Method for improving protein stability of dry white wine

Abstract

The invention discloses a method for improving protein stability of dry white wine, and belongs to the technical field of wine stability treatment. Comprising the following steps: mixing and stirring chitosan powder and an acetic acid solution to obtain a chitosan solution, mixing and stirring the chitosan solution and an egg shell membrane solution to form a mixture, and carrying out alkali treatment on the mixture to obtain chitosan composite large particles; mixing a bromelain solution and the chitosan composite large particles, and performing low-temperature incubation to obtain immobilized bromelain particles; adding the immobilized bromelain particles into the dry white wine, mixing, sequentially performing stability treatment and heat treatment, and then placing in a low-temperature environment to complete the treatment of the dry white wine. Chitosan is modified by using an egg shell membrane to obtain chitosan composite large particles, then bromelain is loaded to obtain the immobilized bromelain, and the stability of fogged protein is improved on the premise that the fragrance and flavor of the dry white wine are reserved.

Description

Technical Field

[0001] The invention belongs to the technical field of wine stability treatment, and in particular relates to a method for improving the protein stability of dry white wine. Background Art

[0002] Wine is a fermented wine with low alcohol content, elegant character, rich nutrients, and health-promoting effects. Wine can also prevent various diseases and enhance human health when consumed in moderation. Clarity is the most intuitive and important sensory quality of wine for consumers. Wine, like all natural foods, contains different amounts of protein. The protein content in red wine is very low because red wine is fermented with skin residue, and the protein will combine with phenolic substances such as tannins leached from the grape skin and flocculate and precipitate. Dry white wine is made from white grape varieties, which are squeezed to obtain grape juice, which is then fermented. Therefore, there is no extraction process of solid parts such as grape skin and pulp during the brewing process. It is made by fermenting grape juice. The content of tannin, pigment, pectin, etc. is lower than that of red wine. Protein cannot combine with phenolic substances in the pomace to form precipitation and is removed during the brewing process. Therefore, this type of finished wine contains higher protein, which makes it easier for dry white wine to produce fog-like precipitation (the main substance of the precipitation is protein), which affects the taste and appearance of the dry white wine, and also reduces consumer acceptance, which in turn has a bad impact on the wine market.

[0003] In the process of making dry white wine, it is essential to ensure the stability of protein. If there is enough time, the unstable protein and other components in the wine can be allowed to condense with each other through electrostatic adsorption and settle naturally under the action of gravity, and finally reach a stable state. However, in actual production, we need a faster and more reliable stabilization treatment method than in the natural state, so various stabilization treatment processes are adopted in the wine brewing process. In wine clarification, bentonite (bentonite) is considered to be the most effective material to reduce protein precipitation. The main component of bentonite is kaolin, and some other substances with lower content include feldspar, gypsum, calcium carbonate, quartz, etc., and also include a very small amount of metals. There are clear regulations on the content standards of some heavy metals. Among them, the content of metal iron in bentonite is an indicator that must be checked, because too high iron will cause the instability of wine and also affect the color of wine. The basic principle of bentonite stabilization of wine is electrostatic adsorption. After being activated by water, bentonite forms a colloidal suspension that can absorb protein in wine or grape juice. During the absorption process, positively charged proteins bind to negatively charged bentonite through electrostatic interactions, or bentonite can bind to protein molecules through hydrogen bonds. The charge state of protein molecules in wine depends on the pH value of the grape juice or wine. The lower the pH value, the higher the positive charge. The use of bentonite to stabilize white wine is mainly affected by the following factors: (1) The swelling capacity of bentonite. The swelling capacity of bentonite is the basis of its ability to absorb protein. High-efficiency bentonite can quickly absorb a large amount of protein and form a light flocculent wine sludge; (2) The choice of the amount of bentonite added. Generally, it is necessary to test the amount of addition, because different wine samples contain different stable states. If the amount is not enough, the clarification effect will be poor. If the amount is too much, although the clarification effect is good, it will also remove other flavor substances in the wine, affecting the quality of the wine and increasing the production cost. (3) The method of preparing bentonite also affects the clarification effect. Generally, hot water activation and vigorous mechanical stirring are selected, and sufficient time is required for hydration and expansion. (4) The acidity of the wine also has a certain effect on the clarification effect. The higher the acidity of the wine, the more positive charges the protein molecules carry, the stronger the electrostatic adsorption of bentonite and protein will be, and the better the effect of removing protein. Many studies have also shown that bentonite can preferentially remove proteins with higher isoelectric points and medium molecular weight proteins. However, these are only a small part of the soluble proteins in wine, and they also happen to exclude sweet proteins and chitinases that can significantly affect the thermal stability of wine. Since bentonite clarification will lose the aroma and flavor components of wine, the wine tastes weak, which has a negative impact on the quality of wine. Moreover, the bentonite used to treat wine cannot be recycled, which not only wastes resources but also pollutes the environment.

[0004] To this end, people have studied a variety of alternatives to bentonite, including ultrafiltration, adding tannins, adding proteolytic enzymes, etc. Ultrafiltration is a pressurized membrane separation technology. Under a certain pressure, small molecular solutes and solvents pass through a membrane with a specific pore size, while large molecular solutes cannot pass through. Added tannins and proteins in wine aggregate into large molecular complexes through electrostatic adsorption and precipitate under gravity to remove proteins from wine. When proteolytic enzymes are applied to wine, proteins are hydrolyzed into small molecular peptide fragments and amino acids, which not only helps to solve the impact of protein fogging on wine, but also facilitates the nitrogen supply of yeast during alcohol fermentation and helps the formation of wine aroma.

[0005] Patent CN102559430A discloses a production process for preparing dry white wine using a composite biological enzyme. The invention irradiates grapes with ultraviolet light, then treats them in darkness and lacks oxygen, freezes them in the early stage of the wine fermentation process, and adds composite yeast to produce composite biological enzymes after thawing, thereby increasing the extraction of grape polyphenol components and thereby improving the clarity of the wine.

[0006] Patent CN108264978A discloses a process for preparing small molecule peptide white wine. The invention uses biologically active enzymes to degrade proteins into small molecule peptide powders, then adds acidic proteases to the white wine liquid for degradation treatment, mixes with the small molecule peptide powders, and obtains small molecule peptide white wine with high stability through post-processing steps such as sterilization filtration and aging.

[0007] Due to the acidic environment of wine, the activity and removal efficiency of proteolytic enzymes will be greatly affected if they are directly treated with proteolytic enzymes. Therefore, how to improve the stability of fogging protein while retaining the aroma and flavor of dry white wine during the process of using proteolytic enzymes to treat dry white wine has become an urgent problem to be solved in the production of dry white wine. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention uses chicken egg shell membrane (ESM) to modify chitosan to obtain chitosan composite large particles, and then loads bromelain to obtain immobilized bromelain, which solves the technical problems raised in the background technology. Specifically, the technical solution of the present invention includes the following contents:

[0009] A method for improving the protein stability of dry white wine, the method comprising the following steps:

[0010] S1. Preparation of large chitosan composite particles:

[0011] Chitosan powder and acetic acid solution are mixed, heated to 45° C. to 55° C. and stirred to obtain a chitosan solution, the chitosan solution and egg shell membrane solution are mixed and stirred to form a mixture, the mixture is treated with a 10% by mass sodium hydroxide solution to a pH of 8.0 to 8.5 to obtain solid large particles, and the solid large particles are sequentially washed and dried to obtain chitosan composite large particles;

[0012] S2. Immobilization of bromelain:

[0013] The bromelain solution and the chitosan composite large particles are mixed and incubated at a low temperature of 3°C to 5°C for 1h to 2h, and then filtered, washed and dried in sequence to obtain immobilized bromelain particles;

[0014] S3, Dry white wine processing:

[0015] The immobilized bromelain particles are added to dry white wine and mixed, and then subjected to stability treatment and heat treatment in sequence, and then placed in a low temperature environment of 2° C. to 6° C. for 14 h to 18 h to complete the treatment of the dry white wine.

[0016] Furthermore, the mass concentration of the acetic acid solution is 1% w / v to 2% w / v.

[0017] Furthermore, the mass ratio of the chitosan powder to the acetic acid solution is 3-4:96-97.

[0018] Furthermore, the mass concentration of the egg shell membrane solution is 6% w / v to 8% w / v.

[0019] Furthermore, the volume ratio of the chitosan solution to the hen's egg shell membrane solution is 10:0 to 5:5.

[0020] Furthermore, the molar concentration of the bromelain solution is 0.04M to 0.08M.

[0021] Furthermore, the bromelain solution is prepared by dissolving bromelain in a phosphate buffer solution with a pH of 6.9 to 7.1.

[0022] Furthermore, the mass ratio of the bromelain solution to the chitosan composite large particles is 4g-6g: 1g-3g.

[0023] Furthermore, the volume ratio of the immobilized bromelain particles to dry white wine is 1:1000-2000.

[0024] Furthermore, the conditions of the stability treatment include a rotation speed of 100 r / min to 140 r / min, a treatment time of 22 h to 26 h and a treatment temperature of 18° C. to 22° C.

[0025] Furthermore, the heat treatment conditions include a treatment temperature of 75° C. to 85° C. and a treatment time of 4 h to 8 h.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The immobilized bromelain particles obtained by fixing bromelain with the chitosan composite large particles constructed by the present invention can significantly improve the stability of protein in dry white wine and improve the clarity of dry white wine.

[0028] (2) The immobilized bromelain particles obtained by the present invention and the corresponding treatment methods can retain the aroma and flavor of dry white wine to the greatest extent.

[0029] (3) The raw materials used to construct the immobilized bromelain particles of the present invention are all natural, green and degradable, and will not cause pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a protein Raman spectrum of the clarified Xinjiang Dynasty semi-dry white wine after being treated in Example 2 of the present invention;

[0031] Figure 2 This is a statistical chart of sensory evaluation results of wine of the present invention;

[0032] Figure 3 This is an electron microscope scanning image of chitosan composite large particles;

[0033] Figure 4 The infrared spectra of the chitosan composite large particles, immobilized bromelain particles and chitosan obtained in Example 2 of the present invention;

[0034] Figure 5 The XRD spectra of chitosan composite large particles and chitosan obtained in Example 2 of the present invention are shown;

[0035] Figure 6 This is a thermogravimetric analysis diagram of the chitosan composite large particles and immobilized bromelain particles obtained in Example 2 of the present invention, wherein Figure 6 The left figure is the thermogravimetric curve. Figure 6 The right figure is the thermogravimetric differential curve;

[0036] Figure 7 This is a test diagram of the enzyme loading of the immobilized bromelain particles and chitosan without hen egg shell membrane obtained in Example 1 and Example 2 of the present invention;

[0037] Figure 8 This is a graph showing the enzyme activity stability results of the immobilized bromelain particles and free bromelain obtained in Example 2 of the present invention;

[0038] Fig. 9This is a graph showing the reusability of the immobilized bromelain obtained in Example 2 of the present invention. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below through the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0041] Embodiment 1:

[0042] A method for improving the protein stability of dry white wine, specifically comprising the following process:

[0043] S1. Preparation of large chitosan composite particles:

[0044] Dissolve 3 g of chitosan powder in 97 g of 1% w / v acetic acid solution, then stir at 45°C until the chitosan powder is completely dissolved, and filter with gauze to obtain a chitosan solution;

[0045] According to the volume ratio of chitosan solution to eggshell membrane solution of 9:1, add 6% w / v eggshell membrane solution to chitosan solution and mix and stir to obtain a mixture, drop 10% sodium hydroxide solution into the mixture and stir until the pH reaches 8.0-8.5, stop dropping sodium hydroxide solution, and let stand for 30 minutes to obtain a suspension. The suspension is centrifuged to obtain large solid particles;

[0046] The solid large particles are washed with deionized water, ethanol and deionized water in sequence until the elution water reaches neutrality, and then the washed solid large particles are dried to obtain chitosan composite large particles, which are stored in a refrigerator at 4°C for later use;

[0047] S2. Immobilization of bromelain:

[0048] A bromelain solution with a molar concentration of 0.04 M was prepared with a phosphate buffer having a buffer pH of 6.9 to 7.1, 4 g of the bromelain solution and 1 g of chitosan composite large particles were mixed, and then gently shaken and incubated for 1 hour in a low temperature environment of 3°C. After the incubation, the particles were collected by filtration, and the particles were washed with a phosphate buffer having a pH of 7.0 until no protein could be detected in the eluent, and then the washed particles were dried to obtain immobilized bromelain particles;

[0049] S3, Dry white wine processing:

[0050] According to the volume ratio of immobilized bromelain particles to dry white wine of 1:1000, the immobilized bromelain particles were added to the Ningxia pigeon dry white wine, and the mixture was subjected to stability treatment at a temperature of 18°C ​​at a stirring speed of 100 r / min. The treatment time was controlled to be 22 hours (10 ml of the sample was taken and the residual protein content of the Ningxia pigeon dry white wine before and after the stabilization treatment was determined by the potassium dodecyl sulfate method, and the results are shown in Table 1). After the stability treatment, the wine was heat treated at a temperature of 75°C for 4 hours. After the heat treatment, the wine was kept at a low temperature of 2°C for 14 hours, thereby completing the treatment of the Ningxia pigeon dry white wine.

[0051] Embodiment 2:

[0052] A method for improving the protein stability of dry white wine, specifically comprising the following process:

[0053] S1. Preparation of large chitosan composite particles:

[0054] Dissolve 4 g of chitosan powder in 96 g of 2% w / v acetic acid solution, then stir at 55°C until the chitosan powder is completely dissolved, and filter with gauze to obtain a chitosan solution;

[0055] According to the volume ratio of chitosan solution to eggshell membrane solution of 5:5, add 8% w / v eggshell membrane solution to chitosan solution and mix and stir to obtain a mixture, drop 10% sodium hydroxide solution into the mixture and stir until the pH reaches 8.0-8.5, stop dropping sodium hydroxide solution, and let stand for 40 minutes to obtain a suspension. The suspension is centrifuged to obtain large solid particles;

[0056] The solid large particles are washed with deionized water, ethanol and deionized water in sequence until the elution water reaches neutrality, and then the washed solid large particles are dried to obtain chitosan composite large particles, which are stored in a refrigerator at 4°C for later use;

[0057] S2. Immobilization of bromelain:

[0058] A bromelain solution with a molar concentration of 0.08 M was prepared with a phosphate buffer having a buffer pH of 6.9 to 7.1, 6 g of the bromelain solution and 3 g of chitosan composite large particles were mixed, and then gently shaken and incubated for 2 h in a low temperature environment of 5°C. After the incubation, the particles were collected by filtration, and the particles were washed with a phosphate buffer having a pH of 7.0 until no protein could be detected in the eluent, and then the washed particles were dried to obtain immobilized bromelain particles;

[0059] S3, Dry white wine processing:

[0060] According to the volume ratio of immobilized bromelain particles to dry white wine of 1:1000, the immobilized bromelain particles were added to the Xinjiang Dynasty semi-dry white wine and mixed, and the mixture was subjected to stability treatment at a temperature of 22°C at a stirring speed of 140r / min, and the treatment time was controlled to be 26h (10ml of the sample was taken and the residual protein content of the Xinjiang Dynasty semi-dry white wine before and after the stabilization treatment was determined by the potassium dodecyl sulfate method. The protein removal rate was the percentage of the residual protein content of the wine with bromelain added compared to the wine without bromelain added. The results are shown in Table 1). Heat treatment was carried out after the stability treatment. The Xinjiang Dynasty dry white wine was placed in a temperature environment of 85°C for 8h, and then kept in a low temperature environment of 6°C for 18h, thereby completing the heat treatment of the Xinjiang Dynasty semi-dry white wine.

[0061] The turbidity of the dry white wines of Example 1 and Example 2 before and after heat treatment was measured using a HD 25.2 turbidimeter. The turbidity was expressed in turbidimeter measurement units (NTU). The results are shown in Table 1 (the calculation method for the stability of the wine is the difference in NTU of the wine before and after heat treatment, i.e., NTU 热处理后 -NTU 热处理前 , which is proportional to the haze potential. If this difference does not exceed 2 NTU, the wine is considered stable. Turbidity reduction rate (TRY(%)) was calculated as the percentage of turbidity reduction in the treated wine with bromelain compared to the wine without bromelain).

[0062] Table 1 Residual protein content and turbidity change values ​​in wine after treatment

[0063]

[0064] As shown in Table 1, compared with the wine without enzyme treatment, the turbidity of the two wines treated with immobilized bromelain particles decreased under stirring and high temperature conditions, and the protein content was reduced after measurement, indicating that the immobilized bromelain particles can effectively remove the protein content in dry white wine, reduce the turbidity of dry white wine under shaking and high temperature conditions, and improve the clarity of dry white wine.

[0065] Changes in protein secondary structure: (Taking Xinjiang Dynasty semi-dry white wine as the research object)

[0066] The protein Raman spectrum of Xinjiang Dynasty semi-dry white wine after being treated with immobilized bromelain particles is shown in Figure 2. Figure 1 As shown. You can see 530cm -1 There is an obvious characteristic peak at 1100cm -1 、1400cm -1The characteristic peaks at 800 cm-1 are assigned to α-helix and β-sheet; -1 The characteristic peak at is assigned to the stretching vibration between CC in the amino acid peptide chain.

[0067] Sensory evaluation:

[0068] Eight professionals with uniform training and experience in wine tasting conducted overall sensory evaluation and olfactory evaluation in a standard wine tasting room using standard wine tasting glasses. During the evaluation process, the eight personnel were required to describe the brightness, clarity, aroma intensity, harmony and purity of the wine before and after treatment with immobilized bromelain particles, and the above indicators were scored on a 10-point scale, with 0 to 10 indicating increasing intensity. In the olfactory evaluation, each taster selected 5 to 15 aroma descriptors to evaluate the aroma of the wine sample. The contribution of aroma compounds is represented by the quantitative intensity value (modified frequency, MF), and the calculation formula is as follows:

[0069] MF=F×I

[0070] Where, F: frequency of aroma descriptors; I: percentage of the average aroma score to the total score (10 points)

[0071] Comparative analysis of the aroma changes of wines with and without immobilized bromelain particles was performed. Figure 2 shown.

[0072] It can be seen that the aroma intensity and coordination of the two wines were improved after being treated with immobilized bromelain particles, and the brightness of the wine was improved. At the same time, the fruity and floral aromas of lychee, lemon, peach, white flowers, etc. of Ningxia Yuge dry white wine were more prominent after treatment, and the baking aroma was weakened. The fruity aromas of pineapple, lychee, apple, lemon, etc. of Xinjiang Dynasty semi-dry white wine were more prominent after treatment, and the baking aroma was also weakened. Overall, the sensory quality of wine treated with immobilized bromelain particles is better.

[0073] (1) Structural characterization of large chitosan particles:

[0074] a.Scanning electron microscope image analysis:

[0075] The chitosan composite large particles obtained in Examples 1 and 2 were subjected to surface electron microscope scanning and cross-sectional scanning to observe the microstructure of chitosan (CS) without any treatment. The results are as follows: Figure 3 As shown, Figure 3 a and Figure 3 d is the surface electron microscope scanning image and cross-section electron microscope scanning image of chitosan (CS); Figure 3 b and Figure 3e is the surface electron microscope scanning image and cross-section electron microscope scanning image of the chitosan composite large particles in Example 1; Figure 3 c and Figure 3 f is the surface electron microscope scanning image and cross-section electron microscope scanning image of the chitosan composite large particles in Example 2.

[0076] Conclusion: From Figure 1 It can be clearly found that with the increase of the proportion of eggshell membrane solution, the macroporous wall changes from a dense sheet to a porous network structure, and a large number of thin meshes are formed at the edge of the eggshell membrane particles (see Figure 3 c and Figure 3 f), which is beneficial to improve the immobilization capacity of chitosan composite large particles.

[0077] b. Infrared spectroscopy analysis:

[0078] The chitosan (CS) without any treatment, the chitosan composite large particles (CSEM) of Example 2 and the immobilized bromelain particles (CSEM+Enzyme) of Example 2 were tested by infrared spectroscopy. The results are shown in Figure 4 .

[0079] Conclusion: From the infrared spectrum, we can see that after loading bromelain, the infrared spectrum of the sample is between 1500-1750cm -1 Many small absorption peaks appear in the range of 1419cm -1 The absorption at 1378 cm is attributed to the NH stretching vibration. -1 and 1321cm -1 The peaks at 2925 cm-1 are attributed to the COH vibrations of the methylene group and the primary alcohol group, respectively. -1 and 2883cm -1 The absorption at is also caused by the NH vibration of the amino group. After bromelain loading, a new peak appears in the spectrum at 1546 cm-1, which can be attributed to the NN deformation and CN stretching of the amide-II region of the bromelain molecule.

[0080] c.XRD spectrum analysis:

[0081] XRD spectroscopy was performed on chitosan (CS) without any treatment and chitosan composite large particles (CSEM) of Example 2. The results are shown in Figure 5 .

[0082] Conclusion: From the XRD spectrum, it can be seen that compared with chitosan CS, the chitosan composite large particles have a sharp crystallization peak at 2θ=46°, indicating that the addition of egg shell membrane (ESM) changes the crystallinity of chitosan.

[0083] d. Thermogravimetric analysis:

[0084] Thermogravimetric analysis was performed on the chitosan composite large particles (CSEM) of Example 2 and the immobilized bromelain particles (CSEM+Enzyme) of Example 2. The results are shown in Figure 6 .

[0085] Conclusion: The thermogravimetric curves show that the first weight loss stage observed at about 100°C for the carrier loaded with bromelain is lower than that for the carrier without bromelain loading, which is attributed to the evaporation of the residual water of the carrier. In the range of 200-400°C, the chitosan composite macroparticles (CSEM) without enzyme loading only lose weight at 289°C, responding to the depolymerization of polymer chains, the oxidative dehydration of sugar rings, and the elimination of pyrolysis products. However, the chitosan composite macroparticles loaded with enzymes (i.e., immobilized bromelain particles) show two consecutive weight loss stages, which are attributed to the thermal degradation of proteins and polysaccharides, respectively (Figure d). The curves show that the addition of bromelain has a great influence on the thermal degradation process of chitosan composite macroparticles, and the immobilized bromelain particles have good thermal stability at 100°C and can fully adapt to the reaction conditions in practical applications.

[0086] The above-mentioned infrared spectrum, XRD spectrum and thermogravimetric analysis proved that bromelain molecules can be successfully loaded on chitosan composite large particles and have good thermal stability.

[0087] (2) Determination of the enzyme loading amount of immobilized bromelain particles:

[0088] The bromelain standard curve was drawn by the Coomassie Brilliant Blue G-250 method, and the enzyme loading of the immobilized bromelain particles (CSEM1) of Example 1 and the immobilized bromelain particles (CSEM2) of Example 2 was measured, and chitosan without adding eggshell membrane (CSEM0) was used as a comparison. The results are as follows: Figure 7 shown.

[0089] Conclusion: With the addition of eggshell membrane (ESM), the loading capacity of chitosan composite large particles on bromelain increased significantly, which shows that ESM has the ability to increase the loading capacity of enzyme molecules in chitosan composite large particles. According to the previous SEM test results, the addition of ESM increased the size of the mesopores and the density of the pores inside the chitosan composite large particles, which is conducive to the diffusion of enzyme molecules inside the carrier, providing sufficient space for them to enter the carrier, which is conducive to the fixation of the enzyme.

[0090] (3) Determination of the enzyme activity stability of the immobilized bromelain particles and free bromelain obtained in Example 2:

[0091] The effects of pH on the activity of free bromelain and immobilized bromelain particles were compared under different pH conditions. Figure 8 As shown in a.

[0092] Conclusion: The pH adaptability of free bromelain is lower than that of immobilized bromelain particles. The optimum pH values ​​of free bromelain and immobilized bromelain particles are 7.3-7.7 and 5.3-5.7, respectively, and the relative enzyme activity of immobilized bromelain particles under acidic and alkaline conditions is higher than that of free bromelain, indicating that immobilized bromelain particles have a higher pH adaptability range and are more adaptable to acidic environments such as alcohol fermentation.

[0093] The effects of temperature on the activity of free bromelain and immobilized bromelain particles were compared under different temperature environments. Figure 8 As shown in Figure b. The optimum temperature of free bromelain is 2-6°C, and the optimum temperature of immobilized bromelain particles is 45-55°C, and the relative activity of immobilized bromelain particles at 20-30°C and 45-55°C is above 90%, which is much higher than that of free bromelain, indicating that immobilized bromelain particles have stronger high temperature resistance and can better adapt to room temperature and higher temperature environments.

[0094] (4) Determination of the reproducibility of the immobilized bromelain particles obtained in Example 2:

[0095] The initial enzyme activity of the immobilized bromelain particles in Example 2 was measured, and then the immobilized bromelain particles were washed 2 to 3 times with a phosphate buffer having a pH of 7.0. This process was repeated 5 times, and the enzyme activity of the immobilized bromelain particles measured each time was recorded. The initial enzyme activity was taken as 100%, and the relative enzyme activity was calculated. The results are shown in FIG. Fig. 9 shown.

[0096] Conclusion: The analysis shows that after five uses, the enzyme activity of the immobilized bromelain particles gradually decreased but still remained above 50% of the initial enzyme activity. This result shows that the immobilized bromelain particles have good reusability.

[0097] The above-described embodiments provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A method for improving the protein stability of dry white wine, characterized in that: The method comprises the following steps: S1. Preparation of large chitosan composite particles: Chitosan powder and acetic acid solution are mixed, heated to 45° C. to 55° C. and stirred to obtain a chitosan solution, the chitosan solution and egg shell membrane solution are mixed and stirred to form a mixture, the mixture is treated with a 10% by mass sodium hydroxide solution to a pH of 8.0 to 8.5 to obtain solid large particles, and the solid large particles are sequentially washed and dried to obtain chitosan composite large particles; S2. Immobilization of bromelain: The bromelain solution and the chitosan composite large particles are mixed and incubated at a low temperature of 3°C to 5°C for 1h to 2h, and then filtered, washed and dried in sequence to obtain immobilized bromelain particles; S3, Dry white wine processing: The immobilized bromelain particles are added to dry white wine and mixed, and then subjected to stability treatment and heat treatment in sequence, and then placed in a low temperature environment of 2° C. to 6° C. for 14 h to 18 h to complete the treatment of the dry white wine.

2. The method for improving the protein stability of dry white wine according to claim 1, characterized in that: The mass concentration of the acetic acid solution is 1% w / v to 2% w / v.

3. A method for improving the protein stability of dry white wine according to claim 1, characterized in that: The mass ratio of the chitosan powder to the acetic acid solution is 3-4:96-97.

4. A method for improving the protein stability of dry white wine according to claim 1, characterized in that: The mass concentration of the egg shell membrane solution is 6% w / v to 8% w / v.

5. The method for improving the protein stability of dry white wine according to claim 1, characterized in that: The volume ratio of the chitosan solution to the hen's egg shell membrane solution is 5-10:0-5.

6. The method for improving the protein stability of dry white wine according to claim 1, characterized in that: The molar concentration of the bromelain solution is 0.04M-0.08M.

7. A method for improving the protein stability of dry white wine according to claim 1, characterized in that: The mass ratio of the bromelain solution to the chitosan composite large particles is 4g-6g:1g-3g.

8. The method for improving the protein stability of dry white wine according to claim 1, characterized in that: The volume ratio of the immobilized bromelain particles to dry white wine is 1:1000.

9. A method for improving the protein stability of dry white wine according to claim 1, characterized in that: The conditions of the stability treatment include a rotation speed of 100 r / min to 140 r / min, a treatment time of 22 h to 26 h and a treatment temperature of 18° C. to 22° C.

10. The method for improving the protein stability of dry white wine according to claim 1, characterized in that: The heat treatment conditions include a treatment temperature of 75° C. to 85° C. and a treatment time of 4 h to 8 h.

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