CN112220031B - Preparation method of conjugated linoleic acid oil-in-water emulsion carried by glycosylated protein hydrolysate - Google Patents
Preparation method of conjugated linoleic acid oil-in-water emulsion carried by glycosylated protein hydrolysate Download PDFInfo
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- CN112220031B CN112220031B CN202011121723.9A CN202011121723A CN112220031B CN 112220031 B CN112220031 B CN 112220031B CN 202011121723 A CN202011121723 A CN 202011121723A CN 112220031 B CN112220031 B CN 112220031B
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Abstract
The invention discloses a preparation method of a novel conjugated linoleic acid oil-in-water emulsion carried by glycosylated protein hydrolysate, which takes hydrolysate of a glycosylation reaction product of galactose and whey protein isolate as an external water phase and Conjugated Linoleic Acid (CLA) as an oil phase, and prepares the CLA oil-in-water emulsion through ultrahigh pressure homogenization, and the specific method comprises the following steps: firstly, whey protein isolate and galactose are prepared into whey protein glycosylation product solution under certain conditions, and then AS1.398 neutral protease is used for hydrolysis to prepare glycosylation protein hydrolysate AS an external water phase; and (3) taking the CLA as an oil phase, mixing the water phase and the oil phase according to a certain proportion, emulsifying at a high speed, and homogenizing at a high pressure to obtain the CLA oil-in-water emulsion product. The invention can directly prepare the oil-in-water emulsion with good stability and can obviously inhibit the oxidation of the grease in the CLA.
Description
Technical Field
The invention belongs to the technical field of food science and engineering, and particularly relates to a method for preparing a conjugated linoleic acid oil-in-water emulsion carried by glycosylated protein hydrolysate.
Background
Oil-in-water (O/W) emulsions are the most common emulsion systems in the food industry, but are liable to cause oxidation, delamination, flocculation and other phenomena of grease during long-term storage, thereby causing the oxidation stability and physical stability of the emulsions to be reduced. An important solution to this problem is to add a natural emulsifier with antioxidant properties to the aqueous phase to improve the stability of the O/W emulsion.
Conjugated Linoleic Acid (CLA) is a conjugated double bond system-containing linoleic acid that has potential biological activities of anticancer, cardiovascular disease prevention, lipid reduction, anti-inflammatory, anti-diabetic, and anti-atherosclerotic, however, CLA is easily oxidized and unstable in food, and gives off unpleasant, rancid odor and taste during processing and storage, and thus CLA is often processed into an emulsion to inhibit lipid oxidation. In recent years, a small molecular surfactant, a surface active protein, or an ionic polysaccharide has been generally used as the external aqueous phase to carry the CLA emulsion, but the above external aqueous phase has low oxidation resistance and it is difficult to store the CLA emulsion for a long period of time.
Glycosylation modification and hydrolytic modification are two ubiquitous modes of food protein modification. Glycosylation reaction products (MRPs) are common in food processing and storage processes, have good oxidation resistance, emulsifying property and safety, and a great deal of research finds that MRPs are applied to O/W emulsion as an emulsifier, can ensure that the emulsion is not layered or flocculated in the long-time storage process, and provides good oxidation stability, thereby slowing down the oxidation of grease; some studies have shown that the hydrolysate has potential as an emulsifier and that the hydrolysate has antioxidant properties, which can inhibit the oxidation of fats and oils of the emulsion. In recent years, glycosylation and hydrolysis combined modification are widely used as novel antioxidants and emulsifiers to be applied to O/W emulsion at home and abroad, however, the oxidation stability and physical stability of CLA emulsion are not reported when glycosylated protein hydrolysate is used as external water to carry the CLA emulsion.
Disclosure of Invention
The invention aims to solve the problem that the glycosylated protein hydrolysate is used as an external water phase to carry CLA emulsion, and the oxidation stability and the physical stability of the emulsion can be improved. The glycosylated protein hydrolysate is prepared by combining glycosylation with a protease hydrolysis technology, and then the glycosylated protein hydrolysate is used as an external water phase to carry the CLA oil-in-water emulsion, so that the oxidation of grease in the CLA can be obviously inhibited, and a method for improving the physical stability and the oxidation stability of the emulsion is provided.
The purpose of the invention is realized by the following technical scheme:
the invention relates to a method for preparing a conjugated linoleic acid oil-in-water emulsion carried by glycosylated protein hydrolysate, which comprises the following steps:
(1) preparation of whey protein isolate glycosylation product solution: dissolving Whey Protein Isolate (WPI) in distilled water to prepare a solution with the concentration of 1-5% (w/v), adding reducing sugar into the solution to enable the ratio of the reducing sugar to WPI powder to be 1: 1(w/w) to prepare a mixed solution, adjusting the pH value of the initial reaction to be 8, reacting for 4 hours at the reaction temperature of 90 ℃, and rapidly cooling after the reaction is finished to prepare a whey protein isolate glycosylation reaction product solution;
(2) preparation of glycosylated protein hydrolysate solution: adjusting the pH value of whey protein isolate glycosylation reaction product solution (1) to 7.0 by using 1mol/L NaOH, adding AS1.398 neutral protease (E/S is 5%), putting the mixture into a water bath kettle at 45 ℃ for hydrolysis, continuously maintaining the pH value of the solution to 7.0 by using 1mol/L NaOH and 6mol/L NaOH during the hydrolysis process, and carrying out hydrolysis for 30min-120min, and when the hydrolysis is finished, putting the reaction solution at 80 ℃ for enzyme deactivation for 10min to obtain glycosylation protein hydrolysate solution;
(3) preparation of oil-in-water emulsion of conjugated linoleic acid: mixing Conjugated Linoleic Acid (CLA) and glycosylated protein hydrolysate solution (2) according to a certain proportion, emulsifying the mixed solution by a high-speed disperser for 4min, preparing crude emulsion under the condition that the dispersion speed is 10000r/min, homogenizing the crude emulsion by an ultrahigh pressure homogenizer, circulating for 4-6 times under the condition of 60-100MPa to form CLA oil-in-water emulsion, and completing the whole ultrahigh pressure homogenization process under the condition of ice water bath to reduce the oxidation of CLA.
In the present invention, it is preferable that the WPI solution concentration in the step (1) is 3%.
In the present invention, it is preferable that the reducing sugar in the step (1) is galactose.
In the present invention, it is preferable that the hydrolysis time in the step (2) is 120 min.
In the present invention, it is preferable that the purity of CLA in the step (3) is 80%.
In the present invention, it is preferable that the ultrahigh-pressure homogenization pressure in the step (3) is 80MPa, and the number of cycles is 6.
In the present invention, it is preferable that the Conjugated Linoleic Acid (CLA) and the glycosylated protein hydrolysate solution (2) are mixed in a ratio of 1: 9(w/w) in the step (3).
Compared with the prior art, the invention has the following technical effects:
(1) the glycosylated protein is hydrolyzed for 120min to expose more hydrophobic groups than the CLA emulsion carried by the unhydrolyzed glycosylation product, the molecular weight is reduced, free amino is generated, and the oxidation resistance is obviously enhanced;
(2) the lipid peroxidation value (POV), thiobarbituric acid reactive substances (TBARS) and Conjugated Diene (CD) of the glycosylated protein are respectively reduced by 42.28%, 48.45% and 19.92% compared with that of CLA emulsion carried by unhydrolyzed glycosylation products after 15 days of storage;
(3) the average particle size of the CLA emulsion carried by glycosylated proteolysis for 120min was reduced by 59.68% compared to the unhydrolyzed glycosylation product, whereas the interfacial adsorption capacity was increased by 16.51%, and the lipid particles of the CLA emulsion carried by glycosylated proteolysis for 120min were the smallest and most uniform as analyzed by the results of the milk analysis index and the microstructure of the emulsion;
(4) the glycosylated protein hydrolysate is used as an emulsifier, so that the oxidation stability, the physical stability and the shelf life of the CLA oil-in-water emulsion can be obviously improved, and the oil oxidation of CLA caused by light, heat and oxygen can be effectively inhibited, thereby exerting the biological activity of CLA.
The CLA oil-in-water emulsion with good physical stability and oxidation stability can be directly obtained by the preparation method, and technical support is provided for the application of CLA in food.
The invention will be further explained with reference to the drawings.
Drawings
FIG. 1 Effect of hydrolysis time on the hydrolyzed free amino content of glycosylated protein
Note: marked with no same letter, the difference is significant (P < 0.05);
FIG. 2 Effect of hydrolysis time on glycosylated proteolytic molecular weight distribution
Note: a is a gel filtration chromatogram of the protein hydrolysate, and B is a gel filtration chromatogram of the glycosylated protein hydrolysate;
FIG. 3 Effect of hydrolysis time on glycosylated proteolytic DPPH radical scavenging Capacity
Note: marked with no same letter, the difference is significant (P < 0.05);
FIG. 4 Effect of hydrolysis time on lipid peroxidation value (POV) of CLA emulsion
FIG. 5 Effect of hydrolysis time on Thiobabituric acid reactive substances (TBARS) of CLA emulsion
FIG. 6 Effect of hydrolysis time on the conjugated diene Content (CD) of CLA emulsion
FIG. 7 Effect of hydrolysis time on the average particle size of CLA emulsion
Note: marked with no same letter, the difference is significant (P < 0.05);
FIG. 8 Effect of hydrolysis time on creaming index and creaming behaviour of CLA emulsions
FIG. 9 Effect of hydrolysis time on the amount of interfacial adsorption of CLA emulsion
Note: marked with no same letter, the difference is significant (P < 0.05);
FIG. 10 the effect of hydrolysis time on the microstructure of CLA emulsions observed using a confocal microscope.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1
A method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate, comprising the steps of:
(1) preparation of whey protein isolate glycosylation product solution: dissolving Whey Protein Isolate (WPI) in distilled water to prepare a solution with the concentration of 3% (w/v), adding galactose into the solution to enable the ratio of the galactose to the WPI powder to be 1: 1(w/w) to prepare a mixed solution, adjusting the pH value of the initial reaction to be 8, reacting for 4 hours at the reaction temperature of 90 ℃, and rapidly cooling after the reaction is finished to prepare a whey protein isolate glycosylation reaction product solution;
(2) preparation of glycosylated protein hydrolysate solution: adjusting the pH value of whey protein isolate glycosylation reaction product solution (1) to 7.0 by using 1mol/L NaOH, adding AS1.398 neutral protease (E/S is 5%), putting the whey protein isolate glycosylation reaction product solution into a water bath kettle at 45 ℃ for hydrolysis, continuously maintaining the pH value of the solution to 7.0 by using 1mol/L NaOH and 6mol/L NaOH during the hydrolysis process, wherein the hydrolysis time is 120min, and when the hydrolysis is finished, putting the reaction solution at 80 ℃ for inactivating enzyme for 10min to obtain glycosylation protein hydrolysate solution;
(3) preparation of oil-in-water emulsion of conjugated linoleic acid: mixing conjugated linoleic acid (CLA, purity 80%) and glycosylated protein hydrolysate solution (2) in a ratio of 1: 9(w/w), emulsifying the mixed solution by using a high-speed disperser for 4min, preparing a coarse emulsion under the condition that the dispersion speed is 10000r/min, homogenizing the coarse emulsion by using an ultrahigh pressure homogenizer, circulating for 6 times under the condition of 80MPa to form CLA oil-in-water emulsion, and completing the whole ultrahigh pressure homogenization process under the condition of ice-water bath to reduce the oxidation of CLA.
Example 2
A method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate, comprising the steps of:
(1) preparation of whey protein isolate glycosylation product solution: dissolving Whey Protein Isolate (WPI) in distilled water to prepare a solution with the concentration of 2% (w/v), adding galactose into the solution to enable the ratio of the galactose to the WPI powder to be 1: 1(w/w) to prepare a mixed solution, adjusting the pH value of the initial reaction to be 8, reacting for 4 hours at the reaction temperature of 90 ℃, and rapidly cooling after the reaction is finished to prepare a whey protein isolate glycosylation reaction product solution;
(2) preparation of glycosylated protein hydrolysate solution: adjusting the pH value of whey protein isolate glycosylation reaction product solution (1) to 7.0 by using 1mol/L NaOH, adding AS1.398 neutral protease (E/S is 5%), putting the whey protein isolate glycosylation reaction product solution into a water bath kettle at 45 ℃ for hydrolysis, continuously maintaining the pH value of the solution to 7.0 by using 1mol/L NaOH and 6mol/L NaOH during the hydrolysis process, wherein the hydrolysis time is 90min, and when the hydrolysis is finished, putting the reaction solution at 80 ℃ for inactivating enzyme for 10min to obtain glycosylation protein hydrolysate solution;
(3) preparation of oil-in-water emulsion of conjugated linoleic acid: mixing conjugated linoleic acid (CLA, purity 80%) and glycosylated protein hydrolysate solution (2) in a ratio of 1: 9(w/w), emulsifying the mixed solution by using a high-speed disperser for 4min, preparing a coarse emulsion under the condition that the dispersion speed is 10000r/min, homogenizing the coarse emulsion by using an ultrahigh pressure homogenizer, circulating for 6 times under the condition of 60MPa to form CLA oil-in-water emulsion, and completing the whole ultrahigh pressure homogenization process under the condition of ice-water bath to reduce the oxidation of CLA.
Example 3
A method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate, comprising the steps of:
(1) preparation of whey protein isolate glycosylation product solution: dissolving Whey Protein Isolate (WPI) in distilled water to prepare a solution with the concentration of 1% (w/v), adding galactose into the solution to enable the ratio of the galactose to the WPI powder to be 1: 1(w/w) to prepare a mixed solution, adjusting the pH value of the initial reaction to be 8, reacting for 4 hours at the reaction temperature of 90 ℃, and rapidly cooling after the reaction is finished to prepare a whey protein isolate glycosylation reaction product solution;
(2) preparation of glycosylated protein hydrolysate solution: adjusting the pH value of whey protein isolate glycosylation reaction product solution (1) to 7.0 by using 1mol/L NaOH, adding AS1.398 neutral protease (E/S is 5%), putting the whey protein isolate glycosylation reaction product solution into a water bath kettle at 45 ℃ for hydrolysis, continuously maintaining the pH value of the solution to 7.0 by using 1mol/L NaOH and 6mol/L NaOH during the hydrolysis process, wherein the hydrolysis time is 60min, and when the hydrolysis is finished, putting the reaction solution at 80 ℃ for inactivating enzyme for 10min to obtain glycosylation protein hydrolysate solution;
(3) preparation of oil-in-water emulsion of conjugated linoleic acid: mixing conjugated linoleic acid (CLA, purity 80%) and glycosylated protein hydrolysate solution (2) in a ratio of 1: 9(w/w), emulsifying the mixed solution by using a high-speed disperser for 4min, preparing a coarse emulsion under the condition that the dispersion speed is 10000r/min, homogenizing the coarse emulsion by using an ultrahigh pressure homogenizer, circulating for 6 times under the condition of 100MPa to form CLA oil-in-water emulsion, and completing the whole ultrahigh pressure homogenization process under the condition of ice-water bath to reduce the oxidation of CLA.
Experimental example 1: effect of hydrolysis time on physicochemical Properties of glycosylated protein hydrolysate
1. Determination of the content of free amino groups
The method for measuring the free amino group by using the OPA method is characterized in that the OPA reagent is prepared at present, and the preparation method comprises the following steps: 125mL of 100mM sodium tetraborate, 12.5mL of 20% (wt/wt) SDS, 0.2g OPA dissolved in 5mL methanol followed by the addition of 500. mu.L of beta-mercaptoethanol. All samples were diluted to 2mg/mL with PBS pH 7.0, 100. mu.L thereof was taken, 3mL of OPA was added thereto, and the reaction was immediately protected from light for 5min, and then the absorbance at 340nm was measured. Taking L-leucine as standard substance, taking its concentration as abscissa and light absorption value as ordinate, measuring in the above manner, and drawing standard curve, wherein the regression equation is that y is 1.667x +0.1397, and R is2＝0.9991。
Calculating the content of free amino group by using the light absorption value of the mixed solution as a y value. Based on the content of the free amino groups of the WPI, the consumption of the free amino groups of other samples is calculated by the following formula:
as can be seen from the test results in FIG. 1, the glycosylated protein hydrolysate (MRPsH) has a significantly lower free amino group content (P > 0.05) than the protein hydrolysate (WPH) with the longer hydrolysis time, the WPH has a free amino group content of 0.49mol/kg and the MRPsH has a significantly lower free amino group content (P > 0.05) than the protein hydrolysate (WPH) with the 120min hydrolysis time, which is 0.35mol/kg, indicating that the hydrolysis consumes the free amino groups, and the free amino groups increase with the increase of the hydrolysis time, and the consumption is the most at the 120min hydrolysis time, because the glycosylation reaction products are further hydrolyzed to release part of the amino groups in the system.
2. Determination of molecular weight distribution
Samples were diluted to 2.5mg/mL, 15. mu.L each, and eluted by isocratic elution with 0.1% TFA in 30% acetonitrile at a flow rate of 0.5 mL/min. The detection wavelength was 214 nm. Respectively selecting bovine serum albumin (66409Da), ovalbumin (44300Da), trypsin inhibitor (20100Da), beta-lactoglobulin (18400Da), alpha-lactalbumin (14147Da), aprotinin (6500Da), oxidized glutathione (612.63Da) and reduced glutathione (307.32Da), preparing standard curves of 8 molecular weights from the peak retention time and the molecular weight distribution, wherein the equation is Log MW-0.1148T +7.865, and R is20.9802 where MW represents molecular weight and T represents retention time.
As can be seen from the test results in FIG. 2, the glycosylated proteins (MRPs) show two independent peaks, the area of peak 1 is larger than that of peak 2, which indicates that after the glycosylation reaction between WPI and galactose, the monomer amount is reduced and the macromolecular aggregates are increased, and peak 1 begins to peak at about 23.05min, which is earlier than that of bovine serum albumin standard (23.67min), so that the corresponding molecular weight is higher than 66409Da, and during the formation of the glycosylation product, a small amount of small molecules and medium molecular weight components are generated, which may be caused by aggregation of some small molecules in WPI, or the small molecules in WPI are combined with a small amount of galactose, or galactose itself is caramelized by long-time high-temperature treatment. Three incompletely separated peaks appear in the spectrum of the MRPsH formed in the hydrolysis time, the molecular weight ranges corresponding to 30min, 60min, 90min and 120min of the MRPsH after hydrolysis are calculated to be 274.52Da-88726.87Da, 239.80Da-88706.26Da, 239.80Da-88685.22Da and 238.63Da-88685.22Da respectively, the molecular weight distribution range of the MRPs hydrolysate is gradually reduced along with the continuous increase of the hydrolysis time, the macromolecular polymer is gradually reduced, and the content of the micromolecular peptides is increased.
3. Determination of DPPH radical scavenging Rate
The sample was diluted to 15mg/mL, 160. mu.L of the diluted sample was mixed with 640. mu.L of a 0.1mM DPPH solution (DPPH dissolved in 95% ethanol), and the mixture was reacted with light at room temperature for 30 min. The absorbance was measured at 517nm using 95% ethanol as a reference solution. The calculation formula is as follows:
a is absorbance of mixed DPPH ethanol solution, AiAbsorbance for mixed 95% ethanol solution, AjAbsorbance of DPPH ethanol solution alone.
As can be seen from the test results in FIG. 3, the DPPH radical scavenging ability of WPH and MRPsH is significantly increased (P < 0.05) compared with that of the control group WPI and MRPs, and gradually increased with the increase of hydrolysis time, whereas the surface hydrophobicity of the MRPsH prepared by longer hydrolysis time can be increased. These peptides are therefore able to localize adjacent DPPH radicals, and thus form small peptides which can readily donate hydrogen or electrons upon hydrolysis. The longer the hydrolysis time of the glycosylated product, the greater the DPPH free radical scavenging ability of the glycosylated protein hydrolysate (MRPsH) because it forms more hydrophobic amino acid residues.
Experimental example 2: CLA oil-in-water emulsion product analysis:
1. determination of lipid peroxidation value (POV)
Sucking 1mL of emulsion, adding 5mL of mixed solution of isopropanol and isooctane (1: 2, v/v), mixing uniformly, and centrifuging at 3000r/min for 2 min. mu.L of supernatant is added with 20 mu.L of potassium thiocyanate and 20 mu.L of ferrous chloride solution, then the solution of normal butanol and methanol (1: 2, v/v) is metered to 5mL, after mixing uniformly, the mixture is kept for 20min in a dark place at room temperature, and the absorbance value at 510nm is measured by taking the mixed solution of methanol and normal butanol (2: 1, v/v) as a control group. The calculation formula is as follows:
POV is the peroxide value (meq/kg) of the emulsion, A is the absorbance of the emulsion, and K is Fe3+And (3) a standard curve slope, wherein m is the content (g) of grease in the weighed emulsion, and n is the volume fraction of the supernatant liquid.
As can be seen from the test results in fig. 4, the lipid peroxidation values of WPI hydrolyzed for 30min, 60min, 90min and 120min were 14.56%, 18.56%, 22.74% and 30.04% respectively lower than the POV of the unhydrolyzed WPI-carried emulsion when stored for 15 days. The MRPs are respectively hydrolyzed for 30min, 60min, 90min and 120min, and the POV of the MRPs is reduced by 7.35 percent, 16.54 percent, 31.03 percent and 42.28 percent compared with the unhydrolyzed MRPs after the MRPs are stored for 15 days, which shows that the MRPsH can obviously reduce the peroxide value content of the emulsion (P is less than 0.05).
2. Measurement of Thiobabituric acid-reactive substance (TBARS)
Prepared by mixing 0.375g of thiobarbituric acid, 15g of trichloroacetic acid, 12M HCl 1.76mL and 82.9mL of water. 0.3mL of the emulsion, 0.7mL of distilled water and 2mL of TBA prepared as described above were mixed well and placed in a centrifuge tube and kept in boiling water for 15min, after that the centrifuge tube was immediately placed in ice water for cooling, and then centrifuged at 3500 Xg for 15 min. The supernatant after centrifugation was taken and absorbance was measured at 532 nm. The concentration of TBARS was calculated from a standard curve prepared using 1, 1, 3, 3-tetraethoxypropane.
From the test results in fig. 5, it is understood that when MRPs are hydrolyzed for 30min, 60min, 90min and 120min after being stored for 15 days, the oxidation resistance thereof is reduced by 11.50%, 16.87%, 19.75% and 48.45% compared with MRPs, which indicates that hydrolysis can significantly reduce the generation of malonaldehyde, thereby improving the oxidation stability of the emulsion, and thus, the oxidation of CLA oil and fat can be inhibited. Under the same hydrolysis time, the malondialdehyde content of the CLA emulsion carried by MRPsH is obviously reduced (P is less than 0.05) compared with that of WPH, and particularly, the TBARS value of the CLA emulsion carried by MRPsH is reduced by 56.07 percent compared with that of WPH when the hydrolysis time is 120min, because MRPsH has stronger oxidation resistance compared with WPH, so that the CLA emulsion prepared by using the MRPsH as an emulsifier has better oxidation stability.
3. Determination of the determination (CD) of the conjugated diolefins
mu.L of the emulsion was added to 1.5mL of a mixed solution of isopropanol and isooctane (1: 3, v/v) and vortexed three times for 10s each. It was then centrifuged at 548 Xg for 5min and 5. mu.L of the supernatant was aspirated into a centrifuge tube and diluted to 5mL with isooctane. Measuring the light absorption value at 234nm, and calculating according to the formula:
CD represents the conjugated diene content (μmol/L), a represents the absorbance of the sample, e represents the molar extinction coefficient of the grease (e 26000), and b represents the optical path length.
From the test results in fig. 6, it is understood that when MRPs were hydrolyzed for 30min, 60min, 90min and 120min, respectively, after 15 days of storage, the CD values were reduced by 7.25%, 13.89%, 22.61% and 50.10% compared with the unhydrolyzed MRPs, and after hydrolysis of the protein, the conjugated diene content was reduced by 3.98%, 7.73%, 13.78% and 19.92% compared with the WPI-carried emulsion, and from the reduction of the conjugated diene, it was found that the reduction of the conjugated diene of MRPsH was higher than that of WPH, indicating that hydrolysis can enhance the oxidation stability of the emulsion. This is because, compared to emulsions carried by MRPs, glycosylated protein hydrolysate (MRPsH) is capable of unfolding protein molecules and releasing amino acid residues to bind to oxidized molecules, and hydrolysis is capable of generating more products with strong antioxidant activity at the oil-water interface, thereby acting to inhibit oxidation of CLA oil.
4. Measurement of particle diameter
The particle size of the emulsion was measured using a particle size distribution instrument. All emulsion samples were diluted to 1mg/mL with PBS buffer at pH 7.0 and the dilutions were then placed in test tubes for testing. All dilutions were assayed at 25 ℃ and in triplicate.
From the experimental results in FIG. 7, it can be seen that the average particle size of the emulsions carried by the hydrolysates WPH and MRPsH was significantly reduced (P < 0.05) and the average particle size of the emulsions carried by WPH and MRPsH was significantly reduced (P < 0.05) with the increase of hydrolysis time, as compared to the emulsions carried by the control groups WPI and MRPs. The average particle size of the MRPsH-carried emulsion was significantly reduced compared to the WPH-carried emulsion over the total hydrolysis time.
5. Milk out index and stratification
10mL of fresh emulsion was placed in a room temperature sample bottle. CI is calculated after 0, 3, 6, 9, 12, 15 days of storage of the emulsion. Meanwhile, a sample of the emulsion which is allowed to stand at room temperature for 15 days is taken to analyze the demixing condition.
The calculation formula of CI is:
wherein Hc is the height (cm) of the supernatant after a certain storage time, and Hs is the total height (cm) of the emulsion.
From the test results of fig. 8, it can be seen that no significant delamination occurred in the hydrolyzed protein molecules within 9 days of storage, all emulsions appeared to have an elutriation rate after 9 days of storage at room temperature, and the elutriation index was close to 0, whereas the emulsions carried by WPH were faster than those carried by MRPsH, and at 15 days of storage, the elutriation indexes of the emulsions carried by WPH-30min and WPH-120min reached 58.76% and 42.31%, respectively, whereas the elutriation indexes of the emulsions carried by MRPsH-30min and MRPsH-120min were 22.86% and 8.47%, respectively, indicating that the emulsions carried by the hydrolysate of the protein could significantly decrease the elutriation index of the emulsions. This is probably because, on the one hand, as the hydrolysis time is prolonged, protein molecules are decomposed into small molecules, and emulsion droplets carried by the small molecules have stronger space repulsion, and on the other hand, MRPsH has higher hydrophobicity, so that the MRPsH is more easily adsorbed on an oil-water interface, thereby increasing the interface adsorption capacity and reducing the elutriation index.
6. Amount of interfacial adsorption
1mL of the emulsion was centrifuged at 15000r/min for 60min in a centrifuge tube, and the supernatant was carefully aspirated using a 1mL syringe and filtered through a 0.22 μm filter. Bovine Serum Albumin (BSA) is used as a standard curve, a Coomassie brilliant blue method is adopted to determine the protein content, and the calculation formula is as follows:
AP represents the amount of interfacial adsorption (mg/mL), C0Denotes the initial concentration (mg/mL) of the protein dispersion, CfThe protein content (mg/mL) in the supernatant is indicated.
From the test results in FIG. 9, it can be seen that the AP of the WPH-carried emulsion formed after hydrolysis is significantly increased (P < 0.05) compared to the untreated WPI-carried emulsion, and the AP of the MRPsH-carried emulsion is also significantly increased (P < 0.05) compared to the AP of the control group MRPs, indicating that the hydrolysis can significantly increase the AP of the emulsion, and that the MRPsH is increased by 13.33%, 13.81%, 15.96%, 17.86% respectively compared to the AP of the WPH-carried CLA emulsion at 30min, 60min, 90min, and 120min in the sequence of hydrolysis time, indicating that the MRPsH-carried emulsion can significantly increase the interfacial adsorption amount of the emulsion compared to the WPH-carried emulsion because the hydrolysis decomposes macromolecular proteins into small molecular substances, the protein molecules are unfolded, resulting in partial exposure of hydrophobic groups, and simultaneously the surface-active polypeptides are exposed, so that most of the surface-active polypeptides move to the non-aqueous phase interface and thus can be adsorbed to the surface of oil droplets, resulting in a significant increase in interfacial protein coverage.
7. Observing the microstructure of the emulsion by confocal microscope
The microstructure of the emulsion was studied with a focusing microscope (CLSM) using a laser. 0.05g of nile blue and 0.005g of nile red are dissolved in 5mL of isopropanol to prepare fluorescent dyes (0.1% (w/v) of nile blue and 0.01% (w/v) of nile red), and the fluorescent dyes are dissolved in dark for 30min and then passed through a 0.45 μm filter. The emulsion was diluted 5-fold with water and 1mL was aspirated, 25. mu.L of Nile blue and 20. mu.L of Nile red were added in that order, and was stained in the dark for 30 min. 5. mu.L of the stained emulsion was used for flaking. The edge of the cover glass was sealed with silicone oil, the slide was placed on a stage, an appropriate field of view was selected under bright field conditions, the functional bonds of the microscope were switched to fluorescence observation, the microstructure of the emulsion was then observed using a 40-fold objective lens, and images were acquired by excitation of the Ar/Kr and He/Ne dual channel laser modes at 488nm and 633 nm.
As can be seen from the test results in fig. 10, all emulsions were stained by Neil Red and Neil Blue, and Neil Red emits Red fluorescence and is used to label the oil phase, Neil Blue emits green fluorescence and is used to label the proteins, showing that the oil droplets are embedded by the proteins, and all microstructures exhibit an oil-in-water structure. The grease particles of the emulsion carried by the hydrolysate are remarkably reduced (P is less than 0.05) compared with the grease particles of the emulsion not carried by the hydrolysate, because the hydrolysis enables macromolecules to be decomposed into micromolecular peptide fragments, the surface hydrophobicity and the molecular flexibility of the protein can be further improved, and the properties are favorable for being adsorbed to the surface of oil drops, so that the interfacial tension is effectively reduced, and the emulsion with smaller particle size and more stability is formed.
Claims (7)
1. A method for the preparation of an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate, characterized in that it comprises the following steps:
(1) preparation of whey protein isolate glycosylation product solution: dissolving whey protein isolate WPI in distilled water to prepare a solution with the concentration of 1% -5% (w/v), adding reducing sugar into the solution to enable the ratio of the reducing sugar to WPI powder to be 1: 1(w/w) to prepare a mixed solution, adjusting the pH value of the initial reaction to be 8, reacting for 4 hours at the reaction temperature of 90 ℃, and rapidly cooling after the reaction is finished to prepare a whey protein isolate glycosylation reaction product solution;
(2) preparation of glycosylated protein hydrolysate solution: adjusting the pH value of whey protein isolate glycosylation reaction product solution (1) to 7.0 by using 1mol/L NaOH, adding AS1.398 neutral protease, wherein the E/S is 5%, putting the whey protein isolate glycosylation reaction product solution into a water bath kettle at 45 ℃ for hydrolysis, continuously maintaining the pH value of the solution to 7.0 by using 1mol/L NaOH and 6mol/L NaOH during the hydrolysis process, wherein the hydrolysis time is 30min-120min, and when the hydrolysis is finished, putting the reaction solution at 80 ℃ for enzyme deactivation for 10min to obtain glycosylation protein hydrolysate solution;
(3) preparation of oil-in-water emulsion of conjugated linoleic acid: mixing Conjugated Linoleic Acid (CLA) and a glycosylated protein hydrolysate solution (2) in a ratio of 1: 9(w/w), emulsifying the mixed solution by a high-speed disperser for 4min, preparing a coarse emulsion under the condition that the dispersion speed is 10000r/min, homogenizing the coarse emulsion by an ultrahigh pressure homogenizer, circulating for 4-6 times at 60-100MPa to form CLA oil-in-water emulsion, and completing the whole ultrahigh pressure homogenization process under the condition of ice water bath to reduce the oxidation of CLA;
the reducing sugar in the step (1) is galactose;
when the glycosylated protein is hydrolyzed for 30-120min in the step (3), the carried CLA oil-in-water emulsion has the lipid peroxidation value, the thiobarbituric acid reactive substance and the conjugated diene of 37.83-27.34 meq/kg, 0.81-0.58mmol/kg and 8.87-5.07 mu mol/L respectively when being stored for 15 days.
2. The method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate of claim 1, wherein: the concentration of the WPI solution in the step (1) is 3% (w/v).
3. The method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate of claim 1, wherein: the molecular weight of the glycosylated protein in the step (1) is in the range of 200Da-90000 Da.
4. The method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate of claim 1, wherein: the hydrolysis time in the step (2) is 120 min.
5. The method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate of claim 1, wherein: the purity of the CLA in the step (3) is 80%.
6. The method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate of claim 1, wherein: in the step (3), the homogenizing pressure is 80MPa, and the cycle number is 6.
7. The method of preparing an oil-in-water emulsion of conjugated linoleic acid carried by a glycosylated protein hydrolysate of claim 1, wherein: when the glycosylated protein is hydrolyzed for 30-120min in the step (3), the carried CLA oil-in-water emulsion has the interface adsorption capacity of 81.76-95.86%.
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Citations (3)
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|---|---|---|---|---|
| CN104906074A (en) * | 2015-06-15 | 2015-09-16 | 湖北工业大学 | Preparation method of stable conjugated linoleic acid emulsion |
| CN105077244A (en) * | 2015-08-26 | 2015-11-25 | 江南大学 | Preparation method of lycopene microcapsules |
| CN110771893A (en) * | 2019-11-12 | 2020-02-11 | 武汉轻工大学 | Method for preparing β -carotene uniform emulsion from whey protein isolate glycosylation reaction product and uniform emulsion |
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| CN104906074A (en) * | 2015-06-15 | 2015-09-16 | 湖北工业大学 | Preparation method of stable conjugated linoleic acid emulsion |
| CN105077244A (en) * | 2015-08-26 | 2015-11-25 | 江南大学 | Preparation method of lycopene microcapsules |
| CN110771893A (en) * | 2019-11-12 | 2020-02-11 | 武汉轻工大学 | Method for preparing β -carotene uniform emulsion from whey protein isolate glycosylation reaction product and uniform emulsion |
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