and finally stabilized at 4.5 (the isoelectric point of the soybean protein); magnetically stirring the quercetin emulsion added with the GDL at room temperature for 30s, and reacting at 40 ℃ for 2h; then, the sample is refrigerated for 12h at 4 ℃ to prepare the quercetin-loaded soybean protein/modified soybean protein emulsion gel.
The properties of the quercetin-containing emulsion gel prepared from modified soy protein obtained by different modification methods were measured:
(1) Texture analysis
The quercetin emulsion gels were analyzed for texture according to the Hu et al (Hu H, wu J, eunice C Y, et al. Effects of ultrasound on structural and systemic properties of Soy Protein Isolate (SPI) dispersions [ J ]. Food Hydrocolloids,2013, 30 (2): 647-655.) study with minor modifications. The hardness, viscosity, elasticity, cohesiveness and the like of the quercetin emulsion gel are tested by using a TA-XT plus texture analyzer. The experimental conditions were: and the speed of the P/0.5 probe before the test is 1mm/s, the test speed is 5mm/s, the speed after the test is 5mm/s, and the trigger force is 5.0g. The sample was compressed to 30% of the original thickness with a 5s time interval between compressions. The results of the texture analysis are shown in Table 1.
TABLE 1 texture Properties of Natural/modified Soy protein Quercetin emulsion gels
The texture properties of the natural soy protein quercetin emulsion gel and the modified soy protein quercetin emulsion gel were measured using a TA-XTplus texture analyzer, and the test results are shown in table 1. As can be seen from table 1, compared to the natural soy protein quercetin emulsion gel, the hardness, elasticity, cohesiveness and chewiness of the modified soy protein (ultrasonic modification, extrusion modification, radical polyphenol covalent bonding modification, alkaline polyphenol covalent bonding modification, extrusion-alkaline polyphenol covalent bonding composite modification) quercetin emulsion gel are all significantly improved. Of the two physical modifications (ultrasonic modification and extrusion modification), the extrusion modification effect is best; of the two chemical modifications (free radical method polyphenol covalent bonding modification and alkaline method polyphenol covalent bonding modification), alkaline method polyphenol covalent bonding modification is the best. However, the single physical modification or the single chemical modification has no good effect of the covalent combination and compound modification of polyphenol by an extrusion-alkali method.
(2) Measurement of Water holding Property
The Water Hold (WHC) measurement of quercetin Emulsion Gels was performed according to Wu et al (Wu M, xiongY L, chen J, et al, rheologicals and Microstructural Properties of Porcine Myofibrillar Protein-Lipid Emulsion compositions Gels [ J ]. Journal of food Science,2009, 74 (4): 77-87.) with minor modifications to the study method. And centrifuging the PE centrifuge tube filled with 5mL of a quercetin emulsion gel sample at 4 ℃ at 10000r/min for 15min. After centrifugation, the tube was inverted to remove water and the remaining water was carefully removed using dry filter paper. Calculating the water holding capacity of the quercetin emulsion gel according to the formula (1):
wherein: w t Is the weight of quercetin emulsion gel before centrifugation, and the unit is g; w r Is the weight of the quercetin emulsion gel after centrifugation and water removal, and the unit is g; w o Is the weight of the PE centrifuge tube in g.
The water holding capacity of the emulsion gel is a key factor of gel stability, and the interaction between a gel network and water molecules can be reflected. The water binding capacity of the quercetin emulsion gel prepared by taking natural soybean protein and modified soybean protein (ultrasonic modification, extrusion modification, free radical method polyphenol covalent bonding modification, alkali method polyphenol covalent bonding modification and extrusion-alkali method polyphenol covalent bonding composite modification) as gel substrates is respectively as follows: 82.50 plus or minus 0.36 percent, 84.20 plus or minus 0.44 percent, 84.90 plus or minus 0.25 percent, 86.13 plus or minus 0.45 percent, 90.26 plus or minus 0.25 percent and 93.17 plus or minus 0.38 percent, therefore, the quercetin emulsion gel obtained by the extrusion-alkali method polyphenol covalent bonding composite modification method has the best water retention property.
(3) Determination of gel encapsulation efficiency of quercetin emulsion
50mg of quercetin emulsion freeze-dried powder is weighed and dissolved in 5mL of mixed solution with ethanol with the volume fraction of 25% and Tween 80 with the volume fraction of 0.5% in equal volume. Centrifuging at 14000r/min for 30min at 4 deg.C, collecting the bottom solution of the centrifuge tube (containing unencapsulated quercetin), diluting with 70% ethanol solution by a proper amount, and measuring the absorbance at 374 nm. Calculating the content of the quercetin by using a quercetin-ethanol standard curve, and calculating the encapsulation rate of the quercetin emulsion gel according to the formula (2):
wherein: a. The total Is the initial amount of quercetin added to the oil phase; a. The outer Is the amount of unencapsulated quercetin present in the external aqueous phase.
The encapsulation rate of the quercetin emulsion gel represents the encapsulation degree of the quercetin by the emulsion gel, and is an important index for measuring the carrying efficiency of an emulsion gel carrying system. The gel encapsulation rate of the quercetin emulsion prepared by taking natural soybean protein and modified soybean protein (ultrasonic modification, extrusion modification, free radical method polyphenol covalent bonding modification, alkaline method polyphenol covalent bonding modification and extrusion-alkaline method polyphenol covalent bonding composite modification) as gel substrates is 73.70 +/-1.23%, 80.17 +/-0.97%, 82.57 +/-0.97%, 87.69 +/-0.87%, 88.57 +/-0.86% and 91.13 +/-0.96% respectively. The result shows that the natural soybean protein and the modified soybean protein quercetin emulsion gel both have good encapsulation capacity, the modified soybean protein quercetin emulsion gel has better encapsulation effect, and the composite modified soybean protein quercetin emulsion gel has the best encapsulation effect.
(4) Determination of gel lipolysis rate of quercetin emulsion
And continuously dropwise adding 0.25mol/L NaOH solution to neutralize free fatty acid released by lipid digestion during the simulated small intestine digestion stage by adopting a pH-stat method. During titration, the pH in the digestion system is maintained to be constantly equal to 7.0, the temperature is constantly equal to 37 ℃, consumption of dropwise adding NaOH is recorded after the quercetin emulsion gel starts in vitro digestion at 1min,3min,5min,10min,20min,60min,90min,120min,150min,180min,210min and 240min, and the lipolysis rate of the quercetin emulsion gel is calculated according to the formula (3):
in the formula: v NaOH The volume of dropping NaOH; m is NaOH Dropping NaOH with molar concentration; m oil Is the average molecular weight, W, of soybean oil oil Is the weight of oil in the digestive system at the beginning of the reaction.
Under the condition of in vitro simulated small intestine digestion, the pH-stat method is adopted to research the digestion condition of the gel lipid of the quercetin emulsion. At the beginning of digestion, the lipolysis rate rapidly increases, and the release rate of free fatty acid rapidly increases; the release rate of free fatty acids gradually flattens out as the digestion time increases. After digestion in small intestine for 240min, the fat decomposition rates of natural soybean protein and modified soybean protein (ultrasonic modification, extrusion modification, free radical method polyphenol covalent bonding modification, alkaline method polyphenol covalent bonding modification, extrusion-alkaline method polyphenol covalent bonding composite modification) quercetin emulsion gel are respectively as follows: 71.20 +/-1.24%, 81.26 +/-1.44%, 83.28 +/-1.53%, 89.57 +/-1.44%, 92.45 +/-1.65% and 93.98 +/-1.22%. The test result of the fat decomposition rate of the quercetin emulsion gel shows that the fat decomposition rate of the compound modified quercetin emulsion gel is highest when the soybean protein is modified, and the fat digestion rate of the grease can be effectively improved.
(5) Determination of biological availability of quercetin emulsion gel
Immediately after the simulated small intestine digestion stage, the digesta was chilled in an ice water bath. Then, centrifugation (15000 r/min,40 min) was carried out at 4 ℃; after centrifugation, the middle micelle layer was aspirated, filtered using a 0.22mm filter, and after dilution to an appropriate multiple with a 70% volume fraction ethanol solution, absorbance was measured at 374 nm. Calculating the content of the quercetin by using a quercetin-ethanol standard curve, wherein the biological availability of the quercetin emulsion gel is calculated according to the formula (4):
in the formula: w is the content of quercetin in the mixed micelle; w is a group of 1 Is the content of quercetin in the original emulsion gel sample.
The biological availability rates of the natural soybean protein and the modified soybean protein (ultrasonic modification, extrusion modification, polyphenol covalent binding modification by a free radical method, polyphenol covalent binding modification by an alkali method, and polyphenol covalent binding composite modification by an extrusion-alkali method) quercetin emulsion gel are respectively as follows: 33.40 plus or minus 0.93 percent, 36.90 plus or minus 0.74 percent, 37.69 plus or minus 0.51 percent, 44.19 plus or minus 0.47 percent, 47.47 plus or minus 0.46 percent and 49.47 plus or minus 0.87 percent. The test result shows that the biological availability of the soybean protein quercetin emulsion gel is in positive correlation with the release rate of Free Fatty Acid (FFA), the biological availability of the bioactive substances in the emulsion gel is highly dependent on the digestion of lipid, and the higher the lipolysis degree is, the higher the biological availability is. Therefore, the biological availability of the compound modified quercetin emulsion gel covalently bonded by the polyphenol through the extrusion-alkali method is the highest.
(6) Determination of anti-oxidation activity of quercetin emulsion gel
In order to study the protective effect of the latex gel on the biological activity of quercetin, the antioxidant activity of a pure quercetin solution before and after digestion and a quercetin latex gel extract after digestion were measured respectively. 7.5mL of undigested pure quercetin solution, digested pure quercetin solution and quercetin emulsion gel were put in a dialysis bag, respectively, and dialyzed with 12mL of a mixed solution of ethanol and methanol (1, v/v), and an extract outside the dialysis bag was collected. The antioxidant activity of the extract was measured as follows.
Determination of DPPH radical scavenging Activity
The DPPH radical scavenging activity of the extracts was determined according to the research protocol of Medina et al (Medina-Perez G, estefes-Duarte J A, afana dor-Barajas L N, et al. Encapsulation precursors inhibitors and Indian identification Activities of cactus Acid group biological units, 2020, 25 (23): 5736.) with minor modifications. 0.1mmol/L DPPH ethanol solution is prepared, and is stored for 2 hours at room temperature in the dark. Then, 1mL of the sample was reacted with 4mL of an ethanol solution of LDPPH at room temperature in the dark for 30min, and then the absorbance was measured at 517 nm. DPPH radical clearance was calculated from formula (5):
in the formula: a is the absorbance of the mixed solution of the sample and DPPH ethanol; a. The i Is the absorbance of the sample with the ethanol solution; aj is the absorbance of the ethanol solution of DPPH alone.
Determination of ABTS free radical scavenging Activity
ABTS free radical scavenging activity of extracts was determined according to the research method of Medina et al (Medina-Perez G, estefes-Duart JA, afana dor-Barajas LN, et al. ABTS stock solution (containing 2.45mmol/L potassium persulfate) with concentration of 7mmol/L was prepared with phosphate buffer solution (0.1 mol/L, pH 7.4), and stored at room temperature in dark for 16h. The ABTS stock solution was diluted to an appropriate ratio with a phosphate buffer solution (0.1 mol/L, pH 7.4) to adjust the absorbance to 0.7. + -. 0.1, and the diluted ABTS solution was used in the subsequent assay reaction. mu.L of the sample was added to a 2mL of the LABTS solution (absorbance: 0.7. + -. 0.1), and the mixture was reacted at room temperature for 5min in the absence of light, and the absorbance was measured at 734 nm. The ABTS radical clearance is calculated from formula (6):
in the formula: a. The 0 Is the absorbance of the ABTS solution; a is the absorbance of the sample after mixing with the ABTS solution.
Quercetin as a natural flavonoid compound has physiological functions of antioxidation, antibiosis, anti-inflammation and the like and medicinal value. Because the oral absorption and utilization rate of the quercetin is low, the research on the physiological activity protection effect of the quercetin by the carrier system has certain significance. The protective effect of the emulsion gel carrying system on the antioxidant activity of quercetin is analyzed by measuring the antioxidant activity of undigested pure quercetin solution (QCT), digested pure quercetin solution (QCT-ED) and quercetin emulsion gel extract.
In a complete in vitro digestion model, DPPH free radical clearance rates of extracts of QCT, QCT-ED, natural soybean protein and modified soybean protein (ultrasonic modification, extrusion modification, polyphenol covalent binding modification by a free radical method, polyphenol covalent binding modification by an alkaline method and polyphenol covalent binding composite modification by an extrusion-alkaline method) after quercetin emulsion gel digestion are respectively 92.51 +/-2.53%, 28.40 +/-1.97%, 32.85 +/-1.53%, 54.35 +/-0.97%, 56.17 +/-1.32%, 63.86 +/-0.77%, 65.42 +/-0.76% and 70.09 +/-1.02%.
In the complete in vitro digestion model, ABTS free radical clearance rates of the extracts of QCT, QCT-ED, natural soybean protein and modified soybean protein (ultrasonic modification, extrusion modification, free radical method polyphenol covalent binding modification, alkaline method polyphenol covalent binding modification and extrusion-alkaline method polyphenol covalent binding composite modification) and quercetin emulsion gel after digestion are respectively 98.72 +/-1.35%, 34.62 +/-2.65%, 44.40 +/-2.33%, 78.49 +/-0.97%, 79.66 +/-1.16%, 83.19 +/-0.77%, 87.89 +/-0.76% and 88.93 +/-0.27%.
By integrating the test results of the DPPH and ABTS free radical clearance rate of the extract after the in vitro digestion of the quercetin emulsion gel, compared with the quercetin which is not embedded, the soybean protein emulsion gel is used for encapsulating the quercetin, and the free radical clearance rate of the quercetin after the complete in vitro digestion is improved. The result shows that the soybean protein emulsion gel has excellent carrying performance and can protect the antioxidant activity of carrying substances.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing an emulsion gel containing quercetin by extruding pretreated soy protein, the method comprising the steps of:
(1) Alkali polyphenol covalent binding modified soy protein: dissolving the soybean protein freeze-dried powder in phosphate buffer solution, stirring for 2h at room temperature to prepare soybean protein solution with the concentration of 2g/100mL, and hydrating at 4 ℃ overnight; adjusting the pH value of the soybean protein solution to 9.0, adding ferulic acid to ensure that the concentration of the ferulic acid in the soybean protein solution is 150 mu mol/g protein, and magnetically stirring at room temperature for 12h; adjusting the pH value of the mixed solution to 7.0, then filling the mixed solution into a dialysis bag, dialyzing the mixed solution for 24 hours by using a phosphate buffer solution, and freeze-drying the dialyzed solution to obtain modified soybean protein;
(2) The extrusion treatment further modifies the soy protein: adding distilled water into the modified soybean protein obtained in the step (1), adjusting the water content of the modified soybean protein to 30% -70%, standing for 24h to balance water, performing extrusion treatment, drying the extruded sample in a drying oven at 45 ℃ to constant weight, grinding into powder, and preparing to obtain the extrusion-alkali polyphenol covalent bonding composite modified soybean protein;
(3) Dissolving the composite modified soybean protein obtained in the step (2) in a phosphate buffer solution, stirring for 2 hours at room temperature, and placing at 4 ℃ for hydration overnight to obtain a composite modified soybean protein solution; dissolving 20mg quercetin in 3mL ethanol solution with volume fraction of 25% at 65 deg.C, adding 10mL soybean oil, heating at 65 deg.C for 30min, and removing ethanol to obtain soybean oil containing quercetin; taking 10mL of the compound modified soybean protein solution, adding 1.76mL of soybean oil containing quercetin, and stirring and homogenizing in a dark place to obtain a coarse emulsion; placing the crude emulsion in an ultrasonic cell pulverizer, treating for 15min under 400W water bath condition at 25 deg.C, centrifuging at low speed to remove air bubbles, adding gluconolactone to adjust pH to 4.5, magnetically stirring, and cold preserving at 4 deg.C for 12 hr to obtain emulsion gel.
2. The method of claim 1, wherein the soy protein lyophilized powder of step (1) is prepared by mixing defatted soy flour with ultrapure water at a feed-liquid ratio of 1g.
3. The method according to claim 1, wherein the phosphate buffer solution of step (1) and step (3) has a concentration of 0.02mol/L and a pH of 7.0.
4. The method of claim 1, wherein the dialysis bag of step (1) has a molecular weight cut-off of 8000-14000Da.
5. The method of claim 1, wherein step (2) is carried out by adjusting the moisture content of the modified soy protein to 40%.
6. The method as claimed in claim 1, wherein the extrusion process of step (2) is carried out under conditions of a main machine screw rotation speed of 300r/min, a feed zone screw rotation speed of 200r/min, and extrusion temperatures of 175 ℃,140 ℃,120 ℃,100 ℃,60 ℃ from the first zone to the fifth zone, respectively.
7. The method as claimed in claim 1, wherein the stock-to-liquid ratio of the complex modified soybean protein and the phosphate buffer solution in step (3) is 1 g.
8. The method of claim 1, wherein the homogenization conditions in step (3) are 10000rpm and 2.5min.
9. The method according to claim 1, wherein the final concentration of gluconolactone of step (3) is 15mg/mL.
10. An emulsion gel obtained by the method of any one of claims 1-9 that protects the antioxidant activity of quercetin.
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