Application of turbidity-causing protein or thaumatin of kiwi fruit wine in prevention and control of post-turbidity

Abstract

The invention discloses application of turbid protein of kiwi fruit wine or thaumatin in prevention and control of post-turbidity. The turbid protein of the kiwi fruit wine is thaumatin-like protein. The key turbidity-causing protein, namely, thaumatin-like protein P83958, of the kiwi fruit wine is found, the binding capacities of the key turbidity-causing protein and different polyphenols are different, thaumatin protein and the key turbidity-causing protein have homology, and the key turbidity-causing protein is difficult to obtain, so that the turbidity-causing effect of a thaumatin protein-polyphenol model is explored by adopting thaumatin protein; therefore, the method is applied to evaluation of the effects of different methods in post-turbidity prevention and control of the kiwi fruit wine, so that an effective method for post-turbidity prevention and control of the kiwi fruit wine is conveniently obtained.

Description

Technical Field

[0001] The invention relates to a kiwi fruit wine turbidity protein, and in particular to an application of a kiwi fruit wine turbidity protein or thaumatin in post-turbidity prevention and control. Background Art

[0002] Kiwi fruit is rich in organic acids and bioactive substances. Kiwi fruit is known as the "king of vitamin C in fruits". In addition, kiwi fruit is rich in polyphenols and flavonoids, which are one of its important natural components and have biological and pharmacological activities. Kiwi fruit wine is a fermented wine made from kiwi fruit juice (pulp) through complete or partial fermentation. Kiwi fruit wine is rich in various nutrients and has a unique flavor. It is deeply loved by consumers and has broad market prospects.

[0003] Fruit wine is a complex mixed solution and a heat-unstable system. It contains a variety of substances such as protein, pectin, cellulose, higher fatty acids, organic acids, metal ions, etc., so it is easy to cause post-turbidity during storage. It can generally be divided into two types: biological turbidity and non-biological turbidity. Biological turbidity refers to the metabolism of microorganisms such as lactic acid bacteria, acetic acid bacteria or other bacteria in fruit wine on the components of the wine, destroying its colloidal balance, resulting in fog, turbidity or precipitation. However, with the innovation of sterilization technology, packaging technology and the substantial improvement of hardware facilities for transportation and storage, it has rarely occurred. Non-biological turbidity mainly refers to the turbidity caused by a series of physical and chemical reactions of substances such as phenols, tannins and proteins, which is the dominant factor causing the overall post-turbidity phenomenon.

[0004] With the change of consumer purchasing habits, the sales volume of fruit wine has been growing steadily in recent years. However, the post-turbidity phenomenon that often occurs in fruit wine during transportation and storage is an obvious defect that damages the physical and chemical stability of fruit wine. The shelf life of the product and the sensory experience of consumers are negatively affected by the post-turbidity phenomenon. Therefore, solving the post-turbidity problem is the key to improving the quality of fruit wine.

[0005] When studying the post-turbidity problem, small molecule gliadin or proline-rich BSA, gelatin and other substances are usually used to construct a simulation system. However, this simulation system may not be completely applicable to kiwi fruit wine. So far, there has been no research report on the direct extraction of protein from kiwi fruit wine. Summary of the invention

[0006] The purpose of the present invention is to provide an application of kiwi wine turbidity-causing protein or thaumatin in the prevention and control of post-turbidity, which solves the problem that the existing simulation system may not be completely applicable to kiwi wine. The application of thaumatin-like protein or thaumatin in the prevention and control of post-turbidity is combined with polyphenols to construct a turbidity model, which can be applied to the prevention and control of post-turbidity in kiwi wine.

[0007] In order to achieve the above-mentioned object, the present invention provides the use of kiwi fruit wine turbidity protein or thaumatin in post-turbidity prevention and control, wherein the kiwi fruit wine turbidity protein is a thaumatin-like protein.

[0008] Preferably, the protein accession number of the thaumatin-like protein is P83958.

[0009] Preferably, the thaumatin is selected from thaumatin derived from African arrowroot.

[0010] Preferably, the application is the evaluation of the effects of kiwi wine haze protein or thaumatin on different methods in preventing and controlling post-production haze in kiwi wine.

[0011] Preferably, the application is: if the clarifier is an adsorbent, different clarifiers are mixed with kiwi wine turbidity protein or thaumatin mother liquor, the pH of the kiwi wine turbidity protein or thaumatin mother liquor is 5, the total protein concentration in the mixed solution at different times is measured, and the clarification effect of the clarifier is expressed by the adsorption rate; if the clarifier is a protease, different clarifiers are mixed with kiwi wine turbidity protein or thaumatin for enzymolysis, after the enzymolysis is completed, the enzyme is inactivated, the pH value of the solution is adjusted back to 5, a portion is taken out and mixed with an equal volume of EGCG or PC solution with pH=5 at room temperature, and then the absorbance is detected at a wavelength of 660nm, and the degree of change of turbidity-causing ability is calculated based on the obtained absorbance; if the clarifier is a polysaccharide, the EGCG or PC solution, thaumatin and polysaccharide are fully mixed under the condition of pH=5, and the mixture is incubated in a constant temperature incubator, and then the mixed solution is taken out, and the absorbance of the reactant is measured at a wavelength of 660nm, and the degree of change of turbidity-causing ability is calculated based on the obtained absorbance.

[0012] Preferably, the kiwi fruit wine turbidity protein or thaumatin mother liquor is prepared with citric acid buffer; and the clarifier is prepared with water to form a clarifier solution.

[0013] Preferably, the concentration of the kiwi wine turbidity protein or thaumatin mother solution is 1-4 mg / mL.

[0014] Preferably, the concentration of the EGCG and PC solution is 1-4 mg / mL.

[0015] Preferably, the EGCG or PC solution, thaumatin and polysaccharide are fully mixed in a ratio of 1:1:1.

[0016] The application of the kiwi fruit wine turbidity protein or thaumatin in the post-turbidity prevention and control of the present invention solves the problem that the existing simulation system may not be completely applicable to kiwi fruit wine, and has the following advantages:

[0017] The present invention discovers the key turbidity-causing protein thaumatin-like protein P83958 of kiwifruit wine, and finds that the protein has different binding abilities with different polyphenols, and thaumatin has homology with the protein, while the key turbidity-causing protein is difficult to obtain. Therefore, thaumatin is used to explore the turbidity-causing effect of the thaumatin-polyphenol model, which is then applied to evaluate the effects of different methods in preventing and controlling the post-turbidity of kiwifruit wine, thereby facilitating the acquisition of an effective method for preventing and controlling the post-turbidity of kiwifruit wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SDS-PAGE image of turbidity caused by different induction methods of kiwi fruit wine in Experimental Example 1 of the present invention; Lane 1 is a marker; Lane 2 is a 1:4 isopropanol precipitated protein; Lane 3 is a 1:5 isopropanol precipitated protein; Lane 4 is the supernatant protein after EGCG-induced turbidity; Lane 5 is the precipitated protein induced by EGCG.

[0019] Figure 2 This is the SDS-PAGE image of the natural precipitation of kiwi fruit wine in Experimental Example 1 of the present invention; lanes 1 and 8 are markers; lanes 2 to 7 are naturally precipitated wine proteins.

[0020] Figure 3 This is the basepeak graph of the EGCG-induced turbidity sample in Experimental Example 1 of the present invention.

[0021] Figure 4 This is the basepeak diagram of the natural precipitation sample in Experimental Example 1 of the present invention.

[0022] Figure 5 Figure 1 is a molecular docking model diagram of P83958 and four polyphenols in Experimental Example 2 of the present invention; A is the interaction model of Thaumatin-like protein (P83958) and EGCG; B is the interaction model of Thaumatin-like protein (P83958) and PC; C is the interaction model of Thaumatin-like protein (P83958) and EC; D is the interaction model of Thaumatin-like protein (P83958) and CGA.

[0023] Figure 6 This is the result of protein homology analysis between the thaumatin-like protein in Experimental Example 3 of the present invention and the thaumatin protein from African arrowroot.

[0024] Figure 7 This is the result of multiple sequence comparison between the thaumatin-like protein in Experimental Example 3 of the present invention and the thaumatin protein from African arrowroot.

[0025] Figure 8This is a protein phylogenetic tree of the thaumatin-like protein in Experimental Example 3 of the present invention and the thaumatin protein from African arrowroot.

[0026] Fig. 9 The effect of different concentration ratios of polyphenols and thaumatin on their absorbance in Experimental Example 4 of the present invention; A: epigallocatechin gallate; B: proanthocyanidins; C: epicatechin; D: chlorogenic acid.

[0027] Fig.10 The fluorescence spectra of the interaction between polyphenols at different concentrations and thaumatin in Experimental Example 4 of the present invention; a: epigallocatechin gallate; b: proanthocyanidins.

[0028] Fig.11 The Stern-Volmer curve of fluorescence quenching of thaumatin by EGCG / PC at room temperature in Experimental Example 4 of the present invention; A: epigallocatechin gallate; B: proanthocyanidins.

[0029] Fig.12 This is a Lineweaver-Burk line graph of EGCG / PC binding to thaumatin at room temperature in Experimental Example 4 of the present invention; A: epigallocatechin gallate; B: proanthocyanidins.

[0030] Fig.13 This is a continuous monitoring experiment of thaumatin and different clarifiers in Experimental Example 5 of the present invention; A: TOP; B: ordinary bentonite; C: diatomaceous earth; D: chitosan; E: bentonite.

[0031] Fig.14 The effects of different protease hydrolysates on thaumatin-polyphenol turbidity in Experimental Example 5 of the present invention; A: epigallocatechin gallate; B: proanthocyanidins.

[0032] Fig.15 It is an SDS-PAGE identification diagram of different enzymatic hydrolysis products in Experimental Example 5 of the present invention; Lane 1 is a marker; Lane 2 is unhydrolyzed thaumatin; Lane 3 is thaumatin hydrolyzed by papain; Lane 4 is thaumatin hydrolyzed by neutral protease; Lane 5 is thaumatin hydrolyzed by alkaline protease.

[0033] Fig.16 This is a continuous monitoring experiment of bovine serum albumin and different clarifiers in Experimental Example 6 of the present invention; A: TOP; B: ordinary bentonite; C: diatomaceous earth; D: chitosan; E: bentonite.

[0034] Fig.17 This is the effect of different clarification methods on the turbidity model of kiwi wine in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0037] Some of the reagents used in the following experimental examples are as follows:

[0038] 1. Citric acid buffer solution: Citric acid-disodium hydrogen phosphate buffer solution is used. The preparation of citric acid-disodium hydrogen phosphate buffer solution is as follows: Solution A (0.2mol / LNa2HPO4·12H2O): weigh 71.64g disodium hydrogen phosphate dodecahydrate solution and add UP water to make up to 1000mL; Solution B (0.1mol / L C6H8O7·H2O): weigh 21.01g citric acid monohydrate and add UP water to make up to 1000mL; pH=5.0 is used to prepare the buffer solution, and the specific ratio is: 10.3mL of Solution A and 9.7mL of Solution B;

[0039] 2. Thaumatin solution: prepared with citric acid buffer;

[0040] 3. Various polyphenol solutions: all are prepared using citric acid buffer.

[0041] Experimental Example 1 Extraction and Identification of Key Turbidity-Causing Proteins in Kiwi Wine

[0042] 1. Preparation of kiwi fruit wine

[0043] Fruit selection (select fresh kiwifruit with high maturity and full fruit) → wash and peel → squeeze juice → enzymatic hydrolysis (use 0.2% complex enzyme, that is, every 100mL juice contains 0.2g complex enzyme, the complex enzyme is pectinase: cellulase with a mass ratio of 1:1.5, and enzymatic hydrolysis at 50℃ for 120 minutes) → adjust sugar (use white sugar to adjust the sugar content of the juice after enzymatic hydrolysis to 20Brix) → pasteurization (70℃ water bath for 30min) → yeast activation (dissolve in warm water at a material-liquid ratio of 1:20, activate at 35℃ for 60min) → fermentation (addition amount is 0.5%, fermentation in a constant temperature incubator at 25℃ for 7 days) → centrifugation (10000r / min, 10min) → bottling for later use.

[0044] The full wavelength of kiwi wine was detected by using an enzyme marker in the visible light wavelength range of 200-1000nm, and it was confirmed that it absorbed light within the visible light range. The absorbance was smaller at 660nm and remained basically stable after this wavelength. Based on this, it was determined that the optimal measurement wavelength for clarity and color was 660nm.

[0045] 2. Extraction of target turbidity-causing protein

[0046] Three different methods were used to extract kiwifruit turbidity proteins from kiwifruit wine, as follows:

[0047] 1) Isopropanol precipitation method

[0048] The kiwifruit wine was divided into 5 mL tubes, and pre-cooled isopropanol was added according to the volume ratio of kiwifruit wine to alcohol solvent of 1:4 and 1:5. After mixing, it was placed in a 4℃ refrigerator for 30 minutes, centrifuged at 12000r / min for 10 minutes, and the precipitate was collected and dissolved with an appropriate volume of solution buffer solution Tirs-HCl (pH=7.4), stored at -20℃ overnight, and ultrasonically lysed for 5 minutes.

[0049] 2) EGCG precipitation method

[0050] Add EGCG to 5 mL of kiwifruit wine, mix thoroughly with a magnetic stirrer, and place in a 4°C refrigerator for 24 hours to allow EGCG and kiwifruit wine to fully react. Centrifuge at 12000 r / min for 10 minutes, collect the precipitate, mix and dissolve with an appropriate volume of buffer solution Tirs-HCl (pH=7.4), store at -20°C overnight, and ultrasonically lyse for 5 minutes.

[0051] 3) Natural precipitation method of kiwi fruit wine

[0052] The prepared kiwifruit wine was placed in a 4°C refrigerator for 4 months. After obvious precipitation appeared, 5 mL of kiwifruit wine with precipitation was taken, centrifuged at 12000 r / min for 10 min, and the precipitate was collected and mixed and dissolved with an appropriate volume of solution buffer solution Tirs-HCl (pH=7.4), kept at -20°C overnight, and ultrasonically lysed for 5 min.

[0053] 3. Analysis of turbidity-causing proteins in kiwifruit

[0054] The differences between the main turbidity-causing proteins of induced turbidity and natural turbidity were compared and analyzed by SDS-PAGE and protein mass spectrometry, as follows:

[0055] 1) SDS-PAGE gel electrophoresis

[0056] The above-extracted turbid protein sample was mixed evenly with SDS-PAGE protein loading buffer (5X) at a ratio of 4:1, and heated in a water bath at 100°C for 5 minutes. After ensuring that the sample is fully denatured, according to the concentration of the protein and the volume of the loading well, a suitable amount of solution was added to the concentrated gel channel with a microsyringe. During the protein concentration process, the initial voltage was set to 75V. After observing that the protein strip is completely separated from the concentrated gel area, the voltage was adjusted to 110V to achieve effective separation of the protein strip. When bromophenol blue runs to the bottom of the gel, turn off the power immediately. After completing the electrophoresis, move the separation gel to a container containing the staining solution and place it on a shaker overnight. Subsequently, the stained gel was transferred to a destaining solution container and also placed on a shaker for destaining until the background is clear. The gel was placed in a scanner and scanned to obtain a picture for result analysis.

[0057] like Figure 1 As shown, it is the SDS-PAGE diagram of turbidity caused by different induction methods of kiwi fruit wine in Experimental Example 1 of the present invention, and lanes 1 to 5 respectively represent marker, 1:4 isopropanol precipitated protein, 1:5 isopropanol precipitated protein, supernatant protein after EGCG-induced turbidity, and EGCG-induced turbidity precipitated protein. Figure 1 It can be seen that the relative molecular mass of the precipitated protein in kiwifruit wine is mainly concentrated in the range of 20 to 27 kDa, but the protein concentration obtained after precipitation by different methods and ratios shows significant differences, and the number of bands, clarity and background color shown in gel electrophoresis are different. Although the isopropanol precipitation method is widely used to try to extract total protein, the concentration of its lane band is significantly low from the SDS-PAGE spectrum, indicating that the extraction effect of this method is not ideal. In addition, given that isopropanol is highly irritating and potentially harmful to the human body, this method is not adopted from the perspective of safety and efficiency. Relatively speaking, the EGCG precipitation method shows a higher extraction efficiency. In the SDS-PAGE spectrum, the lane band corresponding to this method is not only the widest, but also has extremely high clarity and a relatively light background color. In addition, by further analyzing the supernatant after removing the turbidity of kiwifruit wine, it was found that the molecular mass of the protein therein is mainly concentrated in specific ranges such as 20, 14.4, and 6.5 kDa. This indicates that proteins within these molecular weight ranges are closely related to the post-turbidity behavior of kiwifruit wine, which is likely caused by the aggregation of small molecular weight proteins to form complex macromolecular protein aggregates. Comparison of bands 4 and 5 showed that the proteins precipitated by EGCG did not overlap with the proteins in the supernatant, and the sum of the proteins displayed by the two bands overlapped with the total proteins produced by isopropanol, indicating that proteins precipitated by EGCG reaction can be used for proteomic analysis of turbidity proteins in kiwifruit wine.

[0058] Therefore, EGCG-induced turbidity is the best method to precipitate turbidity-causing proteins in kiwi wine, because it can obtain turbidity-causing proteins with high purity and rich protein content. Subsequently, the protein strips (20-27 kDa) precipitated by EGCG reaction were used for QE mass spectrometry identification of kiwi wine turbidity-causing proteins.

[0059] like Figure 2 As shown, it is the SDS-PAGE image of the natural precipitation of kiwi fruit wine in Experimental Example 1 of the present invention, lanes 1 and 8 are markers, and lanes 2 to 7 are naturally precipitated fruit wine proteins. Figure 2 It can be seen that the relative molecular mass of the naturally precipitated protein in kiwi wine is mostly distributed in 20-27 kDa and 4.1-20 kDa, forming two parts of aggregation. Compared with the turbid precipitation induced by EGCG, more 4.1-20 kDa parts were separated. It is speculated that this may be due to the hydrolysis of proteins into smaller peptides distributed in 4.1-20 kDa. Therefore, QE mass spectrometry identification of kiwi wine-induced turbidity proteins was performed on the two parts of the strips.

[0060] 2) QE identification analysis

[0061] In order to identify the types of turbid proteins in kiwi fruit wine, Figure 1 The fifth lane (20-27 kDa) and Figure 2 In the analysis, proteins with molecular weights ranging from 20 to 27 kDa and 4.1 to 20 kDa in lanes 2 to 7 were identified by Q (QExactive & Easy-nLC 1000) mass spectrometry, as follows:

[0062] The lanes corresponding to the three samples were extracted from the SDS-PAGE gel using a tool and placed in a 2 mL centrifuge tube containing an appropriate amount of 10 mM Tirs-HCl buffer solution, and then stored at 4 °C. Proteomics identification was performed on the samples. After enzymatic treatment, the samples were analyzed by mass spectrometry, and the database was searched using the search engine MaxQuant 1.6.14, and the identified protein results were finally obtained. The specific search parameters are as follows:

[0063] Table 1 Database search parameters

[0064]

[0065] Table 2 Mass spectrometry identification of turbid proteins induced by EGCG

[0066]

[0067]

[0068] Table 3 Mass spectrometry identification of turbid proteins in naturally precipitated fruit wine (20-27 kDa)

[0069]

[0070] Table 4 Mass spectrometry identification of turbid proteins in naturally precipitated fruit wine (4.1-20 kDa)

[0071]

[0072] The results are shown in Tables 2 to 4 and Figures 3-4 As shown in the figure, the results of mass spectrometry identification showed that the proteins with higher contents in the turbid precipitate induced by EGCG (20-27kDa) were all thaumatin-like proteins (TLP), the proteins with higher contents in the natural precipitate of kiwi wine (20-27kDa) were thaumatin-like proteins (TLP) and Kiwellin proteins, and the proteins with higher contents in the natural precipitate of kiwi wine (4.1-20kDa) were thaumatin-like proteins (TLP) and 2Ssulfur-rich seed storage proteinlarge chain 2like2Ssulfur-rich seed storage proteinlarge chain. This may be due to the hydrolysis of thaumatin-like proteins (TLP) to smaller peptides existing in the range of 4.1-20kDa. It was found that the highest content of protein in both EGCG-induced turbidity precipitation and kiwifruit wine natural precipitation was thaumatin-like protein (TLP) (P83958·TLP_ACTCC), and its theoretical molecular weight was at 24.267 kDa, which was consistent with the electrophoresis results. It was speculated that thaumatin-like protein (TLP) was the key turbidity-causing protein in kiwifruit wine. After consulting all the identified proteins, it was found that common key turbidity-causing proteins such as chitinase were present in both precipitates. The theoretical isoelectric point of thaumatin-like protein (TLP) is 5.5, and the initial pH of kiwifruit wine is about 3.5. In view of the significant difference in pH value, it is speculated that the reason why proteins in kiwifruit wine participate in post-turbidity is not only due to the precipitation of isoelectric point proteins, but there may be other complex factors that jointly cause post-turbidity. Therefore, thaumatin-like protein (TLP) was selected for the subsequent study of post-turbidity of kiwifruit wine.

[0073] Experimental Example 2 Analysis of the interaction between key turbidity-causing proteins and polyphenols in kiwifruit wine

[0074] According to the above identification results, Protein IDs = P83958·TLP_ACTCC protein was used, and the crystal structure of Thaumatin-like protein used for docking was downloaded from the alphafold database with the retrieval number P83958. The 3D structures of small molecules CGA (chlorogenic acid), EC (epicatechin), EGCG (epigallocatechin gallate), and PC (proanthocyanidins) were downloaded from the PUBCHEM database and energy minimized under the MMFF94 force field.

[0075] AutoDock Vina 1.1.2 software was used for molecular docking. Before docking, PyMol 2.5.5 was used to process the receptor protein, including removing water molecules, salt ions and small molecules. The docking box was then set to wrap the entire protein structure. In addition, ADFRsuite 1.0 was used to convert all processed small molecules and receptor proteins into the PDBQT format required for AutoDock Vina 1.1.2 docking. During docking, the exhaustiveness of the global search was set to 32, and the other parameters remained at the default settings. The docking conformation with the highest score output was considered to be the binding conformation, and finally the docking results were visualized using PyMol 2.5.5.

[0076] Table 5 Molecular docking information of P83958 and four polyphenols

[0077]

[0078] As shown in Table 5, molecular docking was performed on P83958 and the four polyphenols. A negative binding affinity indicates the possibility of binding. Usually, the smaller the value, the greater the possibility of binding. In this complex, the docking software gave the binding affinity scores of EGCG, PC, EC, CGA and P83958 as -7.7, -7.5, -7.1, and -6.9 kcal / mol, respectively, which means that EGCG, PC, EC, CGA and P83958 protein all bind well. It is speculated that the order of binding strength is: EGCG>PC>EC>CGA. In addition, the binding of the four characteristic phenols to P83958 mainly depends on electrostatic interaction, hydrogen bonding, and hydrophobic interaction.

[0079] like Figure 5As shown, it is the molecular docking model of P83958 and four polyphenols in Experimental Example 2 of the present invention (Note: the pink dotted line represents the hydrophobic interaction, the green dotted line represents the hydrogen bonding interaction, and the brown line represents the electrostatic interaction); (A) is P83958 and EGCG; (B) is P83958 and PC; (C) is P83958 and EC; (D) is P83958 and CGA. The four polyphenols studied contain both hydrophobic groups (benzene rings) and hydrophilic groups (hydroxyl groups), and the four polyphenols have different degrees of hydrophobicity. After P83958 was docked with different polyphenols, the binding free energy of different polyphenols and P83958 was calculated by AutoDockVina 1.1.2, and it was found that the binding affinity scores were all less than -6.9 kcal / mol (Table 5), indicating that the complex structure formed by P83958 and the four polyphenols is stable, and P83958 has good affinity for the four polyphenols. The interaction between substances depends on structural compatibility and driving forces, such as hydrogen bonds, T-shaped interactions and hydrophobic interactions. Through molecular docking technology, the binding rules of P83958 and different polyphenols were discovered. The results showed that the benzene ring as a hydrophobic group and the hydroxyl group as a hydrophilic group in the polyphenol structure played a key role in the binding process of P83958. The hydrophobic group benzene ring can form a hydrophobic structure with the amino acid residues of P83958, while the hydrophilic group hydroxyl group can form hydrogen bonds with the amino acid residues of P83958 (see Figure 5 ). At the same time, the basic amino acid residues of P83958 easily interact with polyphenols to form a hydrophobic structure. These forces can mediate the interaction of P83958 and polyphenols and help form a stable complex.

[0080] Experimental Example 3 Homology Analysis of Turbidity-causing Proteins in Kiwi Wine

[0081] The basic information of thaumatin-like proteins (TLP) (P83958, A0A2R6QJH9, L7TRX5) identified by mass spectrometry, thaumatin from African arrowroot (P02883, P02884) and thaumatin-like protein (TLP) from kiwifruit (P81370) were obtained from the NCBI (http: / / www.ncbi.nlm.nih.gov / ) database. The protein sequences were aligned using clustalomega software and the sequence comparison map was beautified using jalview software. Before constructing the phylogenetic tree, the clustaw algorithm was used for sequence alignment and the neighbor-joining algorithm in mega was used to construct the phylogenetic tree.

[0082] The results are as follows Figure 6 and Figure 7As shown in the figure, through sequence comparison, it was found that the thaumatin-like protein (TLP) identified by QE in kiwifruit wine, P83958 and A0A2R6QJH9 had a homology of 92.63%, P83958 and L7TRX5 had a homology of 65.07%, and A0A2R6QJH9 and L7TRX5 had a homology of 73.21%, indicating that the thaumatin-like proteins (TLP) identified by QE all had homology and the two thaumatin-like proteins (TLP) with the highest content, P83958 and A0A2R6QJH9, had the highest homology; the thaumatin-like protein (TLP) P81370 in kiwifruit wine had a homology with P83958 and A0A2R6QJH9. The homology of A0A2R6QJH9 is as high as 96% and 93.64%, and the homology of thaumatin P02883 and P02884 in African arrowroot is 55.87% and 57.28%, indicating that P81370 is homologous to P83958, A0A2R6QJH9, P02883, and P02884, and has a high homology with the thaumatin-like protein (TLP) identified by kiwi wine QE; the homology of thaumatin P02883 and P02884 in African arrowroot with P83958 is 57.28% and 58.69%, indicating that P02883 and P02884 are homologous.

[0083] In summary, all six proteins have homology and are closely related. Figure 8 The phylogenetic tree was consistent. Since the pure thaumatin-like protein P83958 was difficult to obtain, Sigma's thaumatin protein (from African arrowroot) was used as a substitute in subsequent experiments.

[0084] Experimental Example 4 Construction of Thaumatin-Polyphenol Model of Kiwi Fruit Wine

[0085] 1. Determination of the optimal reaction ratio between thaumatin and different polyphenols

[0086] 100 μL of thaumatin protein with different concentrations (1, 2, 3 and 4 mg / mL) was mixed with 100 μL of polyphenol (EGCG, PC, EC, EGA) solution with different concentrations (1, 2, 3 and 4 mg / mL) (i.e., 64 cases of protein and polyphenol binding), and incubated at 25°C for 5 min. The absorbance was measured at 660 nm using a Molecular Devices multi-function microplate reader to determine the optimal reaction ratio of different polyphenols and screen out the best turbidity-causing polyphenols. Each group of experiments was repeated 3 times (n=3).

[0087] like Fig. 9 As shown in A, when the concentration of thaumatin is constant, the turbidity of the system (absorbance A 660 ) increases with the increase of EGCG concentration; when the EGCG concentration is 1, 2, and 3 mg / mL, the turbidity of the system (absorbance A660 ) increased with the increase of thaumatin concentration; however, when the EGCG concentration was 4 mg / mL, the turbidity of the system (absorbance A 660 ) first increased and then decreased. When the concentration of thaumatin was 3 mg / mL, the turbidity of the system (absorbance A 660 ) reached the highest value, that is, when the concentration of thaumatin protein: EGCG concentration = 4:3, the turbidity of the system (absorbance A 660 )Highest.

[0088] like Fig. 9 As shown in B, when the concentration of thaumatin is constant, the turbidity of the system (absorbance A 660 ) shows a trend of first increasing and then decreasing with the increase of PC concentration. When the protein is injected into the pre-mixed PC solution, the polyphenol molecules begin to aggregate on its surface to form a hydrophobic layer, causing the solution to become turbid and precipitate. With the continued addition of protein, when it reaches equilibrium with the polyphenol binding sites, the polyphenols interact with gelatin to form a large network structure, which further promotes the formation of precipitation. However, if the amount of protein added exceeds a certain limit, the relative content of polyphenols decreases, and the binding points on the protein surface are richer than the hydroxyl groups of polyphenols, which destroys the cross-linked network structure formed by polyphenols and proteins and reduces turbidity and precipitation. When the concentration of thaumatin is 1 mg / mL, the turbidity of the system (absorbance A 660 ) did not show an obvious trend with the increase of PC concentration; when the concentration of thaumatin was 2, 3, and 4 mg / mL, the turbidity of the system (absorbance A 660 ) shows a trend of first increasing and then decreasing with the increase of PC concentration. When the PC concentration is 1 mg / mL, the turbidity of the system (absorbance A 660 ) increases with the increase of thaumatin concentration; when the PC concentration is 2, 3 mg / mL, the system turbidity (absorbance A 660 ) showed a trend of first increasing and then decreasing with the increase of thaumatin concentration; when the PC concentration was 4 mg / mL, the turbidity of the system (absorbance A 660 ) did not show an obvious trend with the increase of thaumatin concentration. It can be clearly seen that when the concentration of thaumatin and PC are both 3 mg / mL, that is, when the concentration of thaumatin: PC concentration = 3:3, the turbidity of the system (absorbance A 660 )Highest.

[0089] like Fig. 9 As shown in C, when the concentration of thaumatin is 1, 2 mg / mL, the turbidity of the system (absorbance A 660 ) showed a trend of first increasing and then decreasing with the increase of EC concentration; when the concentration of thaumatin was 3 and 4 mg / mL, the turbidity of the system (absorbance A 660) increases with the increase of EC concentration. When the EC concentration is 1 mg / mL, the turbidity of the system (absorbance A 660 ) increases with the increase of thaumatin concentration; when the EC concentration is 2 mg / mL, the turbidity of the system (absorbance A 660 ) showed a trend of first increasing and then decreasing with the increase of thaumatin concentration; when the EC concentration was 3 and 4 mg / mL, the turbidity of the system (absorbance A 660 ) did not show an obvious trend with the increase of thaumatin concentration. It can be clearly seen that when the thaumatin concentration and EC concentration are both 4 mg / mL, that is, when the thaumatin concentration: EC concentration = 4:4, the system turbidity (absorbance A 660 )Highest.

[0090] like Fig. 9 As shown in D, the system turbidity (absorbance A 660 ) increases with the increase of chlorogenic acid concentration; when the chlorogenic acid concentration is constant, the turbidity of the system (absorbance A 660 ) increases with the increase of thaumatin concentration; when the concentration of thaumatin: chlorogenic acid = 4:4, the turbidity of the system (absorbance A 660 ) is the highest. Compared with the turbidity caused by the reaction of other polyphenols with thaumatin, the absorbance of chlorogenic acid A 660 It is lower, presumably because the chlorogenic acid molecule is smaller, resulting in less obvious turbidity reaction.

[0091] In summary, there is no obvious trend in the reaction between thaumatin and EGCG, PC, EC, and CGA polyphenols. The formation of turbidity and precipitation is indeed affected by the concentration ratio of protein to polyphenols. Only when the protein and polyphenols reach the optimal concentration ratio will the most turbidity and precipitation be produced. In addition, EGCG and PC have significantly stronger turbidity-inducing ability against thaumatin than EC and CGA, which is consistent with the results of the aforementioned experimental docking.

[0092] 2. Effect of kiwifruit wine polyphenols on the fluorescence spectrum of thaumatin

[0093] The endogenous fluorescence of protein is mainly emitted by tryptophan (Trp) and tyrosine (Tyr), both of which can be effectively excited when using an excitation wavelength of 280nm. However, when the polyphenol solution is mixed with the protein solution and a complex is formed, its fluorescence signal will be significantly reduced. According to the above reaction of thaumatin with different polyphenols, EGCG and PC have a strong ability to cause turbidity on thaumatin, so different concentrations of EGCG / PC were selected to react with a certain concentration of thaumatin to detect its fluorescence changes, as follows:

[0094] Under the environment of pH=5, 500μL of 0.1mg / mL thaumatin was mixed with the same volume of different concentrations of EGCG / PC. The concentration range of PC was 0 to 0.4mg / mL (0, 0.1, 0.2, 0.3, 0.4mg / mL), and the concentration range of EGCG was 0 to 0.08mg / mL (0, 0.02, 0.04, 0.06, 0.08mg / mL). After the mixture was incubated for 5 minutes at 25℃, it was scanned using a FluoroMax-4 fluorescence spectrometer. The excitation wavelength was set to 280nm, the emission wavelength range was 300nm to 500nm, the emission slit width was 10nm, and the changes in fluorescence intensity were recorded.

[0095] The results are as follows Fig.10 As shown, at room temperature, thaumatin and EGCG ( Fig.10 a) and PC( Fig.10 b) After the reaction was incubated for 5 min, the fluorescence intensity of the complex formed decreased with the increase of polyphenol concentration, resulting in fluorescence quenching. In this process, the maximum emission wavelength λ of the thaumatin-PC model max An obvious blue shift occurred, indicating that thaumatin interacted with PC, causing changes in the microenvironment and spatial conformation of the fluorescent chromophore of thaumatin, which increased the hydrophobicity of the chromophore microenvironment and reduced the extension of the peptide chain.

[0096] 3. Study on the fluorescence quenching type of thaumatin and characteristic phenols in kiwifruit wine

[0097] Fluorescence quenching refers to the interaction between quencher molecules and fluorescent material molecules in a solution, which leads to a decrease in the fluorescence quantum efficiency of the fluorescent material or a shortened excited state lifetime, thereby weakening the fluorescence intensity. Fluorescence quenching can be divided into two types: dynamic quenching and static quenching. Dynamic quenching originates from direct collisions between substances; static quenching is caused by weaker interaction forces such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions, which cause substances to combine to form a complex with no fluorescence or weak fluorescence. The Sterm-Volmer (SV) equation is used to determine the quenching type, and the calculation is as shown in formula (1):

[0098]

[0099] In formula (1), F represents the fluorescence intensity after adding the quencher; F0 represents the fluorescence intensity without adding the quencher; K sv represents the quenching constant of the SV equation; K q represents the molecular quenching constant; τ0 represents the fluorescence lifetime in the absence of a fluorescence quencher, and the average fluorescence lifetime is generally 10 -8 s.

[0100] The fluorescence lifetime of biological macromolecules is about τ0 = 10-8 , according to K q =K sv / τ0, the protein quenching constant K can be calculated q The order of magnitude can reach 10 12 L / (mol·s), the slope obtained by fitting the SV curve is the molecular quenching constant (K q ), when the fluorescent substance is a biological macromolecule, its diffusion constant is at most 2×10 10 L·mol -1 ·s -1 , if K q >2×10 10 L·mol -1 ·s -1 , then it belongs to static quenching. If K q <2×10 10 L·mol -1 ·s -1 , it belongs to dynamic quenching.

[0101] Table 6 Quenching constants of thaumatin fluorescence quenching by different polyphenols at room temperature

[0102]

[0103] like Fig.11 As shown in FIG. 4 , it is the Stern-Volmer curve of the fluorescence quenching of thaumatin by EGCG / PC at room temperature in Experimental Example 4 of the present invention; A is epigallocatechin gallate; B is proanthocyanidin. Fig.11 It can be seen that the SV curves fitted by the EGCG+Thaumatin protein and PC+Thaumatin protein complexes both showed a good linear relationship, indicating that the two polyphenols EGCG and PC quenched the fluorescence of thaumatin protein in a single way.

[0104] From Table 6, we can see that the K q They are 2.85×10 12 L·mol -1 ·s -1 With 0.51×10 12 L·mol -1 ·s -1 The order of magnitude is 10 12 L·mol -1 ·s -1 , which is much larger than the maximum diffusion constant (2×10 10 L·mol -1 ·s -1), that is, at room temperature within the linear range, the quenching mechanism of EGCG and PC on thaumatin is static quenching, that is, the fluorescence quenching of these thaumatin proteins is due to the formation of new complexes rather than dynamic collisions.

[0105] 4. Study on the binding sites and binding constants of thaumatin and characteristic phenols in kiwifruit wine

[0106] For static quenching, the Lineweaver-Burk (LB) double reciprocal equation can be used for characterization. The LB curve is obtained by linear fitting with lg[Q] as the x-axis and lg[(F0-F) / F] as the y-axis. The binding constant (K) of kiwifruit characteristic phenols for thaumatin can be calculated from the intercept and slope of the LB curve. a ) and the number of binding sites (n), calculated as formula (2):

[0107]

[0108] In formula (2), F represents the fluorescence intensity after adding the quencher; F0 represents the fluorescence intensity without adding the quencher; Q is the concentration of the quencher, i.e., polyphenol, in mol / L; n is the number of binding sites; K a is the binding constant, L / mol.

[0109] Table 7 Binding constants and number of binding sites of different polyphenols binding to thaumatin at room temperature

[0110]

[0111] like Fig.12 As shown in FIG. 4 , it is a Lineweaver-Burk line graph of EGCG / PC binding to thaumatin at room temperature in Experimental Example 4 of the present invention; A is epigallocatechin gallate; B is proanthocyanidin. Fig.12 It can be seen that the LB curves of EGCG, PC and thaumatin all show a good linear relationship. According to the intercept and slope of the linear fitting equation, K a and n, the results are shown in Table 7. At room temperature, the K of the interaction between EGCG and Thaumatin protein a Value 5.27L·mol -1 The number of binding sites n is 1.21, and the K of PC-Thaumatinprotein interaction is a The value is 2.89 L·mol -1 , the number of binding sites n is 0.75, indicating that EGCG has a stronger binding ability with thaumatin and higher stability.

[0112] Experimental Example 5 Application of Thaumatin in the Prevention and Control of Post-Occurrence Turbidity in Kiwi Fruit Wine

[0113] Through the above experiments, we know that the key turbidity protein P83958 of kiwifruit wine is homologous to thaumatin. As the key turbidity protein P83958 is difficult to obtain, thaumatin is used as a replacement. The interaction between thaumatin and polyphenols that cause turbidity in wine is analyzed, and the effects of polyphenols and thaumatin concentrations on the turbidity are studied. According to the above research results, thaumatin or thaumatin-polyphenol model is used to evaluate various control methods in the prevention and control of turbidity in kiwifruit wine, as follows:

[0114] 1. Evaluation of the effects of different adsorbents in the prevention and control of turbidity in kiwi wine

[0115] Take an appropriate amount of thaumatin and mix it with citric acid buffer to prepare a 1 mg / mL thaumatin solution as a mother solution for standby use; take an appropriate amount of imported bentonite (TOPGRAM TM +, referred to as TOP, purchased from Germany's Epsler Group), ordinary bentonite (purchased from Runjufang Food Co., Ltd., Meishan City, Sichuan Province), diatomite, chitosan and bentonite were prepared with UP water to prepare 0.2 mg / mL of adsorbent. According to the addition amount shown in Table 8, the protein was mixed with different adsorbents (thaumatin-imported bentonite TOP; thaumatin-ordinary bentonite; thaumatin-diatomite; thaumatin-chitosan; thaumatin-bentonite), and 6 portions of each added amount of protein and adsorbent system were prepared. After 0.5h, 1h, 2h, 4h, 8h, and 16h, the total protein concentration was determined using a BCA kit (purchased from Beijing Solebold Technology Co., Ltd.), and the specific operation steps were referred to the kit instructions. The clarification effect of the clarifier is expressed by the adsorption rate, in μg / mg.

[0116] Adsorption rate = (initial protein concentration - final protein concentration) / adsorbent concentration

[0117] Standard curve: y = 0.003651x + 0.08616 (thaumatin), R 2 =0.988.

[0118] Table 8 Addition amount of continuous monitoring experimental system

[0119]

[0120] like Fig.13As shown in the figure, the adsorption rate of imported bentonite (TOP) with different added amounts of thaumatin protein increased significantly within 0.5h to 2h of reaction, and then decreased slightly, both showing a trend of first increasing and then basically tending to be unchanged, and the adsorption rate basically reached saturation when the reaction reached 2h, and the more thaumatin protein was added, the higher the adsorption rate was, with the highest adsorption rate of 698.88μg / mg; the adsorption rate of ordinary bentonite with different added amounts of thaumatin protein increased significantly within 0.5h to 2h of reaction, and then decreased slightly, both showing a trend of first increasing and then basically tending to be unchanged, and the adsorption rate basically reached saturation when the reaction reached 2h, and the more thaumatin protein was added, the higher the adsorption rate was, with the highest adsorption rate of 657.79μg / mg; the adsorption rate of diatomaceous earth with different added amounts of thaumatin protein increased significantly within 0.5h to 2h of reaction, and then decreased slightly, both showing a trend of first increasing and then basically tending to be unchanged, and the adsorption rate basically reached saturation when the reaction reached 2h, and the more thaumatin protein was added, the higher the adsorption rate was, with the highest adsorption rate of 657.79μg / mg. The adsorption rate of chitosan and thaumatin increased significantly within 0.5h to 2h of reaction, and then decreased slightly, both showing a trend of first increasing and then basically tending to be unchanged, and the adsorption rate basically reached saturation when the reaction reached 2h, and the more thaumatin was added, the higher the adsorption rate was, with the highest adsorption rate being 503.04μg / mg; the adsorption rate of bentonite and thaumatin added in different amounts increased significantly within 0.5h to 2h of reaction, and then decreased slightly, both showing a trend of first increasing and then basically tending to be unchanged, and the adsorption rate basically reached saturation when the reaction reached 2h, and the more thaumatin was added, the higher the adsorption rate was, with the highest adsorption rate being 544.12μg / mg.

[0121] Therefore, the reaction of thaumatin with different clarifiers showed a trend of first increasing and then basically remaining unchanged, and the adsorption rate basically reached saturation after 2 hours of reaction. The adsorption capacity is: imported bentonite (TOP)> ordinary bentonite> bentonite> diatomaceous earth> chitosan.

[0122] 2. Evaluation of the effects of different proteases in preventing and controlling post-production turbidity in kiwifruit wine

[0123] Select three enzymes: papain (optimum pH 6-7, optimum temperature 55-65°C, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), neutral protease (optimum pH 6.8-7, optimum temperature 45-50°C, purchased from Beijing Solebow Technology Co., Ltd.), and alkaline protease (optimum pH 9-11, optimum temperature 50°C, purchased from Shanghai Yuanye Biotechnology Co., Ltd.); prepare four 2mg / mL thaumatin protein solutions, three of which are adjusted to the optimal pH values ​​of the three enzymes, and the other is used as a control. According to the addition amount of 20U / g of each enzyme, add them to the prepared protein solution respectively, place the solution at the optimal enzymatic hydrolysis temperature for 30 minutes, and immediately perform a boiling water bath to inactivate the enzyme for 10 minutes for all enzymatic solutions (including solutions without protease). Subsequently, the pH value of all samples was adjusted back to 5 using hydrochloric acid solution. A portion was taken from each sample and mixed with equal volumes of 1 mg / mL EGCG and PC solutions at pH = 5 at 25°C, and then the absorbance was detected at a wavelength of 660nm. The above experiment was repeated five times, and the average value was taken. The degree of change in turbidity-causing ability was calculated based on the obtained absorbance. Finally, the undigested thaumatin protein and various enzymatic hydrolysates were identified and compared by SDS-PAGE using precast gels. The change in turbidity-causing ability was calculated using a specific formula:

[0124]

[0125] Depend on Fig.14 It can be seen that after different proteases hydrolyzed thaumatin, there were significant differences in the turbidity-causing ability of the mixture with EGCG solution. Compared with thaumatin before hydrolysis, the turbidity-causing ability of all hydrolyzates was reduced. At the same time, the turbidity-causing ability of the hydrolyzates obtained by different hydrolases also showed differences. The experimental results showed that after thaumatin was hydrolyzed by papain, neutral protease and alkaline protease, the turbidity-causing ability of its products mixed with EGCG solution was reduced by 57.05%, 58.91% and 81.68%, respectively. Among them, the turbidity-causing ability of the product treated with alkaline protease decreased most significantly, followed by neutral protease, and finally papain. This shows that thaumatin is most sensitive to alkaline protease, and its sensitivity to neutral protease and papain is relatively low. Similarly, the turbidity-causing ability of these hydrolyzates after mixing with PC solution is also different, but all are lower than that of thaumatin before hydrolysis. Specifically, the turbidity-causing abilities of papain, neutral protease, and alkaline protease hydrolyzates decreased by 56.29%, 62.24%, and 65.75%, respectively, with the alkaline protease treatment product decreasing the most, followed by the neutral protease, and the papain the least. Therefore, from the perspective of the decrease in turbidity-causing ability after interaction with PC, thaumatin is most sensitive to alkaline protease, followed by neutral protease, and least sensitive to papain.

[0126] Depend on Fig.15 It can be seen that under the action of different hydrolases, thaumatin presents different hydrolysis products. Among them, the hydrolysis product of papain is between 4.1 and 6.5 kDa, the hydrolysis product of neutral protease is between 20 and 27 kDa, and the hydrolysis product of alkaline protease has basically no obvious bands, and it is found that the band of papain hydrolysis product is deeper than that of neutral protease hydrolysis product. According to the above experimental results, the following conclusions are drawn: alkaline protease has the deepest degree of hydrolysis of thaumatin, and the number of polypeptides produced is the largest; neutral protease is second; papain has the smallest hydrolysis effect.

[0127] Experimental Example 6 Comparative Experiment on Effects of Different Turbidity Models

[0128] 1. Comparison of the turbidity model constructed with proline-rich BSA (bovine serum albumin), as follows:

[0129] The experimental process refers to the evaluation of the effects of different adsorbents in the prevention and control of post-turbidity of kiwi wine in Experimental Example 5. BSA and citric acid buffer were mixed to prepare a 1 mg / mL BSA solution as a mother solution for standby use, and BSA was mixed with different adsorbents. The rest was the same as Experimental Example 5.

[0130] Standard curve: y = 0.0048x + 0.0737 (BSA) R 2 =0.997

[0131] like Fig.16 As shown, it is a continuous monitoring experiment of bovine serum albumin and different clarifiers in Experimental Example 6 of the present invention; A is imported bentonite (TOP); B is ordinary bentonite (Runjufang); C is diatomaceous earth; D is chitosan and E is bentonite (Solabo). Fig.16 It can be seen that the reaction of BSA with different clarifiers showed a trend of first increasing significantly, then slowly increasing and finally basically stabilizing, and the adsorption rate basically reached saturation when the reaction lasted for 8 hours. The adsorption capacity was: imported bentonite (TOP) > ordinary bentonite > bentonite > chitosan > diatomaceous earth.

[0132] Compared with the turbidity model constructed by thaumatin in Experimental Example 5, it was found that the adsorption rate showed a trend of first increasing and then basically remaining unchanged. The adsorption rates of thaumatin and BSA were basically saturated at 2h and 8h respectively, and the adsorption capacity was: imported bentonite (TOP)> ordinary bentonite (Runjufang)> bentonite (Solebao). Therefore, the turbidity model constructed by the present invention reacts faster, and can be applied to the evaluation of various prevention and control methods in the prevention and control of turbidity after kiwi fruit wine to improve efficiency.

[0133] 2. The turbidity model constructed by using EGCG and thaumatin-like protein, a key turbidity protein of kiwifruit wine, was compared with the natural turbidity of kiwifruit wine and the existing accelerated turbidity method in the evaluation of various prevention and control methods. Alkaline protease, bentonite, CMC and trehalose were used as clarifiers to verify the effect, as follows:

[0134] Alkaline protease (enzyme addition amount is 20U / g), imported bentonite TOP (TOP concentration is 0.2mg / mL, the preparation is the same as that of Experimental Example 5), CMC (polysaccharide concentration is 1mg / mL), trehalose (polysaccharide concentration is 1mg / mL) were used for the experiment, and the specific addition amount is shown in Table 9. Each clarifier was dissolved in kiwi fruit wine, and 200μL of the supernatant was added to a 96-well plate after being placed at room temperature for 48h. The absorbance value A of the system was determined by Molecular Devices multifunctional microplate reader. 660 A group of blank fruit wines without added clarifiers were set as controls, and different methods were used to accelerate precipitation (hot and cold cycles for 48h, tannins, EGCG).

[0135] The method for accelerating precipitation by hot and cold cycle for 48 hours is as follows: placing the kiwi fruit wine in hot and cold cycle reaction at 4°C and 80°C for 48 hours, for a total of 2 cycles.

[0136] The method for accelerating the precipitation of tannin is as follows: tannin (2 mg / mL) is added to kiwi wine, vortexed and mixed, and then placed in a 4°C refrigerator for 24 hours to allow the tannin to fully react with the kiwi wine.

[0137] The method for accelerating the precipitation of EGCG is as follows: EGCG (2 mg / mL) is added to kiwi wine, vortexed and mixed, and then placed in a 4°C refrigerator for 24 hours to allow EGCG to fully react with the kiwi wine.

[0138] The preparation of CMC (1 mg / mL) is as follows: weigh 10 mg of CMC in a beaker, add appropriate amount of UP water to dissolve, and transfer to a volumetric flask to make up to 10 mL after complete dissolution; the preparation of trehalose (1 mg / mL) is as follows: weigh 10 mg of seaweed in a beaker, add appropriate amount of UP water to dissolve, and transfer to a volumetric flask to make up to 10 mL after complete dissolution.

[0139] Table 9 Kiwi fruit wine application reaction system

[0140]

[0141] Note: All the above clarifiers (alkaline protease, bentonite, CMC, trehalose) are dissolved in kiwi fruit wine.

[0142] like Fig.17As shown, the effect diagram of different clarification methods in different wine turbidity models in Experimental Example 6 of the present invention is shown. Fig.17 It can be seen that under the four turbidity conditions, the four clarification methods all made the absorbance of kiwi fruit wine A 660 There were different degrees of decline. Under the condition of room temperature turbidity, the decline rate was in the order of TOP>alkaline protease>trehalose>CMC. Among them, bentonite TOP had the best clarification effect, with a decrease rate of 67.76%; under the condition of cold and hot cycle turbidity, the four clarification methods also decreased the absorbance of kiwi fruit wine A 660 There were different degrees of decline, and the order of decline rate was: TOP>CMC>trehalose>alkaline protease, among which bentonite TOP had the best clarification effect, and the decrease rate of absorbance was 66.78%; under tannin-induced turbidity, the order of decline rate was: TOP>trehalose>CMC>alkaline protease, among which bentonite TOP had the best clarification effect, and the decrease rate of absorbance was 57.95%; under EGCG-induced turbidity, the order of decline rate was: TOP>alkaline protease>trehalose>CMC, among which bentonite TOP had the best clarification effect, and the decrease rate of absorbance was 57.99%.

[0143] By comparison, it was found that the ranking of the clarification effects of various clarifiers under the EGCG-induced turbidity state was the same as that under the room-temperature turbidity state. Obviously, the turbidity model constructed by the present invention can be applied to the evaluation of different clarification methods in the prevention and control of post-turbidity of kiwi wine.

[0144] In summary, the present invention discovered the key turbidity-causing protein thaumatin-like protein P83958 of kiwi wine, and found that the protein has different binding abilities with different polyphenols, and thaumatin has homology with the protein, while the key turbidity-causing protein is difficult to obtain. Therefore, thaumatin was used to explore the turbidity effect of the thaumatin-polyphenol model, which was then applied to evaluate the effects of different methods in preventing and controlling post-production turbidity of kiwi wine, thereby facilitating the acquisition of an effective method for preventing and controlling post-production turbidity of kiwi wine.

[0145] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. The application of kiwi fruit wine turbidity protein or thaumatin in post-turbidity prevention and control, characterized in that: The turbidity-causing protein in the kiwifruit wine is a thaumatin-like protein.

2. The use according to claim 1, characterized in that: The protein accession number of the thaumatin-like protein is P83958.

3. The use according to claim 1, characterized in that: The thaumatin is selected from thaumatin derived from African arrowroot.

4. The use according to claim 1, characterized in that: The application is to evaluate the effects of kiwi wine turbidity protein or thaumatin on different methods in preventing and controlling post-haze kiwi wine.

5. The use according to claim 1, characterized in that: The application is: If the clarifier is an adsorbent, different clarifiers are mixed with kiwi wine turbidity protein or thaumatin mother liquor, the pH of the kiwi wine turbidity protein or thaumatin mother liquor is 5, and the total protein concentration in the mixed solution at different times is measured, and the clarification effect of the clarifier is expressed by the adsorption rate; If the clarifier is a protease, different clarifiers are mixed with kiwifruit wine turbidity protein or thaumatin for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated, the pH value of the solution is adjusted back to 5, a portion is taken out and mixed with an equal volume of EGCG or PC solution with a pH of 5 at room temperature, and then the absorbance is detected at a wavelength of 660nm, and the degree of change in turbidity-inducing ability is calculated based on the obtained absorbance; If the clarifier is a polysaccharide, the EGCG or PC solution, thaumatin and polysaccharide are fully mixed under the condition of pH = 5, placed in a constant temperature incubator for incubation, and then the mixed solution is taken out, and the absorbance of the reactant is measured at a wavelength of 660nm. The degree of change in turbidity-inducing ability is calculated based on the obtained absorbance.

6. The use according to claim 5, characterized in that: The kiwi fruit wine turbidity protein or thaumatin mother liquor is prepared with citric acid buffer; the clarifier is prepared with water to form a clarifier solution.

7. The use according to claim 1, characterized in that: The concentration of the kiwi fruit wine turbidity protein or thaumatin mother solution is 1-4 mg / mL.

8. The use according to claim 1, characterized in that: The concentration of the EGCG and PC solution is 1-4 mg / mL.

9. The use according to claim 1, characterized in that: The EGCG or PC solution, thaumatin and polysaccharide are fully mixed in a ratio of 1:1:1.

Images