Technology for dissolving out anthocyanin in blueberry juice by ultrahigh pressure-ultrasonic synergistic quaternary complex enzyme

Abstract

The invention discloses a process for dissolving out blueberry juice anthocyanin by ultrahigh pressure-ultrasonic synergistic quaternary complex enzyme, which comprises the following steps: S1, obtaining fresh blueberries, and pretreating the fresh blueberries to obtain blueberry puree; s2, performing ultrasonic treatment on the blueberry primary pulp to obtain blueberry juice I; s3, performing ultrahigh pressure treatment on the blueberry juice I to obtain blueberry juice II; s4, performing quaternary composite enzymolysis treatment on the blueberry juice II to obtain blueberry juice III; and S5, sequentially carrying out enzyme deactivation treatment and filtration treatment on the third blueberry juice to obtain the blueberry juice in which anthocyanin is dissolved out. According to the method, efficient dissolution of anthocyanin and synchronous improvement of juice clarity and storage stability can be realized, and a key technical support is provided for processing of high-quality blueberry juice.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a process for extracting anthocyanins from blueberry juice using a synergistic ultra-high pressure-ultrasound quaternary complex enzyme. Background Technology

[0002] Blueberries, as a functional fruit rich in anthocyanins, primarily contain anthocyanins such as cyanidin-3-glucoside and delphinidin-3-galactoside. These anthocyanins possess physiological functions such as scavenging free radicals, protecting the retina, and regulating intestinal flora. The market demand for blueberry juice has been growing at an average annual rate of over 8.5%. However, the complex cell wall structure of blueberries, composed of cellulose (28%-32%), hemicellulose (22%-26%), pectin (18%-22%), and lignin (6%-9%) forming a dense network, encapsulates over 70% of the anthocyanins within the cell vacuoles. This leads to significant bottlenecks in traditional processing techniques: traditional fresh-pressing processes only mechanically break down the cell wall surface, resulting in an anthocyanin dissolution rate of only 30%-35%, with a large amount of active ingredients being discarded with the fruit pomace, resulting in a raw material utilization rate of less than 45%. Furthermore, fresh-pressed juice is prone to stratification after standing for 24 hours due to incomplete degradation of pectin, with a light transmittance of only 55%-65%, leading to poor sensory quality.

[0003] To address the aforementioned problems, existing technologies mostly employ a single physical field or a single enzymatic hydrolysis treatment, but these have limitations that are difficult to overcome: (1) Single ultra-high pressure treatment: Relying on static pressure of 200-600MPa to destroy the cell wall structure, although it can improve the anthocyanin dissolution rate to a certain extent, it cannot completely degrade cellulose and hemicellulose by physical action alone. The actual dissolution rate is only 50%-55%. Moreover, there is no enzymatic synergistic effect in the treatment process, and the pressure holding time needs to be extended to 15-20min, resulting in energy consumption as high as 100-130kWh / t, significantly increasing the processing cost. At the same time, the inhibition rate of polyphenol oxidase (PPO) by single ultra-high pressure treatment is only 35%-40%. During the storage of juice, browning is easily caused by polyphenol oxidation catalyzed by PPO. After 15 days of storage at 4℃, the anthocyanin retention rate is only 65%-70%.

[0004] (2) Single ultrasonic treatment: The micro-jet and shock wave are generated by cavitation effect of 20-40kHz to break the cell wall. Although nano-level channels can be formed locally, the ultrasonic energy distribution is uneven, which can easily lead to the local temperature rising to 50-55℃, causing anthocyanin oxidation and degradation. The actual dissolution rate is only 45%-50%. Moreover, when ultrasonic is used alone, it cannot activate enzyme activity and needs to be combined with a long enzymatic hydrolysis time (3-4h), resulting in low processing efficiency. At the same time, the local high temperature will cause the loss of terpene characteristic aroma substances (such as linalool) in the juice by 25%-30%, resulting in flavor deterioration.

[0005] (3) Single enzymatic hydrolysis: Existing technologies mostly use a binary complex enzyme of "pectinase + cellulase". Although it can degrade some components of the cell wall, the binary complex enzyme does not have sufficient contact with the substrate. The enzymatic hydrolysis time needs to be 5-6 hours to achieve an anthocyanin dissolution rate of 55%-60%. At the same time, the binary complex enzyme is prone to excessive degradation of pectin, which can easily lead to a drop in juice viscosity to 15-20 mPa•s, resulting in a thin taste and a sensory score of only 65-70 points.

[0006] In summary, existing technologies either focus only on cell wall disruption using a single physical field or rely solely on traditional compound enzymatic hydrolysis, making it difficult to simultaneously improve anthocyanin dissolution rate, processing efficiency, and quality stability. There is an urgent need for a new process to obtain high-quality blueberry juice. Summary of the Invention

[0007] To address the aforementioned problems, this invention aims to provide a process for extracting anthocyanins from blueberry juice using a synergistic ultra-high pressure-ultrasound quaternary complex enzyme.

[0008] The technical solution of the present invention is as follows: A process for extracting anthocyanins from blueberry juice using a combination of ultra-high pressure and ultrasound with a quaternary complex enzyme includes the following steps: S1: Obtain fresh blueberries and pre-process the fresh blueberries to obtain blueberry puree; S2: The blueberry pulp is subjected to ultrasonic treatment to obtain blueberry juice. S3: Perform ultra-high pressure treatment on the blueberry juice one to obtain blueberry juice two; S4: Perform quaternary complex enzymatic hydrolysis on the blueberry juice II to obtain blueberry juice III; S5: The blueberry juice is subjected to enzyme inactivation treatment and filtration treatment in sequence to obtain blueberry juice with dissolved anthocyanins.

[0009] Preferably, in step S1, the fresh blueberries used are fresh blueberries with a ripeness of 85%-90%.

[0010] Preferably, step S1, the pretreatment of the fresh blueberries, specifically includes the following sub-steps: S11: Remove fruit stems, rotten fruit and impurities, rinse several times with deionized water and drain the surface moisture. S12: After draining the surface water, transfer the blueberries to a pulping device for pulping to completely break down the pulp and remove any obvious pits, thus obtaining blueberry puree.

[0011] Preferably, in step S11, the rinsing time is 2 minutes each time, and the water temperature is 20-25℃; in step S12, when pulping, the rotation speed is 3500-4000 r / min, the pulping time is 2-5 minutes, and the obtained blueberry pulp has a solid content of 12%-15% and a pH of 3.2-3.8.

[0012] Preferably, step S2 specifically includes the following sub-steps: mixing the blueberry pulp with purified water in a 1:1 ratio, then transferring the mixture into an ultrasonic treatment container, and ultrasonically treating it for 15-25 minutes at a power of 250-350W and a frequency of 25-35kHz, while controlling the temperature of the mixture to ≤40℃ during the treatment process using a constant temperature water bath.

[0013] Preferably, the temperature of the mixture is controlled at 30-35℃.

[0014] Preferably, step S3 specifically includes the following sub-steps: transferring the blueberry juice into an ultra-high pressure device, sealing it, and maintaining the pressure at 250-350MPa for 6-9 minutes, while controlling the temperature inside the device cavity to be 25-30℃ during the process.

[0015] Preferably, the boost rate is 10 MPa / s and the deboost rate is 15 MPa / s.

[0016] Preferably, step S4 specifically includes the following sub-steps: transferring the blueberry juice II into an enzymatic hydrolysis container, adjusting the pH of the blueberry juice II to 4.0-5.0 with a pH adjuster, then adding 0.1%-0.15% by mass of the blueberry juice II with a quaternary complex enzyme, and enzymatically hydrolyzing for 1.0-1.2 hours at a temperature of 48-52℃ and a stirring rate of 80-100 r / min.

[0017] Preferably, in step S4, the quaternary complex enzyme is composed of pectinase, cellulase, hemicellulase, and arabinofuranylase in a mass ratio of 2:1:1:0.5, and the enzyme activities of the pectinase, cellulase, hemicellulase, and arabinofuranylase are 5000 U / g, 4000 U / g, 3000 U / g, and 2000 U / g, respectively.

[0018] The beneficial effects of this invention are: This invention achieves a three-dimensional synergy of "ultrasound-promoted enzyme substrate contact - ultra-high pressure alteration of cell wall structure - quaternary enzyme degradation of all components": ultrasonic pretreatment forms nanoscale channels through cavitation effect, increasing the contact area between enzyme and cell wall substrate by 40%-45%; ultra-high pressure treatment modifies the cell wall structure in blueberry juice through hydrostatic pressure (increasing the inter-cell wall fiber spacing by 20%-25%, forming potential enzyme action sites); in the quaternary complex enzyme, pectinase degrades pectin, cellulase degrades cellulose, hemicellulase degrades xylan, and arabinofuranosides destroy the cross-linking structure between hemicellulose and pectin, achieving degradation of all cell wall components. The synergistic effect of the three enzymes enables anthocyanin dissolution rate to exceed 85% and shortens the enzymatic hydrolysis time to less than 1 hour. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and technical features described in this application can be combined with each other. It should also be pointed out that, unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms "comprising" or "including" and similar words used in this invention refer to elements or objects preceding the word that encompass the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0020] This invention provides a process for extracting anthocyanins from blueberry juice using a synergistic ultra-high pressure-ultrasound four-component complex enzyme, comprising the following steps: S1: Obtain fresh blueberries and pre-process them to obtain blueberry puree.

[0021] In one specific embodiment, the fresh blueberries used are those with a ripeness of 85%-90%, and the pretreatment of the fresh blueberries specifically includes the following sub-steps: S11: Remove fruit stems, rotten fruit and impurities, rinse several times with deionized water and drain the surface water; optionally, each rinse time is 2 minutes, water temperature is 20-25℃. S12: After draining the surface moisture, transfer the blueberries to a pulping device for pulping to completely break down the pulp and remove any obvious pit residue, thereby obtaining blueberry pulp. Optionally, the pulping speed is 3500-4000 r / min and the pulping time is 2-5 min. The resulting blueberry pulp has a solids content of 12%-15% and a pH of 3.2-3.8.

[0022] S2: The blueberry pulp is subjected to ultrasonic treatment to obtain blueberry juice.

[0023] In a specific embodiment, step S2 specifically includes the following sub-steps: mixing the blueberry pulp with purified water in a 1:1 ratio, then transferring the mixture into an ultrasonic treatment container, and ultrasonically treating it for 15-25 minutes at a power of 250-350W and a frequency of 25-35kHz. During the treatment, the temperature of the mixture is controlled to be ≤40℃ by a constant temperature water bath; optionally, the temperature of the mixture is controlled to be 30-35℃.

[0024] S3: Perform ultra-high pressure treatment on the blueberry juice one to obtain blueberry juice two.

[0025] In a specific embodiment, step S3 specifically includes the following sub-steps: transferring the blueberry juice into an ultra-high pressure device, sealing it, and maintaining the pressure at 250-350MPa for 6-9 minutes, while controlling the temperature inside the device cavity to be 25-30℃ during the process; optionally, the pressure increase rate is 10MPa / s and the pressure decrease rate is 15MPa / s.

[0026] S4: Perform quaternary enzymatic hydrolysis on the blueberry juice II to obtain blueberry juice III.

[0027] In a specific embodiment, step S4 specifically includes the following sub-steps: transferring the blueberry juice II into an enzymatic hydrolysis container, adjusting the pH of the blueberry juice II to 4.0-5.0 with a pH adjuster, then adding 0.1%-0.15% by mass of the blueberry juice II with a quaternary complex enzyme, and enzymatically hydrolyzing for 1.0-1.2 hours at a temperature of 48-52℃ and a stirring rate of 80-100 r / min.

[0028] In one specific embodiment, the quaternary complex enzyme is composed of pectinase, cellulase, hemicellulase, and arabinofuranylase in a mass ratio of 2:1:1:0.5, and the enzyme activities of the pectinase, cellulase, hemicellulase, and arabinofuranylase are 5000 U / g, 4000 U / g, 3000 U / g, and 2000 U / g, respectively.

[0029] Blueberry cell walls have a complex structure, consisting of a dense network of cellulose (28%-32%), hemicellulose (22%-26%), pectin (18%-22%), and lignin (6%-9%), encapsulating over 70% of anthocyanins within the cell vacuoles. In the above embodiments, the pectinase in the quaternary complex enzyme can degrade pectin, the cellulase can degrade cellulose, the hemicellulase can degrade xylan, and the arabinofuranylase can disrupt the cross-linking structure between hemicellulose and pectin, achieving full degradation of the cell wall components and thus accelerating the dissolution of anthocyanins.

[0030] S5: The blueberry juice is subjected to enzyme inactivation treatment and filtration treatment in sequence to obtain blueberry juice with dissolved anthocyanins.

[0031] In one specific embodiment, the enzyme inactivation process includes the following sub-steps: the blueberry juice is immediately transferred into a sterile enzyme inactivation container, and the enzyme inactivation process is carried out by water bath heating, with the enzyme inactivation temperature set at 80-85℃ and the holding time at 3-5 minutes.

[0032] In one specific embodiment, the filtration process includes the following sub-steps: cooling the enzyme-inactivated blueberry juice to 4-8°C, using a 0.45μm hydrophilic composite membrane, cross-flow filtration at a pressure of 0.1-0.12MPa, a flow rate of 1.0-1.2m / s, with nitrogen protection throughout (oxygen partial pressure ≤0.03MPa), and completing the process within 30 minutes.

[0033] Example 1 Using the "Mist" variety of blueberries as raw material, the blueberry juice is processed using the ultra-high pressure-ultrasound synergistic quaternary complex enzyme dissolution process described in this invention to extract anthocyanins.

[0034] In this embodiment, 2 kg of "Mist" blueberries with a maturity of 88% were selected. After manually removing stems, rotten fruit, and impurities, the berries were rinsed three times with deionized water (22°C, 2 min each time). After draining the surface water, the berries were transferred to a pulping device (3800 r / min, 2.5 min) to obtain blueberry pulp with a solid content of 13.5% and a pH of 3.5. The blueberry pulp was mixed with purified water in a 1:1 ratio to form blueberry juice, which was then transferred to an ultrasonic treatment container. The ultrasonic parameters were set as follows: power 280W, frequency 30kHz, time 20 min. The temperature of the blueberry juice was controlled to ≤40°C using a constant temperature water bath (35°C). The cell wall damage rate after treatment was 42%. The blueberry juice was then transferred to an ultra-high pressure device. After sealing, the ultra-high pressure parameters were set as follows: pressure 280 MPa, holding time 7 min, pressure increase rate 10 MPa / s, pressure decrease rate 15 MPa / s. The temperature inside the device was controlled at 28°C. After processing, extract the blueberry juice; adjust the pH of the blueberry juice to 4.0 with 1% citric acid solution or 1% sodium hydroxide solution, add a quaternary complex enzyme (pectinase: cellulase: hemicellulase: arabinofuranoside = 2:1:1:0.5, enzyme activities of 5000 U / g, 4000 U / g, 3000 U / g, and 2000 U / g respectively), with a total enzyme addition of 0.12%; place the enzymatic hydrolysis container in a constant temperature water bath, set the temperature to 50℃ and the stirring speed to 9. The enzyme was hydrolyzed at 0 r / min for 1.1 h, with samples taken every 20 min during the hydrolysis process to determine the anthocyanin dissolution rate. After hydrolysis, the enzyme was incubated in an 82℃ water bath for 4 min (to inactivate PPO and residual enzyme activity), cooled to 6℃, and filtered through a 0.45 μm hydrophilic composite membrane at a cross-flow rate of 1.1 m / s under a pressure of 0.11 MPa, with nitrogen protection throughout (oxygen partial pressure ≤ 0.03 MPa). Filtration was completed within 25 min to obtain clear blueberry juice.

[0035] Example 2 Using the "Zhuixue" variety of blueberries as raw material, the blueberry juice is processed using the ultra-high pressure-ultrasound synergistic quaternary complex enzyme dissolution process described in this invention to extract anthocyanins.

[0036] In this embodiment, 3 kg of "Zhuixue" blueberries with a maturity of 86% were selected. After manually removing stems, rotten fruit, and impurities, the berries were rinsed three times with deionized water (24℃, 2 min each time). After draining the surface water, the berries were transferred to a pulping device (3600 r / min, 2.2 min) to obtain blueberry pulp with a solid content of 12.8% and a pH of 3.6. The blueberry pulp was mixed with purified water in a 1:1 ratio to form blueberry juice, which was then transferred to an ultrasonic treatment container. The ultrasonic parameters were set as follows: power 320W, frequency 32kHz, time 22 min. The temperature of the blueberry juice was controlled to ≤40℃ using a constant temperature water bath (33℃). The cell wall damage rate after treatment was 44%. The blueberry juice was then transferred to an ultra-high pressure device, sealed, and the ultra-high pressure parameters were set as follows: pressure 320MPa, holding time 8 min, pressure increase rate 10MPa / s, pressure decrease rate 15MPa / s. The temperature inside the device was controlled at 29℃. After processing, extract the blueberry juice; adjust the pH of the blueberry juice to 4.5 with 1% citric acid solution or 1% sodium hydroxide solution, add a quaternary complex enzyme (pectinase: cellulase: hemicellulase: arabinofuranoside = 2:1:1:0.5, enzyme activities of 5000 U / g, 4000 U / g, 3000 U / g, and 2000 U / g respectively), with a total enzyme addition of 0.14%; place the enzymatic hydrolysis container in a constant temperature water bath, set the temperature to 51℃ and the stirring speed to 9. Enzymatic hydrolysis was performed at 5 r / min for 1.0 h, with samples taken every 20 min during the hydrolysis process to determine the anthocyanin dissolution rate. After hydrolysis, the mixture was incubated in an 84℃ water bath for 3 min (to inactivate PPO and residual enzyme activity), cooled to 5℃, and filtered using a 0.45 μm hydrophilic composite membrane at a cross-flow rate of 1.2 m / s under a pressure of 0.12 MPa, with nitrogen protection throughout (oxygen partial pressure ≤ 0.03 MPa). Filtration was completed within 28 min to obtain clear blueberry juice.

[0037] Example 3 Using the "rabbiteye" variety of blueberry as raw material, the blueberry juice is processed using the ultra-high pressure-ultrasound synergistic quaternary complex enzyme dissolution process described in this invention to extract anthocyanins.

[0038] In this embodiment, 2.5 kg of "rabbiteye" blueberries with a maturity of 87% were selected. After manually removing stems, rotten fruit, and impurities, the berries were rinsed three times with deionized water (23°C, 2 min each time). After draining the surface water, the berries were transferred to a pulping device (3700 r / min, 2.4 min) to obtain blueberry pulp with a solid content of 14.2% and a pH of 3.4. The blueberry pulp was mixed with purified water in a 1:1 ratio to form blueberry juice, which was then transferred to an ultrasonic treatment container. The ultrasonic parameters were set as follows: power 300W, frequency 28kHz, and time 24 min. The temperature of the blueberry juice was controlled to ≤40°C using a constant temperature water bath (34°C). The cell wall damage rate after treatment was 43%. The blueberry juice was then transferred to an ultra-high pressure device, sealed, and the ultra-high pressure parameters were set as follows: pressure 300 MPa, holding time 8 min, pressure increase rate 10 MPa / s, and pressure decrease rate 15 MPa / s. The temperature inside the device was controlled at 27°C. After processing, extract the blueberry juice; adjust the pH of the blueberry juice to 5.0 with 1% citric acid solution or 1% sodium hydroxide solution, add a quaternary complex enzyme (pectinase: cellulase: hemicellulase: arabinofuranoside = 2:1:1:0.5, enzyme activities of 5000 U / g, 4000 U / g, 3000 U / g, and 2000 U / g, respectively), with a total enzyme addition of 0.13%; place the enzymatic hydrolysis container in a constant temperature water bath, set the temperature to 50℃ and the stirring speed to [missing information]. Enzymatic hydrolysis was performed at 100 r / min for 1.2 h, with samples taken every 20 min during the hydrolysis process to determine the anthocyanin dissolution rate. After hydrolysis, the mixture was incubated in an 83℃ water bath for 5 min (to inactivate PPO and residual enzyme activity), cooled to 7℃, and filtered through a 0.45 μm hydrophilic composite membrane at a cross-flow rate of 1.0 m / s under 0.1 MPa pressure, with nitrogen protection throughout (oxygen partial pressure ≤ 0.03 MPa). Filtration was completed within 26 min to obtain clear blueberry juice.

[0039] Comparative Example 1 Blueberry juice was extracted from three varieties of blueberries: "Mist," "Snow Chaser," and "Rabbiteye," using traditional fresh-pressing techniques as a control group.

[0040] Comparative Example 2 Blueberries of the 'Mist', 'Snow Chaser', and 'Rabbiteye' varieties were used as raw materials and processed using a single ultra-high pressure (UHPP) treatment process. The specific parameters of the UHPP treatment process were 300 MPa for 10 minutes, denoted as the single UHPP group.

[0041] Comparative Example 3 Blueberries of the 'Mist', 'Snow Chaser', and 'Rabbiteye' varieties were used as raw materials and processed using a single ultrasonic treatment process. The specific parameters of the ultrasonic treatment process were 300W, 30kHz, and 20min, denoted as the single ultrasonic group.

[0042] Comparative Example 4 Blueberries of the 'Mist', 'Snow Chaser', and 'Rabbiteye' varieties were used as raw materials and processed using a single-ternary complex enzymatic hydrolysis process. The ternary complex enzyme used in the hydrolysis process consisted of pectinase, cellulase, and hemicellulase in a mass ratio of 2:1:1, with a total enzyme addition of 0.15% and a hydrolysis temperature of 50℃; this group was designated as the single-ternary complex enzymatic hydrolysis group.

[0043] Comparative Example 5 Blueberries of the 'Mist', 'Snow Chaser', and 'Rabbiteye' varieties were used as raw materials and processed using an ultra-high pressure-ultrasound-ternary complex enzymatic hydrolysis process. The ternary complex enzyme in the ternary complex hydrolysis process consisted of pectinase, cellulase, and hemicellulase in a mass ratio of 2:1:1, and was designated as the ultra-high pressure-ultrasound-ternary complex enzymatic hydrolysis group.

[0044] Comparative Example 6 Blueberries of the varieties "Mist," "Snow Chaser," and "Rabbit Eye" were used as raw materials and processed using an ultra-high pressure-quaternary complex enzymatic hydrolysis process, which was designated as the ultra-high pressure-quaternary complex enzymatic hydrolysis group.

[0045] Comparative Example 7 Blueberries of the varieties "Mist," "Snow Chaser," and "Rabbit Eye" were used as raw materials and processed using an ultrasonic-quaternary compound enzymatic hydrolysis process, which was designated as the ultrasonic-quaternary compound enzymatic hydrolysis group.

[0046] Test case The blueberry juices obtained in each embodiment and comparative example were tested, and the results are shown in Table 1: Table 1. Test results of blueberry juice from each embodiment and comparative example.

[0047] As shown in Table 1, the present invention (ultrasound-ultra-high pressure-quaternary complex enzymatic hydrolysis process) exhibits a crushing advantage in terms of anthocyanin dissolution rate. The blank group (traditional fresh-pressed juice) relied solely on mechanical crushing, which failed to break down the dense cell walls of blueberries, resulting in an anthocyanin dissolution rate of only 32.5±1.2%. While the single ultra-high pressure group (52.3±1.5%) and the single ultrasound group (47.8±1.6%) achieved cell wall disruption through physical action, they only partially damaged the cell walls, limiting the improvement in dissolution rate. The single ternary complex enzymatic hydrolysis group (58.5±1.4%) lacked physical field assistance, resulting in insufficient enzyme-substrate contact and an inability to degrade the cross-linked structure of hemicellulose and pectin, thus its dissolution rate still did not exceed 60%. Even in combinations of "two-factor + enzymatic hydrolysis" or "all-factor lack of key enzymes", the dissolution rate still has obvious shortcomings: the ultra-high pressure-ultrasound-ternary complex enzyme group (reduced arabinofuranosides) has a dissolution rate of only 76.2±1.5% due to the lack of a key enzyme that can destroy the cross-linking structure of hemicellulose and pectin; the ultra-high pressure-quaternary complex enzyme group (reduced ultrasound) has a dissolution rate of only 73.5±1.4% due to the lack of ultrasound cavitation effect to facilitate enzyme-substrate contact; and the ultrasound-quaternary complex enzyme group (reduced ultra-high pressure) has a dissolution rate of only 74.8±1.3% due to the lack of the cell wall structure modification effect of ultra-high pressure. This invention, through the three-dimensional synergy of "ultrasonic cell wall disruption - ultra-high pressure modification - tetravalent enzyme complete degradation", achieves an anthocyanin dissolution rate of up to 88.7±1.3%, which is 173.0% higher than the blank group, 51.6% higher than the single ternary enzyme hydrolysis group, and 12.5%-17.1% higher than the above three types of nutrient deficiency groups. This fully verifies the degradation ability of the tetravalent complex enzyme on all components of the cell wall and the synergistic effect of physical field and enzymatic hydrolysis, and highlights the necessity of the three-dimensional synergy of "ultra-high pressure - ultrasound - tetravalent enzyme" to overcome the dissolution rate bottleneck.

[0048] From the perspective of processing efficiency, this invention can significantly shorten the enzymatic hydrolysis time and solve the problem of time consumption in traditional processes. The single ternary enzymatic hydrolysis group, due to its low efficiency, requires 5.5 hours to achieve an anthocyanin dissolution rate of 58.5 ± 1.4%. The comparative group, lacking a single core factor, still struggles to achieve efficient enzymatic hydrolysis even with the combination of some efficient processes: the ultra-high pressure-ultrasound-ternary complex enzyme group (with reduced arabinofuranosylase) has an incomplete enzymatic hydrolysis system, extending the hydrolysis time to 1.6 hours; the ultra-high pressure-quaternary complex enzyme group (with reduced ultrasound) requires 2.0 hours due to the lack of nanoscale channels formed by ultrasound pretreatment (reducing the contact area by 40%-45% compared to the synergistic group); and the ultrasound-quaternary complex enzyme group (with reduced ultra-high pressure) also requires 1.8 hours due to the lack of cell wall fiber spacing expanded by ultra-high pressure (reducing the enzyme action sites by 20%-25% compared to the synergistic group). In contrast, this invention utilizes ultrasonic pretreatment to increase the enzyme-substrate contact area through cavitation effect and ultra-high pressure treatment to increase enzyme action sites. Together, these two methods create a highly efficient action environment for the quaternary complex enzyme, achieving a high dissolution rate of 88.7±1.3% in just 1.1±0.1 hours. The enzymatic hydrolysis time is shortened by approximately 80.0% compared to the single ternary enzyme hydrolysis group, by 31.3%-45.5% compared to the three types of nutrient deficiency groups mentioned above, and by 63.3%-72.5% compared to the "ultrasonic + single enzymatic hydrolysis" process (3-4 hours) reported in existing literature. This significantly improves the time efficiency of industrial production and reduces equipment occupancy costs.

[0049] In terms of quality and stability, the synergistic group of this invention is superior to other processing groups in many key indicators, and "reducing a single core factor" will directly lead to shortcomings in quality or stability. Regarding transmittance, the present invention achieves a transmittance as high as 92.5±1.5%, a 58.7% improvement compared to the blank group (58.3±2.1%) and an 18.1% improvement compared to the single ternary enzyme digestion group (78.3±1.7%). However, the transmittance of the ultra-high pressure-ultrasound-ternary complex enzyme group (reduced arabinofuranosylase) is only 85.7±1.6% (a 7.4% decrease compared to the synergistic group) due to incomplete degradation of the cell wall cross-linking structure. Regarding PPO activity inhibition rate, the present invention achieves a high rate of 87.6±1.6%, 2.9 times that of the blank group (30.2±1.5%) and 2.3 times that of the single ultra-high pressure group (38.6±1.7%). Although the ultra-high pressure-quadrivalent complex enzyme group (reduced ultrasound) has ultra-high pressure assistance, the lack of synergistic activation of enzyme activity by ultrasound results in a PPO inhibition rate of only 76.8±1.6% (a 12.3% decrease compared to the synergistic group). - The quaternary complex enzyme group (reduced ultra-high pressure) had a PPO inhibition rate of 70.5±1.5% (19.5% lower than the synergistic group) due to the lack of physical inhibition from ultra-high pressure. In terms of sensory scores, the present invention scored 91.8±1.3 points, far exceeding the other groups. The ultra-high pressure-quaternary complex enzyme group (reduced ultrasound) had a sensory score of only 80.3±1.7 points (12.5% ​​lower than the present invention) due to the lack of protection of flavor substances by ultrasound. Regarding the anthocyanin retention rate after 30 days of storage at 4℃, the synergistic group reached 90.2±1.5%, which was 41.4% higher than the blank group (63.8±1.9%) and 24.2% higher than the single ternary enzyme hydrolysis group (72.6±1.7%). However, the ultrasound-quaternary complex enzyme group (reduced ultra-high pressure) had a retention rate of only 80.2±1.7% (11.1% lower than the synergistic group) due to the lack of improvement of system stability by ultra-high pressure.

[0050] The above data not only confirms the comprehensive improvement effect of the synergistic process on the clarification effect, browning inhibition, flavor preservation and storage stability of juice, but also proves that the three-dimensional synergy of "ultra-high pressure-ultrasound-quadrivalent complex enzyme" in this invention is the key to achieving simultaneous improvement in quality and stability. Without any one of these elements, it is difficult to meet the requirements of high-quality juice.

[0051] In summary, the ultra-high pressure-ultrasound synergistic quaternary complex enzyme process for dissolving anthocyanins in blueberry juice according to the present invention has the following advantages: (1) Significantly improved dissolution efficiency of active ingredients: Through the synergistic effect of "ultra-high pressure-ultrasound-quadrivalent complex enzyme", the anthocyanin dissolution rate reached 88.7%±1.3, which is 50%-85% higher than the existing single technology. The raw material utilization rate increased from 45% in the traditional process to more than 90%, greatly reducing fruit pomace waste. At the same time, the degradation of all components of the cell wall by the quadrivalent enzyme increased the active ingredients such as polyphenols and vitamin C in the juice. The polyphenol content reached 280-320mg / L, which is 35%-40% higher than the single ultra-high pressure group, and the functional activity was significantly enhanced.

[0052] (2) Processing efficiency and economy are greatly optimized: the enzymatic hydrolysis time is shortened from 3-6h in the existing technology to 1-1.2h, and the processing efficiency is increased by more than 70%; the synergistic effect of ultra-high pressure and ultrasound shortens the ultra-high pressure holding time from 15-20min to 6-9min, and the energy consumption is reduced to 60-70kWh / t, which is 40%-50% lower than that of single ultra-high pressure treatment; at the same time, the total amount of quaternary enzyme added is only 0.1%-0.15%, which reduces the enzyme preparation cost by 20% compared with traditional ternary enzymatic hydrolysis, and the unit product processing cost is reduced by 18%-22%, and the economy is significantly improved.

[0053] (3) Quality and storage stability are improved simultaneously: the light transmittance of the juice is over 92%, and there is no stratification after standing for 30 days, which is 15%-25% higher than the existing technology; the PPO activity inhibition rate is over 85%, and the anthocyanin retention rate is about 90% after 30 days of storage at 4℃, extending the shelf life from 7-10 days of traditional fresh juice to over 60 days; the sensory score is over 90 points, which is superior to existing products in terms of color, taste and clarity, meeting the market demand for high-end functional juices.

[0054] The above description is merely a representative embodiment of the present invention and is not intended to limit the present invention in any way. Any embodiment made by those skilled in the art without departing from the scope of the present invention and utilizing the disclosed technical content is an equivalent embodiment of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme under ultra-high pressure-ultrasound, characterized by, The method comprises the following steps: S1: obtaining fresh blueberries and pretreating the fresh blueberries to obtain blueberry slurry; S2: ultrasonically treating the blueberry slurry to obtain blueberry juice I; S3: ultrahigh pressure treating the blueberry juice I to obtain blueberry juice II; S4: quadruple compound enzymatic hydrolysis treating the blueberry juice II to obtain blueberry juice III; S5: sequentially performing enzyme inactivation treating and filtering treating on the blueberry juice III to obtain blueberry juice with dissolved anthocyanins.

2. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 1, characterized in that, In step S1, the fresh blueberries are fresh blueberries with a maturity of 85%-90%.

3. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 1, characterized in that, In step S1, the pretreating of the fresh blueberries specifically comprises the following substeps: S11: removing fruit stems, rotten fruits and impurities, and rinsing multiple times with deionized water and then draining surface water; S12: transferring the blueberries after draining surface water into a pulping device to perform pulping, so that the fruit flesh is completely broken and no obvious fruit core residues are left, to obtain blueberry slurry.

4. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 3, characterized in that, In step S11, each rinsing time is 2 min, and the water temperature is 20-25 DEG C; in step S12, when performing pulping, the rotating speed is 3500-4000 r / min, the pulping time is 2-5 min, the obtained blueberry slurry has a solid content of 12%-15% and a pH of 3.2-3.

8.

5. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 1, characterized in that, Step S2 specifically comprises the following substeps: mixing the blueberry slurry with pure water at a ratio of 1:1, then transferring the mixed liquid into an ultrasonic treatment container, and ultrasonically treating under the condition of a power of 250-350 W and a frequency of 25-35 kHz for 15-25 min, and controlling the mixed liquid temperature to be ≤40 DEG C through a constant temperature water bath during the treatment.

6. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 5, characterized in that, The mixed liquid temperature is controlled to be 30-35 DEG C.

7. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 1, characterized in that, Step S3 specifically comprises the following substeps: transferring the blueberry juice I into an ultrahigh pressure device, sealing, and then pressurizing to 250-350 MPa and maintaining the pressure for 6-9 min, and controlling the temperature in the device cavity to be 25-30 DEG C during the treatment.

8. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 7, characterized by, The pressure increasing rate is 10 MPa / s, and the pressure decreasing rate is 15 MPa / s.

9. The process for the extraction of blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound according to claim 1, characterized in that, Step S4 specifically comprises the following substeps: transferring the blueberry juice II into an enzymatic hydrolysis container, adjusting the pH of the blueberry juice II to 4.0-5.0 by using a pH regulator, then adding 0.1%-0.15% of quadruple compound enzyme based on the mass of the blueberry juice II, and performing enzymatic hydrolysis under the condition of a temperature of 48-52 DEG C and a stirring speed of 80-100 r / min for 1.0-1.2 h.

10. The process of claim 1-9, wherein the process is a process of extracting blueberry juice anthocyanins by synergistic quaternary composite enzyme of ultra-high pressure-ultrasound, characterized in that, In step S4, the quadruple compound enzyme is composed of pectinase, cellulase, hemicellulase and arabinofuranosidase at a mass ratio of 2:1:1:0.5, and the enzyme activities of the pectinase, cellulase, hemicellulase and arabinofuranosidase are 5000 U / g, 4000 U / g, 3000 U / g and 2000 U / g, respectively.

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