Process method for improving juice yield and active ingredient content of lonicera caerulea juice

Abstract

The invention discloses a process method for improving juice yield and active ingredient content of lonicera caerulea juice, and belongs to the technical field of fruit and vegetable processing. The invention provides a step-by-step enzymolysis process method based on a lonicera caerulea cell wall layered structure (intercellular layer-primary wall-secondary wall) and polyphenol molecular characteristics (hydrogen bond / hydrophobic interaction chelation). By scientifically screening the types and the feeding sequence (first-stage to fourth-stage enzymolysis) of pectinase, xylanase, alpha-galactosidase and acid protease, a cell wall pectin-cellulose-lignin network and a polyphenol-macromolecular compound are degraded in a targeted manner, so that the juice yield is greatly increased, active ingredients such as total polyphenols and anthocyanin are released to the maximum extent, and the quality of the product is improved. The problems of low juice yield, low enzymolysis efficiency, serious component loss and uncontrollable process in the traditional technology are solved, and a solution with theoretical innovation and industrial potential is provided for high-value processing of the lonicera caerulea.

Description

Technical Field

[0001] The invention relates to the technical field of fruit and vegetable processing, in particular to a process for improving the juice yield and active component content of blue loquat fruit juice. Background Art

[0002] The blue indigo fruit (Lonicera caerulea L.), also known as the haskap berry, is a nutritious small cold-region berry containing a variety of vitamins and natural active ingredients such as anthocyanins and flavonoids. Its dry matter content is 10% to 17%, its sugar content is 5% to 10%, and its acid content is 1.5% to 4.5%. The blue indigo fruit is also rich in vitamins such as vitamin B1, vitamin B2, vitamin P, and vitamin C, with vitamin P content nearly 100 times higher than that of ordinary fruits. It also contains essential trace minerals such as zinc, selenium, and iron, as well as 16 amino acids. It exhibits excellent antioxidant, anti-fatigue, and immune-boosting health benefits. It has become a functional berry that has garnered widespread attention in recent years. However, due to its high concentration of polyphenols such as tannins, the blue indigo fruit has a sour and astringent taste, making it unsuitable for fresh consumption. However, due to its unique color and extremely high content of nutrients and functional active ingredients, it is now widely used in food processing, such as juice and wine.

[0003] Traditional juice production methods mostly use physical means such as direct pressing. However, the cell wall structure of blue honeysuckle is relatively tough, and substances such as pectin and cellulose in the fruit are intertwined, forming a tight network structure that hinders the flow of juice. Lignin, as a rigid component of the cell wall, enhances the fruit's resistance to mechanical damage, but also increases the resistance to juicing, making it difficult to extract juice. Therefore, the traditional mechanical pressing method of producing blue honeysuckle juice not only requires high pressure, but also fails to fully destroy the fruit structure, resulting in a large amount of juice remaining in the pomace, resulting in a low juice yield, generally only reaching 60% to 70%. Even though some processes use blanching treatment, which can soften the cell tissue to a certain extent and help improve the juice yield, the blanching process easily leads to the degradation of heat-sensitive active ingredients such as vitamin C and anthocyanins, affecting the nutritional value and color quality of the juice.

[0004] In recent years, efforts to increase juice yield have begun to explore the application of methods such as ultrasonic treatment and bio-enzymatic hydrolysis. Bio-enzymatic hydrolysis, due to its gentleness, high efficiency, and pollution-free properties, has become a key area of ​​research and application. However, due to the complex cell wall structure of blue loquat fruit, single enzyme methods struggle to fully degrade the cell wall, resulting in low efficiency. For example, pectinase treatment only degrades pectin, but is ineffective against cellulose and lignin. This results in an upper limit to juice yield that cannot be exceeded and insufficient release of active ingredients. Furthermore, residual pectin-cellulose complexes increase juice viscosity and clog filtration equipment. Combined enzymatic hydrolysis, to some extent, compensates for the shortcomings of single enzymatic hydrolysis, enhancing cell wall degradation and increasing juice yield. For example, to fully enhance the economic value of blue indigo fruit, Wu Guoxiu and other researchers at Northeast Agricultural University used a technique that used 0.27% pectinase, 0.92% cellulase, and an enzymatic hydrolysis temperature of 47°C. This technique increased the juice yield of blue indigo fruit to 85.67%, and the contents of vitamin C, anthocyanins, and total flavonoids in the juice increased by 1.78, 1.13 mg / mL, and 2.54 mg / mL, respectively. However, combined enzymatic hydrolysis methods (such as the combined use of pectinase and cellulase) often result in incomplete enzymatic hydrolysis or localized over-hydrolysis, producing bitter peptides, due to conflicting enzyme reaction conditions, which affects juice yield and juice quality. Furthermore, polyphenolic active substances such as blue indigo fruit anthocyanins form stable complexes with proteins and polysaccharides within cells through hydrogen bonds or hydrophobic interactions, which hinders the release of polyphenolic active substances. Therefore, selecting the appropriate enzyme system to degrade proteins and polysaccharides is crucial for releasing more polyphenolic active substances.

[0005] Therefore, in view of the low juice yield in the existing blue indigo fruit juice processing (traditional mechanical pressing is only 60% to 70%, and the effect of a single enzyme is limited), insufficient release of active ingredients (more than 30% of polyphenols are retained in the pomace due to the complex cell wall structure and chelation with proteins / polysaccharides), and the problems of conflicting reaction conditions and local over-hydrolysis producing bitter peptides in traditional composite enzymatic hydrolysis, a new enzymatic hydrolysis method to improve the juice yield of blue indigo fruit is urgently needed to solve the current technical problems. Summary of the Invention

[0006] The present invention aims to provide a process for improving the juice yield and active ingredient content of blue honeysuckle fruit juice, so as to solve the problems existing in the above-mentioned prior art. By scientifically screening the types and addition sequences of enzyme preparations, based on secondary enzymatic hydrolysis of pectinase and xylanase, α-galactosidase and acid protease are introduced to achieve efficient decomposition of blue honeysuckle fruit cell wall components and maximum release of internal chelated polyphenols, thereby achieving the purpose of synergistically improving the juice yield and active ingredient extraction efficiency, and at the same time significantly improving the antioxidant capacity of blue honeysuckle fruit juice.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a process for improving the juice yield and active ingredient content of blue loquat fruit juice, comprising the following steps:

[0009] The blue loquat fruit is beaten into pulp, and pectinase is added to perform primary enzymolysis to obtain enzymolysis product I;

[0010] adding xylanase to the enzymatic hydrolysis product I for secondary enzymatic hydrolysis to obtain an enzymatic hydrolysis product II;

[0011] adding α-galactosidase to the enzymatic hydrolysis product II for tertiary enzymatic hydrolysis to obtain an enzymatic hydrolysis product III;

[0012] Acidic protease is added to the enzymatic hydrolysis product III to perform a fourth-stage enzymatic hydrolysis to obtain an enzymatic hydrolysis product IV.

[0013] Preferably, the conditions for the primary enzymatic hydrolysis are: pectinase concentration of 0.1% to 0.3%, w / w; temperature of 30° C. to 50° C.; and enzymatic hydrolysis time of 10 min to 25 min.

[0014] Preferably, the conditions for the secondary enzymatic hydrolysis are: xylanase concentration of 2% to 4%, w / w; temperature of 30° C. to 50° C.; and enzymatic hydrolysis time of 5 min to 15 min.

[0015] Preferably, the conditions for the tertiary enzymatic hydrolysis are: α-galactosidase concentration of 0.5% to 3.5%, w / w; temperature of 30° C. to 50° C.; and enzymatic hydrolysis time of 20 min to 25 min.

[0016] Preferably, the conditions for the four-stage enzymatic hydrolysis are: acidic protease concentration of 5.0% to 9.5%, w / w; temperature of 30° C. to 50° C.; and enzymatic hydrolysis time of 10 min to 15 min.

[0017] The present invention also provides application of the process method in improving the yield of blue loquat fruit juice.

[0018] The present invention also provides application of the process method in increasing the total phenol content of blue loquat fruit juice.

[0019] The present invention also provides an application of the process method in increasing the anthocyanin content of Lonicera edulis fruit juice.

[0020] The present invention also provides the use of the juice prepared by the process method in preparing products that help to improve antioxidant capacity.

[0021] Preferably, the antioxidant indicators are DPPH clearance rate and total reducing power.

[0022] The present invention discloses the following technical effects:

[0023] (1) Hierarchical targeted enzymatic hydrolysis strategy: This invention provides a step-by-step enzymatic hydrolysis technology for Lonicera edulis based on the hierarchical structure of the Lonicera edulis cell wall (intercellular lamella-primary wall-secondary wall) and the molecular characteristics of polyphenols (hydrogen bonding / hydrophobic chelation). This technology breaks through the traditional "synchronous action" model of complex enzymes and designs a four-stage enzymatic hydrolysis sequence based on the structural characteristics of the cell wall and the presence of polyphenols, avoiding problems such as conflicts in enzyme reaction conditions. At the same time, the introduction of α-galactosidase and acid protease specifically solves the degradation problems of the secondary wall and polyphenol-macromolecule complex.

[0024] More specifically, the present invention scientifically screens the types and addition sequence of pectinase, xylanase, α-galactosidase, and acid protease (primary to quadruple enzymatic hydrolysis) to target the degradation of the cell wall pectin-cellulose-lignin network and polyphenol-macromolecular complex, thereby significantly improving the juice yield while maximizing the release of active ingredients such as total polyphenols and anthocyanins. This solves the problems of low juice yield, low enzymatic hydrolysis efficiency, severe ingredient loss, and uncontrollable process in traditional technologies, and provides a solution for the high-value processing of blue indigo fruit that combines theoretical innovation with industrial potential.

[0025] (2) Dual-target synergistic mechanism: Through the dual effects of "structural deconstruction (cell wall) + molecular dissociation (polyphenol complex)", the juice yield and antioxidant activity are simultaneously improved.

[0026] More specifically, the present invention screens enzyme systems suitable for degrading the cell walls and proteoglycans of blue honeysuckle, and constructs a step-by-step enzymatic hydrolysis technology suitable for the efficient degradation of blue honeysuckle. This not only breaks through the efficiency bottleneck of traditional enzymatic hydrolysis in blue honeysuckle processing, but also achieves a synergistic improvement in juice yield and active ingredient release, forming a closed loop from process optimization, component analysis to functional evaluation, and provides a new technical paradigm for the field of cold-region berry processing that combines scientificity and industrialization potential.

[0027] Compared with the juice yield of direct juicing without enzymatic hydrolysis, the method of four-stage enzyme step-by-step enzymatic hydrolysis provided by the present invention can increase the juice yield by 46%; the four-stage enzyme step-by-step enzymatic hydrolysis significantly improves the juice yield and the release rate of active ingredients by destroying the cell wall structure and polyphenol-protein complex, which also means that the antioxidant capacity of the juice should also be improved. The test results show that after the four enzymes step-by-step enzymatic hydrolysis, the total phenol content of the juice increased by 94%, and the anthocyanin content increased by 68%; the DPPH free radical scavenging rate of the juice increased by 37% compared with direct juicing, and the total reducing power increased by 32%. The antioxidant results and the increase in the active ingredient release rate are mutually confirmed. The four-stage enzyme step-by-step enzymatic hydrolysis technology of the present invention is significantly superior to direct juicing and two-stage enzymatic hydrolysis in terms of juice yield, active ingredient content and antioxidant capacity, fully demonstrating the advantages and innovation of the technology.

[0028] (3) Data-driven optimization: Combining single-factor experiments with response surface methodology, the enzyme system ratio and process parameters are accurately determined to provide a quantitative basis for industrial production.

[0029] More specifically, the four-stage enzymatic hydrolysis technology provided by the present invention is expected to promote the deep processing of blue indigo fruit towards the direction of "high value, precision and green" by targeted deconstruction of the multi-layer network of the cell wall and precise breakdown of polyphenol-macromolecule chelates, providing theoretical support and practical path for the technological upgrading of the functional juice industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The effect of pectinase addition on the juice yield of blue loquat fruit;

[0032] Figure 2 The effect of pectinase hydrolysis time on the juice yield of blue loquat fruit;

[0033] Figure 3 The effect of xylanase addition on the juice yield of blue loquat fruit;

[0034] Figure 4 The effect of xylanase enzymatic hydrolysis time on the juice yield of blue loquat fruit;

[0035] Figure 5 The contour map and response surface diagram of the interaction effect of various factors on the juice yield of blue honeysuckle; a: interaction between pectinase addition amount and xylanase addition amount, b: interaction between pectinase addition amount and xylanase enzymolysis time, c: interaction between xylanase addition amount and enzymolysis time;

[0036] Figure 6 The effect of α-galactosidase addition on juice yield; different letters indicate significant differences (P<0.05);

[0037] Figure 7 The effect of α-galactosidase enzymatic hydrolysis time on juice yield; different letters indicate significant differences (P<0.05);

[0038] Figure 8 The effect of the amount of acid protease added on the juice yield; different letters indicate significant differences (P<0.05);

[0039] Figure 9The effect of acid protease enzymatic hydrolysis time on juice yield; different letters indicate significant differences (P<0.05);

[0040] Figure 10 These are the contour plots and response surface diagrams of the response surface experiment; a: interaction between the amount of α-galactosidase added and the enzymatic hydrolysis time of α-galactosidase, b: interaction between the amount of α-galactosidase added and the amount of acid protease added, c: interaction between the enzymatic hydrolysis time of α-galactosidase and the amount of acid protease added;

[0041] Figure 11 The changes in juice yield of blue loquat fruit after enzymatic hydrolysis at different levels; different letters indicate significant differences (P<0.05);

[0042] Figure 12 Gallic acid standard curve was determined for total polyphenols;

[0043] Figure 13 is the standard curve of vitamin C;

[0044] Figure 14 The changes in the content of active substances in Lonicera edulis juice after enzymatic hydrolysis; different letters indicate significant differences (P<0.05);

[0045] Figure 15 The DPPH free radical scavenging ability of blue loquat fruit juice;

[0046] Figure 16 It is the total reducing capacity of blue loquat fruit juice. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] Example 1 Parameter selection of the step-by-step enzymatic hydrolysis system for targeted depolymerization of Lonicera edulis cell walls

[0053] 1. Preparation and enzymatic hydrolysis of blue indigo fruit pulp

[0054] Take an appropriate amount of blue indigo fruit jelly, thaw it and break it into a homogenate with a wall breaking machine, centrifuge it at 8000r / min for 5min, measure the supernatant, and calculate the juice yield of the unhydrolyzed blue indigo fruit.

[0055] Accurately weigh a certain amount of crushed and homogenized blue loquat fruit pulp, weigh the enzymes according to the dosage of the following experimental design, add them to the pulp, mix thoroughly, and then perform enzymatic hydrolysis in a water bath at 30°C to 50°C (50°C was selected in this example) for the duration of the experimental design. After each stage of enzymatic hydrolysis, enzymes from the next stage are added directly without enzyme inactivation, and enzymatic hydrolysis continues until the fourth stage of enzymatic hydrolysis is completed.

[0056] The juice after each stage of enzymatic hydrolysis was centrifuged at 8000 r / min for 5 min, the supernatant was measured, and the juice yield of blue honeysuckle after enzymatic hydrolysis was calculated.

[0057] Juice yield calculation formula:

[0058] Juice yield % = [juice weight (g) × 100%] / pulp mass (g).

[0059] 2. Single factor optimization of enzyme system for step-by-step enzymatic hydrolysis of cell wall

[0060] Experimental Design:

[0061] Accurately weigh a certain amount of crushed and homogenized blue indigo fruit pulp and perform enzymatic hydrolysis in a water bath at 30℃~50℃.

[0062] The enzyme dosage and enzymatic hydrolysis time for the primary and secondary enzymatic hydrolysis are designed as follows:

[0063] Pectinase dosage: 0.1%, 0.2%, 0.3%, 0.4% and 0.5%; the fixed enzymatic hydrolysis time is 20 min.

[0064] Enzyme hydrolysis time: 20min, 30min, 40min, 50min and 60min; the fixed pectinase addition amount is 0.1% to 0.3%.

[0065] Xylanase dosage: 1%, 2%, 3%, 4%, 5%; fixed enzymatic hydrolysis time is 15min.

[0066] Enzyme hydrolysis time: 10min, 20min, 30min, 40min and 50min; the fixed xylanase addition amount was 3%.

[0067] 3. Response surface optimization experiment of enzyme system for step-by-step cell wall degradation

[0068] Based on the results of the single-factor experiment, a multifactor optimization design was conducted using the Box-Behnken response surface methodology, with pectinase dosage, xylanase dosage, and enzymatic hydrolysis time as the factors and juice yield as the response value. The specific factors and levels are shown in Table 1.

[0069] Table 1 Response surface experimental factors and levels

[0070]

[0071] 4. Results and Analysis

[0072] 4.1 Single factor experiment to determine the cell wall degrading enzymes and process parameters of blue loquat

[0073] (1) Pectinase

[0074] like Figure 1 and Figure 2As shown, with the sample without pectinase added as the control, the juice yield of blue honeysuckle initially increases rapidly but then levels off with increasing pectinase dosage. This is because, for a given substrate concentration, a higher pectinase dosage leads to a more complete reaction between the substrate and enzyme, resulting in a higher juice yield. When the pectinase dosage reaches a certain level, the reaction reaches saturation, and the increase in juice yield becomes less noticeable. The juice yield of blue honeysuckle reaches its highest level when the pectinase dosage reaches 0.1% to 0.3%. As the pectinase dosage continues to increase, the increase in juice yield gradually levels off. Using large amounts of pectinase can lead to waste and affect the flavor of blue honeysuckle juice. Therefore, a pectinase dosage of 0.1% to 0.3% was selected for the response surface optimization experiment.

[0075] Taking the pectinase dosage of 0.1% to 0.3% as a fixed value, as the enzymatic hydrolysis time continues to increase, the enzymatic hydrolysis reaction becomes more complete, and the blue indigo fruit juice yield also increases. After 20 minutes of enzymatic hydrolysis, the juice yield tends to be flat. Considering product quality and economic benefits, 10 minutes to 25 minutes is selected as the optimal enzymatic hydrolysis reaction time of pectinase.

[0076] 2. Xylanase

[0077] Xylanase is used to assist pectinase in the reaction, further destroying the plant cell wall structure by decomposing xylan (the main component of hemicellulose in plant cell walls), thereby releasing active substances and water. Xylanase is added to the blue honeysuckle pulp after pectinase enzymolysis. The effect of different xylanase addition amounts and enzymolysis time on the blue honeysuckle juice yield is as follows: Figure 3 and Figure 4 shown.

[0078] like Figure 3 As shown, the juice yield of blueberries continues to increase with increasing xylanase dosage. Using the control group without enzyme addition, the maximum juice yield was achieved at a xylanase concentration of 3%. Further increases in enzyme dosage did not result in further increases in juice yield. Therefore, a xylanase dosage of 2% to 4% was selected for response surface optimization experiments.

[0079] The xylanase addition concentration was 2% to 4%, and the xylanase enzymolysis time experiment was conducted. Figure 4 As shown in the figure, the juice yield increased rapidly within 5-10 min of enzymatic hydrolysis reaction time and gradually stabilized within 10-25 min. Therefore, the enzymatic hydrolysis time of 5-15 min was selected for the response surface optimization experiment.

[0080] 3. Response surface experiment to optimize the process parameters of the blue loquat cell wall degradation enzyme system

[0081] According to the results of the single-factor experiment, the addition of pectinase and xylanase can effectively increase the juice yield of blue loquat. The Box-Behnken response surface methodology was further used to optimize the multi-stage enzymatic hydrolysis process conditions of the cell wall. The experimental results are shown in Table 2.

[0082] Table 2 Response surface design results

[0083]

[0084] (1) Analysis of variance

[0085] The variance analysis of the regression model is shown in Table 3. From the variance analysis in Table 3, it can be seen that the model is relatively significant (P=0.0021<0.01). The variance analysis shows that the model is very effective and the test data is highly reliable. 2 =0.9358 is close to 1, indicating that the model has a good matching degree and a small error. This model is selected to predict the juice yield of blue indigo fruit and analyze the enzymatic hydrolysis process. According to the size of the F value, A, B, C, A 2 、B 2 The P value of BC is less than 0.01, which has a very significant effect on the juice yield; the 0.01<P<0.05 value of BC has a significant effect on the juice yield; the AB, AC, C 2 The P value is greater than 0.05, and the effect on juice yield is not significant. According to the size of the F value, the effect of pectinase addition (A) on juice yield is greater than that of xylanase time (C) and greater than that of xylanase enzymatic hydrolysis amount (B). Among them, the interaction effect of AC is equal to the interaction effect of AB, which is less than the interaction effect of BC. At the same time, the P value of the model lack of fit error is greater than 0.05, and the coefficient of determination of the model R 2 =0.9358, indicating that the model is relatively reliable.

[0086] Table 3 Analysis of variance of regression model

[0087]

[0088] Note: **Extremely significant (P<0.01); *Significant difference (0.01<P<0.05)

[0089] (2) Interaction analysis

[0090] The contour map and response surface diagram of the interaction between pectinase and xylanase on the juice yield of blue loquat are shown in Figure 5 .like Figure 5 There is a relationship between the juice yield of blue indigo fruit and various factors. Figure 5 As shown in Figure a, with the increase of the dosage of pectinase and xylanase, the juice yield of blue honeysuckle first increased and then decreased, and the effect of pectinase on the juice yield of blue honeysuckle was greater than that of xylanase; Figure 5 As can be seen from Figure b, the effect of pectinase dosage on the juice yield of blue loquat fruit is greater than that of xylanase enzymolysis time; Figure 5 As can be seen from Figure c, the effect of xylanase enzymatic hydrolysis time on the juice yield of blue loquat fruit is greater than that of xylanase dosage. This is consistent with the results of variance analysis in Table 2.

[0091] (3) Verification of regression model

[0092] According to the experimental results of response surface analysis, Design-Expert 13.0 was used to conduct regression modeling on the experimental data. The regression equation of blue indigo fruit juice yield was obtained as follows:

[0093] Juice yield / % = 78.9 + 0.0100 × A + 0.0084 × B + 0.0097 × C + 0.0009 × AB + 0.0009 × AC - 0.0028 × BC - 0.0137 × A 2 -0.0137×B 2 -0.0062×C 2 .

[0094] The model predicted the optimal range for enzymatic hydrolysis to be: pectinase addition of 0.1% to 0.3%, xylanase addition of 2% to 4%, and xylanase hydrolysis time of 5 to 15 minutes. More specifically, the optimal conditions were: pectinase addition of 0.2% for 20 minutes, xylanase addition of 3% for 10 minutes, with an estimated juice yield of 79.56%. Under these process conditions, a validation experiment yielded a juice yield of 79.3% for blue indigo fruit, which differed from the predicted value by less than 0.5%, demonstrating that the model can effectively predict the juice yield of blue indigo fruit.

[0095] Example 2 Optimization of the step-by-step enzymatic hydrolysis process for the molecular dissociation of polyphenol active substances in Lonicera edulis cells

[0096] 1. Single-factor optimization of enzyme system for molecular dissociation of polyphenol active substances in edodes fruit cells

[0097] Experimental Design:

[0098] Accurately weigh a certain amount of crushed and homogenized blue honeysuckle pulp, add 0.1% to 0.3% pectinase, and perform enzymatic hydrolysis at 30°C to 50°C for 10 to 25 minutes. Then, add 2% to 4% xylanase and continue enzymatic hydrolysis for 5 to 15 minutes. Based on this, using juice yield as an evaluation indicator, tertiary and quaternary enzymatic hydrolysis is performed on the intracellular active substances, releasing more active substances and further improving the blue honeysuckle juice yield.

[0099] The dosage of each enzyme and the enzymatic hydrolysis time in the three-stage and four-stage enzymatic hydrolysis experiments are designed as follows:

[0100] (1) α-galactosidase dosage: 0.5%, 1%, 1.5%, 2%, 2.5% and 3%;

[0101] Enzyme hydrolysis time: 10min, 20min, 30min, 40min and 50min.

[0102] (2) Acid protease dosage: 2%, 3%, 4%, 5% and 6%;

[0103] Enzyme hydrolysis time: 15min, 30min, 45min, 60min and 75min.

[0104] 2. Response surface optimization experiment of enzyme system for molecular dissociation of polyphenol active substances in edodes fruit

[0105] Response surface optimization experimental design: Based on the results of the previous single-factor experiments, a multi-factor optimization experimental design was conducted using the Box-Behnken response surface methodology, with the α-galactosidase addition, α-galactosidase enzymatic hydrolysis time, and acid protease enzymatic hydrolysis dosage as the factors, and juice yield as the response value. The specific factors and levels are shown in Table 4.

[0106] Table 4 Response surface experimental factors and levels

[0107]

[0108] 3. Single factor experiment to determine the molecular dissociation enzymes and process parameters of blue loquat polyphenol active substances

[0109] 3.1α-galactosidase

[0110] (1) α-galactosidase addition amount

[0111] like Figure 6 As shown, the juice yield of blue honeysuckle after enzymatic hydrolysis with pectinase and xylanase was used as a control. When α-galactosidase was added for enzymatic hydrolysis in the range of 0% to 3%, the juice yield of blue honeysuckle gradually increased with the increase of the addition amount of α-galactosidase, and reached the maximum value after the addition amount was 2.5%. Therefore, it was determined that the optimal addition amount of α-galactosidase was in the range of 2% to 3%.

[0112] (2) α-galactosidase enzymatic hydrolysis time

[0113] like Figure 7As shown, the juice yield of blue loquat fruit increased gradually with increasing enzymatic hydrolysis time, using the juice yield of centrifugation immediately after the addition of α-galactosidase as the control. The rate of increase was fastest between 0 and 10 minutes, reaching over 82% at 15 minutes. After 15 minutes of hydrolysis, the juice yield stabilized with increasing hydrolysis time, confirming that the optimal hydrolysis time for α-galactosidase is between 10 and 30 minutes.

[0114] Experimental results show that the addition of α-galactosidase significantly increases juice yield in blueberries after enzymatic hydrolysis with pectinase and xylanase. This suggests that the presence of galactose in the cell walls of blueberries may play a significant role in their stability, providing a theoretical basis for the use of α-galactosidase to improve juice yield.

[0115] 3.2 Acidic proteases

[0116] (1) Amount of acid protease added

[0117] like Figure 8 As shown in the results, based on the addition of pectinase, xylanase, and α-galactosidase, with 2% acidic protease as the control, increasing the protease addition from 2% to 6% significantly increased the juice yield of blue loquat (P < 0.05). At 6% protease addition, the juice yield exceeded 85%, and no downward trend was observed within this range. Therefore, 6% is considered the optimal protease addition under these experimental conditions. The effect of further increasing the protease addition on juice yield requires further study.

[0118] (2) Acid protease enzymatic hydrolysis time

[0119] like Figure 9 As shown, the juice yield of blue loquat fruit increased gradually with the extension of the acid protease enzymatic hydrolysis time, using the juice yield obtained by centrifugation immediately after the addition of acid protease as the control. The juice yield reached a maximum of approximately 85% at 15 minutes. Thereafter, the juice yield leveled off with increasing enzymatic hydrolysis time, showing no significant change. Therefore, the optimal enzymatic hydrolysis time for acid protease was determined to be 10 to 20 minutes.

[0120] Experimental results show that after enzymatic hydrolysis with pectinase, xylanase, and α-galactosidase, the addition of acid protease significantly increases the juice yield of blue honeysuckle. Acid protease hydrolyzes proteins in the blue honeysuckle cell walls, disrupting the cell wall structure to a certain extent. As a crucial component of the cell wall, protein degradation not only weakens the integrity of the cell wall but also helps release internal cell components, further increasing the juice yield of blue honeysuckle.

[0121] 3.3 Optimization of Process Parameters of the Intracellular Polyphenol Active Substance Molecular Dissociation Enzyme System of Lonicera caerulea by Response Surface Experiment

[0122] According to the results of single-factor experiments, the addition of α-galactosidase and acid protease can effectively improve the juice yield of Lonicera caerulea. To optimize the process conditions, the Box-Behnken response surface method was used for optimization experiments. The specific factors and levels are shown in Table 4, and the specific experimental results are shown in Table 5.

[0123] Table 5 Results of Response Surface Experiment

[0124]

[0125]

[0126] (1) Analysis of Variance

[0127] Table 6 Analysis of Variance of Regression Model

[0128]

[0129] Note: **Extremely significant (P < 0.01); *Significant difference (0.01 < P < 0.05)

[0130] From the results in Table 6, according to the magnitude of the P value, it can be seen that the model has extremely significant significance (P = 0.0007 < 0.01), indicating that the overall regression effect of the model is good. Among them, the P values of C, A 2 , B 2 , C 2 are < 0.01, and the influence on the juice yield is extremely significant. The P values of A, BC are 0.01 < P < 0.05, and the influence on the juice yield is significant. The P values of B, AB, AC are P > 0.05, and the influence on the juice yield is not significant. According to the magnitude of the F value, it can be seen that the influence of the addition amount of acid protease (C) on the juice yield is greater than that of the dosage of α-galactosidase (A) which is greater than the enzymatic hydrolysis time of α-galactosidase (B). Among them, the interaction of BC is greater than the interaction of AC which is greater than the interaction of AB. At the same time, the P value of the lack-of-fit error of the model is > 0.05, and the determination coefficient R 2 = 0.9538, indicating that the model is relatively reliable.

[0131] (2) Analysis of Interaction

[0132] The response surface diagram and contour diagram of this time are as Figure 10 shown.

[0133] From Figure 10 a in, it can be seen that the influence of the addition amount of α-galactosidase on the juice yield is greater than the enzymatic hydrolysis time of α-galactosidase. From Figure 10 b in, it can be seen that the influence of the addition amount of acid protease on the juice yield is greater than the addition amount of α-galactosidase. From Figure 10 From Figure c, we can see that the effect of the amount of acid protease added on the juice yield is greater than the enzymatic hydrolysis time of α-galactose. At the same time, from the shape of the contour map, the interaction of BC is greater than that of AB, and the results are consistent with the results of variance analysis.

[0134] (3) Verification of regression model

[0135] According to the experimental results of response surface analysis, the experimental data were regression modeled using Design-Expert 13.0. The regression equation of the juice yield of blue honeysuckle was as follows: juice yield / % = 87.22 + 0.47 × A + 0.14 × B + 1.17 × C - 0.038 × AB + 0.28 × AC + 0.63 × BC - 0.80 × A 2 -0.77×B 2 -1.15×C 2 The optimal enzymatic hydrolysis conditions predicted by the model are: α-galactosidase addition 1.5-3.5%, enzymatic hydrolysis time 20-25 minutes, acid protease addition 5-9.5%. The expected juice yield is 87.6

[0136] 3%. Under this process condition, the verification test was carried out and the blue indigo fruit juice yield was 89% (see Figure 11 ), the difference from the predicted value is less than 1%, indicating that the model can better predict the juice yield of blue indigo fruit.

[0137] from Figure 11 As can be seen, the juice yields of the various enzymatic hydrolysis groups were: 61% for direct juicing, 70% for primary hydrolysis, 79% for secondary hydrolysis, 83% for tertiary hydrolysis, and 89% for quaternary hydrolysis. This indicates that sequential enzymatic hydrolysis significantly improves the juice yield of blue loquat fruit, with the yield increasing as the hydrolysis level increases. Juices from primary hydrolysis (pectinase), secondary hydrolysis (pectinase + xylanase, with each enzyme added step by step), tertiary hydrolysis (pectinase + xylanase + α-galactosidase, with each enzyme added step by step), and quaternary hydrolysis (pectinase + xylanase + α-galactosidase + acid protease, with each enzyme added step by step) increased juice yield by 15%, 30%, 36%, and 46%, respectively, compared to the direct juicing group.

[0138] Based on the above, the optimal enzymatic hydrolysis process conditions are determined as follows:

[0139] Primary enzymatic hydrolysis: pectinase concentration 0.1% to 0.3% (w / w), action time 10 to 25 minutes. Under these conditions, pectinase breaks down the pectin in the intercellular layer, weakening cell adhesion and increasing juice yield to 70%.

[0140] Secondary enzymatic hydrolysis: Xylanase concentration 2% to 4% (w / w), action time 5 to 15 minutes. Xylanase degrades primary wall hemicellulose, loosens the cell wall skeleton, makes the cell structure looser, and increases juice yield to 79%.

[0141] Tertiary enzymatic hydrolysis: α-galactosidase concentration 0.5% to 3.5% (w / w), action time 20 to 25 minutes. α-galactosidase acts on the galactose side chains of the secondary cell wall, loosening the cell wall structure and further reducing the mechanical strength of the cell wall, increasing the juice yield to 83%.

[0142] Level 4 enzymatic hydrolysis: Acidic protease concentration 5.0% to 9.5% (w / w), action time 10 to 15 minutes. Juice yield increased to 89%.

[0143] Subsequently, functional verification was performed under the optimal process conditions. Specifically, the following conditions were used as an example to perform enzymatic hydrolysis and then determine the function of the enzymatic hydrolysis product:

[0144] The added amount of pectinase was 0.2% and the enzymatic hydrolysis time was 20 minutes; the added amount of xylanase was 3% and the enzymatic hydrolysis time was 10 minutes; the added amount of α-galactosidase was 3.28% and the enzymatic hydrolysis time was 25 minutes; the added amount of acid protease was 9.15% and the enzymatic hydrolysis time was 15 minutes.

[0145] Example 3: Step-by-step enzymatic hydrolysis technology significantly improves the release of polyphenol active ingredients

[0146] 1. Determination of total phenol content

[0147] Preparation method of gallic acid standard stock solution (1 mg / mL): Use an analytical balance to weigh 10 mg of gallic acid standard, add about 8 mL of deionized water, mix thoroughly, then dilute to the 10 mL mark with deionized water, mix again, transfer to a brown reagent bottle and store at 4°C in the dark.

[0148] Preparation of Gallic Acid Standard Working Solution (10 mL): Dispense 0.2, 0.4, 0.6, 0.8, and 1.0 mL of a 1 mg / mL gallic acid stock solution into five 10 mL volumetric flasks. Add deionized water to the mark and mix by inversion to obtain 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL gallic acid standard working solutions. Store at 4°C in the dark and use immediately after preparation.

[0149] Preparation of standard curve: Use a pipette to draw 0.5 mL of each of the 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL gallic acid standard working solutions, add 2.5 mL of Folin-phenol reagent, shake well, and let stand in the dark for 5 minutes. Then add 2 mL of 7.5% sodium carbonate solution, place in a 25°C water bath and keep warm in the dark for 60 minutes. Measure the absorbance at a wavelength of 765 nm and make a standard curve based on the data.

[0150] Standard curve Figure 12 As shown, total polyphenol content: y = 67.271x + 0.0645, R 2 =0.9923.

[0151] Sample determination: Use a pipette to accurately draw 0.02 mL of sample into a 10 mL volumetric flask, add 2.5 mL of Folin-phenol reagent, shake well, and let stand in the dark for 5 minutes. Then add 2.48 mL of 7.5% sodium carbonate solution, place in a 25°C water bath, keep warm in the dark for 60 minutes, and measure the absorbance at a wavelength of 765 nm.

[0152] 2. Determination of anthocyanin content

[0153] Pipette 0.02 mL of blue honeysuckle juice, add 4.98 mL of pH 1.0 potassium chloride buffer and pH 4.5 sodium acetate buffer, measure the sample absorbance at 520 nm and 700 nm, use the corresponding buffer as blank, and calculate the anthocyanin concentration in blue honeysuckle juice according to the following formula:

[0154] Anthocyanin content (mg / g) = (A × D × M × 250) / (ε × L);

[0155] A:A=(A 520nm -A 700nm )pH1.0-(A 520nm -A 700nm )pH4.5;

[0156] M: 449.2 g / mol, molecular weight of cyanidin-3-glucoside;

[0157] D: dilution factor of the test solution (250);

[0158] L: optical path length, cm;

[0159] ε: 26900, M-1·cm -1 (Molar extinction coefficient of cyanidin-3-glucoside).

[0160] 3. Determination of Vitamin C (Ascorbic Acid) Content

[0161] Preparation of vitamin C standard curve: Accurately weigh 100 mg of analytical pure vitamin C, dissolve it with 0.5 mol / L NaCl solution, transfer it to a 100 mL volumetric flask, make up to volume, shake well, and prepare a 1 mg / mL standard stock solution. Take 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of this standard solution, respectively, and inject them into 6 50 mL volumetric flasks, and then make up to the scale with 0.5 mol / L NaCl solution, shake well, and prepare vitamin C standard solutions with concentrations of 0, 8, 16, 24, 32, and 40 μg / mL, respectively. Use 0.5 mol / L NaCl as a blank reference at 245 nm, measure the absorbance of each standard solution, and draw an absorbance-concentration standard curve. The standard curve is as follows: Figure 13 As shown, vitamin C content: y = 0.0025x + 0.0056, R 2 =0.9906.

[0162] Sample pretreatment: After squeezing the juice from the blue indigo fruit, accurately aspirate 0.1 mL (100 μL) of juice with a pipette and add it to a 50 mL volumetric flask. Then, dilute to the mark with 0.5 mol / L NaCl solution, cover the flask with a stopper and shake thoroughly to prepare the sample solution.

[0163] Sample Determination: Using a 0.5 mol / L NaCl solution as a reference, measure the absorbance of the sample solution at a wavelength of 245 nm using a UV spectrophotometer. Based on the measured absorbance, find the corresponding vitamin C concentration on the standard curve and calculate the actual vitamin C content in the original juice based on the sample dilution factor.

[0164] 4. Results and Analysis

[0165] like Figure 14As shown, after each enzymatic hydrolysis treatment, the total phenolic content was 2.95 mg / mL for the direct juice group, 4.32 mg / mL for the primary hydrolysis group, 4.64 mg / mL for the secondary hydrolysis group, 5.23 mg / mL for the tertiary hydrolysis group, and 5.73 mg / mL for the quaternary hydrolysis group. Compared with the direct juice group, the total phenolic content of the primary, secondary, tertiary, and quaternary hydrolysis juices increased by 47%, 57%, 77%, and 94%, respectively. The total anthocyanin content was 2.22 mg / mL for the direct juice group, 3.13 mg / mL for the primary hydrolysis group, 3.36 mg / mL for the secondary hydrolysis group, 3.40 mg / mL for the tertiary hydrolysis group, and 3.74 mg / mL for the quaternary hydrolysis group. Compared with the direct juice group, the anthocyanin content of the primary, secondary, tertiary, and quaternary hydrolysis juices increased by 41%, 51%, 53%, and 68%, respectively. The vitamin C content was 2.88 mg / mL in the direct juicing group, 2.86 mg / mL in the primary enzymatic hydrolysis group, 2.89 mg / mL in the secondary enzymatic hydrolysis group, 2.87 mg / mL in the tertiary enzymatic hydrolysis group, and 2.85 mg / mL in the quaternary enzymatic hydrolysis group. Compared to the direct juicing group, the active substances total polyphenols and anthocyanins in the blue loquat juice increased significantly after each enzymatic hydrolysis step. This indicates that the hierarchical structure of the blue loquat cell wall and the distribution of active ingredients are fully destroyed by the successive enzymatic hydrolysis steps, maximizing the release of various active substances. However, the vitamin C concentration in the juice did not change significantly with each subsequent enzymatic hydrolysis step. This is because the juice yield of the blue loquat increased by 46% after the fourth enzymatic hydrolysis step, which diluted the released vitamin C, creating the illusion that vitamin C release did not increase. However, the absolute content of vitamin C released per unit mass of blue loquat does increase after the fourth enzymatic hydrolysis step.

[0166] Example 4: Step-by-step enzymatic hydrolysis technology significantly enhances the antioxidant capacity of blue loquat fruit juice

[0167] 1. DPPH free radical scavenging ability determination

[0168] Sample determination: Dilute the sample with anhydrous methanol to (2, 4, 6, 8, 10 mg / mL) samples, take 1 mL of the sample with different concentrations into a 10 mL centrifuge tube, add 1 mL of 0.2 mmol / L DPPH methanol solution, measure the absorbance of the reaction system at 517 nm, and use ascorbic acid (V c ) was used as the control, and the free radical scavenging rate of the sample was calculated according to the formula, and the scavenging rate curve was drawn.

[0169] Sample blank control: Take 1 mL of anhydrous methanol in a 10 mL centrifuge tube, add 1 mL of 0.2 mmol / L DPPH methanol solution, and measure the absorbance of the reaction system at 517 nm, which is recorded as A0.

[0170] Sample background control: Take 1 mL of samples of different concentrations into a 10 mL centrifuge tube, add 1 mL of anhydrous methanol, mix thoroughly, and record the absorbance value at 517 nm as A2.

[0171] The calculation formula is as follows:

[0172] DPPH clearance rate (%) = [1-(A1-A2) / A0] × 100%;

[0173] A0: absorbance value without sample, with anhydrous methanol replacing the sample;

[0174] A1: absorbance value after the sample solution reacts with DPPH free radical;

[0175] A2: The absorbance value of the sample solution, i.e., using anhydrous methanol instead of DPPH solution.

[0176] 2. Determination of total reducing capacity

[0177] Sample determination: Dilute the sample with pH 6.6 phosphate buffer to (2, 4, 6, 8, 10 mg / mL) samples, take 1 mL of sample of different concentrations in a 10 mL centrifuge tube, add 0.5 mL of 1% K3[Fe(CN)6], stir evenly, and heat in a 50°C water bath for 20 minutes. After the reaction is completed, add 0.5 mL of 10% trichloroacetic acid, centrifuge at 12000 r / min, 1 mL of supernatant is taken, add 0.3 mL of 0.2% FeCl3 solution, and let it stand at room temperature for 30 minutes. Measure the sample absorbance at 700 nm using a microplate reader.

[0178] Sample blank control: Take 1 mL of pH 6.6 phosphate buffer in a 10 mL centrifuge tube, add 0.5 mL of 1% K3[Fe(CN)6], stir evenly, and heat in a 50°C water bath for 20 minutes. After the reaction is completed, add 0.5 mL of 10% trichloroacetic acid, centrifuge at 12000 r / min for 10 minutes, take 1 mL of the supernatant, add 0.3 mL of 0.2% FeCl3 solution, and let it stand at room temperature for 30 minutes. Measure the sample absorbance at 700 nm using a microplate reader.

[0179] Sample background control: Take 1 mL of sample of different concentrations in a 10 mL centrifuge tube, add 0.5 mL of 1% K3[Fe(CN)6], stir evenly, and heat in a 50°C water bath for 20 min. After the reaction is completed, add 0.5 mL of 10% trichloroacetic acid, centrifuge at 12000 r / min, 10 min, take 1 mL of supernatant, add 0.2 mL of pH 6.6 phosphate buffer, and let it stand at room temperature for 30 min. Measure the sample absorbance at 700 nm using a microplate reader.

[0180] 3. Results and Analysis

[0181] 3.1 DPPH free radical scavenging ability

[0182] With ascorbic acid group as control, the DPPH free radical scavenging ability of each group of juice was tested by enzymatic hydrolysis in the range of 0-10 mg / mL. 50 See Table 7. As shown in the table, with the deepening of the enzymatic hydrolysis, the IC 50 The IC values ​​of the first, second, third and fourth enzymatic hydrolyzed juices were significantly lower than those of the direct juice group. 50 They decreased by 9.5%, 15.6%, 21.6% and 28.3% respectively, which means that as the enzymatic hydrolysis progresses, the ability of the juice to scavenge DPPH free radicals is significantly enhanced.

[0183] Table 7 IC of DPPH radical scavenging rate of each group of juice by stepwise enzymatic hydrolysis 50

[0184]

[0185] The results of the DPPH free radical scavenging rate test of each group of juice by step-by-step enzymatic hydrolysis are as follows Figure 15 As shown in the figure, the DPPH free radical capacity of each group of juices increased significantly with the increase of concentration.

[0186] At a juice concentration of 2 mg / mL, the DPPH radical scavenging rates of the juices in each of the sequential enzymatic hydrolysis groups were: 14% for the fourth-stage hydrolysis group, 10% for the third-stage hydrolysis group, 3.8% for the second-stage hydrolysis group, 1.4% for the first-stage hydrolysis group, and 0% for the direct-juice group. This means that after the fourth-stage hydrolysis, the DPPH radical scavenging rate of the juice at a concentration of 2 mg / mL increased by 14% compared to the direct-juice group without enzymatic hydrolysis.

[0187] At a juice concentration of 10 mg / mL, the scavenging rates of the juices in each group were: 98% for the ascorbic acid group, 73.4% for the four-stage enzymatic hydrolysis group, 70.8% for the three-stage enzymatic hydrolysis group, 65.3% for the two-stage enzymatic hydrolysis group, 60.9% for the one-stage enzymatic hydrolysis group, and 53.6% for the direct-juice group. This means that after the four-stage enzymatic hydrolysis, the DPPH radical scavenging capacity of the juice at a concentration of 10 mg / mL increased by 37% compared to the direct-juice group without enzymatic hydrolysis.

[0188] In summary, within the juice concentration range of 0-10 mg / mL, the DPPH radical scavenging ability of Lonicera edulis juice after step-by-step enzymatic hydrolysis gradually increased with the increase of enzymatic hydrolysis stages, which is consistent with the results in Table 7.

[0189] 3.2 Total reduction capacity

[0190] like Figure 16As shown, the total reducing power was tested in the concentration range of 0-10 mg / mL with ascorbic acid group as control. When the juice concentration was 2 mg / mL, the OD of the total reducing power of each group of juice was obtained by stepwise enzymatic hydrolysis. 700nm The values ​​were as follows: 0.601 for the four-stage enzymatic hydrolysis group, 0.534 for the three-stage enzymatic hydrolysis group, 0.534 for the two-stage enzymatic hydrolysis group, 0.369 for the one-stage enzymatic hydrolysis group, and 0.326 for the direct juice extraction group. The total reducing power of the juice after the four-stage enzymatic hydrolysis was 84.4% higher than that of the juice extracted without enzymatic hydrolysis. When the juice concentration was 10 mg / mL, the OD values ​​of the juices in each group were 700nm The values ​​for the ascorbic acid group were 2.595, the four-stage enzymatic hydrolysis group was 1.833, the three-stage enzymatic hydrolysis group was 1.691, the two-stage enzymatic hydrolysis group was 1.731, the one-stage enzymatic hydrolysis group was 1.524, and the direct juice group was 1.385. The total reducing power of the juice after the four-stage enzymatic hydrolysis was 32.3% higher than that of the direct juice without enzymatic hydrolysis. This indicates that within the concentration range of 0 to 10 mg / mL, the total reducing power of the blue loquat fruit juice after the step-by-step enzymatic hydrolysis was lower than that of the ascorbic acid group, but it gradually increased with the increase in the number of enzymatic hydrolysis stages. The antioxidant capacity of blue loquat fruit juice gradually increased with the increase in the number of enzymatic hydrolysis stages, confirming that the cell wall structure is gradually destroyed during the step-by-step enzymatic hydrolysis process, releasing more active substances (such as polyphenols and anthocyanins), thereby enhancing the antioxidant capacity of the juice.

[0191] The above results show that after the four-stage enzyme hydrolysis, the DPPH free radical scavenging rate of the juice increased by 37% compared with direct juicing, and the total reducing power increased by 32.3%, which is consistent with the increase in the release rate of active ingredients.

[0192] In summary, the four-stage enzymatic hydrolysis technology of the present invention is significantly superior to direct juicing and two-stage enzymatic hydrolysis in terms of juice yield, active ingredient content and antioxidant capacity, fully demonstrating the advantages and innovation of this technology.

[0193] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A process for increasing the yield and active ingredient content of Lonicera edulis fruit juice, characterized in that: The following steps are involved: The blue loquat fruit is beaten into pulp, and pectinase is added to perform primary enzymolysis to obtain enzymolysis product I; adding xylanase to the enzymatic hydrolysis product I for secondary enzymatic hydrolysis to obtain an enzymatic hydrolysis product II; adding α-galactosidase to the enzymatic hydrolysis product II for tertiary enzymatic hydrolysis to obtain an enzymatic hydrolysis product III; Acidic protease is added to the enzymatic hydrolysis product III to perform a fourth-stage enzymatic hydrolysis to obtain an enzymatic hydrolysis product IV.

2. The process according to claim 1, wherein: The conditions for the primary enzymatic hydrolysis are: pectinase concentration of 0.1% to 0.3%, w / w; temperature of 30° C. to 50° C.; and enzymatic hydrolysis time of 10 min to 25 min.

3. The process according to claim 1, wherein: The conditions for the secondary enzymatic hydrolysis are: xylanase concentration of 2% to 4%, w / w; temperature of 30° C. to 50° C.; and enzymatic hydrolysis time of 5 min to 15 min.

4. The process according to claim 1, wherein: The conditions for the tertiary enzymatic hydrolysis are as follows: the concentration of α-galactosidase is 0.5% to 3.5%, w / w; the temperature is 30° C. to 50° C.; and the enzymatic hydrolysis time is 20 min to 25 min.

5. The process according to claim 1, wherein: The conditions for the four-stage enzymatic hydrolysis are: the concentration of acidic protease is 5.0% to 9.5%, w / w; the temperature is 30° C. to 50° C.; and the enzymatic hydrolysis time is 10 min to 15 min.

6. Use of the process according to any one of claims 1 to 5 in improving the yield of Lonicera edulis fruit juice.

7. Use of the process according to any one of claims 1 to 5 in increasing the total phenol content of Lonicera edulis fruit juice.

8. Use of the process according to any one of claims 1 to 5 in increasing the anthocyanin content in Lonicera edulis fruit juice.

9. Use of the juice prepared by the process according to any one of claims 1 to 5 in preparing products that help to improve antioxidant capacity.

10. The use according to claim 9, characterized in that The antioxidant indicators are DPPH clearance rate and total reducing power.

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