Post-turbidity prevention and control method for kiwi fruit wine

Abstract

The invention discloses a post-turbidity prevention and control method for kiwi fruit wine. The method comprises the step of adding polysaccharide or compound polysaccharide and alkaline protease or bentonite into the kiwi fruit wine for post-turbidity prevention and control of the fruit wine. The invention finds that the polysaccharide CMC, the trehalose and the glucan have a better interference effect on a model constructed by the turbid protein and the polyphenol of the kiwi fruit wine, and the compound polysaccharide CMC-trehalose compound polysaccharide has the highest turbid protein decline rate. According to the method, polysaccharide or compound polysaccharide is combined with bentonite or alkaline protease, a synergistic effect is found between the polysaccharide or compound polysaccharide and bentonite or alkaline protease, turbid protein in the kiwi fruit wine can be better removed, and the method can be used for preventing and controlling post-turbidity of the kiwi fruit wine.

Description

Technical Field

[0001] The invention relates to a method for preventing and controlling the turbidity after fruit wine, and particularly relates to a method for preventing and controlling the turbidity after kiwi fruit wine. 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] Heat treatment and bentonite adsorption are the most common processes used to prevent protein instability in white wine. However, heat treatment and bentonite fining may have an impact on the quality of the wine, such as removing color and aromatic compounds or introducing unpleasant cooking or caramel flavors, thus affecting the sensory characteristics of the wine. Summary of the invention

[0006] The purpose of the invention is to provide a method for preventing and controlling the turbidity of kiwi fruit wine, which solves the problem of turbidity of kiwi fruit wine and can well remove turbidity-causing proteins in the wine.

[0007] In order to achieve the above-mentioned object, the present invention provides a method for preventing and controlling post-production turbidity of kiwi fruit wine, which comprises: adding polysaccharide or complex polysaccharide and alkaline protease or bentonite into kiwi fruit wine to prevent and control post-production turbidity of the wine.

[0008] Preferably, the polysaccharide is carboxymethyl cellulose.

[0009] More preferably, the concentration of the polysaccharide is 1 mg / mL.

[0010] Preferably, the complex polysaccharide is selected from carboxymethyl cellulose and trehalose.

[0011] Preferably, the wine post-haze prevention and control is carried out at room temperature.

[0012] Preferably, the alkaline protease or bentonite can decompose or remove turbidity-causing proteins in kiwi wine.

[0013] Preferably, the polysaccharide or complex polysaccharide can interfere with the interaction between the turbidity-causing protein and polyphenols in the fruit wine to achieve the purpose of preventing turbidity.

[0014] More preferably, the turbidity-causing protein is thaumatin.

[0015] Preferably, the volume ratio of the complex polysaccharide, carboxymethyl cellulose and trehalose is 1:1.

[0016] The method for preventing and controlling turbidity of kiwi fruit wine of the present invention solves the problem of turbidity of kiwi fruit wine and has the following advantages:

[0017] The present invention found that polysaccharides CMC, trehalose, and dextran have good interference effects on the model constructed by turbidity proteins and polyphenols in kiwi fruit wine, and the composite polysaccharide CMC-trehalose composite polysaccharide has the highest reduction rate of turbidity proteins. The present invention combines polysaccharides or composite polysaccharides with bentonite or alkaline protease, and finds that they have a synergistic effect, can better remove turbidity proteins in kiwi fruit wine, and can be used for turbidity prevention and control of kiwi fruit wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The turbidity results are shown in Experimental Example 1 of the present invention when different proteases hydrolyze thaumatin and mix it with EGCG (A) and PC (B) solutions.

[0019] Figure 2 The SDS-PAGE results of the thaumatin hydrolyzate under the action of different hydrolases in Experimental Example 1 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.

[0020] Figure 3 The adsorption results of thaumatin by different adsorbents in Experimental Example 2 of the present invention are as follows: A: imported bentonite (TOP); B: ordinary bentonite; C: diatomaceous earth; D: chitosan; E: bentonite.

[0021] Figure 4 This is the result of the turbidity caused by thaumatin-polyphenols in Experimental Example 3 of the present invention.

[0022] Figure 5 This is the result of the effect of polysaccharide concentration on thaumatin-polyphenol turbidity in Experimental Example 3 of the present invention; A: carboxymethyl cellulose; B: trehalose; C: dextran.

[0023] Figure 6 This is the turbidity result of the composite polysaccharide on protein-polyphenol in Experimental Example 3 of the present invention.

[0024] Figure 7 These are the experimental results of applying different clarification methods to fruit wine in Experimental Example 4 of the present invention.

[0025] Figure 8 This is the absorbance of the supernatant of the fruit wine after clarification by different clarification methods in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] The inventors of the present invention adopted EGCG precipitation method and kiwi wine natural precipitation method to cause turbidity, and used the precipitated protein strips to perform QE mass spectrometry identification of the turbidity-causing proteins in kiwi wine. It was found that the key turbidity-causing protein in kiwi wine was thaumatin-like protein (TLP), and the thaumatin-like protein P83958 with a high content was difficult to obtain. Analysis showed that it had homology with the thaumatin protein in African arrowroot. Therefore, the thaumatin protein (derived from African arrowroot) from Sigma Company of the United States was used as a substitute, and a turbidity model of thaumatin-like protein and polyphenols was constructed to verify the effects of different clarification methods on the prevention and control of post-turbidity of kiwi wine.

[0029] Different concentrations of polyphenols EGCG (epigallocatechin gallate), PC (proanthocyanidins), EC (epicatechin) and CGA (chlorogenic acid) were used to react with different concentrations of thaumatin protein. It was found that EGCG and PC had a stronger ability to cause turbidity on thaumatin protein. Therefore, thaumatin protein, EGCG and PC were used to construct turbidity models Thaumatinprotein-EGCG and Thaumatinprotein-PC.

[0030] Some of the reagents used in the experimental examples of the present invention are as follows:

[0031] 1. The citric acid buffer is a citric acid-disodium hydrogen phosphate buffer. The citric acid-disodium hydrogen phosphate buffer is prepared as follows: Solution A (0.2mol / LNa2HPO4·12H2O): weigh 71.64g of disodium hydrogen phosphate dodecahydrate solution and add UP water to make up to 1000mL; Solution B (0.1mol / L C6H8O7·H2O): weigh 21.01g of citric acid monohydrate and add UP water to make up to 1000mL; pH = 5.0 is used to prepare the buffer, and the specific ratio is: 10.3mL of Solution A and 9.7mL of Solution B;

[0032] 2. EGCG and PC solutions: both were prepared using citric acid buffer.

[0033] 3. Thaumatin solution: prepared with citric acid buffer.

[0034] The following experimental examples illustrate the method for preventing and controlling post-production turbidity of kiwi fruit wine provided by the present invention.

[0035] Experimental Example 1 Experiment on the sensitivity of thaumatin to different proteases

[0036] Proteins have different sensitivities to various hydrolases, which results in differences in the number, type and distribution of peptides produced after hydrolysis. The following study explores the differences in sensitivity of thaumatin to different proteases, as follows:

[0037] 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:

[0038]

[0039] Depend on Figure 1 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.

[0040] Depend on Figure 2 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.

[0041] Experimental Example 2 Adsorption experiment of thaumatin by different adsorbents

[0042] 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 Epsler Group), ordinary bentonite (provided by Runjufang Food Co., Ltd., Meishan City, Sichuan Province), diatomite, chitosan and bentonite were prepared with UP water to prepare 0.2 mg / mL adsorbent. According to the addition amount shown in Table 1, the protein was mixed with different adsorbents (thaumatin protein-imported bentonite TOP; thaumatin protein-ordinary bentonite; thaumatin protein-diatomite; thaumatin protein-chitosan; thaumatin protein-bentonite), and 6 portions of each added amount of different 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.). The specific operation steps refer to the kit instructions. The clarification effect of the clarifier is expressed by the adsorption rate, in μg / mg.

[0043] Adsorption rate = (initial protein concentration - final protein concentration) / adsorbent concentration

[0044] Standard curve: y = 0.003651x + 0.08616 (thaumatin), R 2 =0.988.

[0045] Table 1 Addition amount of continuous monitoring experimental system

[0046]

[0047] like Figure 3As 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.

[0048] 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.

[0049] Experimental Example 3 Interference experiment of polysaccharide on thaumatin-EGCG turbidity

[0050] 1. Interference of polysaccharide types on thaumatin-polyphenol turbidity

[0051] Under the condition of pH = 5, 1 mg / mL polyphenols (EGCG, PC), 1 mg / mL thaumatin and 1 mg / mL polysaccharides (Guargum, Acacia senegal, CMC, Salecan, Trehalose, Dextran, prepared with water as solvent) were fully mixed in a volume ratio of 1:1:1 and placed in a 25°C constant temperature incubator for 5 minutes. Then 200 μL of the mixture was taken out and added to a 96-well plate, and the absorbance value A of the reactant was measured at a wavelength of 660 nm using a multifunctional microplate reader.

[0052] At the same time, a group of thaumatin-EGCG mixtures with the same concentration but without added polysaccharides were set up as blank controls for control analysis.

[0053] Based on the measured data, a reaction bar graph of the absorbance decrease rate and the polysaccharide concentration was drawn. To ensure the accuracy of the results, each group of experiments was repeated three times (n=3).

[0054]

[0055] In formula (1), A1 is the absorbance value of polyphenol-protein at the same concentration without adding polysaccharide; A2 is the absorbance value of polyphenol-protein at the same concentration with adding polysaccharide.

[0056] In order to screen out polysaccharides that have a significant effect on the turbidity of the thaumatin-polyphenol model, a relationship between the type of sugar and the rate of decrease in absorbance was plotted. The rate of decrease in absorbance represents the degree of turbidity reduction, that is, the higher the rate of decrease, the more significant the reduction in solution turbidity, which in turn indicates that the interference effect is better. The results are shown in Figure 2. Figure 4 shown.

[0057] Depend on Figure 4 It can be seen that all six sugars can reduce the turbidity of the system and have better interference effects on the thaumatin-EGCG system. The interference effect of the thaumatin-EGCG system is: CMC> dextran> trehalose> gum arabic> solubiloba> guar gum; the interference effect of the thaumatin-PC system is: CMC> trehalose> dextran> solubiloba> gum arabic> guar gum, so CMC, trehalose and dextran were selected for subsequent experiments.

[0058] 2. Selection of optimal polysaccharide concentration

[0059] In view of the fact that the three sugars, CMC, trehalose and dextran, showed significant interference effects on the thaumatin-polyphenol model, we further explored the specific performance of the interference of these three polysaccharides on the turbidity caused by thaumatin-polyphenol at different concentrations, as follows:

[0060] Under the condition of pH=5, 1 mg / mL polyphenols (EGCG, PC), 1 mg / mL thaumatin and polysaccharides with different initial concentrations (0.0005, 0.005, 0.05, 0.5, 1, 2 mg / mL; prepared with water as solvent) were mixed in a volume ratio of 1:1:1, and then placed in a 25°C constant temperature incubator for 5 minutes. 200 μL of the mixture was added to a 96-well plate, and the absorbance A of the system at a wavelength of 660 nm was measured using a Molecular Devices multifunctional microplate reader.

[0061] In order to ensure the accuracy of the experiment, a group of polyphenol-protein mixtures with the same concentration but without disaccharides and polysaccharides was set up as a control group. Each group of experiments was repeated three times (n=3).

[0062] like Figure 5 As shown, A is the interference effect of CMC on the Thaumatin protein-polyphenol model. It can be seen from the figure that the change in CMC concentration has little effect on the interference effect of the Thaumatin protein-PC model, and the decrease rate of absorbance is greater than 40%; when its concentration is 0.5mg / mL, it has the greatest interference with the Thaumatin protein-PC model, and the decrease rate of absorbance is 54%. The decrease rate of absorbance of the Thaumatin protein-EGCG model changes greatly with the change of CMC concentration; when the CMC concentration is 1mg / mL, it has the greatest interference with the Thaumatin protein-EGCG model, and the decrease rate of absorbance is 71%. B is the interference effect of trehalose on the Thaumatin protein-polyphenol model. It can be seen from the figure that the change in trehalose concentration has little effect on the interference effect of the Thaumatin protein-PC model, and the decrease rate of absorbance is greater than 40%; but it has a greater effect on the interference effect of the Thaumatin protein-EGCG model. When its concentration is 1 mg / mL, the interference effect on the Thaumatinprotein-PC model and the Thaumatinprotein-EGCG model is the largest, and the absorbance decrease rate is 46% and 57%, respectively. C is the interference effect of glucan on the thaumatin protein-polyphenol model. As can be seen from the figure, the change of glucan concentration has little effect on the interference effect of the Thaumatinprotein-PC model. When its concentration is 0.05 mg / mL, the interference to the Thaumatinprotein-PC model is the largest, and the absorbance decrease rate is 47%. The decrease rate of the absorbance of the Thaumatinprotein-EGCG model changes greatly with the change of glucan concentration; when the concentration of glucan is 1 mg / mL, the interference to the Thaumatin protein-EGCG model is the largest, and the absorbance decrease rate is 55%.

[0063] Therefore, changes in the concentrations of the three polysaccharides have a significant impact on the interference effect of the Thaumatin protein-EGCG model. Within a certain concentration range, when their concentrations are all 1 mg / mL, the interference effect on the turbidity of the model is better.

[0064] 3. Effect of complex polysaccharides on protein-polyphenol turbidity

[0065] 1 mg / mL polyphenols (EGCG, PC), 1 mg / mL thaumatin and complex polysaccharides were mixed evenly at a volume ratio of 1:1:1, see Table 2. 200 μL of the mixture was added to a 96-well plate, and the absorbance value A of the system was measured at a wavelength of 660 nm using a Molecular Devices multi-function microplate reader.

[0066] To ensure the accuracy of the experimental results, a group of polyphenol-protein models with the same concentration but without the addition of complex polysaccharides was set up as a blank control. Each group of experiments was repeated three times (n=3).

[0067] In this experiment, see Table 2, the composite polysaccharide combinations (the dosage relationship between the components is expressed by volume ratio) are: Combination A: CMC-trehalose (1:1); Combination B: CMC-dextran (1:1); Combination C: trehalose-dextran (1:1), wherein the composite polysaccharide is prepared with 1 mg / mL of polysaccharide.

[0068] Table 2 Reaction system of adding thaumatin-polyphenol to complex polysaccharide

[0069]

[0070] Table 3 Reduction rate of absorbance of disaccharide and polysaccharide on the interaction of thaumatin and polyphenol

[0071]

[0072] Depend on Figure 6As shown in Table 3, the addition of three combinations of complex polysaccharides: CMC-trehalose, CMC-glucan, and trehalose-glucan had interference effects on the two thaumatin protein-EGCG models. Different combinations had a significant effect on the interaction between thaumatin and protein. It is speculated that polysaccharides may play a role by preventing protein-polyphenol precipitation or fog turbidity formation, and their ability and potential mechanism depend not only on the charge characteristics of polysaccharides, but also on the structure of proteins and polyphenols. Taking the interaction between Thaumatin protein and EGCG as an example, CMC single polysaccharide has the best interference effect on the system, and complex polysaccharides do not increase the interference effect on the system; while the three complex polysaccharides have better interference effects on the Thaumatin protein-PC model than the single three polysaccharides, especially CMC-trehalose complex polysaccharide, with a protein reduction rate of up to 71%, indicating that CMC can synergistically inhibit the turbidity caused by protein-polyphenol interaction with trehalose and glucan. Therefore, the subsequent experiments used the CMC-trehalose combination to study its interference effect on fruit wine.

[0073] Experimental Example 4: Kiwifruit wine protein stability test

[0074] 1. Preparation of kiwi fruit wine

[0075] Fruit selection (select fresh kiwifruit with high maturity and full fruit) → washing and peeling → juicing → enzymatic hydrolysis (using 0.2% complex enzyme, that is, 0.2 grams of complex enzyme per 100mL juice, the complex enzyme is pectinase: cellulase with a mass ratio of 1:1.5, and enzymatic hydrolysis at 50℃ for 120 minutes) → sugar adjustment (adjust the sugar content of the juice after enzymatic hydrolysis to 20Brix with white sugar) → pasteurization (70℃ water bath for 30min) → yeast activation (dissolve in warm water at a material-liquid ratio of 1:20, and activate at 35℃ for 60min) → fermentation (the amount of yeast added is 0.5wt%, and fermentation is carried out in a constant temperature incubator at 25℃ for 7 days) → centrifugation (10000r / min, 10min) → bottling for later use.

[0076] 2. Fruit wine protein stability test

[0077] According to Experimental Examples 1 to 3, the protease, clarifier and polysaccharide with the best clarification effect on thaumatin were selected and applied to kiwi fruit wine. All clarifiers (alkaline protease, bentonite, CMC, trehalose) were dissolved in kiwi fruit wine, and EGCG was used to accelerate precipitation, as follows:

[0078] Alkaline protease (enzyme addition amount was 20U / g), imported bentonite TOP (TOP concentration was 0.2mg / mL, water was used as solvent), CMC (polysaccharide concentration was 1mg / mL), CMC-trehalose composite polysaccharide (each polysaccharide concentration was 1mg / mL, mixed at a volume ratio of 1:1) were selected for the experiment. The specific addition amount is shown in Table 4. The clarifier was added to the kiwi fruit wine, and after being placed at room temperature for 48h, 200μL of the supernatant was added to a 96-well plate, and the absorbance value A of the system was determined using a Molecular Devices multifunctional microplate reader. 660 EGCG can react with protein to produce precipitation. To verify the removal effect of different clarification methods on protein, the treated supernatant was reacted with EGCG (2 mg / mL) and placed at room temperature for 48 hours. After that, 200 μL of the supernatant was added to a 96-well plate to measure the absorbance value A. 660 .

[0079] To further explore the adsorption effect of different methods on proteins, 0.2 mg / mL bentonite, 0.2 mg / mL imported bentonite TOP, 1 mg / mL CMC, and 1 mg / mL trehalose were prepared, and 12 methods including alkaline protease, bentonite, TOP, alkaline protease + CMC, alkaline protease + trehalose, alkaline protease + composite polysaccharide, bentonite + CMC, bentonite + trehalose, bentonite + composite polysaccharide, TOP + CMC, TOP + trehalose, and TOP + composite polysaccharide were used to treat fruit wine. The treated supernatant was reacted with EGCG (2 mg / mL) and placed at room temperature for 48 hours. After that, 200 μL of the supernatant was added to a 96-well plate, and the absorbance value A of the system was determined using a Molecular Devices multifunctional microplate reader. 660 , and a group of blank fruit wine without adding clarifier was set as control, and the specific addition amount is shown in Table 5.

[0080] Table 4 Kiwi fruit wine application reaction system

[0081]

[0082] Note: All the above clarifiers (alkaline protease, bentonite, CMC, trehalose) are dissolved in kiwi fruit wine.

[0083] Table 5 Kiwi fruit wine supernatant reaction system

[0084]

[0085] Note: All the above clarifiers (alkaline protease, bentonite, bentonite, CMC, trehalose) are dissolved in kiwi fruit wine.

[0086] Depend on Figure 7 It can be seen that under the two conditions, the five clarification methods all made the kiwifruit wine absorbance A 660 There were different degrees of decline. Under normal temperature, the decline rate was in the order of TOP>alkaline protease>CMC+trehalose>trehalose>CMC, among which bentonite TOP had the best clarification effect, and the absorbance value decline rate was 67.76%; under EGCG-induced turbidity, the decline rate was in the order of TOP>alkaline protease>trehalose>CMC+trehalose>CMC, among which bentonite TOP had the best clarification effect, and the absorbance value decline rate was 57.99%. By comparison, it was found that TOP had the best clarification effect under both conditions, and the clarification effects of CMC, trehalose, and CMC+trehalose composite polysaccharide were not much different. Compared with the normal temperature state, the clarification effect of alkaline protease was significantly reduced. It was speculated that the inactivation of protease may have caused the failure to continue the reaction after induced turbidity.

[0087] like Figure 8 As shown in Figure 2, when the fruit wine supernatant reacted with EGCG, the absorbance A of the fruit wine supernatant treated with alkaline protease + CMC, alkaline protease + compound polysaccharide, TOP + compound polysaccharide, and TOP + CMC was 660 The smallest, indicating that these four methods have the best effect on protein removal. These four methods all involve the complex reaction of polysaccharides, indicating that the complex of polysaccharides, alkaline protease and TOP is more effective and can better remove proteins.

[0088] In summary, under normal temperature and accelerated turbidity conditions, the four methods of alkaline protease + CMC, alkaline protease + compound polysaccharide, TOP + compound polysaccharide, and TOP + CMC can better remove protein from fruit wine. At the same time, the combination of polysaccharide, alkaline protease and TOP can more effectively precipitate protein.

[0089] 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. A method for preventing and controlling turbidity of kiwi fruit wine, characterized in that: The method includes: Polysaccharide or complex polysaccharide and alkaline protease or bentonite are added to kiwi fruit wine to prevent and control post-production turbidity of the wine.

2. The method for preventing and controlling turbidity of kiwi fruit wine according to claim 1, characterized in that: The polysaccharide is selected from carboxymethyl cellulose.

3. The method for preventing and controlling turbidity of kiwi fruit wine according to claim 1, characterized in that: The complex polysaccharide is selected from carboxymethyl cellulose and trehalose.

4. The method for preventing and controlling turbidity of kiwi fruit wine according to claim 1, characterized in that: The fruit wine post-haze prevention and control is carried out at room temperature.

5. The method for preventing and controlling turbidity of kiwi fruit wine according to claim 1, characterized in that: The polysaccharide or the composite polysaccharide and the alkaline protease or bentonite can remove the turbidity-causing protein in the kiwi fruit wine.

6. The method for preventing and controlling turbidity of kiwi fruit wine according to claim 5, characterized in that: The turbidity-causing protein is thaumatin.

7. The method for preventing and controlling post-turbidity of kiwi fruit wine according to claim 1, characterized in that: The volume ratio of the complex polysaccharide, carboxymethyl cellulose and trehalose is 1:1.

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