Preparation method of natural grape seed protein-polysaccharide compound

Abstract

The invention discloses a method for extracting a natural grape seed protein-polysaccharide compound, which does not need to additionally use a cross-linking agent or a cross-linking means, and comprises the following steps of: taking peel residue product particles after grape wine brewing, crushing, degreasing by using supercritical CO2, adding the treated grape seed powder into a buffer solution with the pH value of 7.0-13.0, stirring, filtering, washing, and drying to obtain the natural grape seed protein-polysaccharide compound. After extracting for a period of time, removing grape seed residues, and adjusting the pH value of an extracting solution to 4.0-4.5 to obtain a compound precipitate; the method has the advantages that the high-quality industrial raw material of the protein-polysaccharide compound in the grape seeds is obtained at one time under the condition that a cross-linking method is not used, and the method has the advantages that resources are saved, the process is simple and convenient, and the cost is low; the compound is extracted by adopting an acid-base precipitation method, the extraction rate reaches 90%, the protein content in the extract reaches 50%, and the polysaccharide content reaches 30%.

Description

Technical Field

[0001] The invention relates to a method for preparing a natural grape seed protein-polysaccharide complex, and belongs to the field of natural raw material extraction. Background Art

[0002] In 2018, the global production of wine grapes was about 49.4 million tons. Assuming that the amount of grape pomace produced per kilogram of wine grapes accounts for 20% of the total weight, the annual production of grape pomace is as high as 9.88 million tons. my country has become the country with the largest grape production in the world. At present, the wine grape industry only uses grape seeds as livestock feed. At the same time, my country has not yet formed a complete grape pomace comprehensive utilization industry, which also limits the large-scale utilization of grape seed by-products. Therefore, according to the characteristics of grape pomace, a by-product of wine, establishing a preparation method for its characteristic products can play a role in multi-stage production, quality improvement and efficiency enhancement.

[0003] Previous studies have found that grape skins and seeds are rich in proteins and polysaccharides. After being crushed and squeezed during the winemaking process, a close interaction is formed between the protein and the polysaccharide, thus forming a stable complex. This complex can not only be used as a composite raw material that supplies both protein and polysaccharide, but also has the dual physical and chemical properties of protein and polysaccharide (such as significantly improved emulsification properties). It can be further developed and utilized as a production raw material to enhance its market value.

[0004] This technology does not use physical or chemical cross-linking methods. It uses grape seed by-product pomace as raw material to prepare natural grape seed protein-polysaccharide complexes. This method can not only obtain high-quality industrial raw materials, but also solve the problem of comprehensive utilization of by-products in the wine grape industry. Summary of the invention

[0005] (I) Purpose of the invention

[0006] The purpose of the present invention is to obtain a protein-polysaccharide complex directly from grape pomace without using an additional cross-linking method, thereby making full use of by-products of the wine grape industry, reducing environmental pollution and maximizing the residual value of grape seeds.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for preparing a grape seed protein-polysaccharide complex comprises the following steps:

[0009] Step 1: crush the grape seeds, and then degrease the powder obtained by supercritical CO2;

[0010] Step 2: adding defatted grape seed powder into alkaline buffer solution, stirring and leaching, to obtain an extract;

[0011] Step 3: Centrifuge the extract to separate the solid and liquid phases;

[0012] Step 4: Filter the extract through a water filter membrane;

[0013] Step 5: Adjust the pH of the extract to be acidic to obtain a protein-polysaccharide complex precipitate.

[0014] Preferably, in step 1, the grape seed solid powder is not overheated during the crushing process, the temperature is lower than 40° C., the crushed powder is sieved through a 10-60 mesh sieve, the fat content of the defatted grape seed powder is lower than 5%, and the work is carried out in a sterile environment.

[0015] Preferably, in step 2, the pH of the buffer solution is in the range of 7.0-13.0, and is adjusted using a sodium hydroxide solution with a concentration of 1 mol / L. The mass ratio of solid to solution is 20:1-30:1, and the extraction time is 20-48 hours. Within the first 2 hours, the pH of the extract is corrected every 0.5 hours.

[0016] Preferably, in step three, the centrifugal speed is 5000-2000 rpm / L, and the centrifugal time is 20-60 min.

[0017] Preferably, in step 4, the filter membrane through which the extract passes is a tubular water filter membrane with a pore size of 0.45 μm, and the transmembrane pressure does not exceed 0.75 MPa.

[0018] Preferably, in step five, a 1 mol / L hydrochloric acid solution is used to adjust the pH of the extract to 4.0-4.5, and the extract is allowed to stand for 24-48 hours in a sterile environment.

[0019] (III) Beneficial effects

[0020] The present invention provides a process for preparing a protein-polysaccharide complex. It has the following beneficial effects:

[0021] (1) Compared with the existing protein-polysaccharide complex preparation technology, the present invention utilizes grape pomace, a by-product of the red wine industry, to prepare high-quality protein-polysaccharide complexes without chemical cross-linking, thus providing a new industrial raw material and food ingredient.

[0022] (2) This process avoids the use of ultrasound, high static pressure and other methods to prepare the composite, and has the advantages of saving time and labor, low cost, etc.

[0023] The compound produced by this process comes from the by-products of the wine grape industry, has low cost, easy-to-obtain materials and high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a schematic diagram of the composition of proteins and polysaccharides in the present invention;

[0025] Figure 2 Schematic diagram of protein bands of the complex solution of the present invention in SDS-PAGE electrophoresis;

[0026] Figure 3 This is a schematic diagram of the particle size distribution of the composite solution of the present invention;

[0027] Figure 4 Schematic diagram of the investigation of the hydrophobicity of the complex of the present invention. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention. Specific implementation method one:

[0030] Step 1: Take wine grape skin residue, wash it thoroughly, dry it, and crush it, and use supercritical carbon dioxide to defatted the crushed grape seed powder to obtain a powder with a fat content of less than 5%;

[0031] Step 2: Take 100 g of defatted grape seed powder, add it to 2000 mL of an extract adjusted to a pH of 7.0-13.0 using a 1 mol / L NaOH solution, place it at 4° C. and stir at a speed of 1000 rpm for 20 hours to obtain an extract;

[0032] Step 3: Centrifuge the extract at 10,000 g, 4 °C for 20 min to obtain the supernatant;

[0033] Step 4: Add 1 mol / L HCl to the supernatant, adjust the pH of the supernatant to 4.0-4.5, and obtain a protein-polysaccharide complex precipitate. Specific implementation method 2:

[0035] Step 1: Take wine grape skin residue, wash it thoroughly, dry it, and crush it, and use supercritical carbon dioxide to defatted the crushed grape seed powder to obtain a powder with a fat content of less than 5%;

[0036] Step 2: Take 150 g of defatted grape seed powder, add it to 2500 mL of an extract adjusted to a pH of 7.0-13.0 using a 1 mol / L NaOH solution, place it at 4°C and stir at a speed of 800 rpm for 25 hours to obtain an extract;

[0037] Step 3: Centrifuge the extract at 10,000 g, 4 °C for 25 min to obtain the supernatant;

[0038] Step 4: Add 1 mol / L HCl to the supernatant and adjust the pH of the supernatant to 4.3-4.5 to obtain a protein-polysaccharide complex precipitate. Specific implementation method three:

[0040] Step 1: Take wine grape skin residue, wash it thoroughly, dry it, and crush it, and then defatted the crushed grape seed powder by supercritical carbon dioxide to obtain a powder with a fat content of less than 5%;

[0041] Step 2: Take 50 g of defatted grape seed powder, add it to 1000 mL of an extract adjusted to a pH of 7.0-13.0 using a 1 mol / L NaOH solution, place it at 4°C and stir at a speed of 1100 rpm for 30 hours to obtain an extract;

[0042] Step 3: Centrifuge the extract at 10,000 g, 4 °C for 15 min to obtain the supernatant;

[0043] Step 4: Add 1 mol / L HCl to the supernatant, adjust the pH of the supernatant to 4.0-4.3, and obtain a protein-polysaccharide complex precipitate.

[0044] Comparative Example 1

[0045] Step 1: Take wine grape skin residue, wash it thoroughly, dry it, and crush it, and then defatted the crushed grape seed powder by supercritical carbon dioxide to obtain a powder with a fat content of less than 5%;

[0046] Step 2: Take 50 g of defatted grape seed powder, add it to 1000 mL of an extract adjusted to a pH of 6.0-7.0 using a 1 mol / L HCl solution, place it at 4°C and stir at a speed of 1100 rpm for 30 hours to obtain an extract;

[0047] Step 3: Centrifuge the extract at 10,000 g, 4 °C for 15 min to obtain the supernatant;

[0048] Step 4: 1 mol / L HCl was added to the supernatant to adjust the pH of the supernatant to 4.0-4.3. The solution was clear and transparent, and no precipitation of protein-polysaccharide complex or any other substances was observed.

[0049] Comparative Example 2:

[0050] Step 1: Take the whole grape seed particles, wash them thoroughly, dry them, and crush them. Degrease the crushed grape seed powder with supercritical carbon dioxide to obtain a powder with a fat content of less than 5%;

[0051] Step 2: Take 50 g of defatted grape seed powder, add it to 1000 mL of an extract adjusted to a pH of 7.0-13.0 using a 1 mol / L NaOH solution, place it at 4°C and stir at a speed of 1100 rpm for 30 hours to obtain an extract;

[0052] Step 3: Centrifuge the extract at 10,000 g, 4 °C for 15 min to obtain the supernatant;

[0053] Step 4: Add 1 mol / L HCl to the supernatant and adjust the pH of the supernatant to 4.0-4.3 to obtain protein-polysaccharide complexes with different contents and low yields. For detailed results, see the performance investigation.

[0054] Comparative Example 3:

[0055] Step 1: Take the whole grape seed particles, wash them thoroughly, dry them, and crush them. Degrease the crushed grape seed powder with supercritical carbon dioxide to obtain a powder with a fat content of less than 5%;

[0056] Step 2: Take 5 g of defatted grape seed powder, add it to 1000 mL of an extract adjusted to a pH of 7.0-13.0 using a 1 mol / L NaOH solution, place it at 4°C and stir at a speed of 1100 rpm for 30 hours to obtain an extract;

[0057] Step 3: Centrifuge the extract at 10,000 g, 4 °C for 15 min to obtain the supernatant;

[0058] Step 4: Add 1 mol / L HCl to the supernatant to adjust the pH of the supernatant to 4.0-4.3. The solution becomes clear and transparent, and a protein-polysaccharide complex precipitate that is invisible before microcentrifugation is obtained.

[0059] Performance inspection

[0060] 1. Determination of the content of complex proteins and polysaccharides extracted from wine grape pomace and whole grape seeds

[0061] The prepared complex was ground, and the powder passed through a 60-sieve was placed in a 55° C. oven and dried for 10 h to obtain a dry complex powder. 200 mg of the powder was dissolved in a pH 10.0 buffer solution, and ultrasonically assisted dissolution was performed at 25° C. and 100 W for 15 min to obtain a test solution.

[0062] The standard solution was prepared using bovine serum albumin, and the sample solution and the standard solution were stained by Bradford staining. After incubation at 25°C for 5 minutes, the samples were scanned at a wavelength of 595 nm using an ELISA reader, the absorbance was recorded, and the protein content in the complex was calculated.

[0063] The polysaccharide content in the sample was determined using the phenol-sulfuric acid method. A standard solution was prepared with standard glucose. Another sample solution was taken. 1 mL of 6% phenol was added to every 2 mL of the test solution. 5 mL of concentrated sulfuric acid was quickly added. The solution was mixed on a vortex and allowed to stand for 30 min. The absorbance was measured at 490 nm, the absorbance value was recorded, and the polysaccharide content in the complex was calculated.

[0064] The experimental results are as follows Figure 1 As shown, the protein content in the complex is 51.94%, and the polysaccharide content is 32.33%, which proves that this process successfully produces a protein-polysaccharide complex from grape seeds.

[0065] 2. SDS-PAGE electrophoresis of proteins in the complex extracted from wine grape pomace

[0066] The obtained complex was subjected to SDS-PAGE electrophoresis. Figure 2 As shown, it can be determined from the reported literature that the protein in the protein-polysaccharide complex prepared by this process is grape seed 11S storage protein, which is a heterogenous hexameric protein containing acidic subunits and basic subunits in its natural state. In its amino acid sequence, the most abundant amino acids in a single subunit are lysine (13.18%) and leucine (13.18%), two essential amino acids for the human body, and it has extremely high nutritional value.

[0067] 3. Particle size distribution of aqueous solution of the complex

[0068] The particle size of the solution was measured using a Malvern particle size analyzer. Figure 3 As shown, the particle size of the complex is monodisperse with a peak value of 193nm; the particle size distribution in the complex solution shows a highly uniform unimodal distribution, which indicates that its structure is stable and can avoid aggregation or agglomeration. Due to the unimodal distribution characteristics, most of the particles are concentrated in the area close to 193nm. It reflects the precise control of the particle size of the complex, greatly reduces the possibility of its aggregation, and ensures its stable dispersion in the solution, which is very critical for its efficient transmission in biological applications or other technical fields. Compared with the shortcomings of traditional polysaccharide complexes with uneven particle size distribution or easy agglomeration, the complex of the present invention has better solvent stability.

[0069] 4. Hydrophobicity of the complexes extracted from wine grape pomace and whole grape seeds

[0070] The three-phase contact angle of two composite powders dropped into a water drop in an oil phase solution was obtained using the sessile drop method, as shown in Figure 4 shown.

[0071] The contact angle is an important indicator for measuring the hydrophobicity of a material surface. The larger the angle, the stronger the hydrophobicity. The complex extracted from wine grape pomace exhibited a larger contact angle (88.8°), indicating that its hydrophobicity was stronger than that of the complex extracted from a single whole grape seed (72.0°). The enhanced hydrophobicity indicates that the material has stronger compatibility in the oil phase and can be more stably dispersed in an oily environment.

[0072] The emulsifying properties of materials are closely related to their hydrophobic / hydrophilic balance. Protein-polysaccharide complexes with moderate hydrophobicity can be more effectively adsorbed to the oil-water interface, reduce interfacial tension, and thus form a stable emulsified system. Since the complex extracted from wine grape pomace is highly hydrophobic, its interfacial activity in the oil-water emulsion system will be higher, so it can provide better emulsifying properties and is suitable as a food emulsifier. Strong hydrophobicity and good emulsifying properties make the complex extracted from wine grape pomace more suitable for use in emulsified foods (such as salad dressings, emulsified beverages, and creams). These foods require the material to remain stable in a complex interfacial environment while providing an ideal texture and taste. In addition, the material extracted from the pomace may also contain more polyphenols and other functional ingredients, further enhancing its application value in the food industry.

[0073] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a natural grape seed protein-polysaccharide complex, comprising the following steps: Step 1: crush the grape winemaking byproduct, the skin residue, and degrease the powder obtained by supercritical CO2; Step 2: adding defatted grape skin residue powder into alkaline buffer solution and stirring and leaching to obtain an extract; Step 3: Centrifuge the extract to separate the solid and liquid phases; Step 4: Filter the extract through a water filter membrane; Step 5: Adjust the pH of the extract to be acidic to obtain a protein-polysaccharide complex precipitate.

2. The preparation method according to claim 1, characterized in that: In step 1, the temperature of crushing the grape skin residue is lower than 40° C., the crushed powder is passed through a 10-60 mesh sieve, and the fat content of the defatted grape skin residue powder is lower than 5%.

3. The preparation method according to claim 1, characterized in that: In step 2, the pH of the buffer solution is in the range of 7.0-13.0, and is adjusted using a 1 mol / L sodium hydroxide solution. The mass ratio of solid to solution is 20:1-30:1, and the extraction time is 20-48 hours. Within the first 2 hours, the pH of the extract is corrected every 0.5 hours.

4. The preparation method according to claim 1, characterized in that: In step three, the centrifugal speed is 5000-2000rpm / L, and the centrifugal time is 20-60min.

5. The preparation method according to claim 1, characterized in that: In step 4, the filter membrane through which the extract passes is a tubular water filter membrane with a pore size of 0.45 μm, and the transmembrane pressure does not exceed 0.75 MPa. The method for extracting grape seed protein-polysaccharide complex according to claim 1, characterized in that In step 5, a 1 mol / L hydrochloric acid solution is used to adjust the pH of the extract to 4.0-4.5, the extract is allowed to stand for 24-48 hours, and the precipitate is obtained by centrifugation.

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