Key turbidity-causing protein ASR1 of green plum fruit wine and application of key turbidity-causing protein ASR1 in post-turbidity prevention and control

Abstract

The invention discloses a key turbid-causing protein ASR1 of green plum fruit wine and application of the key turbid-causing protein ASR1 in post-turbid prevention and control. The amino acid sequence of the key turbid-causing protein ASR1 is shown as SEQ ID NO.1, and the application of the key turbid-causing protein ASR1 is selected from any one of the following applications: (a) post-turbid prevention and control are realized by targeting the key turbid-causing protein ASR1 of the green plum fruit wine; or (b) evaluating the post-turbidity prevention and control effect of the clarifying agent on the green plum fruit wine through the key turbidity-causing protein ASR1 of the green plum fruit wine or the key turbidity-causing protein ASR1 of the green plum fruit wine and a polyphenol turbidity-causing model. The key turbidity-causing protein ASR1 of the green plum fruit wine is found, the key turbidity-causing protein ASR1 is applied to turbidity prevention and control of the green plum fruit wine, prevention and control are carried out through the target ASR1, and the clarification effects of different clarification methods on the fruit wine can be evaluated through a constructed recombinant ASR1 protein-polyphenol turbidity-causing model.

Description

Technical Field

[0001] The invention relates to a technology for preventing and controlling post-turbidity of greengage wine, and in particular to a key turbidity-causing protein ASR1 of greengage wine and an application thereof in preventing and controlling post-turbidity. Background Art

[0002] During the storage period, fruit wine will produce turbid precipitation. This has a negative impact on the appearance and sales of the wine. Protein is the main cause of the post-turbidity of fruit wine, mainly because protein can interact with polyphenols to produce turbidity. The occurrence of post-turbidity is also related to pH and microbial species. Studies have shown that when the pH value is acidic, the particle size of the precipitate is smaller. Beverages contaminated by microorganisms will produce turbidity, and the metabolites produced by microorganisms will also cause changes in the taste of the wine.

[0003] The post-turbidity phenomenon of wine has attracted the attention of scholars in the early days, but the research on fruit wine at home and abroad is mainly focused on wine, especially white wine. The clarity of white wine is one of the most intuitive and important sensory quality indicators. Since the current research is limited to wine, which restricts the development of the fruit wine industry, it is necessary to study other types of fruit wine.

[0004] Bentonite adsorption has been proven to be an effective method for removing unstable proteins from white wine. When bentonite binds to proteins, these complexes are deposited in a loose manner at the bottom of the wine storage tank or fermentation tank, occupying 3% to 10% of the total volume of the wine. However, the effectiveness of bentonite is not limited to removing proteins associated with precipitation, but may also cause loss of wine aroma and color.

[0005] Greengage wine is a kind of fruit wine that is made from greengage fruit as the main raw material and carefully brewed through a specific winemaking process. Greengage wine is bright in color, usually amber or light yellow, and the wine is clear and transparent. In terms of taste, greengage wine has both the sweet and sour taste of greengage and the soft taste of alcohol, and the overall flavor is mellow and refreshing. When drinking, the aroma of greengage wine is intoxicating and brings a pleasant sensory experience. Greengage wine is divided into fermented greengage wine and soaked greengage wine. Fermented greengage wine is made from greengage or greengage juice as the main raw material, and is fermented under the action of yeast to form a greengage wine with a certain alcohol content, usually 5-14% vol.

[0006] However, due to the turbidity caused during storage and transportation, fruit wine has a considerable quality loss. This problem has become a common technical problem with the continuous development of the global beverage industry. Although researchers have made great progress in related research in this field, its mechanism has not yet been fully clarified, and there are still many basic issues that need to be further explored and studied.

[0007] Although the protein content in fruit wine is relatively low, protein instability is still one of the main reasons for turbidity. This turbidity is not caused by microorganisms, but by the gradual aggregation of proteins. As the storage time increases, it eventually leads to turbidity and precipitation in bottled fruit wine. At present, there is no specific report on the types of turbidity proteins in green plum wine. Therefore, it is necessary to study the turbidity proteins in green plum wine so as to prevent and control the turbidity of fruit wine. Summary of the invention

[0008] The purpose of the present invention is to provide a key turbidity protein ASR1 of greengage wine and its application in post-turbidity prevention and control, discover the key turbidity protein ASR1 in greengage wine, and inhibit the post-turbidity of wine by targeting ASR1 prevention and control technology.

[0009] In order to achieve the above object, the present invention provides a key turbidity protein ASR1 of greengage wine, and the amino acid sequence of the key turbidity protein ASR1 is shown in SEQ ID NO.1.

[0010] Another object of the present invention is to provide a gene encoding the key turbidity protein ASR1 of greengage wine, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] Another object of the present invention is to provide a key turbidity protein ASR1 of greengage wine and its application in post-turbidity prevention and control, wherein the application is selected from any one of the following:

[0012] (a) To achieve post-turbidity control by targeting the key turbidity protein ASR1 in greengage wine; or,

[0013] (b) The key turbidity protein ASR1 of green plum wine or the key turbidity protein ASR1 and polyphenol turbidity model of green plum wine was used to evaluate the effect of clarifier on the prevention and control of post-turbidity of green plum wine.

[0014] The amino acid sequence of the key turbidity-causing protein ASR1 is shown in SEQ ID NO.1.

[0015] Preferably, the key turbidity protein ASR1 of the greengage wine is a recombinant ASR1 protein; the recombinant ASR1 protein is obtained by constructing a pET-30a(+)-ASR1 expression vector expressing the ASR1 protein, transferring it into an Escherichia coli expression system, inducing expression, breaking the bacteria, and separating and purifying it through a 6×His affinity purification system.

[0016] Preferably, the polyphenols are selected from epigallocatechin gallate and / or proanthocyanidins.

[0017] Preferably, if the clarifier is an adsorbent, the clarifier is mixed with the key turbidity protein ASR1 of green plum wine, the pH of the mixed solution of the clarifier and the key turbidity protein ASR1 of green plum wine is 4.2, and the clarification effect is reflected by measuring any one or more of the total protein concentration in the mixed solution, the transmittance or absorbance at a wavelength of 540nm, and the chromaticity at a wavelength of 480nm; if the clarifier is a protease, the clarifier is mixed with the key turbidity protein ASR1 of green plum wine for enzymolysis, and after the enzymolysis is completed, the clarifier is inactivated. The method comprises the following steps: treating the solution with enzyme, adjusting the pH value of the solution back to 4.2, taking out a portion of the solution and mixing it with a polyphenol solution with a pH value of 4.2 in equal volumes at room temperature, and detecting the transmittance or absorbance at a wavelength of 540 nm to reflect the clarification effect; if the clarifier obtains a soluble complex by combining with the key turbidity protein ASR1 of greengage wine and the polyphenol turbidity model, then mixing the polyphenol solution, the key turbidity protein ASR1 of greengage wine and the clarifier under the condition of pH 4.2, and detecting the transmittance or absorbance at a wavelength of 540 nm to reflect the clarification effect.

[0018] Another object of the present invention is to provide a method for constructing a turbidity model of the key turbidity protein ASR1 and polyphenols of greengage wine, the method comprising: constructing a pET-30a(+)-ASR1 expression vector expressing the ASR1 protein, and transferring it into an Escherichia coli expression system, inducing expression, breaking the bacteria, and separating and purifying the recombinant ASR1 protein through a 6×His affinity purification system; mixing the recombinant ASR1 protein and polyphenols in a pH 4.2 environment to construct a turbidity model.

[0019] Preferably, the recombinant ASR1 protein and polyphenols are mixed at room temperature and pH 4.2 to construct a turbidity model.

[0020] Preferably, the polyphenols are selected from epigallocatechin gallate and / or proanthocyanidins; the concentration of epigallocatechin gallate is 0.4-1 mg / mL; the concentration of proanthocyanidins is 0.4-0.6 mg / mL; the concentration of the recombinant ASR1 protein is 0.8-1 mg / mL; and the solutions of the ASR1 protein and polyphenols are mixed in equal volumes.

[0021] Another object of the present invention is to provide a method for constructing the recombinant ASR1 protein, which comprises: based on the amino acid sequence of the key turbidity protein ASR1 of green plum wine as shown in SEQ ID NO.1, constructing a pET-30a(+)-ASR1 expression vector expressing the ASR1 protein, and transferring it into an Escherichia coli expression system, inducing expression, breaking the bacteria, and separating and purifying it through a 6×His affinity purification system; wherein the induction expression temperature is 37°C for 4 hours.

[0022] Preferably, based on the amino acid sequence of the key turbidity protein ASR1 of greengage wine as shown in SEQ ID NO.1, codon optimization is performed, and the optimized amino acid sequence is shown in SEQ ID NO.2; Nde I and HindIII restriction enzyme sites are added on both sides of the optimized amino acid sequence.

[0023] The key turbidity protein ASR1 of greengage wine of the present invention and its application in post-turbidity prevention and control have the following advantages:

[0024] The present invention discovers the key turbidity protein ASR1 of greengage wine, and applies it to the prevention and control of post-production turbidity of greengage wine. The prevention and control is carried out by targeting ASR1, and the clarification effects of different clarification methods on wine can be evaluated through the constructed recombinant ASR1 protein-polyphenol turbidity model. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the result of the hydrophilicity / hydrophobicity analysis of the ASR1 protein in Experimental Example 1 of the present invention; the peak with score < 0 is a high hydrophilicity region, and the peak with score > 1.5 is a high hydrophobicity region; the horizontal axis represents the sequence number of the amino acid residues of the protein, and the vertical axis represents the hydrophilicity characteristics of the residues.

[0026] Figure 2 : This is the secondary structure of the ASR1 protein in Experimental Example 1 of the present invention; Note: the lines of different colors in the figure represent different structural elements, the blue curve represents the α-helix, the green curve represents the β-turn, the red curve represents the extended chain, and the purple curve represents the random coil.

[0027] Figure 3 This is the prediction result of the ASR1 protein signal peptide in Experimental Example 1 of the present invention.

[0028] Figure 4 This is the prediction result of the transmembrane structure of ASR1 in Experimental Example 1 of the present invention; Note: Purple lines represent the transmembrane domain of the protein; Blue lines represent the intracellular protein distribution; Gold lines represent the extracellular protein distribution.

[0029] Figure 5 This is the analysis result of ASR1 phosphorylation sites in Experimental Example 1 of the present invention.

[0030] Figure 6 This is the tertiary structure prediction model of ASR1 in Experimental Example 1 of the present invention.

[0031] Figure 7 This is the comparison between the amino acid sequence of Prunus mume (NP_001306732.1) in Experimental Example 1 of the present invention and sequences of different species.

[0032] Figure 8This is the phylogenetic tree of Prunus mume in Experimental Example 1 of the present invention.

[0033] Fig. 9 This is the SDS-PAGE of the recombinant ASR1 protein expressed in BL21 (DE3) in Experimental Example 2 of the present invention; Lane M1: Protein marker; PC1: BSA 1μg; PC2: BSA2μg; NC: uninduced whole cells; 1: whole cells induced at 15°C for 16h; 2: whole cells induced at 37°C for 4h; NC1: uninduced cell lysis supernatant, 3: cell lysis supernatant induced at 15°C for 16h; 4: cell lysis supernatant induced at 37°C for 4h; NC2: uninduced cell lysis precipitate; 5: cell lysis precipitate induced at 15°C for 16h; 6: cell lysis precipitate induced at 37°C for 4h.

[0034] Fig.10 This is the SDS-PAGE of the purified recombinant ASR1 protein in Experimental Example 2 of the present invention; M1: Protein marker; BSA: 2.00 μg; ASR1: purified recombinant ASR1 protein.

[0035] Fig.11 It is the WB identification result of the recombinant ASR1 protein in Experimental Example 2 of the present invention; Lane M2: Western blot Protein marker; Lane 3: cell lysis supernatant induced at 15°C for 16 hours; Lane 4: cell lysis supernatant induced at 37°C for 4 hours; Lane 5: cell lysis precipitate induced at 15°C for 16 hours; Lane 6: cell lysis precipitate induced at 37°C for 4 hours.

[0036] Fig.12 The reaction of proteins with different polyphenols in Experimental Example 3 of the present invention; a: reaction of BSA with different polyphenols; b: reaction of ASR1 with different polyphenols.

[0037] Fig.13 : The fluorescence spectrum of the recombinant ASR1 protein in Experimental Example 3 of the present invention; a: fluorescence spectrum of ASR1 at different concentrations; b: fluorescence spectrum of ASR1 after high temperature induction.

[0038] Fig.14 The fluorescence spectra of the interaction between polyphenols of different concentrations and the recombinant ASR1 protein in Experimental Example 3 of the present invention; a: epigallocatechin gallate; b: proanthocyanidins.

[0039] Fig.15 This is the effect of different polysaccharides on the recombinant ASR1 protein-polyphenol turbidity model in Experimental Example 4 of the present invention.

[0040] Fig.16 The figures are the actual effects of different clarifiers in greengage wine in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] The reagents used in the following experimental examples were prepared as follows:

[0044] 0.1mol / L citric acid-sodium citrate buffer (pH 4.2, hereinafter referred to as citric acid buffer): Pipette 1.6mL of solution A and 18.4mL of solution B, and mix well. Solution A (0.1mol / L citric acid): weigh 19.213g of citric acid, dissolve, and dilute to 1000mL. Solution B (1mol / L sodium citrate): weigh 25.813g of sodium citrate, dissolve, and dilute to 1000mL.

[0045] Gum arabic, sodium alginate, carboxymethyl cellulose, guar gum, Solubleco, dextran: weigh 0.08 g of each polysaccharide and dissolve it in 0.02 M pH 4.2 citric acid buffer to obtain a polysaccharide stock solution with a concentration of 8.0 mg / mL. Dilute the working solution to the required concentration for the experiment and set aside.

[0046] Experimental Example 1 Analysis of the key turbidity protein ASR1 in greengage wine

[0047] In the early stage of greengage wine research, the inventors of the present invention analyzed the precipitation components of natural turbidity and polyphenol-induced turbidity of greengage wine by peptide mass spectrometry, and identified the key turbidity protein ASR1 of greengage wine. This experimental example predicts and analyzes the biological information of ASR1, as follows:

[0048] 1. Bioinformatics analysis

[0049] 1) Analysis of the physicochemical properties of ASR1 protein

[0050] The physicochemical properties of the ASR1 amino acid sequence were predicted and analyzed using the ExPASy (http:web.expasy.org / protparam / ) online software. The prediction results showed that the protein molecular formula was C 931 H 1351 N 283 O322 S1, protein molecular mass (MW / Da) is 22691.73, total number of atoms is 205, theoretical isoelectric potential (PI) is 5.57, total number of negatively charged amino acid residues (Asp+Glu) is 39, total number of positively charged amino acid residues (Arg+Lys) is 21.

[0051] Table 1 shows the average amino acid content of ASR1 protein

[0052]

[0053]

[0054] Table 1 shows the average amino acid content of ASR1 protein. It can be seen that glycine is the largest, with a content of 22.4%, followed by histidine, which is a turbidity-causing protein rich in hydrophobic amino acids. The lowest content of tryptophan and cysteine ​​is 0. Generally, turbidity-causing proteins are mainly divided into two categories according to the characteristics of their amino acid composition. One type of protein or polypeptide is rich in proline (Pro) or hydroxyproline (Hyp) residues, and the other type of protein is rich in hydrophobic amino acids - histidine (His), arginine (Arg), glycine (Gly) and glutamic acid (Glu) residues. The appearance of high proportions of His, Gly, etc. will lead to irregular curling of proteins.

[0055] 2) Analysis of hydrophilicity and hydrophobicity of ASR1 protein

[0056] ProScaLe (http:web.ecpasy.org / protscale / ) was used to predict the hydrophobicity and hydrophilicity of the ASR1 protein amino acid sequence. The results are as follows: Figure 1 As shown, ASR1 has 4 highly hydrophilic regions and 1 highly hydrophobic region, that is, the protein is a highly hydrophilic protein. A highly hydrophilic protein means that ASR1 is easily dissolved in water or exists stably in an aqueous environment.

[0057] 2. Protein structure prediction

[0058] 1) Prediction of ASR1 protein secondary structure

[0059] The secondary structure of protein is composed of E (extended strand), H α-helix), C (coil) and T (β-turn), which may reflect certain specific biological activities of the protein. Multiple adjacent amino acid residues in the polypeptide chain can form the three-dimensional structure of its local peptide chain.

[0060] The present invention uses SOPMA software to predict and analyze the secondary structure of the ASR1 protein amino acid sequence. The results are as follows: Figure 2 As shown, the protein in ASR1 protein is composed of α-helix, extended chain, β-turn and random coil. The structural element with the highest proportion in the secondary structure of this protein is random coil, reaching 49.27%. The remaining structural elements occupy the following proportions: α-helix (Hh) is 28.29%, extended chain (Ee) is 11.22%, and β-turn (Tt) is 11.22%.

[0061] 2) Prediction of protein signal peptide of ASR1 protein

[0062] In addition to the expression host and promoter, the secretory signal peptide also significantly affects the secretory expression of recombinant heterologous proteins. The signal peptide is a small hydrophobic peptide fragment located at the N-terminus of the newly synthesized peptide chain, generally composed of 5 to 30 amino acids, including 10 to 15 hydrophobic amino acids. The main function of the signal peptide is to guide the newly synthesized polypeptide to pass through the endoplasmic reticulum membrane and enter the lumen, and then be secreted outside the cell.

[0063] The present invention uses the software SignaLP5.0 to predict and analyze the ASR1 signal peptide. The results are as follows: Figure 3 As shown, the prediction result shows that the ASR1 protein has no signal peptide function, indicating that the protein is not a secretory protein.

[0064] 3) Analysis of the transmembrane domain of ASR1 protein

[0065] Usually membrane proteins cannot be expressed in prokaryotic expression systems. If the transmembrane region is predicted and found in the sequence, a eukaryotic expression system will be needed for expression. If a prokaryotic expression system is used for expression, the transmembrane region sequence will need to be deleted.

[0066] The present invention uses online transmembrane region prediction software TMHMM analysis, the results are as follows Figure 4 As shown, it was found that the protein did not have a transmembrane structure and was an extramembrane protein, so it could be expressed using the Escherichia coli prokaryotic expression system.

[0067] 4) Analysis of phosphorylation sites of ASR1 protein

[0068] Protein phosphorylation is mainly concentrated on the tyrosine, serine and threonine residues in the peptide chain. These residues have free hydroxyl groups and are not charged themselves. After phosphorylation, the protein has a charge, which changes the structure and further causes changes in protein activity. This is the significance of protein phosphorylation.

[0069] The present invention uses the software Netphos3.1 to analyze the potential phosphorylation sites of the ASR1 amino acid sequence. Figure 5 As shown, the ASR1 protein has 8 serine (Ser) phosphorylation sites, 2 threonine (Thr) phosphorylation sites, and 10 tyrosine (Tyr) phosphorylation sites.

[0070] 5) Prediction of the tertiary structure of ASR1 protein

[0071] The present invention uses the software SWISS-MODLE to analyze the three-dimensional structure of ASR1 protein and predicts the structural changes of the protein through three-dimensional modeling of the protein structure. The results are as follows: Figure 6 As shown in the figure, the ASR1 protein itself has a relatively loose structural characteristic. This structural change increases the possibility of the ASR1 protein interacting with polyphenols, which easily causes turbidity. By analyzing the structure of the ASR1 protein, it was found that it is mainly composed of α-helices and random coils, which is consistent with the secondary structure prediction and the basic structure of turbidity-causing proteins.

[0072] 3. Protein sequence alignment and phylogenetic tree construction

[0073] By analyzing the reference sequence in the NCBI protein database, the gene (SEQ ID NO.2) and protein (SEQ ID NO.1) sequences of ASR1 are as follows:

[0074]

[0075] The present invention uses blast to compare the sequence of Prunus mume (NP_001306732.1) and selects species with high similarity for analysis (see Figure 7), apple Malus domestica (XP_008340103.1), peach Prunus persica (XP_007201299.1), strawberry Fragaria x ananassa (AEO86811.1), wild soybean Glycine soja (7AG4984084.1), Abrus precatorius (XP_027364991.1), tea Camellia sinensis (AHJ09608.1), camphor Cinnamomum micranthumf.kanehirae (RWR82263.1), soybean Glycine max (XP_003536424.1), wild peanut Senna tora (KAF7812824.1) and sequences with high similarity were found in these species. Further analysis revealed that the ASR1 gene family is an ABA-WDS domain. The domain is one of the most useful levels for understanding protein function. Analysis based on the domain family has a profound impact on the functional characterization and annotation of recently released genomic data. Currently, there is little research on the ABA-WDS domain. Only one study found that the domain is an intrinsically disordered functional domain, and its conformational plasticity may help support the multiple functions of the ASR family, including its role in abiotic stress tolerance (Structural and functional characterization of the aba-water deficit stress domain from wheat and barley: An intrinsically disordered domain behind the versatile functions of the plant abscissic acid, stress and ripening protein family[J]. International Journal of Molecular Sciences, 2021, 22(5): 2314-2339).

[0076] The phylogenetic tree of these species sequences was constructed using MEGA11 software (see Figure 8), the similarity of ASR1 protein sequence with the amino acid sequences of 9 different species ranged from 59.35% to 92.68%. The highest similarity was 92.68% with the amino acid sequence of peach Prunuspersica, and the lowest similarity was 59.35% with the amino acid sequence of strawberry Fragaria x ananassa. Since this sequence is a turbidity-causing protein in green plum beverages, it is speculated that it may also be a key turbidity-causing protein in beverages of the above-mentioned similar species.

[0077] Experimental Example 2 Expression of recombinant ASR1 protein

[0078] Based on the bioinformatics analysis of ASR1 protein, the pET-30a(+)-ASR1 expression vector was constructed, and the recombinant ASR1 protein was expressed, separated and purified using the Escherichia coli expression system and 6×His affinity purification system.

[0079] 1. Synthesis of ASR1 expression sequence

[0080] According to the ORF of the target gene ASR1 reference sequence NM_001319803, with the help of GenSmart TM CodonOptimization online codon optimization software was used to optimize the ORF (open reading frame), and Nde I and Hind III restriction enzyme sites were added on both sides, and finally sequence synthesis was performed. The designed ASR1 expression sequence (SEQ ID NO.3) is as follows (Note: the shaded part is the ORF, and the underline indicates the Nde I and Hind III restriction enzyme sites):

[0081]

[0082] 2. Construction of pET-30a(+)-ASR1 expression vector

[0083] Double digestion with restriction endonucleases NdeI and HindIII, the reaction system is as follows:

[0084] Table 2 shows the ASR1 double enzyme digestion reaction system

[0085]

[0086] Prepare an ice box, melt the reagents on ice, perform the experiment on ice, mix well, and set the reaction conditions at 37°C for 1 hour. Pipette the digested product and spot it into 1% agarose gel for electrophoresis. If the target fragment is completely digested with the vector, purify and recover the product and store it at -2°C or directly connect it.

[0087] After the digestion products were separated by gel electrophoresis, the DNA sequence to be expressed and the cut pET30a(+) plasmid were cut from the gel and recovered by the method provided by the agarose gel recovery kit, and ligated and transferred into competent E. coli (BL21(DE3)) according to the experimental method provided by the T4 ligase kit, and positive single clones were screened for DNA sequencing verification. The plasmid was extracted according to the method provided by the plasmid mini-extraction kit and stored at -20°C for use.

[0088] The recombinant plasmid was sent to Sangon Biotech Co., Ltd. for sequencing identification. After the sequencing results were returned, the sequence was compared using NCBI online software. The results showed that the cloned ASR1 ORF sequence was consistent with the actual ASR1 sequence, indicating that the cloned fragment was correct.

[0089] 2. Inducible expression of recombinant ASR1 protein

[0090] The correctly identified pET-30a(+)-ASR1 recombinant plasmid was transferred into the expression strain Escherichia coli BL21(DE3). The positive clones of Escherichia coli BL21(DE3) containing the pET-30a(+)-ASR1 recombinant plasmid were selected and placed in LB medium containing kanamycin, and cultured overnight at 37°C in a constant temperature shaker. The next day, the cells were inoculated into fresh LB medium containing kanamycin at a ratio of 1:100 between the volume of the cultured cells the night before and the volume of the fresh LB medium to expand the culture. At the same time, the Escherichia coli BL21(DE3) strain containing the pET-30a(+) plasmid was used as a blank control. Culture until OD 600nm When the concentration reaches about 0.6, add IPTG with a final concentration of 0.5mM, and then induce expression at 37℃ for 4h and 15℃ for 16h in a 28℃ constant temperature shaker. At the same time, set up a group without IPTG as an uninduced control. After induction, centrifuge at 8000rpm and 4℃ for 10min to collect the bacteria. Wash the bacteria with 1 / 2 and 1 / 4 volume of pre-cooled PBS in turn, and collect the bacteria by centrifugation. Finally, resuspend the bacteria with 1 / 10 volume of PBS for use.

[0091] 1) Small-scale expression of ASR1 protein

[0092] Pick from a single colony and inoculate into 5 ml LB medium containing the corresponding antibiotics, then place in a shaker at 37°C and culture overnight at 220rpm. Set aside a non-induced control group, then add IPTG with a final concentration of 0.5mM to the culture, induce at different temperatures, and culture at 200rpm for the corresponding time (15°C, 16h; or 37°C, 4h). Take 10μL of bacterial solution and add protein loading buffer and store at -20°C. Transfer the remaining bacterial solution to a centrifuge tube, centrifuge at 4°C, 10000rpm for 5min, and collect the bacteria. Resuspend the bacteria with 10mL PBS buffer, add PMSF solution at a volume ratio of 1:1000 of PBS bacterial solution and PMSF, and ultrasonically break it for 20min under the cooling of an ice-water mixture. Take 2mL of the broken liquid, centrifuge at 4°C, 9000rpm for 20min, take 10μL of the supernatant and add the protein loading buffer. Pour off the remaining supernatant, resuspend the precipitate with an appropriate amount of deionized water, and take 10 μL to add protein loading buffer. Heat the supernatant after induction at 95°C for 10 minutes, and then perform SDS-PAGE electrophoresis to detect protein expression.

[0093] 2) Inducible expression of ASR1 protein

[0094] After determining the optimal induction conditions for in vitro expression of the target protein, large-scale expression of the target protein can be performed as follows:

[0095] Place the culture medium containing the target protein in a 3L conical flask and place it in a temperature-controlled shaker for shaking culture. Dissolve the glycerol bacteria of the strain stored at -80℃ and inoculate it into 10mL of sterile LB culture medium containing the corresponding antibiotics, and shake (220rpm) in a 37℃ shaker overnight. Transfer the overnight culture to 1L of sterile LB culture medium containing the corresponding antibiotics at a ratio of 1:1000, and shake (220rpm) in a 37℃ shaker until OD 600nm When the p value reaches 0.8, add IPTG with a final concentration of 0.5mM and induce expression at 37℃ (200rpm) for 5h or 22℃ (200rpm) for 16h. After induction, centrifuge the bacterial solution at 4℃ and 9000rpm for 10min to collect the bacteria. The bacteria can be used directly for protein purification or immediately frozen at -80℃.

[0096] 3. Extraction of recombinant protein

[0097] Use a cell ultrasonic disruptor to disrupt the bacteria, and the whole process is carried out on ice. The working conditions are: power 200W, working time 5s, interval time 9s, and the whole time depends on the amount of bacteria. Generally, ultrasonication is performed until the liquid is clear. After ultrasonic disruption, centrifuge at 12000rpm and 4℃ for 15min, collect the supernatant and precipitate respectively, and resuspend the precipitate in PBS for subsequent protein expression analysis.

[0098] 4. Purification of recombinant ASR1 protein

[0099] The target protein was dialyzed into buffer overnight after one-step nickel column affinity chromatography. The protein was filtered with a 0.22μm filter membrane and then aliquoted and placed on crushed ice for later use or temporarily stored at -20°C. The His-tagged protein purification pre-packed column was properly installed on the natural product purification instrument that had been cleaned with deionized water; the flow rate was adjusted (5mL / min), and the pre-packed column was washed with 3 to 5 column volumes of deionized water, and then the column was balanced with 10 to 15 column volumes of Washbuffer to stabilize the baseline and load the sample; 2min / mL, 5 column volumes of Elutionbuffer were used for one-step elution and the eluate was collected; all pipelines were washed with pH8.0, 10mM PBS buffer, and the eluate was loaded according to the operating requirements of the G25 desalting column, and the desalting was performed at a flow rate of 1mL / min and the desalted product was collected. The desalted product was aliquoted and stored at -70°C or freeze-dried into powder and stored for later use. A small amount of all components such as uninduced whole cells, unpurified whole cells, puncture peaks (elution with wash buffer), and elution products (elution with elution buffer) during the purification process were reserved for subsequent identification and analysis. Finally, about 10 mL of 20% ethanol was added to the column to prevent microbial growth and placed at 4°C to protect the Ni affinity column.

[0100] 5. SDS-PAGE identification of recombinant protein

[0101] Preparation of protein samples: Take 20 μL of blank sample before induction, supernatant and precipitate after induction, mix them thoroughly in SDS-PAGE protein loading buffer (5×) at a ratio of 4:1, then put them in a boiling water bath for denaturation for 10 min, centrifuge at 12000 rpm for 1 min at room temperature, and add samples in sequence.

[0102] Preparation of SDS-PAGE gel: Refer to the instructions of the SDS-PAGE gel preparation kit of Solebow Biotechnology Co., Ltd. to select and prepare a vertical electrophoresis gel with a 5% concentrated gel and a 12% separation gel. The gel electrophoresis gel ratio is according to Table 3. The speed of pouring the gel should not be too fast, otherwise bubbles will easily appear.

[0103] Sample loading and electrophoresis: correctly load the vertical electrophoresis film into the vertical electrophoresis tank, add 1× electrophoresis buffer, and gently pull out the comb teeth above the colloid; add Maker, supernatant and loading buffer mixture, precipitation solution and loading buffer mixture to the comb slot in sequence; use 80V for protein electrophoresis in the stacking gel, and when indophenol blue reaches the boundary between the stacking gel and the separation gel, increase the voltage to 160V, and stop the electrophoresis when the indophenol blue electrophoresis reaches 1cm from the bottom of the film.

[0104] Coomassie Brilliant Blue Staining: Take out the glass slide and gently place the colloid sandwiched between them into the staining tray. Add an appropriate amount of Coomassie Brilliant Blue staining solution to the staining tray, then place the staining tray on a decolorizing shaker, and after appropriate slight shaking, let it stand overnight. The next day, recover the staining solution and add an appropriate amount of decolorizing solution for the corresponding decolorization treatment.

[0105] Development: When the protein bands are clearly visible after destaining, take out the film and place it in a gel imager for observation, recording and photographing.

[0106] Table 3 is the gel electrophoresis gel ratio table

[0107]

[0108] The results of SDS-PAGE are shown in Figures 9-10 The molecular weight of the purified recombinant protein ASR1 is about 30 kDa. The purified sample band is very single, and the purity of the sample is greater than 90% after grayscale scanning quantification.

[0109] 6. Western-blot identification of recombinant protein

[0110] To further verify the accuracy of the induced expression of recombinant ASR1, Western blot analysis of the above protein products was performed using an antibody that specifically recognizes the 6×His tag, as follows:

[0111] After the SDS-PAGE electrophoresis was completed, the SDS-PAGE strips were removed from the device, rinsed with water, and immersed in membrane transfer buffer. The purified ASR1 protein was tested for antibodies using Western-blot.

[0112] Transfer: Cut two filter papers and a PVDF membrane. Activate the PVDF membrane in methanol for 10 seconds and then soak it in clean water for 3 minutes. Soak the filter paper and PVDF membrane in transfer buffer for several minutes. Install them in the order of positive electrode-sponge-filter paper-PVDF membrane-protein gel-filter paper-sponge-cathode and place them in the transfer electrophoresis tank. Use a glass rod as a roller to gently squeeze out bubbles in each layer. Connect the gel surface to the negative electrode, the PVDF membrane to the positive electrode, and turn on the power supply. The transfer conditions are set to a constant current of 250mA for 70min.

[0113] Blocking: remove the PVDF membrane and wash the membrane with PBST on a horizontal shaker for 5 minutes; Blocking: 5% skim milk, put it in an incubation box, and shake it gently on a shaker for 2 hours.

[0114] Primary antibody incubation: dilute the antibody to an appropriate concentration with skim milk solution. Place the PVDF membrane in the primary antibody solution (anti-6×His tag antibody, concentration 1:2000), incubate overnight at 4°C, and then wash the PVDF membrane 3 times with TBS buffer, 5 minutes each time.

[0115] Secondary antibody incubation: Take out the membrane, wash it three times with PBST, place the PVDF membrane in the secondary antibody solution (goat anti-mouse, concentration of 1:5000), incubate at room temperature for 2 to 3 hours, then wash the PVDF membrane three times with TBST buffer, each time for 10 minutes.

[0116] Development: After the reaction is completed, take out the PVDF membrane and place it in a clean box. Rinse it in water, take a photo and store it.

[0117] The results are as follows Fig.11 As shown, compared with the results of SDS-PAGE, the protein band specifically recognized by the antibody is around 30 kDa, which is consistent with the induced expression fragment in SDS-PAGE, proving that the present invention successfully induced the expression of recombinant ASR1 protein, and the optimal temperature for the expression of recombinant ASR1 protein is 37°C for 4 hours.

[0118] 7. Determination of the secondary structure of recombinant ASR1 protein

[0119] Circular dichroism (CD) spectroscopy can be used to study the secondary conformation of proteins and the composition of their secondary structures. In the far-ultraviolet region of the circular dichroism spectrum (190-240 nm), peptide bonds are the main absorbing groups, so the CD spectrum in this region can reflect the conformational information of the protein main chain, such as α-helix, β-fold, β-turn and random curl.

[0120] The recombinant purified ASR1 (4.49 mg / mL) was scanned at a wavelength range of 180-260 nm, a bandwidth of 1 nm, and a response time of 2 s, using ultrapure water as a blank control. Each sample was scanned 3 times, and the average value was taken for graphical analysis. The circular dichroism results are expressed as the average ellipticity [θ], in deg cm 2 / dmol. CDNN software was used to analyze the secondary structure of the protein.

[0121] The results showed that ASR1 had a negative peak at a wavelength of 203nm, which is usually a hallmark of irregular curling. CDNN was used to further calculate that the proportion of antiparallel helices in ASR1 was 42.7%. This is consistent with the expectations in Experimental Example 1, and the main original structure of ASR1 is irregular curling.

[0122] Experimental Example 3 Characterization of the turbidity-causing activity of recombinant ASR1 protein

[0123] 1. Verification of the turbidity-causing activity of recombinant ASR1 protein and different types of polyphenols

[0124] By examining the changes in the active ingredients of greengage wine under different process conditions, the characteristic phenols of greengage wine, chlorogenic acid (CA), syringic acid (SA), epicatechin (EC) and catechin ((+)-Catechin) were screened out. Therefore, the common plant polyphenols epigallocatechin gallate (EGCG), proanthocyanidins (PC) and greengage wine characteristic phenols chlorogenic acid (CA), syringic acid (SA), epicatechin (EC) and catechin ((+)-Catechin) were selected for the experiment.

[0125] The polyphenols were prepared with pH 4.2 citric acid buffer to concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively (the following experiments were all prepared with pH 4.2 citric acid buffer, which will not be described in detail); ASR1 protein solution (pH 4.2) and BSA (bovine serum albumin) solution (pH 4.2) were prepared into solutions with a concentration of 1 mg / mL; the protein solution and the polyphenol solution were mixed in equal proportions, and after keeping them at 25°C for 5 minutes, the absorbance was detected at a wavelength of 540 nm.

[0126] See also Fig.12 a, with the increase of EGCG concentration, BSA turbidity gradually increased; with the increase of PC concentration, the system turbidity also increased significantly, and after the concentration of 0.6 mg / mL, it was much greater than the turbidity of EGCG and CA; for the characteristic phenols of green plum wine, chlorogenic acid, epicatechin, syringic acid, and catechin, there was no obvious turbidity effect in the BSA system. At the same time, within the selected polyphenol concentration range, the interactive turbidity system of CA and BSA did not change significantly. See Fig.12b. With the increase of EGCG concentration, the turbidity of ASR1 gradually increased; with the increase of PC, the turbidity of ASR1 showed a trend of first rising and then falling. The absorbance of PC was the largest at 0.4 mg / mL, proving that the most turbidity was produced at this time, and the turbidity was greater than that of EGCG and CA at 0.4 mg / mL and 0.6 mg / mL. When catechin was 0.4-1.0 mg / mL, the absorbance gradually increased, proving that the turbidity formed was increasing, and the absorbance was the highest at 1 mg / mL, which was 0.116. However, the overall turbidity-inducing ability of the characteristic phenols of green plum wine was not as good as that of EGCG and PC. Similar to the BSA reaction, there was no obvious change in the turbidity of the interaction between ASR1 and CA. In subsequent experiments, EGCG and PC were selected for the next step of the experiment.

[0127] Under certain polyphenol concentration conditions, the turbidity-causing ability of ASR1 was significantly better than that of BSA, proving that the ASR1 protein has a strong turbidity-causing ability.

[0128] 2. Changes in fluorescence spectra after the combination of recombinant ASR1 protein and different types of polyphenols

[0129] 1) ASR1 fluorescence spectrum analysis

[0130] The ASR1 protein was dissolved into a 1 mL / mg mother solution with pH 4.2 buffer, and then diluted with buffer to 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL and 400 μg / mL, and then 1 mL of the corresponding protein solution was taken in a microcentrifuge tube and analyzed by a fluorescence spectrophotometer at room temperature. 300 μg / mL ASR1 protein solution was selected for heating for 10 min denaturation treatment and then measured again. The intrinsic fluorescence of the protein mainly comes from Trp and Tyr residues. When the excitation wavelength is 280 nm, the luminescence of the protein is the content of Trp residues. The scanning conditions are set as follows: the excitation wavelength is 280 nm, the emission wavelength is 300 nm to 500 nm, and the emission slit is 10 nm.

[0131] like Fig.13 As shown in a, the fluorescence intensity of ASR1 increases with the increase of protein concentration. The structural integrity of ASR1 was further studied by detecting the fluorescence spectrum of ASR1 before and after denaturation. Fig.13From b, we can see that the undenatured ASR1 shows the maximum fluorescence emission intensity at 325nm. After heating at 100℃ for 10min, the fluorescence intensity of ASR1 increases, and the maximum fluorescence absorption peak blue-shifts. Blue-shift means that the absorption peak or emission peak in the fluorescence spectrum moves toward higher energy, corresponding to light with shorter wavelength. This indicates that the protein structure has changed, and the high temperature condition induces protein depolymerization, resulting in an increase in fluorescence intensity. It is speculated that during the process of protein denaturation, the Trp residues inside the protein are gradually exposed to the solvent, thereby enhancing the hydrophobicity of the loosely structured protein complex.

[0132] 2) Fluorescence spectroscopy of the interaction between polyphenols and ASR1

[0133] After a certain concentration of polyphenol solution is mixed with ASR1 solution to form a complex, the intensity of its endogenous fluorescence signal decreases, that is, fluorescence quenching. The complex formed by mixing two polyphenols of different concentrations with ASR1 and incubating for a certain period of time is analyzed by fluorescence spectrum, as follows:

[0134] ASR1 protein (pH 4.2) at a concentration of 400 μg / mL was mixed with EGCG or PC at different concentrations (0, 200, 400 μg / mL) in equal volumes, incubated at 25°C for 5 min, and then the fluorescence spectrum was scanned using FluoroMax-4. Scanning conditions: the excitation wavelength was set to 280 nm, the emission wavelength was 300 nm to 500 nm, and the emission slit was 10 nm.

[0135] The results are as follows Fig.14 As shown in the figure, the fluorescence intensity of the complex formed after ASR1 was incubated with different polyphenols decreased with the increase of polyphenol concentration, and the fluorescence quenching phenomenon was consistent with expectations. Different polyphenol concentrations have different effects on the fluorescence quenching of ASR1. Under the condition of constant temperature, after adding EGCG and PC solutions, the fluorescence intensity of ASR1 weakened, and the fluorescence intensity of the composite system also weakened, indicating that the higher the concentration of polyphenols, the stronger the fluorescence quenching effect of ASR1. It is speculated that it is the interaction between polyphenol molecules and proteins. Polyphenol molecules have multiple phenolic hydroxyl groups, which can bind to hydrophobic groups on the surface of proteins. Therefore, the addition of polyphenols leads to a reduction in the binding sites of fluorescent probes and proteins, thereby reducing the fluorescence intensity. In addition, with the increase of polyphenol concentration, the overall peak shape of the fluorescence emission peak of ASR1 remains basically unchanged.

[0136] Experimental Example 4 Construction of a recombinant ASR1 protein-polyphenol turbidity model for the prevention and control of post-turbidity in green plum wine

[0137] 1. Determination of the wavelength for measuring clarity of the recombinant ASR1 protein-polyphenol turbidity model

[0138] 1 mg / mL EGCG and 1 mg / mL recombinant ASR1 protein were mixed in equal proportions and incubated for 5 minutes, and an appropriate amount of 1M HCl was added to adjust the pH to 2.6 to simulate the pH of green plum wine. 2 mL of sample was taken, and the full wavelength of the turbid system was measured with an enzyme marker to determine the absorption light of the turbid simulation system in the visible light region, and the optimal measurement wavelength of clarity and color was determined based on the absorption curve.

[0139] The results showed that the green plum wine turbidity system had a maximum absorption peak at 300nm, and there was no obvious absorption peak in the visible light range of 420nm to 800nm, which reflected that the simulated fruit wine turbidity system had no color change in the visible light range. At 540nm, the measured absorbance was small and remained basically unchanged after 540nm, so 540nm was selected as the determination wavelength of the clarity of the fruit wine turbidity system in subsequent experiments.

[0140] 2. Recombinant ASR1 protein-polyphenol turbidity model to evaluate the effect of polysaccharide interference

[0141] At pH 4.2, 1 mg / mL EGCG, 1 mg / mL ASR1 protein, and different concentrations of polysaccharides were added to a centrifuge tube (phenolic substances 100 μL + protein 100 μL + polysaccharide 100 μL), and a vortex was used to mix them thoroughly. The tube was placed in a 25°C constant temperature incubator for 5 minutes, and 200 μL was taken from each tube and added to a 96-well plate for determination. The absorbance value A of the reactant was determined using an enzyme marker. 540 At the same time, a group of polyphenol-protein with the same concentration without adding polysaccharides was set as a blank control, and the absorbance change value-polysaccharide concentration response curve was drawn. Each group of experiments was repeated three times (n=3). The six polysaccharides were: gum arabic, sodium alginate, carboxymethyl cellulose, guar gum, β-glucan, and dextran. The polysaccharide concentration gradient was set as 0, 0.5, 1, 2, 4, and 8 mg / mL.

[0142] Depend on Fig.15 It can be seen that the interference effect is: sodium alginate> dextran> gum arabic; the interference effects are all greater than 30%, and the interference effects are better.

[0143] 3. The actual application effect of polysaccharides in fruit wine

[0144] 1) Preparation of greengage wine

[0145] The primary fermented greengage wine was prepared by turbid juice fermentation and only a simple clarification treatment was performed. The main process route is as follows:

[0146] Pretreatment: Select fresh fruits that are highly mature, plump and not rotten. Wash them repeatedly and put them into a wall-breaking machine to break them into green plum pulp.

[0147] Adjust sugar and acid: Add sugar to make the soluble solid content reach 20°Brix.

[0148] Pasteurization: Place the green plum juice in a constant temperature water bath at 60℃ for 30 minutes.

[0149] Enzymatic hydrolysis: 0.02% (w / w) of complex enzyme (pectinase: cellulase = 1:1.5) was added.

[0150] Yeast activation: first dissolve the yeast in warm water at a solid-liquid ratio of 1:20, and activate the bacteria at 35°C for 60 minutes; under a sterile environment, transfer the green plum juice to a sterilized 1L triangular bottle, with the volume of the fermentation liquid in each bottle not exceeding 70%, inoculate 0.5% SY Angel Yeast, and seal the bottle with a sealing film for later use.

[0151] Fermentation: Transfer the flask containing the fermentation liquid to a constant temperature shaker set to a certain temperature and ferment for 12 days.

[0152] 2) Practical application effect of polysaccharides in fruit wine

[0153] In order to accelerate the precipitation, four models were established in different ways: a 48-hour hot and cold cycle model, a tannin system model, an EGCG system model, and a set of fruit wine control system models. TM+ (0.2 mg / mL), gum arabic (0.005 mg / mL), and sodium alginate (0.005 mg / mL) were used as experimental reagents. The additions were made as follows:

[0154] Group 1: 1000 μL green plum wine;

[0155] Group 2: 500 μL alkaline protease + 500 μL green plum wine;

[0156] Group 3: 500 μL bentonite + 500 μL green plum wine;

[0157] Group 4: 500 μL gum arabic + 500 μL green plum wine;

[0158] Group 5: 500μL sodium alginate + 500μL green plum wine.

[0159] 48-hour hot and cold cycle model: Define the temperature range of hot and cold cycles, freezing temperature -5°C and heating temperature 50°C, set the cycle period (cycle every 12 hours). Store the sample at the set freezing temperature for 12 hours, then transfer to the set heating temperature for 12 hours, for a total of 48 hours of cycles.

[0160] Tannin system model: prepare a 2 mg / mL tannin solution and dissolve the tannin with pH 4.2 citric acid buffer; divide the fruit wine samples into several groups, add 2 mg / mL tannin solution to each group, and fully mix the tannin solution and the fruit wine samples to ensure uniform distribution.

[0161] EGCG system model: prepare 2 mg / mL EGCG solution, the solvent is pH 4.2 citric acid buffer; divide the fruit wine samples into several groups, add 2 mg / mL EGCG solution to each group, and mix the EGCG solution and the fruit wine samples thoroughly.

[0162] All the above-mentioned clarifiers (alkaline protease, bentonite, gum arabic, sodium alginate) were dissolved in green plum wine. The reaction system was centrifuged at 10000rpm for 5min. After centrifugation, 200μL of the supernatant was taken and added to a 96-well plate. After sufficient reaction, the absorbance of the system was measured at 540nm using a Molecular Devices multifunctional microplate reader. A group of room temperature blank green plum wines without any added substances was set up as a control.

[0163] Depend on Fig.16 It can be seen that in practical applications, gum arabic and sodium alginate also show good clarification effects.

[0164] In summary, the present invention discovered the key turbidity protein ASR1 of greengage wine, and applied it to the prevention and control of post-production turbidity of greengage wine by targeting ASR1. The clarification effects of different clarification methods on wine can be evaluated through the constructed recombinant ASR1 protein-polyphenol turbidity model.

[0165] 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. ASR1, a key turbidity protein in greengage wine, is characterized by: The amino acid sequence of the key turbidity protein ASR1 is shown in SEQ ID NO.

1.

2. The gene encoding the key turbidity protein ASR1 of greengage wine according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The use of the key turbidity protein ASR1 of greengage wine as claimed in claim 1 in the prevention and control of post-turbidity, characterized in that: The application is selected from any of the following: (a) To achieve post-turbidity control by targeting the key turbidity protein ASR1 in greengage wine; or, (b) The key turbidity protein ASR1 of green plum wine or the key turbidity protein ASR1 and polyphenol turbidity model of green plum wine was used to evaluate the effect of clarifier on the prevention and control of post-turbidity of green plum wine.

4. The use according to claim 3, characterized in that: The key turbidity-causing protein ASR1 of the greengage wine is a recombinant ASR1 protein; the recombinant ASR1 protein is obtained by constructing a pET-30a(+)-ASR1 expression vector for expressing the ASR1 protein, transferring the vector into an Escherichia coli expression system, inducing expression, breaking the bacteria, and separating and purifying the vector through a 6×His affinity purification system.

5. The use according to claim 3, characterized in that: The polyphenols are selected from epigallocatechin gallate and / or proanthocyanidins.

6. The use according to claim 3, characterized in that: If the clarifier is an adsorbent, the clarifier is mixed with the key turbidity protein ASR1 of greengage wine, the pH of the mixed solution of the clarifier and the key turbidity protein ASR1 of greengage wine is 4.2, and the clarification effect is reflected by measuring any one or more of the total protein concentration in the mixed solution, the transmittance or absorbance at a wavelength of 540nm, and the chromaticity at a wavelength of 480nm; If the clarifier is a protease, the clarifier is mixed with the key turbidity protein ASR1 of green plum wine for enzymatic hydrolysis, and the enzyme is inactivated after the enzymatic hydrolysis is completed, and the pH value of the solution is adjusted back to 4.2, and a portion is taken out and fully mixed with an equal volume of a polyphenol solution with a pH of 4.2 at room temperature, and the clarification effect is reflected by measuring the transmittance or absorbance at a wavelength of 540nm; If the clarifier obtains a soluble complex by combining with the key turbidity protein ASR1 of green plum wine and the polyphenol turbidity model, the polyphenol solution, the key turbidity protein ASR1 of green plum wine and the clarifier are mixed under the condition of pH 4.2, and the clarification effect is reflected by measuring the transmittance or absorbance at a wavelength of 540nm.

7. A method for constructing a key turbidity protein ASR1 and polyphenol turbidity model of greengage wine as claimed in claim 1, characterized in that: The method includes: The pET-30a(+)-ASR1 expression vector for expressing ASR1 protein was constructed and transferred into the E. coli expression system. After induction of expression and bacterial cell disruption, the recombinant ASR1 protein was separated and purified by 6×His affinity purification system. The recombinant ASR1 protein and polyphenols were mixed at pH 4.2 to construct a turbidity model.

8. The construction method according to claim 7, characterized in that: The recombinant ASR1 protein and polyphenols were mixed at room temperature and pH 4.2 to construct a turbidity model.

9. The construction method according to claim 7, characterized in that: The polyphenol is selected from epigallocatechin gallate and / or proanthocyanidins; the concentration of the epigallocatechin gallate is 0.4-1 mg / mL; the concentration of the proanthocyanidins is 0.4-0.6 mg / mL; The concentration of the recombinant ASR1 protein is 0.8-1 mg / mL; The ASR1 protein and polyphenol solutions were mixed in equal volumes.

10. A method for constructing a recombinant ASR1 protein as claimed in claim 7, characterized in that: The method includes: Based on the key turbidity protein ASR1 of greengage wine as shown in SEQ ID NO.1, the pET-30a(+)-ASR1 expression vector expressing the ASR1 protein was constructed and transferred into the Escherichia coli expression system. After induction of expression, the bacteria were broken and separated and purified by 6×His affinity purification system. The induction temperature for expression is 37°C for 4 hours.

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