Coupling sterilization method of fruit juice and application thereof

Abstract

This invention relates to a coupled sterilization method for fruit juice and its application, primarily targeting heat-sensitive fruit juices such as lychee juice. The method first adds food-grade quercetin as a photosensitizer to the fruit juice, adjusting its color, sugar content, and pH value before incubation in the dark. Subsequently, it undergoes light treatment to induce the generation of reactive oxygen species (ROS), which disrupt the microbial structure. Finally, it undergoes low-temperature pasteurization. This technology achieves a synergistic sterilization effect of "1+1≫2" through the combined action of photodynamic sterilization and mild heat treatment, effectively killing Escherichia coli, molds, and yeasts while significantly reducing the intensity of heat treatment, thus maximizing the preservation of the fruit juice's natural flavor, color, and nutritional components.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a sterilization method that couples low-temperature pasteurization with photodynamic technology. Background Technology

[0002] Lychee juice is highly favored by consumers for its unique sweet flavor and rich nutritional components (such as vitamin C and polyphenols). However, the near-neutral pH and nutrient-rich nature of lychee juice provide an ideal substrate environment for the growth and reproduction of microorganisms (including bacteria, yeast, and mold), making it highly susceptible to spoilage during production and storage. Therefore, effective sterilization is a key technology for ensuring the safety of lychee juice and extending its shelf life.

[0003] Currently, the most widely used sterilization technology in the fruit juice industry is traditional heat sterilization, such as high temperature short time (HTST) and ultra-high temperature instantaneous (UHT) sterilization. While these methods can effectively kill microorganisms and enzymes, they have significant drawbacks for heat-sensitive products like lychee juice: high-intensity heat treatment inevitably leads to the volatilization of the original fresh flavor substances in lychee juice, the development of a cooked taste, darkening of color (browning), and significant degradation of heat-sensitive nutrients (such as vitamin C and antioxidant active substances), thereby significantly reducing the sensory quality and nutritional value of the product.

[0004] To overcome the drawbacks of thermal processing, non-thermal processing technologies have been extensively researched, such as high-pressure processing (HPP), pulsed light, ultrasound, and ultraviolet (UV). While each of these non-thermal sterilization technologies has its advantages, they also have significant drawbacks. High-pressure processing (HPP) has extremely high equipment investment and operating costs; it places special requirements on packaging materials (requiring high pressure resistance and flexibility); and it is difficult to effectively kill bacterial spores. Pulsed light (PL) has weak penetrating power and is only suitable for surface or transparent liquid sterilization; it is ineffective on uneven or opaque materials. Ultrasound (US) has low sterilization efficiency when used alone and usually needs to be combined with other technologies; and high-intensity, long-term treatment may affect food flavor. Ultraviolet (UV) has extremely poor penetrating power and cannot treat turbid, opaque foods or foods containing solid particles; it also has blind spots, resulting in uneven sterilization.

[0005] On the other hand, low-temperature pasteurization (usually referring to treatment at 50-75°C) is a mild heat treatment method that can reduce the microbial load and has a much smaller negative impact on product quality than high-temperature treatments such as UHT. However, low-temperature pasteurization alone is usually insufficient to kill all spoilage microorganisms and pathogens, especially heat-resistant bacteria and spores.

[0006] Therefore, there is a lack of efficient and energy-saving sterilization methods in the existing technology that can take into account both the microbial safety of lychee juice and its high quality (flavor, color, and nutrition). Summary of the Invention

[0007] To address the aforementioned technical problems, a first aspect of the present invention provides a coupled sterilization method for fruit juice, comprising the following steps:

[0008] Step S1, Add quercetin: Add food-grade quercetin to the juice to adjust the juice's color L value to 45-55, color a value to 1.0-1.5, color b value to 4.5-5.5, sugar content to 15-20 Brix, and pH value to 5.02-5.07; after adjustment, incubate in the dark.

[0009] Step S2, light-coupled low-temperature pasteurization: The sample obtained in step S1 is light-treated; the light-treated sample is immediately subjected to low-temperature pasteurization, the temperature of which is 50-60℃ and the treatment time is 5-20 minutes.

[0010] As a preferred technical solution, the final concentration of quercetin in the fruit juice is 100-300 μM.

[0011] As a preferred technical solution, the wavelength of the illumination is 400-420 nm.

[0012] As a preferred technical solution, the wavelength of the illumination is 405 nm.

[0013] As a preferred technical solution, the light treatment time is 15-20 minutes.

[0014] In a second aspect, the present invention provides a method for preparing fruit juice, the method comprising the coupled sterilization method described in the first aspect.

[0015] In a third aspect, the present invention provides a fruit juice prepared by the preparation method of the second aspect.

[0016] A fourth aspect of the present invention provides the application of the method described in the second aspect in the preparation of fruit juice products, such as lychee juice, apple juice, pear juice, grape juice, etc.

[0017] A fifth aspect of the present invention provides a method for improving the flavor of fruit juice, the method employing the coupled sterilization method described in the first aspect.

[0018] In a sixth aspect, the present invention provides a method for improving the color of fruit juice, the method employing the coupled sterilization method described in the first aspect.

[0019] The principle of photodynamic inactivation (PDI) is as follows: under irradiation with a specific wavelength of light, quercetin is excited, converting ground-state oxygen into highly reactive reactive oxygen species (ROS), including singlet oxygen and free radicals. These strong oxidizing substances can irreversibly attack the cell membranes, proteins, and nucleic acids of microorganisms, leading to their inactivation or death.

[0020] Despite the advantages of PDI (Prepared Direct Intake) such as being non-thermal, environmentally friendly, and highly targeted, its application alone in liquid foods with complex compositions and a certain degree of turbidity, such as lychee juice, still faces significant challenges:

[0021] (1) Suspended particles, pigments and other endogenous components in the juice will strongly scatter and absorb light, greatly weakening the penetration depth of the light source and causing uneven sterilization;

[0022] (2) To achieve commercial sterility requirements, extremely high light intensity or extremely long processing time is often required, resulting in high energy consumption and low efficiency, which makes it difficult to meet the needs of industrial production.

[0023] (3) For resistant spores or dormant microorganisms, PDI alone may not be able to completely inactivate them.

[0024] To address the aforementioned bottlenecks, the inventors creatively proposed that, under the premise of limiting the color and sugar content of the juice, PDI should be used as a pretreatment, followed by low-temperature pasteurization. The synergistic effect of the two can produce a significant synergistic sterilization effect.

[0025] Its core mechanism is as follows: PDI first induces extensive sublethal oxidative damage in microorganisms—disrupting cell membrane permeability, oxidizing key proteins, and damaging DNA / RNA, causing severe impairment of their physiological functions and placing them in a "sensitized" state; the subsequent application of mild heat treatment (low-temperature pasteurization) precisely "finishes off" the damage, accelerating the denaturation and inactivation of the damaged biomolecules and completely blocking the stress repair pathways of microorganisms, thereby achieving efficient and irreversible inactivation.

[0026] Thanks to this synergistic strategy, the present invention achieves comprehensive sterilization efficiency far exceeding that of a single technology while significantly reducing the processing intensity of each unit (such as lower sterilization temperature, shorter light exposure time, and lower photosensitizer dosage), and preserves the fresh flavor, natural color, and heat-sensitive nutrients of lychee juice to the greatest extent.

[0027] As a result, the present invention achieves the following outstanding technical effects:

[0028] Significant synergistic bactericidal effect: The combined treatment is not a simple additive, but presents a "1+1 ≫ 2" synergistic effect. The inactivation effect on Escherichia coli, mold, yeast and total number of native colonies is close to or even better than traditional high temperature sterilization at 80–90℃.

[0029] Excellent product quality retention: Due to the significant reduction in the intensity of heat and light treatment, the amount of characteristic flavor substances (such as volatile sulfur compounds) generated in lychee juice is significantly reduced, and the color difference (L*, a*, b* values) changes are minimal.

[0030] Energy-efficient and economical: The synergistic effect significantly improves processing efficiency, effectively shortens the illumination time, reduces heat consumption, and significantly reduces the overall processing cost, showing good prospects for industrial application.

[0031] Safe and clean, with no risk of residue: The photosensitizer used is food-grade or GRAS (Generally Recognized As Safe) substance, and the reaction process does not produce harmful chemical residues, which is in line with the development trend of clean label products. Attached Figure Description

[0032] Figure 1 This refers to the total bacterial count test results in Example 1; among which, Figure 1 A: Pasteurize at 50℃ for 20 min, coupled with photodynamic therapy for 20 min; Figure 1 B: Pasteurize at 60℃ for 5 min, coupled with photodynamic therapy for 20 min; Figure 1 C: Pasteurize at 50℃ for 20 min, coupled with photodynamic therapy for 15 min; Figure 1 D: Pasteurize at 60℃ for 5 minutes, then couple photodynamic therapy for 15 minutes.

[0033] Figure 2 This refers to the total Escherichia coli count test results in Example 2; among which, Figure 2 A: Pasteurize at 50℃ for 20 min, coupled with photodynamic therapy for 20 min; Figure 2 B: Pasteurize at 60℃ for 5 min, coupled with photodynamic therapy for 20 min; Figure 2 C: Pasteurize at 50℃ for 20 min, coupled with photodynamic therapy for 15 min; Figure 2 D: Pasteurize at 60℃ for 5 minutes, then couple photodynamic therapy for 15 minutes.

[0034] Figure 3 This is to detect the total number of molds and yeasts in Example 3; among which, Figure 3 A: Pasteurize at 50℃ for 20 min, coupled with photodynamic therapy for 20 min; Figure 3 B: Pasteurize at 60℃ for 5 min, coupled with photodynamic therapy for 20 min; Figure 3 C: Pasteurize at 50℃ for 20 min, coupled with photodynamic therapy for 15 min; Figure 3 D: Pasteurize at 60℃ for 5 minutes, then couple photodynamic therapy for 15 minutes. Detailed Implementation

[0035] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the applicant has conducted a comparative analysis through specific embodiments and comparative examples.

[0036] Example 1: Quercetin-mediated photodynamic therapy combined with low-temperature pasteurization (Condition A: pasteurization at 50°C for 20 min; light irradiation for 20 min)

[0037] Take 9.82 mL of litchi juice concentrate, add 80 μL of 25 mM quercetin stock solution (to a final concentration of 200 μM) and 100 μL of Escherichia coli suspension, and mix well. Incubate the mixture at room temperature (25±1℃) in the dark for 10 minutes. Then, irradiate it under a 405 nm LED light source (output light power density of 180-200 mW / cm²) for 20 minutes. After the irradiation, immediately transfer the sample to a 50℃ constant temperature water bath for 20 minutes.

[0038] Example 2: Quercetin-mediated photodynamic therapy combined with low-temperature pasteurization (Condition B: pasteurization at 60°C for 5 min; light irradiation for 20 min)

[0039] Take 9.82 mL of litchi juice concentrate, add 80 μL of 25 mM quercetin stock solution (to a final concentration of 200 μM) and 100 μL of Escherichia coli suspension, and mix well. Incubate the mixture at room temperature (25±1℃) in the dark for 10 minutes. Then, irradiate it under a 405 nm LED light source (output light power density of 180-200 mW / cm²) for 20 minutes. After the irradiation, immediately transfer the sample to a 60℃ constant temperature water bath for 5 minutes.

[0040] Example 3: Quercetin-mediated photodynamic therapy combined with low-temperature pasteurization (Condition C: pasteurization at 50°C for 20 min; light irradiation for 15 min)

[0041] Take 9.82 mL of litchi juice concentrate, add 80 μL of 25 mM quercetin stock solution (to a final concentration of 200 μM) and 100 μL of Escherichia coli suspension, and mix well. Incubate the mixture at room temperature (25±1℃) in the dark for 10 minutes. Then, irradiate it under a 405 nm LED light source (output light power density of 180-200 mW / cm²) for 15 minutes. After the irradiation, immediately transfer the sample to a 50℃ constant temperature water bath for 20 minutes.

[0042] Example 4: Quercetin-mediated photodynamic therapy combined with low-temperature pasteurization (Condition D: pasteurization at 60°C for 5 min; light irradiation for 15 min)

[0043] Take 9.82 mL of litchi juice concentrate, add 80 μL of 25 mM quercetin stock solution (to a final concentration of 200 μM) and 100 μL of Escherichia coli suspension, and mix well. Incubate the mixture at room temperature (25±1℃) in the dark for 10 minutes. Then, irradiate it under a 405 nm LED light source (output light power density of 180-200 mW / cm²) for 15 minutes. After the irradiation, immediately transfer the sample to a 60℃ constant temperature water bath for 5 minutes.

[0044] Comparative Example 1: High-temperature sterilization control group (80℃, 10min)

[0045] Take 9.82 mL of lychee juice concentrate, add 80 μL of physiological saline (in place of photosensitizer) and 100 μL of Escherichia coli suspension, and mix well. The sample was not subjected to light treatment and was directly transferred to an 80℃ constant temperature water bath for 10 minutes.

[0046] Comparative Example 2: High-temperature sterilization control group (90℃, 10 min)

[0047] Take 9.82 mL of lychee juice concentrate, add 80 μL of physiological saline (in place of photosensitizer) and 100 μL of Escherichia coli suspension, and mix well. The sample was not subjected to light treatment and was directly transferred to a 90℃ constant temperature water bath for 10 minutes.

[0048] Comparative Example 3: Blank Control Group (CK)

[0049] Take 9.82 mL of lychee juice concentrate, add 80 μL of physiological saline (in place of photosensitizer) and 100 μL of Escherichia coli suspension, and mix well.

[0050] Comparative Example 4: Single PDI treatment group (20 min)

[0051] Take 9.82 mL of lychee juice concentrate, add 80 μL of 25 mM quercetin stock solution (to a final concentration of 200 μM) and 100 μL of Escherichia coli suspension, and mix well. Incubate the mixture at room temperature (25±1℃) in the dark for 10 minutes. Then, irradiate it under a 405 nm LED light source (output light power density of 180-200 mW / cm²) for 20 minutes.

[0052] Comparative Example 5: Single pasteurization group (50℃, 20min)

[0053] Take 9.82 mL of lychee juice concentrate, add 80 μL of physiological saline (in place of photosensitizer) and 100 μL of Escherichia coli suspension, and mix well. The sample was not subjected to light treatment and was directly transferred to a 50℃ constant temperature water bath for 20 minutes.

[0054] Comparative Example 6: Single pasteurization group (60℃, 5 min)

[0055] Take 9.82 mL of lychee juice concentrate, add 80 μL of physiological saline (in place of photosensitizer) and 100 μL of Escherichia coli suspension, and mix well. The sample was not subjected to light treatment and was directly transferred to a 60℃ constant temperature water bath for 5 minutes.

[0056] Comparative Example 7: Single PDI treatment group (15 min)

[0057] Take 9.82 mL of litchi juice concentrate, add 80 μL of 25 mM quercetin stock solution (to a final concentration of 200 μM) and 100 μL of Escherichia coli suspension, and mix well. Incubate the mixture at room temperature (25±1℃) in the dark for 10 minutes. Then, irradiate it under a 405 nm LED light source (output light power density of 180-200 mW / cm²) for 15 minutes.

[0058] In the above examples and comparative examples, the lychee extract had a color L value of 45-55, a color a value of 1.0-1.5, a color b value of 4.5-5.5, a sugar content of 15-20 Brix, and a pH value of 5.02-5.07.

[0059] Detection Example 1: Detect the total number of colonies in Examples 1-4 and Comparative Examples 1-7.

[0060] Test results are shown Figure 1The initial total number of native bacterial colonies in the litchi juice of the blank control group (CK) was (4.81±0.08) logCFU / mL. After PDI treatment for 15 min and 20 min, the total number of colonies decreased to (4.40±0.01) and (1.55±0.05) logCFU / mL, respectively. The bactericidal effect increased significantly with the extension of light exposure time, indicating that longer exposure time is crucial for bactericidal efficiency, consistent with the trend of Escherichia coli inactivation. In contrast, the total number of colonies in the single pasteurization treatment groups at 60℃ for 5 min and 50℃ for 20 min decreased to (3.07±0.02) and (4.11±0.03) logCFU / mL, respectively. It can be seen that the short-term pasteurization at higher temperature (60℃ for 5 min) is more effective than the long-term treatment at lower temperature (50℃ for 20 min). However, the maximum reduction of single pasteurization is only 1.8 logCFU / mL, which still has certain limitations. After treatment at 80℃ for 10 min, the concentration decreased to (1.31±0.03) logCFU / mL, and after treatment at 90℃ for 10 min, it further decreased to (0.38±0.08) logCFU / mL. Although the high-temperature sterilization effect of 90℃ for 10 min was significant, the high temperature easily caused the loss of flavor and nutrients in the lychee juice. Coupling PDI with pasteurization significantly improved the sterilization effect, with the following order from strongest to weakest: PDI 20min + 60℃ 5min (1.24 logCFU / mL) > PDI 20min + 50℃ 20min (1.43 logCFU / mL) > PDI 15min + 60℃ 5min (2.53 logCFU / mL) > PDI 15min + 50℃ 20min (3.95 logCFU / mL). The coupled treatment was not only significantly better than pasteurization alone, but its effect (1.24~3.95 logCFU / mL) was also close to the level of high-temperature sterilization at 80℃ for 10min (1.13 logCFU / mL). At the same time, the treatment temperature (≤60℃) was much lower than that of high-temperature sterilization (80~90℃), thus more effectively preserving the original sensory characteristics and nutritional components of lychee juice.

[0061] Detection Example 2: Detection of the total number of Escherichia coli in Examples 1-4 and Comparative Examples 1-7.

[0062] Test results are shown Figure 2For *E. coli*, experimental data showed that PDI treatment alone could only reduce the initial viable bacterial count from (5.10 ± 0.10) logCFU / mL to (4.40 ± 0.09) logCFU / mL within 15 min, indicating a limited inactivation effect. When the treatment time was extended to 20 min, the viable bacterial count further decreased to (1.60 ± 0.08) logCFU / mL, indicating that the treatment time had a significant impact on the bactericidal efficiency of PDI, but still could not achieve complete eradication. Regarding PS treatment, the combined effect of temperature and time was very significant: at 60℃, only 5 min was needed to achieve a bacterial reduction of 4.30 logCFU / mL (with a viable bacterial count of 0.80 ± 0.07 logCFU / mL), while after 20 min of treatment at 50℃, (2.80 ± 0.11) logCFU / mL of viable bacteria remained. This result suggests that higher temperatures can achieve a more significant bactericidal effect in a shorter time. The combined treatment of PDI and PS showed a strong synergistic effect. When PDI (15 min or 20 min) was combined with 60℃ for 5 min, the number of viable bacteria dropped below the detection limit, achieving complete eradication. In contrast, PDI (15 min) combined with 50℃ for 20 min still resulted in the detection of approximately 2.55 log CFU / mL of viable bacteria. Although the combined treatment of PDI (20 min) and 50℃ for 20 min still resulted in the number of viable bacteria below the detection limit, the best treatment (especially PDI (20 min) + 60℃ for 5 min) achieved a level of sterilization comparable to that of traditional high-temperature heat treatment at 80℃ or 90℃ for 10 min, both of which reduced the number of E. coli below the detection limit.

[0063] Test Example 3: Detect the total number of molds and yeasts in Examples 1-4 and Comparative Examples 1-7.

[0064] Test results are shown Figure 3The study on the antibacterial and bactericidal effects of PDI on dominant spoilage microorganisms, molds and yeasts, showed that single PDI treatment significantly reduced the number of viable molds and yeasts in the system. When the PDI treatment time was 15 min, the initial viable number of molds and yeasts in litchi juice was effectively reduced from (5.10±0.10) log CFU / mL to (2.70±0.10) log CFU / mL, achieving a decrease of 2.40 log. As the treatment time was extended to 20 min, the bactericidal effect of PDI on molds and yeasts was further enhanced, and the viable number was stably reduced to (1.60±0.08) log CFU / mL, with a bactericidal range of 3.50 log. This indicates that extending the PDI treatment time can significantly enhance its inactivation efficiency on molds and yeasts in litchi juice, exhibiting a good time-dependent bactericidal law. Mild heat treatment (PS) alone showed a significant dependence on treatment parameters in its inactivation effect on molds and yeasts in lychee juice, with temperature having a much greater impact on sterilization than treatment time. Under the test conditions, treatment at 50 ℃ for 20 min only achieved a (2.00±0.11) log reduction in viable cell count, while short-term high-temperature treatment at 60 ℃ for 5 min reduced the viable cell count to (0.50±0.10) log, demonstrating a significantly better sterilization effect than the 50 ℃ for 20 min treatment group. This indicates that in PS treatment, appropriately increasing the treatment temperature and shortening the treatment time is more conducive to achieving efficient control of molds and yeasts in lychee juice, and also better meets the quality protection requirements of low-heat treatment for heat-sensitive juices such as lychee juice. Further research showed that when PDI and PS were combined, the two sterilization methods exhibited a significant synergistic sterilization effect on molds and yeasts in lychee juice, and the complete sterilization effect that was difficult to achieve with a single treatment could be easily achieved under combined treatment. Especially under the mild treatment conditions of 60℃ for 5 minutes, the synergistic effect of PDI and PS can completely kill molds and yeasts in lychee juice, with no viable bacteria detected. Its bactericidal efficacy is comparable to that of conventional high-temperature heat treatment at 80℃ and 90℃ for 10 minutes. This result fully demonstrates that the synergistic sterilization technology of PDI and PS can achieve the same microbial control effect as traditional high-temperature sterilization while significantly reducing the treatment temperature and shortening the treatment time. It can effectively control the spoilage of lychee juice by molds and yeasts while preserving the original flavor, color, and nutritional quality of lychee juice to the greatest extent.

[0065] Test Example 4: Test the color difference of lychee juice in Examples 1-4 and Comparative Examples 1-7.

[0066] A colorimeter was used to measure the lightness (L*), red-green hue (a*), and yellow-blue hue (b*) of the samples. A standard white plate was used for calibration before measurement.

[0067] The results of L, a, and b values ​​of litchi juice in each treatment group are shown in Table 1. Compared with the control group (CK), the L value of the PDI-treated group for 15 min dropped sharply to 4.61, indicating a significant decrease in brightness; the L values ​​of the other treatment groups ranged from 45.84 to 50.63, with the 90℃ for 10 min treatment group showing the most significant decrease in brightness. Regarding the a value, the control group was 1.29, and the a* value decreased in most treatment groups, with the lowest value (0.11) in the 20 min + 60℃ for 5 min group, indicating an enhanced green hue; the a* value of the 90℃ for 10 min group rose to 1.43, indicating a recovery in red hue. The b* value of the control group was 5.10, and the b* values ​​of the PDI-treated and PDI+PS combined treatment groups generally increased, reaching 10.58 in the 15 min + 60℃ for 5 min group, indicating a significant increase in yellowness; the b* value of the heat-treated groups increased slightly. In summary, PDI and its combined treatments have a significant impact on color. Mild heat treatment (60℃ for 5 min, 80℃ for 10 min) preserves color well, while high-temperature treatment (90℃ for 10 min) results in obvious browning.

[0068] Table 1: Results of Lychee Juice Color Detection

[0069]

[0070] Test Example 5: Test the sulfide content of litchi in Examples 1-4, Comparative Example 2, and Comparative Example 3.

[0071] Quantitative analysis of sulfides was performed using a combination of internal and external standards. Five concentration gradients of mixed sulfide standards (DMS, DMDS, DMTS) were prepared, and ethyl hexanoate was added as the internal standard. A standard curve was plotted based on the ratios of the internal standard to the standard concentrations and the ratios of the peak areas of the internal standard to the standard, and the sulfide concentrations were calculated. The inhibition rate represents the change in sulfide concentration in the litchi juice relative to the heat treatment (90℃ for 10 min) in the treatment group.

[0072] Table 2: Results of Sulfide Concentration Detection in Lychee Juice

[0073]

[0074] Sulfides such as dimethyl sulfide (DMS), dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS) are commonly associated with undesirable flavors (such as cooked taste or off-flavors) produced during fruit juice processing and are important indicators of fruit juice quality. Example 5 compared the control effects of the "coupled sterilization method" and the "traditional high-temperature sterilization method" on sulfide formation. The results showed that the sulfide formation using the coupled sterilization method of this invention (Examples 1-4) was significantly lower than that of traditional high-temperature sterilization. The quercetin-mediated photodynamic coupled low-temperature pasteurization technology used in this invention produces a lower concentration of sulfides during sterilization than traditional high-temperature sterilization (90℃ for 10 min), demonstrating that this method better preserves the fresh flavor of lychee juice and reduces the generation of heat-processed off-flavors.

[0075] The chemical reagents and bacterial strains used in this application are as follows:

[0076] Quercetin standard (purity ≥95%) and anhydrous ethanol (analytical grade) were purchased from Xilong Scientific Co., Ltd. Escherichia coli ATCC 43889 was stored in the laboratory at -20℃ with glycerol. LB broth medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd., and LB agar medium was purchased from Beijing Luqiao Technology Co., Ltd.

[0077] The experimental instruments used in this application are as follows:

[0078] 405 nm LED light source (Zhongshan Zigu Lighting & Electrical Factory), ultrasonic cleaning machine (Chunlin Cleaning Equipment Co., Ltd.), constant temperature water bath (Quanzhou Shengxing Technology Co., Ltd.), ultra-clean workbench (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), constant temperature shaker (Shanghai Zhichu Instrument Co., Ltd.), constant temperature incubator (Ningbo Jiangnan Instrument Factory).

[0079] The preparation method of the photosensitizer solution in this application is as follows:

[0080] Accurately weigh 0.0756 g of quercetin solid into a 10 mL volumetric flask. First, add approximately 2-3 mL of anhydrous ethanol, and dissolve the solid completely with ultrasonic assistance. Then, bring the volume to 10 mL with anhydrous ethanol to obtain a 25 mM quercetin stock solution. Before use, filter the stock solution through a 0.22 μm sterile microporous membrane and store it in a 4°C refrigerator protected from light.

[0081] The method for preparing the bacterial suspension in this application is as follows:

[0082] The preserved *Escherichia coli* ATCC 43889 glycerol culture was taken from a -20°C freezer and thawed at room temperature. In a clean bench, 100 μL of the thawed bacterial suspension was inoculated into an Erlenmeyer flask containing 100 mL of LB broth and placed in a constant-temperature shaking incubator at 37°C and 120 r / min for 24 h to activate the culture. After incubation, the OD value of the bacterial suspension was measured, and the culture was stored at 4°C for subsequent experiments.

[0083] The method for evaluating the bactericidal effect in this application is as follows:

[0084] The bactericidal effect was evaluated using the plate count method. All treated and control samples were serially diluted, and 2-3 suitable dilutions were selected for plating. For the bacterial suspension system, 100 μL of the diluted solution was plated onto LB agar plates, with two replicates for each dilution. After incubating the plates upside down at 37°C for 24-48 h, the colony-forming units (CFU) on the plates were counted. The number of viable bacteria per milliliter of sample (CFU / mL) was calculated based on the dilution factor, and this was used to evaluate the bactericidal efficiency of different treatments. For the litchi juice stock solution system, the pour method was used: 1 mL of the diluted solution was poured into a sterile Petri dish, and then approximately 15-20 mL of LB agar medium cooled to 46-50°C was poured in, mixed well, and allowed to solidify. After incubating all plates upside down at 37°C for 24-48 h, the colony-forming units (CFU) on the plates were counted. The number of viable bacteria (CFU / mL) per milliliter of sample was calculated based on the dilution factor, and the sterilization efficiency of different treatments was evaluated accordingly.

Claims

1. A coupled sterilization method of fruit juice, characterized by, The method comprises the following steps: Step S1, adding quercetin: adding food-grade quercetin into the juice, adjusting the L value of the juice to be 45-55, the a value to be 1.0-1.5, the b value to be 4.5-5.5, the sugar degree to be 15-20 Brix, and the pH value to be 5.02-5.07; and then incubating in the dark after the adjustment; Step S2, light coupling low-temperature pasteurization: light treating the sample obtained in step S1; and immediately performing low-temperature pasteurization on the light-treated sample, wherein the temperature of the low-temperature pasteurization is 50-60 DEG C, and the treatment time is 5-20 minutes.

2. The coupling sterilization method according to claim 1, wherein The final concentration of the quercetin in the juice is 100-300 μM.

3. A method of preparing a fruit juice, characterized by, The preparation method comprises the coupling pasteurization method of claim 1.

4. A fruit juice, characterized by, The juice is prepared by the preparation method of claim 3.

5. Use of the process according to claim 3 for the preparation of a fruit juice product, characterized in that, The juice is litchi juice.

6. A method of improving the flavor of fruit juice, characterized by, The method adopts the coupling pasteurization method of claim 1.

7. A method of improving the color of fruit juice, characterized by, The method adopts the coupling pasteurization method of claim 1.

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