Xylanase mutant with improved enzymatic properties and application thereof

Abstract

According to the xylanase mutant with the improved enzymatic property and the application of the xylanase mutant, sites of D116 and E119 of xylanase XynASP are mutated, the amino acid sequence of the obtained mutant is shown as SEQ ID NO.1-SEQ ID NO.3, or the amino acid sequence has at least 90% of homology with the amino acid sequence, and the function of clarified fruit juice is reserved. In the fruit juice clarification application, the clarification rates of the wild type mango juice and the dragon fruit juice are increased to the maximum values of 53.34% and 60.16% in 90 min and 105 min, and the XynASP-SV reaches the maximum values of 65.73% and 75.61% in 75 min. The xylanase mutant obtained by introducing two mutation sites in a cord region has significantly improved enzymatic properties compared with a wild type enzymatic property, and shows a high application prospect in clarification experiments of mango juice and pitaya juice.

Description

Technical Field

[0001] The invention belongs to the technical field of xylanase, and particularly relates to a xylanase mutant with improved enzymatic properties and application thereof in fruit juice clarification. Background Art

[0002] Endo-β-1,4-xylanases, commonly referred to as xylanases (EC 3.2.1.8), are a class of glycoside hydrolases that specialize in the hydrolysis of xylan. Their unique function is that they can accurately break the β-1,4-glycosidic bonds in the xylan backbone to generate functional oligosaccharides, such as xylobiose, xylotriose, and higher-order xylo-oligosaccharides. Based on differences in structural characteristics, catalytic mechanisms, and physicochemical properties, xylanases are subdivided into multiple families, including 5, 7, 8, 10, 11, and 43. Among them, the GH10 and GH11 families are particularly prominent, and the GH11 family is defined as a typical representative of xylanases in the CAZy database. GH11 family xylanases are known for their excellent catalytic activity and substrate specificity, and can efficiently convert xylan into oligosaccharide products such as xylobiose and xylotriose.

[0003] However, although the GH11 family xylanases have excellent catalytic efficiency, most natural members are mesophilic enzymes. They are not stable enough in high temperature environments and are difficult to adapt to extreme conditions such as high temperature, strong acid or strong alkali often encountered in industrial production. Therefore, the optimization and improvement of their performance is particularly urgent. Studies have shown that although enhancing the rigidity of the enzyme structure can effectively improve its thermal stability, it is often accompanied by a significant decrease in enzyme activity. How to improve thermal stability while maintaining high catalytic activity has become a major challenge in current research. In particular, the cord region in xylanase, as a highly flexible peptide chain, can adjust its conformation according to the substrate morphology during the catalytic process to promote substrate binding. This property is crucial for the efficient catalytic effect of the enzyme. Qin et al. introduced three mutants (SrxynF, SrxynM, SrxynFM) into the cord region of the xylanase Srxyn from Streptomyces rochei L10904 through protein engineering technology, and successfully increased the enzyme activity to 2.1, 3.2 and 5.3 times that of the wild type, respectively. This finding highlights the potential impact of the modification of the cord region on the catalytic performance of xylanase. Nevertheless, there is still a lack of comprehensive and systematic exploration of how the mutation of the cord region specifically affects the thermal stability and enzyme activity of xylanase.

[0004] Xylanase occupies an important position in the fields of textile industry, bioenergy development and food processing due to its wide application prospects. Especially in juice processing, freshly squeezed juice contains a large amount of polysaccharide components such as cellulose and hemicellulose, which not only increase the viscosity of the juice, but also hinder the precipitation of solid particles, resulting in turbidity of the juice. Xylanase can specifically decompose hemicellulose and convert it into small molecules such as oligosaccharides and xylose, effectively reducing the viscosity of the juice and promoting the sedimentation of solid particles, thereby improving the clarity of the juice. Existing studies have shown that the optimal application conditions for xylanases from different sources in juice clarification are temperature 40-60℃, time 60-150 minutes, and pH 3.0-5.0. In view of the application limitations of medium-temperature xylanases, it is of great significance to develop xylanases that are resistant to high temperature, acid and have higher catalytic efficiency through rational or semi-rational design strategies for the improvement of juice clarification process. Summary of the invention

[0005] Technical problem to be solved: In order to solve the above problems, the present invention provides a xylanase mutant with improved enzymatic properties and its application, involving two potential beneficial mutation sites, and combining the mutations to form a xylanase mutant XynASP-SV with improved enzymatic properties, wherein the thermal stability and catalytic efficiency are improved, and the optimum temperature of the mutant XynASP-SV is increased by 5°C to 50°C compared with the wild type; T m The value increased by 6℃ to 52℃, and the half-life at 50℃ increased by 4.62 times to 24.51min; the conversion efficiency (kcat / Km) increased by 27.7 times to 1765.80mL mg -1 s -1 The specific enzyme activity was 156U / mg, which was 3.04 times higher than that of the wild type. In a low-acid environment (pH 3.0), XynASP-SV still maintained a high catalytic ability, and the residual enzyme activity was 30% higher than that of the wild type enzyme. In the juice clarification application, the improvement of the clarification rate of mango juice and pitaya juice by XynASP-SV reached the maximum at 75min, with the clarification rate improvement values ​​reaching 65.73% and 75.61%.

[0006] Technical solution: A xylanase mutant, wherein the D116 and E119 sites of the xylanase XynASP are mutated, and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.1-SEQ ID NO.3, or an amino acid sequence having at least 90% homology with the above amino acid sequence, and retains the function of clarifying juice.

[0007] Among them, the amino acid sequence of the xylanase mutant D116S is shown in SEQ ID NO.1; the amino acid sequence of the xylanase mutant E119V is shown in SEQ ID NO.2; and the amino acid sequence of the xylanase mutant XynASP-SV is shown in SEQ ID NO.3.

[0008] The gene encoding the above xylanase mutant has a nucleotide sequence as shown in SEQ ID NO.4-SEQ ID NO.6, or a nucleic acid sequence having at least 90% homology with the above nucleic acid sequence, and can encode a protein with the function of clarifying juice.

[0009] Among them, the nucleotide sequence of the xylanase mutant D116S is shown in SEQ ID NO.4; the nucleotide sequence of the xylanase mutant E119V is shown in SEQ ID NO.5; and the nucleotide sequence of the xylanase mutant XynASP-SV is shown in SEQ ID NO.6.

[0010] An expression vector containing the above nucleic acid sequence.

[0011] Preferably, the above expression vector is a plasmid.

[0012] A host cell containing the above expression vector.

[0013] Preferably, the host cell is Escherichia coli or yeast.

[0014] A method for producing xylanase using the host cell comprises culturing the host cell and collecting the xylanase mutant expressed by the cell.

[0015] The use of the above xylanase mutants in the fruit juice clarification process.

[0016] Preferably, the fruit juice is apple juice, orange juice, peach juice, mango juice or pitaya juice.

[0017] Beneficial effects: The present invention has found through determination that the optimum pH of the mutant XynASP-SV is 5.0, which is slightly lower than that of the wild type (pH 6.0). When kept at 40°C for 1 hour under pH 3.0-9.0 conditions, the residual enzyme activity of XynASP-SV under various pH conditions is greater than that of the wild type; the optimum temperature of the mutant XynASP-SV is increased by 5°C to 50°C compared with the wild type; when treated at 50°C and pH 5.0 for 60 minutes, the wild type only retains 16.84% of the enzyme activity, while the enzyme activity of the mutant XynASP-SV is 55.16%, which is 3.27 times higher than that of the wild type; mThe value increased by 6℃ to 52℃; the half-life at 50℃ increased by 4.62 times to 24.51min; the conversion efficiency (kcat / Km) increased by 27.7 times to 1765.80mL mg -1 s -1 ; The specific enzyme activity was increased by 3.04 times compared with the wild type, reaching 156U / mg. XynASP-SV showed good acid tolerance, with residual enzyme activities of 75.86%, 89.01%, and 83.92% at pH 3.0-5.0, which were improved compared with the residual enzyme activities of 45.86%, 65.69%, and 60.10% of the wild type. In the application of juice clarification, the maximum improvement of the clarification rate of mango juice and pitaya juice by the wild type was 53.34% and 60.16% at 90min and 105min, respectively, while XynASP-SV reached the maximum at 75min, which was 65.73% and 75.61%. The xylanase mutant obtained by introducing two mutation sites in the cord region of the present invention has significantly improved enzymatic properties compared with the wild type, and shows a high application prospect in the clarification experiments of mango juice and pitaya juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the three-dimensional model of xylanase XynASP.

[0019] Figure 2 These are the optimum temperature and optimum pH for xylanases XynASP, D116S, E116V and XynASP-SV, where A is the optimum temperature and B is the optimum pH.

[0020] Figure 3 The pH thermostability of xylanases XynASP, D116S, E116V and XynASP-SV.

[0021] Figure 4 The thermostability of xylanases XynASP, D116S, E116V and XynASP-SV at 50°C.

[0022] Figure 5 The clarification rate of mango juice and pitaya juice treated with xylanase XynASP and XynASP-SV. DETAILED DESCRIPTION

[0023] The present invention discloses a xylanase mutant, a preparation method and application thereof, a DNA molecule encoding the xylanase mutant, a vector, and a host cell. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0024] Materials required for the experiment in Example 1:

[0025] The xylanase XynASP gene was derived from Aspergillus saccharolyticus JOP1030-1. Escherichia coli DH5α, Escherichia coli BL21 (DE3) and pET-28a (+) were purchased from Novagen. The recombinant plasmid pET-28a-xynASP was fully synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. High-fidelity DNA polymerase, deoxyribonucleoside triphosphates (dNTPs) and restriction endonuclease DpnI were purchased from New England Biolabs. Isopropyl-β-D-thiogalactopyranoside (IPTG) and kanamycin (Kan) were purchased from Takara. The beech xylan used in this study was purchased from Megazyme. SYPRO dye was purchased from Sigma-Aldrich, and other reagents were domestic analytical grade.

[0026] Preparation of main reagents:

[0027] LB medium:

[0028] Weigh 0.5 g yeast powder, 1 g peptone, and 1 g NaCl, fully dissolve in 70 mL deionized water, dilute to 100 mL, and sterilize at 121°C for 20 min.

[0029] Solid LB medium: Add 2% agar powder to the liquid medium.

[0030] TB medium recipe

[0031] 6 g of tryptone, 12 g of yeast extract, 2 mL of glycerol, add distilled water to 450 mL, divide into 500 mL Erlenmeyer flasks, 90 mL per bottle, for a total of 5 bottles.

[0032] 3,5-Dinitrosalicylic acid (DNS reagent)

[0033] First weigh 91g of potassium sodium tartrate and dissolve it in 300mL of hot water. Then add 10.48g of NaOH, 3.15g of 3,5-dinitrosalicylic acid, 2.5g of phenol, and 2.5g of anhydrous sodium sulfite in sequence. Stir and heat to dissolve. After cooling, make up to 500mL. Protect from light and store at room temperature. Use after about a week.

[0034] Disodium hydrogen phosphate-citrate buffer (pH 5.0)

[0035] Weigh 33.5 g of disodium hydrogen phosphate and 11.2 g of citric acid, dissolve in 800 mL of ddH2O, make up to 1000 mL, and adjust the pH to 5.0.

[0036] Preparation of 0.5% beech wood xylan solution

[0037] Take 100 mL of disodium hydrogen phosphate-citric acid buffer (pH 4.6), weigh 0.5 g of beech wood xylan, put it into a conical flask, heat to boiling, boil for 5 minutes, cool, dilute to 100 mL with distilled water, and store at 4°C.

[0038] Construction of mutant model and determination of mutation sites

[0039] The amino acid sequence of the recombinant xylanase XynASP was submitted to Alphafold2 (https: / / alphafold2.biodesign.ac.cn / ) for homology modeling, and the 5 models obtained were evaluated by Predicted Local Distance Difference Test (PLDDT) and the model quality distribution map was drawn to determine the most reasonable model structure. The specific steps were performed according to the default program of the software. After obtaining a high-quality model, the protein sequence was analyzed and the structure of the model was divided. The cord region is 12 amino acids connecting B5 and B6 in β-sheet B (Asp116, Tyr117, Gly118, Glu119, Tyr120, Asn121, Pro122, Gly123, Ser124, Ala125, Gly126, and Thr127).

[0040] Enzyme activity assay

[0041] DNS method was used. 1.5 mL of 0.5% beechwood xylan and 1 mL of appropriately diluted enzyme solution were reacted at an appropriate temperature for 15 min, 2.5 mL of DNS was added to terminate the reaction, and the reaction was boiled in a water bath for 7 min; after cooling, 5 mL of ddH2O was added, oscillated and mixed, and the absorbance was measured at a wavelength of 540 nm. The activity unit (U) of xylanase is defined as the amount of enzyme required to hydrolyze xylan to produce 1 μmol of xylose per min under the above-mentioned activity determination conditions.

[0042] Construction of saturation library of mutation sites and library screening

[0043] Using pET-28a-xynASP as template, D116X-F, Y117X-F, G118X-F, E119X-F, Y120X-F, N121X-F, P122X-F, G123X-F, S124X-F, A125X-F, G126X-F, and T127X-F as upstream primers (Table 1), and T7-R as downstream primer, saturation mutation was performed on Asp116, Tyr117, Gly118, Glu119, Tyr120, Asn121, Pro122, Gly123, Ser124, Ala125, Gly126, and Thr127 codons by whole-plasmid PCR. The PCR products were digested with DpnI and transformed into E. coli BL21 (DE3) to construct a saturation mutation library. For each mutation site, 97 transformants ( E. coli / xynD116X, E. coli / xyn Y117X, E. coli / G118X, E. coli / xyn E119X, E. coli / xyn Y120X, E. coli / xyn N121X, E. coli / xynP122X, E. coli / xyn G123X, E. coli / S124X, E. coli / xynA125X, E. coli / xynG126X, and E. coli / xyn T127X, where X represents any amino acid) were selected from LB solid medium, inoculated into 1 mL of LB medium containing Kan (final concentration of 50 μg / mL), cultured at 37°C, 220 r / min for 12 h, and transferred to 1 mL of the same medium at a 2% inoculum volume fraction, and cultured until OD 600When the concentration is about 0.6-0.8, add 0.5mmol / L IPTG and induce expression at 28℃ for 5h. Centrifuge at 4℃ and 8000r / min for 5min to collect the bacteria. Add 200μL, 1mg / mL lysozyme solution, place in an incubator at 37℃ for 2h to lyse the bacteria. Collect the supernatant after centrifugation as the crude enzyme solution. Dilute the enzyme solution appropriately and take 5μL to add to 95μL 0.5g / mL beech wood xylan solution (pH 5.5, 50mmol / L citric acid-sodium dihydrogen phosphate buffer). Use a PCR instrument to carry out catalytic and color development reaction (75℃ for 10min, add 50μL DNS reagent, 95℃ for 5min, cool to 10℃), add to a 96-well plate, and measure OD with an enzyme reader. 540 The mutants corresponding to the mutant xylanase with an enzyme activity greater than 30% of the original enzyme were defined as positive mutants, and DNA sequencing was performed on them. The sites where the enzyme activity was significantly improved were combined with mutations, and the DNS method was used to screen the optimal mutants.

[0044] Table 1 Mutation primer design

[0045]

[0046] Mutant expression and purification

[0047] The recombinant plasmid was transformed into E.coli BL21 (DE3) cells. Positive colonies were selected and inoculated into 2 mL of LB liquid medium containing Kan+ (final concentration of 50 μg / mL). The culture medium was then cultured overnight at 37°C and 180 rpm. Subsequently, 1 mL of the bacterial solution was transferred to TB medium, cultured at 37°C and 180 rpm for 3 hours, and then 200 μL of IPTG (final concentration of 2 mmol / L) was added to induce at 30°C and 180 rpm for 2 hours. After completion, the cells were broken and the supernatant was extracted as a crude enzyme solution. The recombinant xylanase was purified using a 10 cm nickel agarose gel (Ni Sepharose HP) column. The column was filled with 50 mmol / L phosphate buffer (pH 7.8, containing 300 mmol / L NaCl and imidazole at a concentration of 40.00-500.00 mM). Use Fast protein liquid chromatography (FPLC) purification system. The purified recombinant xylanase was subjected to SDS-PAGE analysis using 10% separation gel and 5% stacking gel to evaluate the purity. The protein concentration was quantified by Coomassie brilliant blue staining using bovine serum albumin (BSA) as a standard.

[0048] Characterization of mutant enzymatic properties

[0049] The optimum pH of xylanase was determined at 37°C in the pH 3.0-9.0 range. The pH stability of xylanase was determined by incubating the enzyme at 45°C in a pH 3.0-9.0 buffer for 15 min and measuring the residual enzyme activity. The optimum temperature of xylanase was determined by measuring the activity in 50 mM citric acid buffer at 5°C intervals for 15 min in the range of 35°C-60°C. The pH stability of xylanase was determined by incubating the enzyme at 40°C at pH 3.0-9.0 for 60 min. The thermostability of xylanase was evaluated by incubating the enzyme at different times and temperatures. The protein concentration of pure enzyme was appropriately diluted with pH 5.0 citric acid buffer. The enzyme was kept at 35°C-50°C for 60 min and then rapidly cooled in an ice-water bath. The untreated xylanase activity was defined as 100%. The residual enzyme activity of mutants and XynASP was calculated. Half-life t 1 / 2 Equal to ln2 / k, the formula is y=Ae -kt (A is the initial enzyme activity, “t” is the time, and “k” is the decay constant), and the half-lives of the mutant and XynASP at 45°C and 50°C were calculated according to the formula.

[0050] The conformational stability of the enzyme was determined by differential scanning fluorimetry (DSF) using the CFX Touch 96-well system. The melting temperature Tm was measured. Based on the DSF analysis, the enzyme was used at a concentration of 0.8-1.0 mg / mL and SYPRO dye was used. The sample was heated from 10°C to 95°C with a gradient of 0.5°C / s. It was monitored by measuring the fluorescence at 480 / 580nm (excitation / emission).

[0051] The enzyme was incubated with different concentrations of metal ions and chemical reagents at 40°C for 1 hour to detect the residual enzyme activity. The initial enzyme activity without any treatment was set as 100% to calculate the enzyme's tolerance to these substances. The metal ions (including K+, Ca+, etc.) at a concentration of 5 mM were detected. 2 +, Na+, Mg2+, Al3+, Mn2+, Fe2+, Fe3+, Ni 2 +, Cu2+, Zn2+ and Ba 2 +). And the resistance to the chemical reagents sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA) at a concentration of 5 mM. In addition, the resistance to methanol, ethanol and isopropanol at a final concentration of 5% (vol / vol) was also studied.

[0052] Kinetic parameters were measured using different concentrations of beechwood xylan under the optimal reaction conditions for each enzyme. Km and Vmax values ​​were calculated using GraphPad Prism software according to the Michaelis-Menten equation. All experiments were repeated three times.

[0053] Application of mutants in juice clarification

[0054] Fresh fruits (apples, mangoes, oranges, peaches and pitaya) were thoroughly washed and peeled, and then blended into a uniform pulp using a high-speed blender. The pulp was then mixed with xylanase in a 1:1 volume ratio (v / v) and incubated at 40°C for 150 min. During this period, samples were collected every 15 min. Each sample was boiled in boiling water for 5 min to inactivate the enzyme and then centrifuged at 4000g for 10 min. The transmittance (Tt) of the resulting supernatant at 640 nm was analyzed. The transmittance (Tc) of the control group not treated with xylanase was also analyzed. The percentage increase in clarity was calculated as follows: percentage increase in clarity = (Tt-Tc) / Tc×100; Tt = transmittance of the test sample; Tc = transmittance of the control sample.

[0055] Results

[0056] Some enzymatic properties are shown in Table 2. The optimum pH value of D116S, E119V and XynASP-SV is 5.0. This is slightly lower than the optimum pH value of the wild type (WT) (6.0). When incubated at 40°C for 1 hour in the pH range of 3.0 to 9.0, the pH fluctuations of the wild-type and mutant enzymes were relatively stable, indicating that the enzyme has a wide pH adaptation range. In particular, in the pH range of 3.0 to 5.0, XynASP-SV exhibited 75.86%, 89.01% and 83.92% residual enzyme activity, respectively. In contrast, the activities of the wild-type enzyme at the same pH level were 45.86%, 65.69% and 60.10%, respectively. It shows that within this pH range, the activity of XynASP-SV is higher than that of the wild type; the optimum temperature of D116S, E119V and XynASP is 50°C. Compared with the optimum temperature of 45°C for the wild-type (WT) enzyme, it is 5°C higher. After treatment at 50°C and pH 5.0 for 60 minutes, both the wild-type and mutant enzymes retained more than 50% of their activity after 10 minutes of heat treatment. However, after 20 minutes of heat treatment, the activity of the wild-type enzyme dropped rapidly, retaining only 16.84% of its original activity. In contrast, the mutant enzymes D116S, E119V and XynASP-SV retained 41.75%, 44.44% and 55.16% of their activity, respectively. Compared with the wild type, these were increased by 2.47 times, 2.64 times and 3.27 times, respectively. The melting temperature (Tm) values ​​of the enzymes and the half-life of the enzyme activity are listed in Tables 2 and Figure 5The Tm value of the wild-type enzyme was 46°C, while the Tm values ​​of D116S, E119V, and XynASP-SV increased to 50°C, 50°C, and 52°C, respectively. At 45°C, the half-life of the wild-type enzyme was 22.34 minutes, while all mutants had longer half-lives. At 50°C, the half-life of the wild-type enzyme was only 5.31 minutes, but the half-life of XynASP-SV was 24.51 minutes, which was 4.62 times longer than that of the wild-type. When beech xylan was used as a substrate for the determination of the wild-type (WT), D116S, E119, and XynASP-SV, their kinetic parameters are shown in Table 2. Compared with the wild type, the mutants have a lower Michaelis constant (Km) value, and the mutant enzyme has an enhanced affinity for the substrate; the catalytic efficiency has been significantly improved, and the ratio of the catalytic constant to the Michaelis constant (kcat / Km) of D116S, E119 and XynASP-SV is 6.93 times, 15.65 times and 27.69 times higher than that of the wild type, respectively. XynASP-SV presents the lowest Km value and higher catalytic efficiency (kcat / Km), which greatly increases the substrate release rate. In the application of juice clarification, the maximum improvement of the clarification rate of mango juice and pitaya juice by the wild type is 53.34% and 60.16% at 90min and 105min, respectively, while XynASP-SV reaches the maximum value at 75min, which is 65.73% and 75.61%. Compared with the data of xylanases from different sources in juice clarification that have been published so far, it can be found in Table 4 that our xylanase shows a better effect in juice clarification.

[0057] Table 2 Enzyme property parameters

[0058]

[0059] Table 3 Clarification effect of various juices

[0060]

[0061] Table 4 Application of xylanases from different sources in juice clarification

[0062]

[0063] In summary, the present invention screened 10 potential beneficial mutants in the cord region of xylanase XynASP, and constructed an optimal mutant XynASP-SV through rational combination, which significantly improved thermal stability, acid tolerance and catalytic activity, and showed wide application potential in juice clarification, among which the clarification effect on mango juice and pitaya juice was the best.

Claims

1. A xylanase mutant, characterized in that The amino acid sequences of the mutants obtained by mutation of the D116 and E119 sites of the xylanase XynASP are shown in SEQ ID NO.1-SEQ ID NO.3, or amino acid sequences having at least 90% homology with the above amino acid sequences, and retaining the function of clarifying juice.

2. A gene encoding the xylanase mutant according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.4-SEQ ID NO.6, or a nucleic acid sequence having at least 90% homology with the above nucleic acid sequence, and can encode a protein having the function of clarifying juice.

3. An expression vector containing the nucleic acid sequence of claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a plasmid.

5. A host cell containing the expression vector according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli or yeast.

7. A method for producing xylanase using the host cell according to claim 5, characterized in that: The method comprises culturing the host cell and collecting the xylanase mutant expressed by the cell.

8. Use of the xylanase mutant according to claim 1 in a juice clarification process.

9. The use according to claim 8, characterized in that: The fruit juice is apple juice, orange juice, peach juice, mango juice or pitaya juice.

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