Method for optimizing preparation process of lime fruit vinegar by double enzymes through response surface methodology

Abstract

The application provides a method for optimizing a double-enzyme preparation lime fruit vinegar process by a response surface method, and the process integrates the key advantages of efficient resource utilization, deep strengthening of core strains, double-strain synergistic optimization fermentation, response surface optimization process and the like. Not only the low-value raw materials are effectively utilized, but also through the strengthening of yeast fermentation technology and precise process control, high-quality lime fruit vinegar with unique flavor, stable quality and potential health value is produced, and the market competitiveness and economic benefits of the lime fruit vinegar are significantly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of lime fruit vinegar preparation, and particularly relates to a method for optimizing a lime fruit vinegar preparation process by using a double-enzyme response surface method. BACKGROUND

[0002] Lime (Citrus aurantium L; CAL) is a plant of the Rutaceae family and the Citrus genus. The mature fruit is bitter and has poor taste. The mature lime fruit after the harvest period is rich in functional components such as dietary fiber (DF) and flavonoids, and has potential application value. The peel accounts for 30%-50% of the whole fruit, and unreasonable use of the CAL peel will cause great waste of resources, so it is necessary to improve the added value of the CAL peel.

[0003] Liquid fermentation has the advantages of short fermentation period, high yield and easy control, but still has some shortcomings, and the most obvious disadvantage is that the single strain has a strong sour taste. To ensure that the growth state of the yeast is not changed, the use of mixed strains for fermentation as much as possible can provide rich flavor substances for the fermentation stage. The lime and by-products such as peel are fermented into fruit vinegar, which has outstanding significance in resource utilization and value enhancement. The low-value raw materials such as small fruits and peels can be effectively converted, and the waste can be greatly reduced, which is in line with the concept of sustainable development. The lime fruit vinegar obtained by fermentation not only has unique flavor and rich layers, but also far exceeds ordinary vinegar, and further integrates the health benefits of vinegar acid, such as digestion and metabolism regulation. The nutritional value is doubled, and has stronger antioxidant, anti-inflammatory and potential health promotion functions. This deep processing method creates high-value characteristic products, expands market applications, improves the overall efficiency of the industry, and can realize resource recycling and value innovation.

[0004] During the fermentation of fruit vinegar, yeast and acetic acid bacteria are usually used for combined fermentation. The yeast primarily completes the efficient conversion of sugar to ethanol to provide the necessary substrate for acetic acid bacteria, and generates ester and other flavor precursor substances to lay the foundation for the aroma of fruit vinegar. The acetic acid bacteria are responsible for oxidizing ethanol to acetic acid and enriching functional components to achieve flavor setting and health efficacy enhancement. Due to the naturally high-acid environment of lime juice, screening and enhancing acid-tolerant yeast is the key to this process. It can resist acid stress and maintain high ethanol yield, ensuring seamless connection between the two-stage fermentation and avoiding metabolic interruption or flavor deterioration, which is a decisive factor for improving product quality and process stability. SUMMARY

[0005] The application discloses a method for optimizing a lime fruit vinegar preparation process by using a double-enzyme response surface method, to solve the above and any potential problems in the prior art. To solve the above technical problems, the application provides the following process:

[0006] Raw material pretreatment: select the diameter of less than 6 cm of lime, and lime peel, clean after mixing, according to the quality ratio of pulp: peel 4:6, add water according to the ratio of material to liquid 1:6, use the pulper to break it into pulp;

[0007] Enzymolysis, sugar adjustment: add 0.3% pectinase and 0.2% cellulase to the above slurry, and carry out enzymolysis at 55°C for 1h, add citric acid or sodium bicarbonate to adjust the initial pH value to 4.0, and place in water bath at 70°C for 30min for pasteurization, and cool to room temperature;

[0008] Fermentation: through the setting of response surface optimization test, the strengthened yeast and acetic acid bacteria liquid are fermented according to the optimal fermentation process conditions obtained by response surface optimization;

[0009] Filtration and clarification: after fermentation is completed, the fruit residue is separated out with filter cloth, then 0.5% chitosan is added for treatment for 1h, and then filtration is carried out with filter cloth; after the filtrate is sterilized in water bath at 70°C for 30min, it is cooled to room temperature; centrifugation is carried out at room temperature at 4000r / min for 20min, the supernatant is collected, boiled for 10min, and hotly filled, to obtain lime fruit vinegar.

[0010] The fermentation is: adding sucrose to adjust the sugar degree to 16-20% in the adjusted lime fruit slurry, adding 8-12% of bacteria liquid, wherein the inoculation ratio of the strengthened yeast and acetic acid bacteria liquid is 1:1-3, and the fermentation is carried out at 28-32°C for 4-6d.

[0011] The culture of the strengthened yeast includes:

[0012] Prepare 1078.11-1091.61 portions of basic medium, adjust the initial pH to 5.0-5.5 with hydrochloric acid or sodium hydroxide, sterilize at 90°C for 15min, cool to 30°C, inoculate 20-30 portions of yeast dry powder under sterile conditions, the temperature is 30°C±1°C, the shaking speed is 150-200rpm, culture for 2h, collect the bacteria by sterile centrifugation at 4000rpm for 10min at 4°C; wash the bacteria once with sterile 0.85% NaCl solution to remove residual basic medium components;

[0013] Resuspend the washed wet bacteria in the transformation medium, the resuspended cell density OD600 is 5-20 concentrated suspension, the electric field pulse treatment field strength is 8kV / cm, the pulse length is 1μs, the pulse number is 2-5 times / min, the pulse interval is 2min, the temperature is 28-31°C, the time is 1h, after the transformation culture is completed, immediately perform sterile centrifugation at 4000rpm for 10min, discard the supernatant;

[0014] The wet bacteria collected after 1-2 times of gentle washing of the bacteria with sterile 0.85% mass fraction NaCl solution are the fortified yeast bacteria.

[0015] The components of the basic culture medium are: 1000 parts of water, 50-60 parts of glucose, 10 parts of yeast extract powder, 10 parts of peptone, 5-6 parts of NH4Cl, 2-4 parts of KH2PO4, 0.5 parts of MgSO4·7H2O, 0.1-0.3 parts of CaCl2·2H2O, 0.01 parts of MnSO4·H2O, 0.5-0.8 parts of Tween 80.

[0016] The conversion culture medium formula is: 1000 parts of water, 20-30 parts of glucose, 0.2 parts of MgSO4·7H2O, 5-10 parts of inducer, and the pH is controlled to be 5.5±0.1 by using 2.56% mass fraction sodium citrate buffer.

[0017] The inducer is L-arginine-alpha-ketoglutarate.

[0018] The advantages and beneficial effects of the present application are:

[0019] 1. The response surface method for preparing lime fruit vinegar provided by the present application significantly improves the quality, production efficiency and resource utilization rate of fruit vinegar by precisely controlling fermentation through raw material treatment, core strain strengthening and response surface optimization, and realizes the dual optimization of product value and process efficiency.

[0020] 2. The process reduces the waste of processing byproducts, more fully integrates the sugar of the pulp and the unique aromatic oils, flavonoids and other active ingredients of the peel, and the pectinase and cellulase double enzyme synergistic fermentation reduces the viscosity by degrading pectin through pectinase, improves the juice yield by hydrolyzing fiber bundles through cellulase, deeply releases terpene aroma substances and phenolic active ingredients, and at the same time completely removes pectin and fiber impurities, improves the light transmittance, and lays a solid foundation for the final fruit vinegar with rich complex flavor and potential health value.

[0021] 3. The culture method of the fortified yeast bacteria significantly enhances the tolerance of the yeast cells by using specific basic culture combined with electric field pulse treatment technology and selecting L-arginine-alpha-ketoglutarate as a key inducer, so that the yeast cells can effectively resist the subsequent high-sugar and high-acid fermentation environment pressure. Compared with ordinary additives that only adjust the pH value, the inducer can more naturally activate the stress response mechanism of the yeast by acting as a specific precursor or signal molecule, improve the survival vitality and fermentation performance of the yeast, and avoid the risk of chemical residues.

[0022] 4. Using L-arginine-α-ketoglutarate as an inducer has significant advantages. It is not only an acidic substance, but also a combination of amino acids with physiological activity. During the intensification process, it can be effectively absorbed and utilized by yeast, not only significantly enhancing the tolerance of the bacterial body to high acidity and high osmotic pressure, ensuring its vigorous activity under harsh conditions for efficient production of acid precursors such as alcohol, but more importantly, it can enhance the ability of yeast to secrete or activate related enzyme systems by regulating specific metabolic pathways, thereby more effectively acting on the abundant flavonoids, limonin and other beneficial ingredients in the acid orange peel, promoting the release, transformation or stabilization of these substances, and ultimately contributing to the more excellent biological activity, flavor complexity and potential health value of the finished fruit vinegar.

[0023] 5. In the double-enzyme fermentation, the yeast is intensified by its excellent tolerance and high-efficiency alcohol conversion ability, which can quickly produce stable and high-quality wine in an acidic high-sugar environment, providing a substrate basis for acetic acid fermentation. The synergistic effect of the two bacteria under the optimized conditions of the response surface ensures not only the high conversion rate of alcohol to acetic acid, but also the formation of complex flavor substances, especially esters, making the fruit vinegar more rich in flavor levels and better in quality. DETAILED DESCRIPTION

[0024] The application will be further described in detail below in conjunction with examples. The yeast dry powder used below is a dry yeast specially for fruit wine, which is purchased from Angel Yeast Co., Ltd.; the acetic acid bacteria is Acetobacter pasteurianus subsp. (Hulian 1.01) purchased from Shandong Hezhong Kangyuan Biological Technology Co., Ltd.

[0025] Activation and expansion of acetic acid bacteria: take a single colony of Acetobacter pasteurianus subsp., inoculate into a triangular flask containing 100 mL of activation medium (components: glucose 10 g / L, yeast extract 10 g / L, ethanol 20 mL / L, pH 5.5) under sterile operation, and cultivate at 30°C, 160 r / min constant temperature oscillation for 24 h, until the culture solution presents uniform turbidity and slight bacterial film;

[0026] Primary expansion: take 1 mL of activated bacterial solution, inoculate into 250 mL of fermentation base medium (components: acid orange juice 30% v / v, yeast extract 5 g / L, ethanol 40 mL / L, MgSO40.2 g / L, pH 5.0), and maintain 30°C, 160 r / min oscillation for 24 h, during which the acidity is monitored to ≥2.0 g / 100 mL (calculated as acetic acid).

[0027] Secondary propagation: take 10 mL of the primary bacterial solution (4% v / v inoculation amount) and transfer it to 500 mL of the reinforced fermentation medium (composition: lime juice 50% v / v, ethanol 60 mL / L, yeast autolysate 3 g / L, ammonium acetate 1 g / L, pH 4.5), cultivate at 30°C and 160 r / min for 24 h. When the viscosity of the bacterial solution significantly increases and the acidity reaches ≥4.5 g / 100 mL, terminate the cultivation, and obtain the high-activity acetic acid bacterial solution.

[0028] Example 1

[0029] Culture of the reinforced yeast: basic medium: 1000 parts of water, 50 parts of glucose, 10 parts of yeast extract powder, 10 parts of peptone, 6 parts of NH4Cl, 2 parts of KH2PO4, 0.5 parts of MgSO4·7H2O, 0.3 parts of CaCl2·2H2O, 0.01 parts of MnSO4·H2O, 0.5 parts of Tween 80, adjust the initial pH to 5.5 using hydrochloric acid or sodium hydroxide, sterilize at 90°C for 15 min, cool to 30°C, inoculate 20 parts of yeast dry powder under sterile conditions, temperature 31°C, shaking speed 150 rpm, cultivate for 2 h, collect the bacterial cells by sterile centrifugation at 4000 rpm for 10 min at 4°C; gently wash the bacterial cells once with sterile normal saline (0.85% mass fraction NaCl solution) to remove residual basic medium components;

[0030] Resuspend the washed wet bacterial cells in the transformation medium: 1000 parts of water, 30 parts of glucose, 0.2 parts of MgSO4·7H2O, 5 parts of L-arginine-α-ketoglutarate, control the pH to 5.6 with 2.56% mass fraction sodium citrate buffer, the concentration of the resuspended cell suspension is OD600 of 20, electric field pulse treatment field strength 8 kV / cm, pulse length 1 μs, pulse frequency 2 times / min, pulse interval 2 min, temperature 31°C, time 1 h, after the transformation culture is completed, immediately perform sterile centrifugation at 4000 rpm for 10 min, discard the supernatant;

[0031] Gently wash the bacterial cells twice with sterile normal saline (0.85% mass fraction NaCl solution), and collect the wet bacterial cells as the reinforced yeast.

[0032] Raw material pretreatment: select limes with a diameter of less than 6 cm and lime peels, wash them clean, mix them according to a fruit pulp: peel mass ratio of 4:6, add water according to a material: liquid ratio of 1:6, and use a pulper to crush them into a pulp;

[0033] Enzymolysis and sugar adjustment: add 0.3% pectinase and 0.2% cellulase to the above pulp solution, perform enzymolysis in a constant temperature water bath at 55°C for 1 h, add citric acid or sodium bicarbonate to adjust the initial pH value to 4.0, place it in a water bath at 70°C for 30 min for pasteurization, and cool it to room temperature.

[0034] Fermentation: add sucrose to the adjusted lime pulp to adjust the sugar content to 20%, add 8% of the bacteria liquid, inoculate the bacteria liquid with the acetic acid bacteria at a ratio of 1:3, and ferment at 32°C for 6 days;

[0035] Filtration and clarification: after the fermentation is completed, separate the fruit residue with filter cloth, then add 0.5% of chitosan by mass fraction to treat for 1 h, and then filter with filter cloth; sterilize the filtrate at 70°C for 30 min in a water bath, and then cool to room temperature; centrifuge at 4000 r / min at room temperature for 20 min, collect the supernatant, boil for 10 min, and then fill while hot to obtain lime vinegar.

[0036] Example 2

[0037] Culture of the enhanced yeast: the basic culture medium: 1000 parts of water, 60 parts of glucose, 10 parts of yeast extract powder, 10 parts of peptone, 5 parts of NH4Cl, 4 parts of KH2PO4, 0.5 parts of MgSO4·7H2O, 0.1 parts of CaCl2·2H2O, 0.01 parts of MnSO4·H2O, and 0.8 parts of Tween 80; use hydrochloric acid or sodium hydroxide to adjust the initial pH to 5.0, sterilize at 90°C for 15 min, cool to 30°C, inoculate 30 parts of yeast dry powder under sterile conditions, the temperature is 29°C, the shaking speed is 200 rpm, culture for 2 h, collect the bacteria by sterile centrifugation at 4000 rpm for 10 min at 4°C; gently wash the bacteria once with 0.85% NaCl solution by mass fraction to remove residual basic culture medium ingredients;

[0038] Resuspend the washed wet bacteria in the transformation culture medium: 1000 parts of water, 20 parts of glucose, 0.2 parts of MgSO4·7H2O, 5 parts of L-arginine-α-ketoglutaric acid, and 2.56% sodium citrate buffer by mass fraction to control the pH to 5.4; the concentration of the resuspended cell suspension is OD600 of 5; the electric field pulse treatment field strength is 8 kV / cm, the pulse length is 1 μs, the pulse frequency is 5 times / min, the pulse interval is 2 min, the temperature is 28°C, and the time is 1 h; after the transformation culture is completed, immediately perform sterile centrifugation at 4000 rpm for 10 min, and discard the supernatant;

[0039] Gently wash the bacteria once with 0.85% NaCl solution by mass fraction, and collect the wet bacteria as the enhanced yeast.

[0040] Raw material pretreatment: select lime with a diameter of less than 6 cm, and lime peel, wash them clean, mix them according to a mass ratio of 4:6 of fruit pulp to peel, add water at a material to liquid ratio of 1:6, and crush them into pulp with a beater;

[0041] Enzymolysis, sugar adjustment: 0.3% pectinase and 0.2% cellulase were added to the above slurry, and the slurry was subjected to enzymolysis at 55°C for 1h. Citric acid or sodium bicarbonate was added to adjust the initial pH value to 4.0. The slurry was subjected to pasteurization at 70°C for 30min and cooled to room temperature;

[0042] Fermentation: sucrose was added to the adjusted lime pulp to adjust the sugar content to 16%, and 12% of the bacterial liquid was added, wherein the inoculation ratio of the strengthened yeast and acetic acid bacteria was 1:1, and the fermentation was carried out at 28°C for 4d.

[0043] Filtration and clarification: after the fermentation was completed, the fruit residue was separated out with filter cloth, and then 0.5% chitosan was added for treatment for 1h, and then the filtrate was filtered with filter cloth. The filtrate was sterilized at 70°C for 30min, and then cooled to room temperature. The supernatant was collected by centrifugation at 4000r / min at room temperature for 20min, and then boiled for 10min. The lime vinegar was obtained by hot filling.

[0044] Test 1: optimization of fermentation conditions of lime vinegar

[0045] The effects of different fermentation temperatures, inoculation ratios of yeast and acetic acid bacteria, and initial sugar contents on the acidity were investigated. According to the principle of Box-Behnken test design, on the basis of the single factor test, a three-factor three-level influence surface analysis method was adopted, and a total of 15 test points were designed. The test factors and level design are shown in Table 1.

[0046] Table 1 factor level table

[0047] Horizontal Inoculation ratio of yeast and acetic acid bacteria (v / v) Fermentation temperature (℃) Initial sugar content (%) -1 1：1 28 16 0 1：2 30 18 1 1：3 32 20

[0048] Determination method:

[0049] The most suitable conditions for lime double-enzyme fermentation were explored, and the response value was set as the total acid content (calculated as acetic acid). Three-factor level analysis test was designed by response surface software. The results are shown in Table 2.

[0050] Table 2

[0051] Test No. Inoculation ratio (A) Fermentation temperature (B, ℃) Sugar content (C, %) Total acid content (g / 100 mL) 1 1：1 28 18 5.72 2 1：3 28 18 6.25 3 1：1 32 18 6.38 4 1：3 32 18 6.95 5 1：1 30 16 6.05 6 1：3 30 16 6.52 7 1：1 30 20 5.88 8 1：3 30 20 6.18 9 1：2 28 16 6.31 10 1：2 32 16 6.87 11 1：2 28 20 5.95 12 1：2 32 20 6.42 13 1：2 30 18 7.15 14 1：2 30 18 7.22 15 1：2 30 18 7.18

[0052] Quadratic polynomial regression equation

[0053] The regression model of total acid content (Y) and each factor: Y=7.18+0.21A+0.32B+0.05C-0.12AB-0.08AC-0.05BC-0.42A²-0.28B²-0.35C²

[0054] Table 3 significance test of regression model and variance analysis

[0055] Variation source Sum of squares Degrees of freedom Mean square F value P value Significance Model 4.876 9 0.542 42.15 ＜0.0001 ** A-Inoculation ratio 0.529 1 0.529 41.13 0.0003 ** B-Temperature 1.229 1 1.229 95.57 ＜0.0001 ** C-Sugar content 0.030 1 0.030 2.33 0.1687 - AB 0.058 1 0.058 4.51 0.1688 - AC 0.026 1 0.026 2.20 0.1962 - BC 0.010 1 0.010 0.78 0.4056 - A 2 ]] 0.754 1 0.754 58.64 ＜0.0001 ** B 2 ]]> 0.332 1 0.332 25.82 0.0011 ** [C 2 ]]> 0.518 1 0.518 40.28 0.0003 ** Residual error 0.090 7 0.013 Nonsignificant 0.062 3 0.021 2.41 0.1984 Pure error Total variation 0.028 4 0.007 Group 1.966 16

[0056] R² = 0.982

[0057] Adjusted R² = 0.959

[0058] Predicted R² = 0.917

[0059] CV = 1.76%

[0060] Significance mark: **P < 0.01 (very significant); *P < 0.05 (significant); - (not significant);

[0061] According to the above response surface optimization, the optimal fermentation process conditions are: the inoculation ratio of yeast bacteria to acetic acid bacteria = 1:2.56, the fermentation temperature is 31.26°C, and the initial sugar content is 17.9%. According to the model equation, the total acid content of the fermentation liquor is predicted to be 7.34 g / 100 mL. Considering the operability of actual production, the optimized conditions are: the inoculation ratio of yeast bacteria to acetic acid bacteria = 1:2.5, the fermentation temperature is 31°C, and the initial sugar content is 18%. To test the reliability of the results obtained by the experimental design, fermentation experiments were conducted under these conditions. The acidities of three parallel experiments were 7.35, 7.33, and 7.39 g / 100 mL, respectively, with an average acidity of 7.37 g / 100 mL. Compared with the theoretical predicted value, the relative error is about 0.004%. Therefore, the process parameters optimized by response surface analysis method are reliable and have practical value.

[0062] According to the above response surface optimization data, the following is implemented according to the controllable range of actual production:

[0063] Example 3

[0064] Strengthening the culture of yeast bacteria: basic medium: 1000 parts of water, 55 parts of glucose, 10 parts of yeast extract powder, 10 parts of peptone, 5.5 parts of NH4Cl, 3 parts of KH2PO4, 0.5 parts of MgSO4·7H2O, 0.2 parts of CaCl2·2H2O, 0.01 parts of MnSO4·H2O, 0.6 parts of Tween 80, adjust the initial pH to 5.2 with hydrochloric acid or sodium hydroxide, sterilize at 90°C for 15 min, cool to 30°C, inoculate 25 parts of yeast dry powder under sterile conditions, temperature 30°C, shaking speed 180 rpm, culture for 2 h, collect the bacterial cells by sterile centrifugation at 4000 rpm for 10 min at 4°C; gently wash the bacterial cells once with sterile physiological saline (0.85% mass fraction NaCl solution) to remove residual basic medium components;

[0065] The washed wet bacteria were resuspended in a transformation medium: 1000 parts of water, 25 parts of glucose, 0.2 parts of MgSO4·7H2O, 8 parts of L-arginine-alpha-ketoglutarate, a 2.56% mass fraction of sodium citrate buffer to control pH at 5.5, a resuspended cell density OD600 of 12 concentrated suspension, an electric field pulse treatment field strength of 8 kV / cm, a pulse length of 1 μs, a pulse number of 3 times / min, a pulse interval of 2 min, a temperature of 29°C, and a time of 1 h. After the transformation culture was completed, sterile centrifugation was immediately performed at 4000 rpm for 10 min, and the supernatant was discarded.

[0066] The bacteria were gently washed twice with sterile normal saline of 0.85% mass fraction of NaCl solution, and the collected wet bacteria were the fortified yeast bacteria.

[0067] Raw material pretreatment: acid oranges with a diameter of less than 6 cm and acid orange peels were selected, washed clean, mixed according to a mass ratio of pulp:peel of 4:6, added with water at a material-liquid ratio of 1:6, and crushed into a pulp shape by a pulper;

[0068] Enzymolysis and sugar adjustment: 0.3% pectinase and 0.2% cellulase were added to the above slurry, and the slurry was subjected to enzymolysis at 55°C for 1 h in a constant temperature water bath, and then pasteurized at 70°C for 30 min. The initial pH value was adjusted to 4.0 by adding citric acid or sodium bicarbonate, and the slurry was cooled to room temperature.

[0069] Fermentation: 10.5% of the bacteria liquid was added to the adjusted acid orange pulp, in which the inoculation ratio of the fortified yeast bacteria to acetic acid bacteria liquid was 1:2.5, and the mixture was subjected to fermentation at 31°C for 5 d.

[0070] Filtration and clarification: after the fermentation was completed, the fruit residue was separated by filter cloth, and then treated with 0.5% mass fraction of chitosan for 1 h, and then filtered by filter cloth. The filtrate was sterilized at 70°C for 30 min, and then cooled to room temperature. The supernatant was collected by centrifugation at 4000 r / min for 20 min at room temperature, boiled for 10 min, and then filled in a bottle while hot, to obtain the acid orange vinegar.

[0071] Comparative Example 1

[0072] The difference between the present comparative example and Example 3 is that the present comparative example does not add L-arginine-alpha-ketoglutarate to the transformation medium; the rest is the same as Example 3.

[0073] Comparative Example 2

[0074] The difference between the present comparative example and Example 3 is that the present comparative example does not add L-arginine-alpha-ketoglutarate to the transformation medium; the rest is the same as Example 3.

[0075] Comparative Example 3

[0076] The difference between the present comparative example and Example 3 is that the present comparative example replaces L-arginine-alpha-ketoglutarate with citric acid; the rest is the same as Example 3.

[0077] Comparative Example 4

[0078] The difference between the present comparative example and Example 3 is that the present comparative example strengthens the culture of the yeast: base medium: 1000 parts of water, 55 parts of glucose, 10 parts of yeast extract powder, 10 parts of peptone, 5.5 parts of NH4Cl, 3 parts of KH2PO4, 0.5 parts of MgSO4·7H2O, 0.2 parts of CaCl2·2H2O, 0.01 parts of MnSO4·H2O, 0.6 parts of Tween 80, adjust the initial pH to 5.2 using hydrochloric acid or sodium hydroxide, sterilize at 90°C for 15 min, cool to 30°C, inoculate 25 parts of yeast dry powder under sterile operation, temperature 30°C, shaking speed 180 rpm, culture for 2 h, collect the bacteria by sterile centrifugation at 4000 rpm for 10 min at 4°C; gently wash the bacteria once with sterile normal saline 0.85% mass fraction NaCl solution to remove residual base medium components; the rest is the same as Example 3.

[0079] The washed wet bacteria were resuspended in a transformation medium: 1000 parts of water, 25 parts of glucose, 0.2 parts of MgSO4·7H2O, 8 parts of L-arginine-α-ketoglutaric acid, a 2.56% mass fraction of sodium citrate buffer to control pH at 5.5, a resuspended cell density OD600 of 12, a concentration of the suspension, a temperature of 29°C, and a time of 1 h. After the transformation culture was completed, the bacteria were immediately subjected to sterile centrifugation at 4000 rpm for 10 min, and the supernatant was discarded.

[0080] The bacteria were gently washed twice with sterile normal saline (0.85% mass fraction of NaCl solution), and the collected wet bacteria were the fortified yeast bacteria. The rest was the same as in Example 3.

[0081] Comparative Example 5

[0082] The difference between this comparative example and Example 3 is that only acetic acid bacteria were added in this comparative example.

[0083] Raw material pretreatment: Select limes with a diameter of less than 6 cm and lime peels, wash them clean, mix them according to a fruit pulp: peel mass ratio of 4:6, add water at a material: liquid ratio of 1:6, and use a pulper to break them into a pulp;

[0084] Enzymolysis and sugar adjustment: 0.3% pectinase and 0.2% cellulase were added to the above slurry, which was subjected to enzymolysis at 55°C for 1 h, 4.0 was added as the initial pH value by adding citric acid or sodium bicarbonate, and pasteurization was performed at 70°C for 30 min, and the slurry was cooled to room temperature.

[0085] Fermentation: 10.5% acetic acid bacteria solution was added to the adjusted lime pulp to adjust the sugar content to 18%, and fermentation was performed at 31°C for 5 d.

[0086] Filtration and clarification: After the fermentation was completed, the dregs were separated out using filter cloth, 0.5% chitosan was added for treatment for 1 h, and then the mixture was filtered using filter cloth. The filtrate was sterilized at 70°C for 30 min, and then cooled to room temperature. Centrifugation was performed at 4000 r / min for 20 min at room temperature, the supernatant was collected, boiled for 10 min, and then filled while hot to obtain lime vinegar.

[0087] Test 2: Property determination

[0088] After the fermentation was completed, samples were taken for detection of alcohol content, total acid (calculated as acetic acid) was determined by direct titration, total sugar was determined by direct titration, and alcohol content was determined by an alcohol meter.

[0089] The results are shown in Table 4 below.

[0090] Table 4

[0091] Total sugar (g / L) Alcohol content (%) Total acid (g / 100 ml) Example 1 Example 2 1.24 2.84 7.16 Example 3 1.35 2.67 7.20 Comparative Example 1 0.87 2.59 7.38 Comparative Example 2 4.75 5.12 5.12 Comparative Example 3 4.13 4.63 5.34 Comparative Example 4 3.56 4.09 5.68 Comparative Example 5 2.41 3.68 7.87 Group 5.46 5.48 4.87

[0092] The total flavonoid content was determined by aluminum nitrate colorimetry. The obtained fruit vinegar was extracted with 60% ethanol, and the clear liquid was obtained by filtration. 1 mL of sample extract was taken, 5 mL of distilled water was added, and then 1 mL of 5% sodium nitrite solution was added. After shaking, it was placed for 6 min, then 1 mL of 10% aluminum nitrate solution was added, shaken and placed for 6 min. 10 mL of 5% sodium hydroxide solution was added, and the volume was made to 25 mL with 60% ethanol, shaken and mixed, placed in a 45°C water bath for 10 min, then taken out and cooled. Centrifugation was performed at 3000 r / min for 5 min, and the supernatant was taken to measure the absorbance at 505 nm.

[0093] The total phenol content was determined by directly diluting the fruit vinegar sample, taking 100 μL of sample liquid, adding 7 mL of distilled water, adding 0.5 mL of Folin reagent, shaking for 1 min, then adding 1.5 mL of 20% sodium carbonate solution, making the volume to 10 mL, placing in the dark for 1 h, and measuring the absorbance at 760 nm.

[0094] The results are shown in Table 5 below.

[0095] Table 5

[0096] Total phenol (mg / L) Total flavonoids (mg / L) Example 1 Example 2 408.32 528.74 Example 3 410.65 519.65 Comparative Example 1 420.94 528.06 Comparative Example 2 368.48 385.49 Comparative Example 3 379.24 405.83 Comparative Example 4 386.62 423.18 Comparative Example 5 397.51 462.75 ​ 355.67 369.41

Claims

1. A method for optimizing the process of preparing lime fruit vinegar by dual enzyme in response surface methodology characterized in that, The process is: Raw material pretreatment: select the diameter of less than 6 cm of lime, and lime peel, clean after mixing, according to the quality ratio of 4:6, adding water, with the beating machine, it is broken into pulp; Enzymolysis, sugar adjustment: add 0.3% pectinase and 0.2% cellulase to the above slurry, and then carry out enzymolysis at 55°C for 1h. Add citric acid or sodium bicarbonate to adjust the initial pH value to 4.0, and then carry out pasteurization at 70°C for 30min. Cool to room temperature; Fermentation: set the response surface optimization test, and then ferment the strengthened yeast and acetic acid bacteria liquid according to the optimal fermentation process conditions obtained by the response surface optimization test; Filtering and clarification: after the fermentation is completed, separate the fruit residue by using filter cloth, then add 0.5% chitosan to the fruit residue, and then carry out treatment for 1h. Then filter the fruit residue by using filter cloth. After sterilization at 70°C for 30min, cool to room temperature. Then centrifuge at 4000r / min under the condition of room temperature for 20min. Collect the supernatant, and then boil for 10min. Hotly fill the bottle, and then lime vinegar is obtained; The fermentation is: add sucrose to the adjusted lime fruit slurry to adjust the sugar degree to 16-20%, and then add 8-12% bacteria liquid. The inoculation ratio of the strengthened yeast and acetic acid bacteria liquid is 1:1-3. Then carry out fermentation at 28-32°C for 4-6d; The culture of the strengthened yeast includes: Prepare 1078.11-1091.61 portions of basic culture medium, and then adjust the initial pH to 5.0-5.5 by using hydrochloric acid or sodium hydroxide. Sterilize at 90°C for 15min, and then cool to 30°C. Inoculate 20-30 portions of yeast dry powder by using sterile operation. The temperature is 30°C±1°C. The rotation speed of the shaking table is 150-200rpm. Culture for 2h. Collect the bacteria by using sterile centrifugation at 4000rpm for 10min at 4°C. Wash the bacteria once by using sterile normal saline with 0.85% mass fraction of NaCl solution to remove the residual basic culture medium ingredients; Resuspend the washed wet bacteria in the transformation culture medium. The resuspended cell density OD600 is 5-20 concentrated suspension. The electric field pulse treatment field strength is 8kV / cm. The pulse length is 1μs. The pulse number is 2-5 times / min. The pulse interval is 2min. The temperature is 28-31°C. The time is 1h. After the transformation culture is completed, immediately carry out sterile centrifugation at 4000rpm for 10min. Discard the supernatant. Wash the bacteria 1-2 times by using sterile normal saline with 0.85% mass fraction of NaCl solution. The collected wet bacteria are the strengthened yeast. The transformation culture medium formula is: 1000 portions of water, 20-30 portions of glucose, 0.2 portions of MgSO4·7H2O, 5-10 portions of inducer, and 2.56% mass fraction of sodium citrate buffer solution to control the pH to 5.5±0.

1. The inducer is L-arginine-α-ketoglutaric acid.

2. The method of optimizing the process of preparing lime fruit vinegar by dual enzyme using response surface methodology as claimed in claim 1 wherein, The components of the basic culture medium are: 1000 parts of water, 50-60 parts of glucose, 10 parts of yeast extract powder, 10 parts of peptone, 5-6 parts of NH4Cl, 2-4 parts of KH2PO4, 0.5 parts of MgSO4·7H2O, 0.1-0.3 parts of CaCl2·2H2O, 0.01 parts of MnSO4·H2O, 0.5-0.8 parts of Tween 80.