Method for efficiently degrading tannin in fruit juice

Abstract

The invention discloses a method for efficiently degrading tannin in fruit juice, which comprises the following steps: taking the fruit juice, adding tannase, carrying out enzymolysis for 12-24 hours, carrying out 100-600Mpa ultrahigh pressure treatment for 10-20 minutes, and then continuing to carry out enzymolysis for 12-24 hours. According to the method disclosed by the invention, an ultrahigh pressure combined tannase treatment technology is adopted, and ultrahigh pressure treatment is arranged after the enzymolysis reaction is performed for a period of time, so that the enzymolysis reaction period of tannase is prolonged by utilizing the activation effect of the ultrahigh pressure treatment on enzyme, and the degradation efficiency of tannin can be remarkably improved; meanwhile, the content of heat-sensitive active ingredients such as polyphenol, flavone and vitamin C in the fruit juice can be well retained, the method is suitable for roxburgh rose juice and other fruit juice rich in tannin and heat-sensitive active ingredients, and the taste and quality of the fruit juice can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing and relates to a method for degrading tannin in fruit juice. Background Art

[0002] Tannin, also known as tannin and tannic acid, is a secondary metabolite of plants with a relative molecular mass of 500 to 3000 Da. Tannin is widely distributed and is usually found in the roots, stems, leaves, flowers, fruits, seeds and bark of higher plants. Rice, sorghum, barley, grapes, persimmons and berries are rich in tannins. Tannins in food are anti-nutritional and will affect people's digestion of starch, fat, protein and cellulose, reducing the nutritional value of food. In the processing of fruit juice products, tannins will combine with proteins and other substances to form water-insoluble precipitation, affecting product quality. Tannin is the main substance that causes the sour taste of fruits. Tannin content is 0.03% to 0.1%, which can enhance the sour taste of fruits and give fruits a cool taste, but if the content is higher than 1%, the fruit will have a strong astringent taste. Therefore, reducing the tannin content is an effective measure to solve the taste of fruit juice products.

[0003] Rosa roxburghii Tratt, belonging to the Rosaceae family, is a deciduous shrub with a common distribution range in southwest, south-central and northwest China. Rosa roxburghii is rich in a variety of bioactive substances, such as flavonoids, polyphenols, polysaccharides, triterpenes and organic acids, and its vitamin C and superoxide dismutase (SOD) content ranks first among all fruits and vegetables. These substances give rosa roxburghii a variety of functional activities, such as antioxidant, anti-cancer, and hypoglycemic. Rosa roxburghii has a strong aroma, but due to its rough texture and sour taste, it is not suitable for fresh consumption and is mainly used for juice processing. The tannin content in rosa roxburghii juice is usually above 0.6%, and some varieties can even reach 2.2%. Excessive tannin content will have an adverse effect on the stability, clarity, flavor, color, taste, etc. of the juice product, making it difficult for consumers to accept it. At the same time, the active ingredients in rosa roxburghii juice are easily degraded under thermal processing conditions.

[0004] Therefore, establishing a non-thermal processing method that can efficiently degrade tannins in juice while better retaining the heat-sensitive active ingredients in the juice is of great significance to improving the quality of sea buckthorn juice and other juices rich in tannins and heat-sensitive active ingredients. Summary of the invention

[0005] The object of the present invention is to provide a method for efficiently degrading tannins in fruit juice, which can effectively degrade tannins in fruit juice and better retain heat-sensitive active ingredients in the juice, thereby improving the quality of roxburghii juice and other fruit juices rich in tannins and heat-sensitive active ingredients.

[0006] After research, the present invention provides the following technical solutions:

[0007] A method for efficiently degrading tannin in fruit juice comprises the following steps: taking fruit juice, adding tannin enzyme, first enzymolysis for 12-24 hours, ultra-high pressure treatment at 100-600Mpa for 10-20 minutes, and then continuing enzymolysis for 12-24 hours.

[0008] Furthermore, the method for efficiently degrading tannin in juice comprises the following steps:

[0009] a. Take the juice, add tannin, first enzymolyze for 12-24 hours, treat with 100-600Mpa ultra-high pressure for 10-20min, and then continue enzymolyze for 12-24 hours;

[0010] b. Take the juice processed in step a, add lactic acid bacteria suspension and MRS broth medium, ferment for 12-24 hours, and centrifuge to obtain the supernatant.

[0011] In some embodiments of the present invention, the juice is roxburghii juice.

[0012] In some embodiments of the present invention, the lactic acid bacteria is Lactobacillus casei subsp. casei.

[0013] In the above method, preferably: taking fruit juice, adding tannin, first enzymolysis for 24 hours, treating at 500 MPa ultrahigh pressure for 15 minutes, and then continuing enzymolysis for 24 hours.

[0014] In the above method, it is preferred that: take the juice treated in step a, add lactic acid bacteria suspension and MRS broth culture medium, the volume ratio of the juice, lactic acid bacteria suspension and MRS broth culture medium is 4:1:5, ferment for 24 hours, centrifuge and take the supernatant.

[0015] The beneficial effect of the present invention is that the present invention provides a method for efficiently degrading tannin in fruit juice.

[0016] (1) Ultrahigh pressure combined with tannase treatment is adopted, and creative research is focused on the timing of ultrahigh pressure intervention. Compared with ultrahigh pressure treatment immediately at the beginning of enzymolysis, ultrahigh pressure treatment is arranged after the enzymolysis reaction has been carried out for a period of time, which can significantly improve the degradation efficiency of tannin. Because the activity of tannase has been reduced after the enzymolysis reaction for a period of time, the ultrahigh pressure treatment at this time can be used to activate the enzyme and extend the enzymolysis reaction cycle of tannase, thereby promoting the enzymatic degradation of tannin. The ultrahigh pressure combined with tannase treatment technology of the present invention can effectively degrade tannins in roxburghii juice, and at the same time, the content of heat-sensitive active ingredients such as polyphenols, flavonoids, vitamin C and other substances in roxburghii juice can be well retained, and the influence of tannin degradation on its own lipid-lowering activity can be reduced as much as possible.

[0017] (2) On the basis of ultra-high pressure combined with tannin enzyme treatment, it can be further combined with lactic acid bacteria fermentation treatment. Lactic acid bacteria fermentation treatment has the ability to degrade tannins in sea buckthorn juice. The combined ultra-high pressure and tannin enzyme treatment has a better degradation effect on tannins, which can effectively improve the astringency of sea buckthorn juice and enhance its taste. Although lactic acid bacteria fermentation treatment is not conducive to the retention of active components such as polyphenols, flavonoids, and vitamin C in sea buckthorn juice, the combined ultra-high pressure and tannin enzyme treatment can better retain these active components. At the same time, lactic acid bacteria fermentation has no significant effect on the smell of sea buckthorn juice.

[0018] (3) The ultrahigh pressure combined with tannase treatment technology and the lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment technology of the present invention are also applicable to other fruit juices rich in tannins and heat-sensitive active ingredients besides sea buckthorn juice. While efficiently degrading tannins in the fruit juice, they can also better retain the heat-sensitive active ingredients in the fruit juice, thereby improving the taste and quality of the fruit juice.

[0019] (4) As a green and efficient degradation method, the method of the present invention meets the requirements of sustainable development. Compared with the traditional chemical degradation method, it does not produce harmful byproducts during the degradation process, thus reducing the pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To investigate the effect of ultrahigh pressure intervention on the degradation of tannins in roxburghii juice by tannase.

[0021] Figure 2 To investigate the effect of ultrahigh pressure intervention timing on the degradation of tannins in roxburghii juice by tannase.

[0022] Figure 3 The effect of the timing of ultrahigh pressure intervention on tannase activity.

[0023] Figure 4 Effects of ultrahigh pressure combined with tannase treatment on the polyphenol content in roxburghii juice.

[0024] Figure 5 This study aimed to investigate the effects of ultrahigh pressure combined with tannase treatment on the flavonoids content in roxburghii juice.

[0025] Figure 6 This study aimed to investigate the effect of ultrahigh pressure combined with tannase treatment on the content of vitamin C in roxburghii juice.

[0026] Figure 7 To investigate the effects of ultrahigh pressure combined with tannase treatment on the lipid-lowering activity of roxburghii juice in vitro.

[0027] Figure 8 To investigate the effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the tannin content in roxburghii juice.

[0028] Fig. 9 To investigate the effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the polyphenol content in roxburghii juice.

[0029] Fig.10 To investigate the effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the flavonoids content in roxburghii juice.

[0030] Fig.11 To investigate the effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the content of vitamin C in roxburghii juice.

[0031] Fig.12 To study the effect of lactic acid bacteria fermentation combined with ultrahigh pressure and tannin enzyme treatment on the activity of SOD enzyme in sea buckthorn juice.

[0032] Fig.13 To investigate the effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the soluble solids content in sea buckthorn juice.

[0033] Fig.14 To investigate the effect of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the binding capacity of roxburghii juice with sodium glycocholate.

[0034] Fig.15 To investigate the effect of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the binding capacity of roxburghii juice with sodium taurocholate.

[0035] Fig.16 Radar chart of the response values ​​of the electronic nose sensor to the sea buckthorn juice processed by different methods.

[0036] In the above figures, different lowercase letters indicate significant differences among the groups (P < 0.05). DETAILED DESCRIPTION

[0037] In order to make the objectives, technical solutions and beneficial effects of the present invention more clear, the preferred embodiments of the present invention are described in detail below.

[0038] The main materials and instruments in the preferred embodiment are as follows:

[0039] The roxburghii juice was freshly squeezed juice of roxburghii fruit, provided by Jinweiyi Health Industry Development Co., Ltd.; tannin enzyme was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; lactic acid bacteria was Lactobacillus casei subsp. casei ATCC334, purchased from Guangdong Huankai Biotechnology Co., Ltd.; the ultrahigh pressure processing equipment was HPP3-5 L600MPa high pressure food processing equipment, purchased from Baotou Kefa High Pressure Technology Co., Ltd., Inner Mongolia, China.

[0040] The ultra-high pressure treatment method in the preferred embodiment is as follows:

[0041] The sample was placed in a polyethylene plastic bag, sealed, and placed in an ultra-high pressure treatment device. It was treated at a certain pressure for a period of time. The pressure change during the pressure maintenance period did not exceed 10MPa. The pressure increase rate was 100MPa / min, and the pressure was released instantly.

[0042] The active ingredient analysis method in the preferred embodiment is as follows:

[0043] 1. Determination of tannin content

[0044] The determination was carried out in accordance with NY / T 1600-2008 “Determination of tannin content in fruits, vegetables and their products – Spectrophotometric method”.

[0045] Accurately weigh 0.1100g of monohydrated gallic acid, dissolve it in water and dilute to 100mL to obtain a gallic acid standard stock solution with a concentration of 1000mg / L. Pipette 0.0, 1.0, 2.0, 3.0, 4.0, 5.0mL of the gallic acid standard stock solution into a 100mL volumetric flask, dilute to the scale with water, and obtain gallic acid standard working solutions with concentrations of 0.0, 10.0, 20.0, 30.0, 40.0, 50.0mg / L, respectively. Take 1.0 mL of the gallic acid standard solution of different concentrations, add 5.0 mL of water, 1.0 mL of sodium tungstate-sodium molybdate mixed solution and 3.0 mL of 75 g / L sodium carbonate solution, mix well, and obtain gallic acid standard solutions with concentrations of 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L, respectively. Color development, place for 2 hours, take the standard curve 0.0 mg / L as blank, measure the absorbance of the standard solution at a wavelength of 765 nm, take the gallic acid concentration as the horizontal coordinate and the absorbance value as the vertical coordinate, draw the standard curve, and obtain the regression equation y=0.1509x-0.0015(R 2 =0.9996).

[0046] Take the sample to be tested, dilute it with water, absorb 1.0mL of sample diluent, add 5.0mL of water, 1.0mL of sodium tungstate-sodium molybdate mixed solution and 3.0mL of 75g / L sodium carbonate solution, mix well, develop color, and place for 2h. Use the standard curve 0.0mg / L as blank, measure the absorbance at a wavelength of 765nm, and calculate the tannin content of the sample according to the standard curve, calculated as gallic acid.

[0047] The tannin content (in terms of gallic acid) of the sample is calculated according to the following formula: ω = (ρ × 10 × A) / m; where: ω is the tannin content of the sample, mg / L; ρ is the concentration of gallic acid in the sample test solution, mg / L; 10 is the fixed volume of the sample test solution, mL; A is the sample dilution multiple; m is the sample volume, mL.

[0048] 2. Determination of total phenol content

[0049] Weigh 25.00 mg of gallic acid, dissolve and dilute with methanol to prepare gallic acid standard solutions with concentrations of 0, 20, 60, 100, 200, 300, 400, and 500 μg / mL, respectively. Take 0.2 mL of the gallic acid standard solutions of different concentrations, add water to 1 mL, add 0.2 mL of Folin phenol reagent, mix well, let stand for 6 min, add 2 mL of 7% sodium carbonate solution and 1.6 mL of water, mix well, let stand at room temperature in the dark for 90 min, use the standard curve 0 μg / mL as blank, measure the absorbance of the standard solution at a wavelength of 760 nm, use the gallic acid concentration as the horizontal coordinate and the absorbance value as the vertical coordinate, draw the standard curve, and obtain the regression equation y=0.1461x+0.0042(R 2 =0.9991).

[0050] Take the sample to be tested, dilute it, draw 0.2mL of sample diluent, make up to 1mL with water, add 0.2mL of Folin phenol reagent, mix well, let stand for 6min, add 2mL of 7% sodium carbonate solution and 1.6mL of water, mix well, let stand at room temperature in the dark for 90min, use the standard curve 0μg / mL as blank, measure the absorbance at a wavelength of 760nm, and calculate the total phenol content of the sample according to the standard curve, calculated as gallic acid.

[0051] The total phenol content (calculated as gallic acid) of the sample is calculated according to the following formula: x = (c×A) / m; wherein: x is the total phenol content of the sample, mg GAE / mL; c is the concentration of gallic acid in the sample assay solution, mg / mL; A is the sample dilution multiple; and m is the sample volume, mL.

[0052] 3. Determination of flavonoid content

[0053] Accurately weigh 10 mg of rutin standard, dissolve it in 70% ethanol and dilute to 50 mL to prepare a rutin standard solution with a concentration of 0.2 mg / mL. Take 0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL of rutin standard solution respectively, add 1 mL of 5% sodium nitrite solution, 1 mL of 10% aluminum nitrate solution, and 10 mL of 4% sodium hydroxide solution. Each reagent needs to react for 6 minutes. Finally, dilute to 25 mL with 70% ethanol, let it stand for 30 minutes, use 70% ethanol as blank, measure the absorbance at a wavelength of 510 nm, use rutin concentration as the horizontal coordinate and absorbance value as the vertical coordinate, draw a standard curve, and get the regression equation y=8.6914x-0.0066(R 2 =0.9992).

[0054] Take the sample to be tested, dilute it, aspirate 1.0mL of sample diluent, add 1mL of 5% sodium nitrite solution, 1mL of 10% aluminum nitrate solution and 10mL of 4% sodium hydroxide solution, each addition of a reagent requires reaction for 6 minutes, finally dilute to 25mL with 70% ethanol, let it stand for 30 minutes, use 70% ethanol as blank, measure the absorbance at a wavelength of 510nm, and calculate the flavonoid content of the sample according to the standard curve, calculated as rutin.

[0055] The flavonoid content (in terms of rutin) of the sample is calculated according to the following formula: x = (m×d) / (v×1000); where: x is the flavonoid content of the sample, mg / mL; m is the mass of rutin in the sample test solution, μg; v is the sample volume, mL; d is the sample dilution multiple.

[0056] 4. Determination of vitamin C content

[0057] The determination was carried out using the third method 2,6-dichloroindophenol titration method in GB 5009.86-2016 “Determination of ascorbic acid in foods”.

[0058] Weigh 100 mg of L(+)-ascorbic acid standard, dissolve it with oxalic acid solution and dilute to 100 mL to prepare an ascorbic acid standard solution with a concentration of 1.000 mg / mL. Accurately pipette 1 mL of ascorbic acid standard solution into a 50 mL conical flask, add 10 mL of oxalic acid solution, shake well, and titrate with 2,6-dichloroindophenol solution until it turns pink and does not fade for 15 seconds. At the same time, take another 10 mL of oxalic acid solution for a blank test.

[0059] The titer of 2,6-dichloroindophenol solution is calculated according to the following formula: T = (c × V) / (V1-V0); where: T is the titer of 2,6-dichloroindophenol solution, that is, the number of milligrams of ascorbic acid equivalent to each milliliter of 2,6-dichloroindophenol solution, mg / mL; c is the concentration of ascorbic acid standard solution, mg / mL; V is the volume of ascorbic acid standard solution absorbed, mL; V1 is the volume of 2,6-dichloroindophenol solution consumed in titrating ascorbic acid standard solution, mL; V0 is the volume of 2,6-dichloroindophenol solution consumed in titrating blank, mL.

[0060] Take the sample to be tested, dilute it, pipette 10mL of the sample dilution into a 50mL conical flask, and titrate it with the calibrated 2,6-dichloroindophenol solution until the solution turns pink and does not fade for 15 seconds. Perform a blank test at the same time.

[0061] The ascorbic acid content of the sample is calculated according to the following formula: X = [(V-V0) × T × A] / m; where: X—ascorbic acid content of the sample, mg / mL; V—volume of 2,6-dichloroindophenol solution consumed by titrating the sample diluent, mL; V0—volume of 2,6-dichloroindophenol solution consumed by titrating the blank, mL; T—titer of 2,6-dichloroindophenol solution, mg / mL; A—sample dilution multiple; m—sample volume, mL.

[0062] 5. Determination of SOD enzyme activity

[0063] The determination was carried out according to the Marklund method modified from the first method in GB / T5009.171-2003 “Determination of superoxide dismutase (SOD) activity in health foods”.

[0064] At about 25℃, add 2.35mL of solution A, 2.00mL of distilled water, and 0.15mL of solution B to a 10mL colorimetric tube. Add solution B and mix immediately and pour into a colorimetric dish. Measure the absorbance at the initial time and after 1 minute at a wavelength of 325nm. The difference between the two is the self-oxidation rate of pyrogallol △T. According to the above steps, add a certain amount of diluent of the sample to be tested to inhibit the self-oxidation rate of pyrogallol to about 1 / 2△T, that is, △T'.

[0065] The SOD activity of the sample was calculated according to the following formula: SOD activity (U / mL) = [(△T-△T') / △T] × 100% × 4.5 × (1 / V) × D / 50%; where: △T is the rate of pyrogallol self-oxidation; △T' is the rate of pyrogallol self-oxidation inhibited by the sample; V is the volume of the added sample diluent, mL; D is the sample dilution factor; 4.5 is the total volume of the reaction solution, mL.

[0066] 6. Determination of soluble solids content

[0067] Use a handheld saccharimeter (0-32%) for determination. Calibrate the saccharimeter in a well-lit room, open the cover, drop 2-3 drops of distilled water on the surface of the detection prism, gently close the cover to allow the water to cover the prism surface, aim the front of the saccharimeter at the light, and use a screwdriver to turn the adjustment screw to make the blue-white dividing line consistent with the zero scale line. Open the cover, carefully wipe the water on the prism surface with lens paper, drop 2-3 drops of sample on the prism, cover the cover and gently flatten it to cover the surface, look in through the eyepiece, the scale value of the blue-white dividing line is the soluble solid content of the sample.

[0068] 7. In vitro lipid-lowering activity assay

[0069] Take 1 mL of standard bile salt solution of different concentrations (0.03, 0.06, 0.12, 0.18, 0.24, 0.30, 0.36 mmol / L of sodium glycocholate; 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 mmol / L of sodium taurodeoxycholate), add 3 mL of 60% sulfuric acid solution, bathe at 70°C for 40 min, ice bath for 5 min, and measure the absorbance at 387 nm. Draw a standard curve with bile salt concentration as the horizontal axis and absorbance as the vertical axis, and obtain the regression equation of sodium glycocholate y=0.1615x+0.0517(R 2 =0.9963), regression equation of sodium taurocholate y = 0.1403x + 0.0529 (R 2 =0.995).

[0070] Take the sample to be tested, dilute it, take two sample dilutions, 200 μL each, and put them into a conical flask, add 200 μL 10 mg / mL pepsin solution (prepared with 0.1 mol / L phosphate buffer at pH 6.3) and 600 μL 0.01 mol / L HCL solution, and digest at 37°C with shaking for 1 hour to simulate the gastric environment; then adjust the pH to 6.3 with 0.1 mol / L NaOH solution, add 800 μL 10 mg / mL trypsin solution (prepared with 0.1 mol / L phosphate buffer at pH 6.3), and digest at 37°C with shaking for 1 hour to simulate the intestinal environment; after the end, add 800 μL 0.4 mmol / L sodium glycocholate (prepared with 0.1 mol / L phosphate buffer at pH 6.3) and 800 μL 0.5 mmol / L sodium taurocholate (prepared with 0.1 mol / L phosphate buffer at pH 6.3) 6.3 of 0.1 mol / L phosphate buffer), shake at 37°C for 1 h, centrifuge at 4000 r / min for 6 min, take 1 mL of supernatant, add 3 mL of 60% sulfuric acid, and measure the absorbance at a wavelength of 387 nm.

[0071] The binding capacity of sodium glycocholate and sodium taurocholate was calculated according to the following formula: R1 = [(N1-N2) / V1] × A; R2 = [(N3-N4) / V2] × A; where: R1 is the binding capacity of sodium glycocholate, mmol / mL; N1 is the amount of sodium glycocholate added, mmol; N2 is the remaining amount of sodium glycocholate, mmol; V1 is the volume of sample diluent added, mL; A is the sample dilution multiple; R2 is the binding capacity of sodium taurocholate, mmol / mL; N3 is the amount of sodium taurocholate added, mmol; N4 is the remaining amount of sodium taurocholate, mmol; V2 is the volume of sample diluent added, mL.

[0072] 8. Electronic nose analysis

[0073] The headspace aspiration method was used to collect volatile substances in the sample to be tested: 5 mL of sample was accurately drawn into a 25 mL small beaker, covered with food plastic wrap and sealed with a rubber band, and left to stand for 30 minutes to allow the odor molecules in the sample to evaporate and reach a steady state. The Bosin cNose electronic nose was turned on and connected to the computer equipment, the instrument was preheated for 30 minutes, the sensor was cleaned before injection, and the detection conditions were: injection needle temperature 25 ° C, injection flow rate 150 mL / min, injection volume 3 mL, injection time 5 s, data acquisition delay time 120 s, data acquisition time 80 s, and cleaning time 60 s. The sensor performance description of the electronic nose is shown in Table 1.

[0074] Table 1 Description of electronic nose sensing performance

[0075]

[0076] The data processing and analysis method in the preferred embodiment is as follows:

[0077] SPSS21 software was used for one-way ANOVA test to analyze significant differences, P < 0.05 indicated a significant difference, and P < 0.001 indicated an extremely significant difference. Origin 2021 and prism9.5.1 were used for drawing. The experiment was carried out in triplicate, and the results were expressed as Mean ± SD.

[0078] (I) Ultrahigh pressure combined with tannin enzyme treatment

[0079] 1. Degradation of tannins in roxburghii juice by ultrahigh pressure combined with tanninase treatment

[0080] 1. Effect of ultrahigh pressure on the degradation of tannins in roxburghii juice by tanninase

[0081] The experimental groups and treatment methods of each group are as follows:

[0082] Control 1: Seabuckthorn juice stored at 4℃.

[0083] Control 2: Take the sea buckthorn juice stored at 4°C and treat it at 600MPa ultra-high pressure for 15min.

[0084] Control 3: Take the sea buckthorn juice stored at 4°C and place it at 37°C for 24 hours.

[0085] Control 4: Take the roxburghii juice stored at 4°C, add tannase at a ratio of 50mL:0.1g, and enzymolyze at 37°C for 24h.

[0086] 100 MPa group, 200 MPa group, 300 MPa group, 400 MPa group, 500 MPa group, 600 MPa group: Take the sea buckthorn juice stored at 4°C, add tannase according to 50 mL: 0.1 g, and immediately treat it under ultrahigh pressure for 15 min under 100 MPa, 200 MPa, 300 MPa, 400 MPa, 500 MPa, and 600 MPa conditions, and then enzymolyze it at 37°C for 24 h.

[0087] Take the samples from each group after treatment and measure the tannin content. Figure 1 As shown. When ultrahigh pressure is involved, within a certain pressure range, the degree of degradation of tannin by tannase increases. Ultrahigh pressure combined with tannase treatment is superior to simple enzymatic treatment and simple ultrahigh pressure treatment.

[0088] Ultrahigh pressure treatment can produce strong physical damage to the cell structure and molecular structure in the roxburghii juice. The destruction of cell membranes and cell walls leads to the release of cell contents, and more tannin molecules are released from the cells into the extracellular liquid environment, which increases the possibility of reacting with other molecules, thereby promoting the degradation of tannins. At the same time, tannin molecules contain a large number of phenolic hydroxyl groups and aromatic ring structures, which are easily destroyed under high pressure conditions. For example, high pressure may cause the CO bonds and CC bonds in tannin molecules to break, decomposing the complex polyphenol structure into smaller molecular fragments, thereby reducing the tannin content. Ultrahigh pressure treatment is connected on the basis of tannin enzymatic hydrolysis. Ultrahigh pressure treatment may activate or enhance the activity of certain enzymes in roxburghii juice, thereby promoting the enzymatic degradation of tannins. For example, ultrahigh pressure treatment may cause structural changes in the active center of enzymes such as tannase, making it easier to bind to tannin molecules and catalyze their degradation reactions. In addition, ultrahigh pressure treatment may also change environmental factors such as pH value and ionic strength of roxburghii juice, thereby providing more suitable conditions for enzymatic reactions and further improving the degradation efficiency of tannins.

[0089] 2. Effect of ultrahigh pressure intervention timing on the degradation of tannins in roxburghii juice by tanninase

[0090] The present invention further explores the effect of the timing of ultrahigh pressure intervention on the tannin degradation efficiency, and implements ultrahigh pressure treatment at the initial stage of the enzymatic hydrolysis reaction and after the enzymatic hydrolysis reaction has been carried out for a period of time to evaluate the difference in the effect on tannin degradation.

[0091] The experimental groups and treatment methods of each group are as follows:

[0092] Control 1: Seabuckthorn juice stored at 4℃.

[0093] Control 2: Take the sea buckthorn juice stored at 4°C and treat it at 600MPa ultra-high pressure for 15min.

[0094] Control 3: Take the sea buckthorn juice stored at 4°C and place it at 37°C for 48 hours.

[0095] Control 4: Take the roxburghii juice stored at 4°C, add tannase at a ratio of 50mL:0.1g, and enzymolyze at 37°C for 48h.

[0096] Samples 1 to 6: Take the sea buckthorn juice stored at 4°C, add tannase according to 50mL:0.1g, and immediately treat it under ultra-high pressure for 15min at 100MPa (sample 1), 200MPa (sample 2), 300MPa (sample 3), 400MPa (sample 4), 500MPa (sample 5), and 600MPa (sample 6), and then enzymolyze it at 37°C for 48h.

[0097] Samples 7 to 12: Take sea buckthorn juice stored at 4°C, add tannase at a ratio of 50 mL:0.1 g, first enzymatically hydrolyze at 37°C for 24 h, then treat with ultra-high pressure at 100 MPa (sample 7), 200 MPa (sample 8), 300 MPa (sample 9), 400 MPa (sample 10), 500 MPa (sample 11), and 600 MPa (sample 12) for 15 min, and then continue enzymatic hydrolysis at 37°C for 24 h.

[0098] Take the samples from each group after treatment and measure the tannin content. Figure 2 The tannin content of the samples was measured after the ultra-high pressure treatment was immediately started and then the enzymatic hydrolysis was continued for 48 hours. Figure 1 Under the same conditions, the tannin content measured by enzymatic hydrolysis for only 24 hours and simple enzymatic hydrolysis for 24 hours and 48 hours was basically the same. The ultra-high pressure treatment can significantly improve the degradation efficiency of tannin after the enzymatic reaction for a period of time. When the pressure reaches 500MPa, the tannin degradation rate reaches a peak of about 77%. This shows that ultra-high pressure treatment may activate or enhance the activity of certain enzymes in roxburghii juice and prolong its reaction cycle, thereby promoting the enzymatic degradation of tannin.

[0099] 3. Effect of the timing of ultrahigh pressure intervention on tannase activity

[0100] The control group 4 (roxburgh juice + tannase, 37°C enzymatic hydrolysis for 48h) in the above "2. Effect of the timing of ultrahigh pressure intervention on the degradation of tannins in roxburgh juice by tannase" was taken as the control group, as well as 6 groups of samples (samples 1 to 6) that were immediately subjected to ultrahigh pressure treatment at the initial stage of the enzymatic hydrolysis reaction and 6 groups of samples (samples 7 to 12) that were subjected to ultrahigh pressure treatment 24h after the enzymatic hydrolysis reaction, and the activity of tannase was measured respectively. The results are as follows Figure 3 As shown in Figure 2, the activity of tannase was stronger in the sample that was treated with ultrahigh pressure after 24 hours of enzymatic hydrolysis.

[0101] When ultra-high pressure treatment of 100-600 MPa was applied immediately at the initial stage of the enzymatic hydrolysis reaction, the activity of tannase in the sample gradually increased with the increase of pressure in the range of 100-300 MPa, and then the enzyme activity decreased as the pressure continued to increase. This shows that ultra-high pressure treatment can activate enzyme activity within a certain pressure range, and excessive pressure may cause the destruction of the active center structure of the enzyme, causing the enzyme to lose its activity.

[0102] After 24 hours of enzymatic hydrolysis, ultra-high pressure treatment at 100-600 MPa was applied. The activity of tannase in the sample showed an upward trend in the range of 100-500 MPa, reaching a peak at 500 MPa. After that, the pressure was increased to 600 MPa, and the enzyme activity dropped to the same level as that at 300 MPa. This shows that after a period of enzymatic hydrolysis, the activity of tannase has decreased. At this time, ultra-high pressure treatment can utilize its activation effect on the enzyme and prolong the enzymatic hydrolysis cycle. However, excessive pressure may also cause the destruction of the active center structure of the enzyme, resulting in a decrease in enzyme activity.

[0103] From the above experimental results, it can also be seen that the sensitivity of tannase to pressure after a period of enzymatic reaction is different from that of newly added tannase. The tannase after 24 hours of enzymatic reaction is most sensitive to 500MPa, while the newly added tannase is most sensitive to 300MPa.

[0104] Based on the above experimental results, the optimal process for degrading tannins in sea buckthorn juice by ultrahigh pressure combined with tannin enzyme treatment is as follows: take the juice, add tannin enzyme, first enzymolyze at 37°C for 24 hours, then treat at 100-600MPa ultrahigh pressure for 15 minutes, and then continue enzymolyze at 37°C for 24 hours.

[0105] 2. Effects of ultrahigh pressure combined with tannase treatment on active components of roxburghii juice

[0106] 1. Effect of ultrahigh pressure combined with tannase treatment on polyphenol content in roxburghii juice

[0107] The effect of the optimal process of ultra-high pressure combined with tannase treatment to degrade tannins in sea buckthorn juice on the polyphenol content in sea buckthorn juice was further explored.

[0108] Take control group 1 (sea roxburghii juice placed at 37°C for 48h), control group 2 (sea roxburghii juice stored at 4°C) and samples 7 to 12 in the above "2. Effect of the timing of ultrahigh pressure intervention on the degradation of tannins in sea roxburghii juice by tanninase" (sea roxburghii juice subjected to ultrahigh pressure treatment at 100 to 600 MPa after 24h of enzymatic hydrolysis and then continued to be enzymatically hydrolyzed for 24h), and determine the polyphenol content respectively.

[0109] The results are as follows Figure 4As shown, the polyphenol content of samples 7 to 12 was significantly higher than that of control groups 1 and 2. As the ultrahigh pressure treatment pressure increased, the polyphenol content of samples 7 to 12 also gradually increased.

[0110] Tannins in sea buckthorn juice belong to bound polyphenols, whose molecules are tightly bound to molecules of other substances such as sugar to form macromolecular complexes, which are difficult to be directly absorbed and utilized by the human body. When tannins are degraded, these bound polyphenols will be released from the complexes and exist in the sea buckthorn juice in a free state, thereby directly increasing the content of free polyphenols in the sea buckthorn juice. In addition, during the degradation of tannins, a series of chemical reactions may be triggered, prompting other polyphenol precursors in sea buckthorn juice to be converted into polyphenols. For example, some phenolic acids may undergo decarboxylation, dehydrogenation and other reactions during the degradation process to generate new polyphenol compounds, which ultimately increase the polyphenol content.

[0111] 2. Effect of ultrahigh pressure combined with tannase treatment on the flavonoid content in roxburghii juice

[0112] The effect of the optimal process of ultra-high pressure combined with tannin enzyme treatment on the flavonoid content in roxburghii juice was further explored. Flavonoids are a class of polyphenolic secondary metabolites with multiple activities such as antibacterial, anticancer, anti-inflammatory, antiviral, anti-aging and anti-diabetic.

[0113] The control group 1 (sea roxburghii juice placed at 37°C for 48 hours), the control group 2 (sea roxburghii juice stored at 4°C) and the samples 7 to 12 in the above "2. Effect of the timing of ultrahigh pressure intervention on the degradation of tannins in sea roxburghii juice by tanninase" (sea roxburghii juice subjected to ultrahigh pressure treatment at 100 to 600 MPa after 24 hours of enzymatic hydrolysis and then further enzymatic hydrolysis for 24 hours) were taken, and the flavonoid content was determined respectively.

[0114] The results are as follows Figure 5 As shown. The flavonoid content of samples 7 to 12 was significantly higher than that of control groups 1 and 2. With the increase of ultrahigh pressure treatment pressure, the flavonoid content of samples 7 to 10 gradually increased slightly, the flavonoid content of sample 11 (500MPa) increased significantly to reach a peak, and the flavonoid content of sample 12 (600MPa) decreased but was still higher than that of sample 10 (400MPa).

[0115] Tannins in roxburghii juice may combine with flavonoids to form complexes. Tanninase can degrade these complexes and release flavonoids, which may increase the content of free flavonoids in roxburghii juice. At the same time, ultra-high pressure treatment can destroy the cell structure, increase the contact between tannin molecules and the extracellular environment, improve their reactivity with other components, and promote the degradation of tannins and the release of flavonoids.

[0116] 3. Effect of ultrahigh pressure combined with tannase treatment on the content of vitamin C in roxburghii juice

[0117] The effect of the optimal process of ultra-high pressure combined with tannin enzyme treatment on the vitamin C content in roxburghii juice was further explored. Vitamin C is a water-soluble vitamin with strong reducing properties. In the complex metabolic process of the body, vitamin C can promote growth, enhance the body's resistance to disease, and can be used as a nutritional supplement, antioxidant, etc.

[0118] Take control group 1 (sea roxburghii juice placed at 37°C for 48h), control group 2 (sea roxburghii juice stored at 4°C) and samples 7 to 12 in the above "2. Effect of the timing of ultra-high pressure intervention on the degradation of tannins in sea roxburghii juice by tanninase" (sea roxburghii juice subjected to ultra-high pressure treatment at 100 to 600 MPa after 24h of enzymatic hydrolysis and then continued to be enzymatically hydrolyzed for 24h), and measure the vitamin C content respectively.

[0119] The results are as follows Figure 6 As shown. The vitamin C content of samples 7 to 12 is significantly higher than that of control groups 1 and 2. With the increase of ultra-high pressure treatment pressure, the vitamin C content of samples 7 to 12 shows a trend of first increasing and then fluctuating slightly. Sample 11 (500MPa) has the highest content, and the contents of samples 10 (400MPa) and sample 12 (600MPa) are comparable.

[0120] Tannins and other antioxidant components in sea buckthorn can effectively protect vitamin C and reduce its loss during processing and storage. Under certain circumstances, tannins and vitamin C may form complexes, and the degradation of tannins may cause these complexes to dissociate and release vitamin C. In addition, ultra-high pressure processing helps maintain the nutrients in food, including vitamin C, especially after the degradation of tannins, which can further enhance the stability of vitamin C in sea buckthorn juice.

[0121] 4. Effects of ultrahigh pressure combined with tannin treatment on the lipid-lowering activity of roxburghii juice in vitro

[0122] The effect of the optimal process of ultra-high pressure combined with tannin enzyme treatment on the in vitro lipid-lowering activity of roxburghii juice was further explored. Bile acid is a cholesterol-derived product in the liver. It is secreted into the dynamic equilibrium bile acid pool and then returns to the liver through the enterohepatic circulation. Studies have shown that some food ingredients can bind to bile acid, accelerate the decomposition of cholesterol in the body, and effectively reduce the cholesterol content in human serum and liver; and bile acid is an emulsifier of fat in the intestine, which can promote lipid hydrolysis and absorption. If bile acid is bound by other substances, the absorption and utilization of dietary fat will be inhibited, thereby achieving the purpose of lowering blood lipids. 90% of the bile acid in normal people is conjugated bile acid. Bile acid in the liver mainly exists in the form of bile salts, and is mainly combined with glycocholic acid and taurocholic acid to form sodium glycocholate or sodium taurocholate. Therefore, the ability to bind bile salts can be used as one of the evaluation criteria for lipid-lowering activity.

[0123] Take control group 1 (sea roxburghii juice placed at 37°C for 48 hours), control group 2 (sea roxburghii juice stored at 4°C) and samples 7 to 12 in the above "2. Effect of the timing of ultrahigh pressure intervention on the degradation of tannins in sea roxburghii juice by tanninase" (sea roxburghii juice subjected to ultrahigh pressure treatment at 100 to 600 MPa after 24 hours of enzymatic hydrolysis and then continued to be enzymatically hydrolyzed for 24 hours), and measure the sodium glycocholate binding rate and sodium taurocholate binding rate of the sea roxburghii juice respectively.

[0124] The results are as follows Figure 7 As shown. The binding rates of samples 7 to 12 to sodium glycocholate were lower than those of control groups 1 and 2, among which the binding rate of sample 8 (200MPa) was relatively the lowest, and the binding rate of sample 11 (500MPa) was relatively the highest. The binding rates of samples 7 (100MPa), 8 (200MPa) and 11 (500MPa) to sodium taurocholate were higher than those of control groups 1 and 2, which was mainly because the binding ability of roxburghii juice itself to sodium taurocholate was weak.

[0125] The tannin component in sea buckthorn juice has multiple mechanisms of action in lowering blood lipids. First, tannin can inhibit the activity of pancreatic lipase and cholesterol esterase, thereby reducing the digestion and absorption of fat and cholesterol. In addition, tannin can also bind to bile acid, reduce the reabsorption of bile acid, and then reduce the intestinal absorption rate of lipids, break the enterohepatic circulation of bile acid, and promote the excretion of cholesterol. At the same time, tannin activates the AMPK signaling pathway, downregulates the expression of lipid synthesis-related genes (such as SREBP1, ACC, FAS), inhibits the synthesis of fatty acids, reduces fat synthesis in the liver and adipose tissue, and promotes the decomposition and oxidation of fat. Therefore, the degradation of tannins in sea buckthorn juice will reduce its blood lipid-lowering level to a certain extent. However, flavonoids and polyphenols produced during the degradation of tannins also have blood lipid-lowering functions. Flavonoids can bind to bile acids, reduce the reabsorption of bile acids, reduce the intestinal absorption rate of lipids, and improve blood lipid profiles by regulating lipoprotein metabolism, increasing high-density lipoprotein cholesterol (HDL-C) levels, reducing low-density lipoprotein cholesterol (LDL-C) levels. Polyphenols have strong antioxidant capacity, which can remove free radicals in the body, reduce the interference of oxidative stress on lipid metabolism, and help lower blood lipids. Polyphenols can also reduce fat synthesis in the liver and adipose tissue by inhibiting the activity of lipid synthesis-related enzymes (such as fatty acid synthase), while promoting the decomposition and oxidation of fat, and reducing total cholesterol and triglyceride levels in serum. Therefore, the flavonoids and polyphenols added after the degradation of tannins in sea buckthorn juice make up for the decrease in blood lipid levels caused by tannin degradation to a certain extent.

[0126] According to the above experimental results, the optimal process for degrading tannins in roxburghii juice by ultrahigh pressure combined with tannin enzyme treatment is as follows: take juice, add tannin enzyme, first hydrolyze at 37℃ for 24h, then treat at 500MPa ultrahigh pressure for 15min, and then continue hydrolyzing at 37℃ for 24h. Under this process, the degradation degree of tannins in roxburghii juice is the highest, and the contents of polyphenols, flavonoids, vitamin C and other substances are all at excellent levels. At the same time, the effect of tannin degradation on its own lipid-lowering level is minimized as much as possible.

[0127] In addition to roxburghii juice, the ultra-high pressure combined with tannin enzyme treatment technology is also applicable to other juices rich in tannins and heat-sensitive active ingredients. It can efficiently degrade the tannins contained therein and improve the taste and quality of the juice. In addition, as a green and efficient degradation method, this technology meets the requirements of sustainable development. Compared with traditional chemical degradation methods, it does not produce harmful by-products during the degradation process, reducing pollution to the environment.

[0128] (II) Lactic acid bacteria fermentation combined with ultra-high pressure and tannin treatment

[0129] Based on the above-mentioned optimal process for degrading tannins in roxburghii juice by ultrahigh pressure combined with tannase treatment, the present invention further explores the effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the contents of tannins and other active ingredients in roxburghii juice.

[0130] The experimental groups and treatment methods of each group are as follows:

[0131] Original juice group: The raw material was the roxburghii juice without any treatment.

[0132] Ultrahigh pressure group: the raw material is the roxburghii juice treated according to the above-mentioned optimal process of ultrahigh pressure combined with tannin enzyme treatment to degrade tannin in the roxburghii juice.

[0133] According to the different subsequent processing methods of raw materials, the above two groups each include 4 subgroups:

[0134] 4 degree fresh juice: store the raw materials at 4°C.

[0135] 37 degree juice: Place the raw materials at 37℃ for 24h.

[0136] Juice + MRS: Add MRS broth culture medium to the raw materials, mix well, filter and obtain the filtrate.

[0137] Juice + MRS + bacteria: Add MRS broth culture medium and lactic acid bacteria suspension to the raw materials, ferment for 24 hours, and centrifuge to obtain the supernatant; the volume ratio of the raw materials, lactic acid bacteria suspension and MRS broth culture medium is 4:1:5.

[0138] 1. Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on tannin content in roxburghii juice

[0139] Take the samples from each group above and determine the tannin content respectively.

[0140] The results are as follows Figure 8 As shown in the figure, the tannin content of the roxburghii juice treated with ultrahigh pressure combined with tanninase was significantly reduced compared with that of the untreated roxburghii juice. The addition of MRS broth medium alone to the original juice group and the ultrahigh pressure group could significantly reduce the tannin content of the roxburghii juice, and after adding lactic acid bacteria for fermentation, the tannin content of the roxburghii juice was reduced again.

[0141] Tannin is an acidic substance, which is divided into hydrolyzed tannin and condensed tannin. Tannin contains many hydroxyl groups, which can have strong non-covalent interactions with proteins (hydrogen bonds, hydrophobic interactions and van der Waals forces). After adding MRS broth culture medium, the tannin content in sea buckthorn juice decreased significantly, which may be because the hydrolyzed tannin in it reacted with the protein in the culture medium for precipitation. After adding lactic acid bacteria, lactic acid bacteria grow and reproduce in the environment provided by the culture medium, and degrade the tannin in sea buckthorn juice through the enzyme system and metabolites produced in its metabolic activities. On the basis of ultra-high pressure and tannin enzyme treatment, combined with probiotic fermentation treatment, the tannin in sea buckthorn juice can be further degraded.

[0142] Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the total phenolic content in roxburghii juice

[0143] Take the above samples and measure the total phenol content. Phenolic compounds are secondary metabolites widely present in fruits and vegetables, which are divided into polyphenolic compounds and monophenolic compounds. Polyphenolic compounds include flavonoids, phenolic acids and tannins. Phenolic compounds are one of the main substances that determine the antioxidant capacity of fruit juice.

[0144] The results are as follows Fig. 9 As shown. The total phenol content in the UHP group increased slightly compared with the original juice group, indicating that UHP treatment has a synergistic effect on the total phenol content, which may be because the cell permeability increases under the action of UHP, causing the polyphenols in the cells to dissolve. After adding MRS broth medium, the total phenol content in both groups decreased significantly. After adding lactic acid bacteria for fermentation, the total phenol content continued to decrease, but overall, the decrease in the total phenol content in the UHP group was not as great as that in the original juice group.

[0145] Phenolic compounds can bind to proteins due to non-covalent interactions, forming water-insoluble precipitates. After adding MRS broth medium, the phenolic compounds in the roxburghii juice and the proteins in the medium precipitated due to binding, which significantly reduced the total phenol content (P < 0.05). The enzymes and metabolites produced by the growth and reproduction of lactic acid bacteria further decompose and utilize polyphenols, which reduces the total phenol content again. Because low temperature treatment in ultra-high pressure treatment helps to retain phenols in roxburghii juice, the total phenol content of the ultra-high pressure group did not decrease as much as that of the original juice group.

[0146] Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the flavonoid content in roxburghii juice

[0147] Take the samples from each group above and determine the flavonoid content respectively.

[0148] The results are as follows Fig.10 As shown. The flavonoid content of the 4-degree fresh juice in the ultra-high pressure group was reduced compared with that in the original juice group, indicating that ultra-high pressure combined with tannin treatment had an effect on the flavonoid content of the roxburghii juice. After being placed at 37°C for 24 hours, the flavonoid content of both groups decreased, indicating that temperature may have an effect on the flavonoid content. After adding MRS broth medium, the flavonoid content of both groups decreased significantly. After adding lactic acid bacteria for fermentation, the flavonoid content of both groups decreased again. But overall, the degree of decrease in flavonoid content in the ultra-high pressure group was smaller than that in the original juice group.

[0149] After adding MRS broth culture medium, the flavonoid content of both groups decreased significantly, which is speculated to be caused by the interaction and binding of proteins in the culture medium with flavonoid compounds. Studies have shown that lactic acid bacteria produce a large amount of glycosidase during fermentation, which can hydrolyze the glycosidic bonds of isoflavones, and separate glycoside isoflavones into free isoflavones, resulting in a decrease in flavonoid content. Therefore, adding lactic acid bacteria fermentation is not conducive to the retention of flavonoids in sea buckthorn juice. Since ultra-high pressure treatment can destroy cell structure and release cell contents including flavonoid compounds, the ultra-high pressure group has a higher flavonoid content than the original juice group.

[0150] Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the content of vitamin C in roxburghii juice

[0151] Take the above-mentioned groups of samples and determine the content of vitamin C respectively.

[0152] The results are as follows Fig.11 As shown. Compared with the original juice group, the vitamin C content of the 4-degree fresh juice in the ultra-high pressure group was slightly increased. After being placed at 37°C for 24 hours, the vitamin C content of both groups decreased, which is speculated to be due to the fact that vitamin C itself is easily oxidized, resulting in a decrease in its content. After adding MRS broth medium and lactic acid bacteria fermentation, the vitamin C content of both groups decreased significantly, but compared with the original juice group, the vitamin C in the ultra-high pressure group was better retained. This may be because the ultra-high pressure treatment reduces the content of dissolved oxygen in the sea buckthorn juice, reducing the oxidation rate of vitamin C; and ultra-high pressure partially degrades polysaccharides and polyphenols, which have antioxidant activity, thereby alleviating the degradation of vitamin C.

[0153] 5. Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannin treatment on the activity of SOD enzyme in roxburghii juice

[0154] Take the samples from each group and measure the SOD enzyme activity. SOD enzyme is an active protein with biological catalysis, and has anti-oxidation, anti-inflammatory and other effects.

[0155] The results are as follows Fig.12 As shown. By comparing the ultra-high pressure group with the original juice group, it can be found that the temperature and culture medium have little effect on the activity of SOD enzyme, while lactic acid bacteria fermentation and ultra-high pressure treatment can greatly improve the activity of SOD enzyme. The increase in SOD enzyme activity during the fermentation period may be related to the growth of lactic acid bacteria, because SOD enzyme is an intracellular enzyme that can be released by the cell lysis of lactic acid bacteria. The increase in SOD enzyme activity caused by ultra-high pressure treatment may be due to the increase in the hydrophobicity of the SOD enzyme surface, or it may be due to the rearrangement of the enzyme conformation, resulting in changes in the active site of the enzyme reaction.

[0156] Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannase treatment on the soluble solids content in roxburghii juice

[0157] Take the above samples and measure the soluble solid content. Soluble solids are a general term for all compounds dissolved in water in liquid or fluid food, including sugar, acid, vitamins and minerals.

[0158] The results are as follows Fig.13 When no culture medium and lactic acid bacteria were added for fermentation, the soluble solids content of the ultra-high pressure group and the original juice group was about (9.89±0.05%), indicating that ultra-high pressure combined with tannase treatment had no significant effect on the soluble solids content in the roxburghii juice (P>0.05), while lactic acid bacteria fermentation significantly reduced the soluble solids content of the two groups (P<0.05), which may be due to the fact that lactic acid bacteria used the sugars in the roxburghii juice for growth and fermentation.

[0159] Effects of lactic acid bacteria fermentation combined with ultrahigh pressure and tannin treatment on the lipid-lowering activity of roxburghii juice in vitro

[0160] The samples in the above groups were taken to determine the binding capacity of Rosa roxburghii juice with sodium glycocholate and sodium taurocholate.

[0161] The results are as follows Fig.14 and Fig.15As shown. The ability of the ultra-high pressure group and the original juice group to bind sodium glycocholate was improved after lactic acid bacteria fermentation. The ultra-high pressure combined with tannin treatment had no significant effect on the ability of the roxburghii juice to bind bile salts (P>0.05). The binding effect of lactic acid bacteria on bile salts is due to the fact that under anaerobic conditions, the bile salt hydrolase secreted by lactic acid bacteria converts the bound bile salts into the unbound type, thereby enhancing the binding ability of bile salts. The ultra-high pressure group and the original juice group had no binding ability to sodium taurocholate before fermentation, and the binding ability of roxburghii juice to sodium taurocholate was significantly improved after lactic acid bacteria fermentation (P<0.05). This may be because lactic acid bacteria secrete extracellular polysaccharides during the fermentation process, which reduces the absorption of bile salts by roxburghii juice.

[0162] 8. Analysis of the response of the electronic nose sensor to the roxburghii juice treated by different methods

[0163] Take the above groups of samples and analyze the response of the electronic nose sensor. The intensity change of the volatile components of the sample can be represented by the change of the response value of different sensors of the electronic nose at a specific time point (60s in this experiment).

[0164] The results are as follows Fig.16 As shown. The response values ​​of the roxburghii juice samples treated by different methods on sensor W3C did not change significantly, and the value was about 1.0; the response values ​​on sensors W2S, W2W and W3S also did not change significantly, and the values ​​were all around 0.9, indicating that ultrahigh pressure combined with tannase treatment had no significant effect on the content of volatile components of roxburghii juice such as ammonia, alcohols and aldehydes, organic sulfides and long-chain alkane compounds. The sensor response values ​​of the 4-degree fresh juice group, the 4-degree ultrahigh pressure fresh juice group, and the original juice + MRS group did not change significantly. Compared with the 4-degree fresh juice group, the response values ​​of the four sensors W1C, W5S, W6S and W1W in the 37-degree original juice group increased significantly, indicating that temperature has a significant effect on the content of volatile components of roxburghii juice such as benzene, nitrogen oxides, hydrides and sulfides. Compared with the fresh juice at 4 degrees, the response values ​​of the five sensors W1C, W6S, W5C, W1S, and W1W in the original juice + MRS + lactic acid bacteria group and the ultrahigh pressure + MRS group increased significantly, indicating that lactic acid bacteria fermentation, ultrahigh pressure combined with tannin treatment have a significant effect on the content of volatile components of sea buckthorn juice, such as benzene, hydride, short-chain alkane, methyl and sulfide. The results of electronic nose analysis showed that lactic acid bacteria had no significant effect on the smell of sea buckthorn juice.

[0165] According to the above experimental results, lactic acid bacteria fermentation has the ability to degrade tannins in roxburghii juice. The combined ultrahigh pressure and tannin enzyme treatment has a better degradation effect on tannins, which can effectively improve the astringency of roxburghii juice and enhance its taste. However, lactic acid bacteria fermentation is not conducive to the retention of active components such as polyphenols, flavonoids, and vitamin C in roxburghii juice. The combined ultrahigh pressure and tannin enzyme treatment can better retain these active components in roxburghii juice. At the same time, lactic acid bacteria fermentation has no significant effect on the smell of roxburghii juice.

[0166] In addition to sea buckthorn juice, lactic acid bacteria fermentation combined with ultra-high pressure and tannase treatment technology is also applicable to other fruit juices rich in tannins and heat-sensitive active ingredients. It can efficiently degrade the tannins contained therein and improve the taste and quality of the juice.

[0167] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for efficiently degrading tannin in fruit juice, characterized in that: The method comprises the following steps: taking juice, adding tannin enzyme, performing enzymolysis for 12-24 hours, performing ultrahigh pressure treatment at 100-600Mpa for 10-20 minutes, and then continuing enzymolysis for 12-24 hours.

2. A method for efficiently degrading tannin in fruit juice as claimed in claim 1, characterized in that: The following steps are involved: a. Take the juice, add tannin, first enzymolyze for 12-24 hours, treat with 100-600Mpa ultra-high pressure for 10-20min, and then continue enzymolyze for 12-24 hours; b. Take the juice treated in step a, add lactic acid bacteria suspension and MRS broth medium, ferment for 12-24 hours, and centrifuge to obtain the supernatant.

3. A method for efficiently degrading tannin in fruit juice as claimed in claim 1 or 2, characterized in that: The fruit juice is roxburghii juice.

4. A method for efficiently degrading tannin in fruit juice as claimed in claim 2, characterized in that: The lactic acid bacteria is Lactobacillus casei subsp. casei.

5. A method for efficiently degrading tannin in fruit juice as claimed in claim 1 or 2, characterized in that: Take the juice, add tannin, first enzymolyze at 37℃ for 24 hours, treat at 500Mpa ultra-high pressure for 15 minutes, and then continue enzymolyze at 37℃ for 24 hours.

6. A method for efficiently degrading tannin in fruit juice as claimed in claim 2, characterized in that: Take the juice treated in step a, add lactic acid bacteria suspension and MRS broth culture medium, the volume ratio of the juice, lactic acid bacteria suspension and MRS broth culture medium is 4:1:5, ferment for 24 hours, centrifuge and take the supernatant to obtain.

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