Preparation method of fructan-rich jujube juice, fructan-rich jujube juice and application of fructan-rich jujube juice

Abstract

The invention provides a preparation method of jujube juice rich in fructan, the jujube juice rich in fructan and application of the jujube juice rich in fructan, and particularly belongs to the technical field of fruit juice preparation. The preparation method of the fructan-rich jujube juice comprises the following steps: mixing immobilized fructan sucrase with jujube juice, carrying out sugar conversion, centrifuging, and discarding precipitate, so as to obtain the fructan-rich jujube juice, the nucleotide sequence of the gene for coding the fructan sucrase is as shown in SEQ ID NO. 1. According to the preparation method disclosed by the invention, the sucrose in the jujube juice can be efficiently converted into the fructan, and the sucrose is converted into the fructan while the content of the easily utilized sugar (sucrose) is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit juice preparation, and particularly relates to a preparation method of jujube juice rich in fructan, the jujube juice rich in fructan and applications thereof. Background Art

[0002] Jujube is a food that can be used as both medicine and food, and has a good health-care effect. The sucrose content in jujube is 15-66%. Sucrose is composed of one molecule of glucose and one molecule of fructose. It is a sugar that is easily and quickly utilized. The high sugar content makes jujube unusable for people with high blood sugar. Fructans are a water-soluble dietary fiber that can promote the growth of probiotics and has the function of lowering blood lipids and blood sugar. At present, there is a lack of a method that can effectively convert sucrose in jujube into fructans. Summary of the invention

[0003] The purpose of the present invention is to provide a preparation method of jujube juice rich in fructan, jujube juice rich in fructan and application. The preparation method of the present invention can efficiently convert sucrose in jujube juice into fructan while reducing the content of readily available sugar (sucrose).

[0004] The present invention provides a method for preparing jujube juice rich in fructan, comprising the following steps:

[0005] The immobilized fructan sucrase is mixed with jujube juice, sugar conversion is performed, and the precipitate is discarded by centrifugation to obtain jujube juice rich in fructan; the nucleotide sequence of the gene encoding the fructan sucrase is shown in SEQ ID NO.1.

[0006] Preferably, the temperature of the sugar conversion is 37-50° C.; the time of the sugar conversion is 8-24 hours; and the enzyme concentration of the sugar conversion is 12 U / ml.

[0007] Preferably, the immobilized levansucrase is expressed and immobilized based on a bacterial enhancer matrix (BEM) and a Lactococcus lactis peptidoglycan hydrolase (AcmA) expression system.

[0008] Preferably, the method for preparing the immobilized levansucrase comprises the following steps:

[0009] constructing the gene encoding the levansucrase into a plasmid with a Lactococcus lactis peptidoglycan hydrolase tag to obtain a recombinant plasmid;

[0010] The recombinant plasmid is transformed into Escherichia coli, cultured, induced, the bacteria are collected, ultrasonically broken, and centrifuged to obtain the supernatant to obtain a crude levansucrase enzyme solution;

[0011] The obtained crude levansucrase enzyme solution is immobilized by using a bacteria-enhanced matrix purification method to obtain immobilized levansucrase.

[0012] Preferably, the plasmid comprises pET-28a.

[0013] Preferably, the immobilization method comprises: mixing the bacterial enhancement matrix with the crude levansucrase enzyme solution, centrifuging to obtain a precipitate after combining, and washing.

[0014] Preferably, the bonding time is 10 to 60 minutes; the bonding temperature is 0 to 4°C.

[0015] Preferably, the bacteria used in preparing the bacteria-enhanced matrix include Lactococcus lactis.

[0016] The present invention also provides jujube juice rich in fructan prepared by the preparation method described in the above technical scheme.

[0017] The present invention also provides the use of immobilized levansucrase in increasing the levan content in jujube juice. The nucleotide sequence of the gene encoding the levansucrase is shown in SEQ ID NO.1.

[0018] The present invention provides a method for preparing jujube juice rich in fructan. The present invention can catalyze the synthesis of sucrose into fructan in vitro through heterologously expressed fructan sucrase SacB3, and realize the synthesis of fructan in jujube juice. Compared with the control, the jujube juice of the present invention has no significant difference in tissue state, surface color, aroma and taste, and maintains the original flavor of jujube juice, and also has beneficial properties. At the same time, through the observation of the morphological characteristics of fructan, it is found that its surface presents a smooth and porous structural feature, indicating that it has excellent water absorption properties, and can be used as a moisturizer to maintain the freshness of juice and extend the shelf life during the production and storage of juice. The present invention lays a solid foundation for the future industrial large-scale production of jujube juice containing fructan. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative labor.

[0020] Figure 1 The electrophoresis result diagram provided by the present invention; wherein a is the electrophoresis diagram of the amplified gene; b is the electrophoresis diagram of the colony PCR product;

[0021] Figure 2The SDS-PAGE result diagram of the recombinant SacB3 expression and BEM purification and immobilization SacB3 provided by the present invention; wherein, in a, band M: protein marker; 1: whole cell protein distribution before induction; 2: whole cell protein distribution after induction; 3: protein distribution of supernatant after ultrasonic crushing and centrifugation; 4: protein distribution of precipitate after ultrasonic crushing and centrifugation; in b, 5: protein distribution of supernatant after ultrasonic crushing and centrifugation; 6: protein distribution of BEM as a control; 7: protein distribution after BEM purification and immobilization of precipitate; 8: protein distribution of supernatant after BEM purification and immobilization;

[0022] Figure 3 This is a picture of SacB3 provided by the present invention catalyzing the production of fructan in a jujube juice reaction system; the left picture is a picture of the result without adding SacB3, and the right picture is a picture of the result with adding SacB3;

[0023] Figure 4 The ultraviolet absorption spectrum of fructan catalyzed in the jujube juice reaction system provided by the present invention;

[0024] Figure 5 This is a graph showing the effect of adding the levansucrase provided by the present invention to jujube juice on its sensory quality. DETAILED DESCRIPTION

[0025] The present invention provides a method for preparing jujube juice rich in fructan, comprising the following steps:

[0026] The immobilized fructan sucrase is mixed with jujube juice, sugar conversion is performed, and the precipitate is discarded by centrifugation to obtain jujube juice rich in fructan; the nucleotide sequence of the gene encoding the fructan sucrase is shown in SEQ ID NO.1.

[0027] In the present invention, the immobilized levansucrase is expressed and immobilized based on the BEM and AcmA expression systems.

[0028] In a specific embodiment, the preparation method of the immobilized fructan sucrase comprises the following steps: constructing the gene encoding the fructan sucrase into a plasmid with an AcmA label to obtain a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli, culturing, inducing, collecting the bacteria, ultrasonically disrupting, centrifuging and taking the supernatant to obtain a crude fructan sucrase enzyme solution; and immobilizing the obtained crude fructan sucrase enzyme solution using the BEM method for purifying immobilized enzymes to obtain immobilized fructan sucrase.

[0029] The present invention constructs the gene encoding the levansucrase into a plasmid with an AcmA tag to obtain a recombinant plasmid. In a specific embodiment, the plasmid includes pET-28a. The present invention does not specifically limit the source of the plasmid with the AcmA tag, and it can be constructed by conventional methods. The article "Study on the purification and immobilization method of recombinant protein based on BEM and AcmA expression system" published by Zhao Fangkun in Tianjin University can be used to construct pET28a-Z-AcmA. In a specific embodiment, the present invention designs a forward primer 5'-CGCGGATCCAAAGGAAACGACAGCAAAG-3' (SEQ ID NO.2) and a reverse primer 5'-ATAGAATTCGTCTTCGTCTACTGTTAAC-3' (SEQ ID NO.3) based on the levansucrase gene of Priesteria gigantea (GenBank ID: KFM98004.1); the levansucrase gene is obtained by polymerase chain reaction (PCR) amplification. In a specific embodiment, the nucleotide sequence of the gene encoding the levansucrase is shown in SEQ ID NO. 1. In a specific embodiment, restriction endonucleases BamHI and EcoRI can be used for cleavage, ligation and transformation.

[0030] After obtaining the recombinant plasmid, the present invention transforms the recombinant plasmid into Escherichia coli, cultures, induces, collects the bacteria, ultrasonically breaks, centrifuges and takes the supernatant to obtain a crude enzyme solution of levansucrase. In a specific embodiment, the Escherichia coli includes Escherichia coli BL21. In a specific embodiment, the Escherichia coli is an Escherichia coli BL21 competent cell. In a specific embodiment, the culture inoculation amount can be 0.5-2%, or 1%; the culture temperature can be 25-50°C, or 45°C; the culture speed can be 150-200rpm, or 200rpm; the culture time can be 2-3h, or 2.5h. In a specific embodiment, IPTG is used to induce expression. In a specific embodiment, the induction temperature can be 16-37°C, or 16°C, 25°C or 37°C. In a specific embodiment, the ultrasonic breaking time is 10-30min, or 20min. In a specific embodiment, the ultrasonic breaking conditions are 60w, on 2s, off 2s. In a specific embodiment, the centrifugation condition is 7500-9000×g, 5-20 min, and can be 8400×g, 10 min. In a specific embodiment, the centrifugation temperature is 4°C.

[0031] After obtaining the crude enzyme solution of levansucrase, the present invention uses the method of purifying immobilized enzymes with BEM to immobilize the obtained crude enzyme solution of levansucrase to obtain immobilized levansucrase. In a specific embodiment, the immobilization method comprises: mixing the bacterial enhanced matrix with the crude enzyme solution of levansucrase, centrifuging to obtain a precipitate after combining, and washing. In a specific embodiment, the combining time is 10 to 60 minutes, which can be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes; the combining temperature is 0 to 4 degrees Celsius. In a specific embodiment, the centrifugation conditions can be 12000×g, 1 minute, 4 degrees Celsius. In a specific embodiment, after centrifugation to remove the supernatant, the levansucrase combined with BEM is resuspended and washed using Tris-HCl buffer, and the number of washings can be 2 times, and the obtained precipitate is immobilized levansucrase. In a specific embodiment, the bacteria used to prepare the bacterial enhanced matrix include Lactococcus lactis. In a specific embodiment, the lactococcus lactis is lactococcus lactis subspecies lactis, lactococcus lactis NZ9000. Other bacteria that can prepare BEM include Escherichia coli BL21, lemon Leuconostoc NM105, lemon Leuconostoc B2 sake Lactobacillus L3, etc. The present invention does not specifically limit the preparation method of the bacterial enhanced matrix, and reference can be made to the article "Study on the purification and immobilization method of recombinant protein based on BEM and AcmA expression system" published by Zhao Fangkun in Tianjin University. In a specific embodiment, the lactococcus lactis is cultured using MRS culture medium. In a specific embodiment, the lactococcus lactis is cultured overnight. In a specific embodiment, after the overnight culture, the cells are collected by centrifugation. In a specific embodiment, the centrifugation conditions can be 8400×g, 15min, room temperature. In a specific embodiment, the room temperature is 15-30°C. In a specific embodiment, after the cells are collected by centrifugation, they are washed and boiled. In a specific embodiment, the washing includes washing with Tris-HCl buffer (50mM, pH=8.4). In a specific embodiment, after the washing, 0.1M HCl is added to resuspend the bacteria. In a specific embodiment, the boiling time is 20 to 40 minutes, and can be 30 minutes. Boiling can remove protein and DNA. In a specific embodiment, after boiling, centrifugation is performed. In a specific embodiment, the centrifugation conditions can be 8400×g, 15 minutes, and 4°C. The precipitate obtained by centrifugation is a bacterial enhanced matrix. In a specific embodiment, the precipitate is obtained and can also be washed to remove excess HCl. In a specific embodiment, Tris-HCl buffer is used for washing, and the number of washes is 2 times. The essence of BEM is peptidoglycan, and the AcmA tag, as a peptidoglycan hydrolase, can specifically bind to the cell wall of Lactococcus lactis, thereby achieving protein purification and immobilization in one step.

[0032] In a specific embodiment, the temperature of the sugar conversion is 25-50°C; the time of the sugar conversion is 8-24h; the enzyme concentration of the sugar conversion is 12U / ml. In a specific embodiment, the temperature of the sugar conversion can be 37°C, 40°C, 45°C or 50°C. In a specific embodiment, the time of the sugar conversion can be 6h, 8h, 10h, 12h or 16h. The present invention also provides jujube juice rich in fructan prepared by the preparation method described in the above technical scheme. The present invention uses an immobilized enzyme to convert sucrose in jujube into fructan, and removes the enzyme by centrifugation after the reaction is completed. The jujube juice of the present invention increases the content of prebiotic / dietary fiber fructan while reducing the sucrose content.

[0033] The present invention also provides the use of immobilized levansucrase in increasing the levan content in jujube juice. The nucleotide sequence of the gene encoding the levansucrase is shown in SEQ ID NO.1.

[0034] In order to further illustrate the present invention, a preparation method of jujube juice rich in fructan, jujube juice rich in fructan and applications provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] The sucrose content was determined by HPLC.

[0036] Conditions: Deionized water was used as the mobile phase with a flow rate of 1 mL / min. A differential refractometer was used as the detector. The injection volume was 6 uL. The column temperature and detector temperature were both 30 °C. The chromatographic column was Shodex Asahipak NH2P-504E. The changes in the sucrose content in jujube were determined.

[0037] Preparation method of jujube juice

[0038] The Ruoqiang red dates were washed, pitted, dried and ground to obtain red date powder, which was then mixed with deionized water at a ratio of 1 g:20 mL (w / v). The mixture was extracted in a boiling water bath for 4 h using a hot water extraction method to reduce the volume of the red date juice to half of its original volume. The red date juice was obtained by centrifugation at room temperature (8000×g, 15 min) and discarding the jujube residue precipitate.

[0039] Example 1

[0040] Selection of Levansucrase

[0041] The levansucrase genes SacB1, SacB2 and SacB3 of Bacillus subtilis, Bacillus amyloliquefaciens and Priesteria megaterium were selected, and the nucleotide sequences were as follows:

[0042] Nucleotide sequence of SacB1, GenBank ID: MH178234.1:

[0043]

[0044] Nucleotide sequence of SacB2, GenBank ID: KC477262.1:

[0045]

[0046] Nucleotide sequence of SacB3, GenBank ID: KFM98004.1:

[0047]

[0048] The recombinant expression plasmid was constructed (the construction method was the same as that in Example 2) for recombinant expression, and the immobilized fructan sucrase was purified by BEM (the immobilization method was the same as that in Example 3, and the BEM and crude enzyme solution were combined for 30 min) and then added to the jujube juice for sugar conversion. According to the national food safety standard GB5009.8-2016, the sucrose content before and after conversion was determined by high performance liquid chromatography. Under the conditions of sugar conversion reaction temperature of 45 ° C, time of 12 h, and enzyme concentration of 12 U / ml (the amount of enzyme required to catalyze the synthesis of 1 μmol of glucose per 1 min was 1 U), the results showed that Bacillus subtilis fructan sucrase SacB1 could reduce the sucrose in jujube by 40%, Bacillus amyloliquefaciens fructan sucrase SacB2 could reduce the sucrose in jujube by 45%, and Priesteria gigantea fructan sucrase SacB3 could reduce the sucrose in jujube by 60%. Compared with the other two strains, the levansucrase from Priesteria gigantea can reduce more sucrose content in date juice, so the levansucrase SacB3 from Priesteria gigantea was selected for subsequent experiments.

[0049] Example 2

[0050] Construction of pET-28a-AcmA-SacB3 recombinant plasmid

[0051] Gene synthesis was performed according to the levansucrase gene (GenBank ID: KFM98004.1, SEQ ID NO.1) published in the National Center for Biotechnology Information (NCBI) database, and specific primers were designed: forward primer 5'-CGCGGATCCAAAGGAAACGACAGCAAAG-3' (SEQ ID NO.2) and reverse primer 5'-ATAGAATTCGTCTTCGTCTACTGTTAAC-3' (SEQ ID NO.3). The levansucrase gene was amplified by polymerase chain reaction (PCR) using the synthesized gene as a template, and a band of about 1.5 kb was obtained by gel electrophoresis detection, which was consistent with the theoretical value. The results are as follows: Figure 1 The target gene fragment was recovered, and the recovered PCR product was subjected to BamHI and EcoRI double restriction digestion reaction with the plasmid pET28a-Z-AcmA, and then the plasmid was connected with the target gene fragment and transferred into the E. coli cloning host DH5α for colony verification to screen out positive clones. The full length of the SacB gene is 1456 bp. The electrophoresis results are shown in Figure 1As shown in b, the band size is correct. The sequencing results were compared and analyzed to prove that the recombinant plasmid pET28a-Z-AcmA-SacB3 was successfully constructed. The cloned DNA sequence is the Priestia megaterium SacB gene fragment, named SacB3.

[0052] Recombinant expression of levansucrase with AcmA tag

[0053] The recombinant plasmid pET28a-Z-AcmA-SacB3 was heat-shock transformed into Escherichia coli BL21 (DE3) competent cells, and cultured in a shaker at an inoculum size of 1%, 37° C., and 200 rpm in an LB medium containing 50 mg / mL of kanamycin for 2.5 h. Expression was induced using 0.5 mM IPTG, and 37° C., 25° C., and 16° C. were selected as induction temperatures for optimization. After the expression, the bacteria were collected and ultrasonically disrupted (power 60 w, on 2 s, off 2 s) for 20 min, and centrifuged (8400×g, 10 min, 4° C.) to collect the supernatant. The crude fructan sucrase enzyme solution in the supernatant was purified and immobilized using the BEM method (same as in Example 3) (the combination time of BEM and the crude enzyme solution was 30 min), and then sugar conversion in jujube juice was performed (conditions were the same as in Example 1), and the sucrose content before and after the conversion was determined. The test results showed that the sucrose content in jujube juice decreased by 15% at 37°C, 30% at 25°C, and 60% at 16°C. Compared with the two induction temperatures of 37°C and 25°C, levansucrase could reduce more sucrose content in jujube juice under the induction condition of 16°C.

[0054] Example 3

[0055] Purification and immobilization of levansucrase by BEM

[0056] The preparation method of the crude levansucrase solution was the same as that in Example 2. The induction temperature was set to 16° C., and the levansucrase was successfully induced to express.

[0057] Lactococcus lactis was cultured overnight in MRS medium, and the cells were collected by centrifugation (8400×g, 15 min, room temperature), washed once with Tris-HCl buffer (50 mM, pH=8.4), and then resuspended with 0.1 M HCl, boiled for 30 min to remove protein and DNA, and the precipitate obtained after centrifugation (8400×g, 10 min, 4°C) was bacterial enhancermatrix (BEM), which was washed twice with Tris-HCl buffer to remove excess HCl to obtain BEM particle precipitate.

[0058] The crude enzyme solution of levansucrase was purified using BEM technology for purifying immobilized enzymes.

[0059] After combining an appropriate amount of BEM particle precipitate with crude enzyme solution on ice (0°C) for 10min, 30min, and 60min, respectively, the mixture was centrifuged at 12000×g for 10min at 4°C, the supernatant was removed at 4°C, and the precipitate of fructan sucrase after BEM combination was washed with 50mM Tris-HCl (i.e., the precipitate was resuspended with 50mM Tris-HCl and then centrifuged (12000×g, 4°C, 2min)). The precipitate obtained was the purified immobilized fructan sucrase, and the sugar in jujube juice was converted using the immobilized fructan sucrase (the conditions were the same as those in Example 1), and the sucrose content before and after the conversion was determined. The results showed that the sucrose in jujube decreased by 50% after 10min of combination, 60% after 30min of combination, and 60% after 60min of combination.

[0060] like Figure 2 As shown in Figure 2, the SDS-PAGE electrophoresis results of levansucrase before and after induced expression are as follows: Figure 2 As shown in a in the figure. Compared with lane 1 before induction, lane 2 after induction showed a protein band with a molecular weight of about 70KDa, which is consistent with the molecular weight of the target protein. The E. coli BL21 (DE3) cells were broken by ultrasound to release the proteins in the cells. The molecular weight of the main proteins in the supernatant and precipitate after ultrasonic cell disruption proved that the target protein was successfully induced. The results of purifying levansucrase based on the BEM immobilized enzyme method (binding time of 30min) are shown in Figure 2 As shown in b. Lane 7 shows, BEM has a good purification effect on levansucrase, and a single protein band appears, showing that the molecular weight is close to that of the target protein, proving that BEM particles have a good purification effect on levansucrase, and compared with the traditional 6×His tag using Ni filler to purify protein, it can improve purification efficiency and reduce costs.

[0061] Example 4

[0062] Sucrose-to-fructan conversion in jujube juice

[0063] The immobilized fructan sucrase obtained in Example 3 under the conditions of 30 min was added to the jujube juice, and the sucrose in the jujube juice was converted at 37°C. The reaction time was 6 h, 12 h, and 18 h, respectively. After the reaction was completed, the immobilized enzyme was removed by centrifugation at 12000×g for 10 min (i.e., the precipitate was discarded). The sucrose content before and after the conversion was measured. The measurement results showed that the sucrose in the jujube decreased by 40% after 6 h of reaction, 60% after 12 h of reaction, and 60% after 18 h of reaction.

[0064] Sensory evaluation of jujube juice samples

[0065] The sensory evaluation of jujube juice samples was carried out with sensory score as the evaluation index. 30 sensory evaluators were invited to score the jujube juice samples from five aspects, including tissue state, surface color, taste, aroma, and acceptability. The score was scored on a 100-point scale. The sensory evaluators were evaluated according to the evaluation criteria in Table 1 before the sensory evaluation. The results were collected, and the average value was taken as the final sensory evaluation result after removing the highest and lowest scores.

[0066] Table 1 Sensory evaluation standards for jujube juice samples

[0067]

[0068] The results are as follows Figure 3 to Figure 5 shown.

[0069] according to Figure 3 It can be seen that by comparing with the jujube juice sample without SacB3 added, it was found that the jujube juice sample with SacB3 added had obvious milky turbidity, which proved that the heterologously expressed fructansucrase SacB3 can catalyze the synthesis of sucrose into fructan in vitro.

[0070] Ultraviolet absorption spectroscopy is a method for analyzing the composition of substances based on the different degrees of absorption of ultraviolet rays of different wavelengths by substances. The jujube juice before and after the addition of fructan sucrase was subjected to polysaccharide purification (the jujube juice was centrifuged at room temperature (8000×g, 15min) and the jujube residue precipitate was discarded. The protein was removed by sevag method. Three volumes of 95% ethanol were added to the jujube juice sample and precipitated overnight at 4°C. After centrifugation (8000×g, 15min), the supernatant was discarded. The precipitate was washed 3 times with 75% ethanol and centrifuged and the supernatant was discarded. The precipitate was fully dried and re-dissolved in deionized water and dialyzed (molecular weight cutoff, 3500Da) to remove small molecular weight sugars in the jujube juice sample. The polysaccharides in the jujube juice sample were freeze-dried to constant weight using a vacuum freeze dryer to obtain purified jujube juice polysaccharides) and then full wavelength scans were performed in the range of 190-700nm. The results are as follows Figure 4 The results showed that after the two groups of jujube juice samples were diluted by the same multiple, obvious peaks appeared at around 220nm, the characteristic absorption peak of polysaccharides. The maximum UV spectrophotometric value of the jujube juice sample with fructan sucrase was significantly higher than that of the blank group jujube juice, indicating that after fructan sucrase was added to the jujube juice, the total polysaccharide content in the system increased. The results showed that in addition to the inherent polysaccharides in the jujube juice, additional fructans were synthesized in the jujube juice samples.

[0071] Sensory evaluation was conducted on two groups of jujube juice samples; there were no significant differences in tissue state, surface color, aroma and taste between the jujube juice control group (without SacB3) and the jujube juice experimental group (with SacB3), and the sensory evaluators' acceptance of the two jujube juice samples was basically the same. This shows that compared with the jujube juice in the control group, the addition of fructan sucrase to jujube juice has no effect on the morphology, color, smell and taste of the original jujube juice, and fructan sucrase successfully converts sucrose in jujube juice into fructan with probiotic properties. In this experiment, fructan sucrase was successfully applied to jujube juice without affecting the original flavor of jujube juice, so that fructan with probiotic properties was produced in jujube juice, laying a solid foundation for the future industrial large-scale production of jujube juice containing fructan.

[0072] The present invention uses an immobilized enzyme to convert sucrose in jujube into fructan, and removes the enzyme by centrifugation after the reaction is completed. The sucrose content is reduced while the content of prebiotic / dietary fiber fructan is increased. The present invention can achieve the effect on the change of sucrose content in jujube juice, and at the same time, realize the functional application of fructan.

[0073] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing jujube juice rich in fructan, characterized in that: The following steps are involved: The immobilized fructan sucrase is mixed with jujube juice, sugar conversion is performed, and the precipitate is discarded by centrifugation to obtain jujube juice rich in fructan; the nucleotide sequence of the gene encoding the fructan sucrase is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that: The temperature of the sugar conversion is 25-50°C; the time of the sugar conversion is 8-24h; the enzyme concentration of the sugar conversion is 12U / ml.

3. The preparation method according to claim 1, characterized in that: The immobilized levansucrase is expressed and immobilized based on a bacteria-enhanced matrix and a Lactococcus lactis peptidoglycan hydrolase expression system.

4. The preparation method according to claim 1 or 3, characterized in that: The preparation method of the immobilized levansucrase comprises the following steps: constructing the gene encoding the levansucrase into a plasmid with a Lactococcus lactis peptidoglycan hydrolase tag to obtain a recombinant plasmid; The recombinant plasmid is transformed into Escherichia coli, cultured, induced, the bacteria are collected, ultrasonically broken, centrifuged and the supernatant is taken to obtain a crude levansucrase enzyme solution; The obtained crude levansucrase enzyme solution is immobilized by using a bacteria-enhanced matrix purification method to obtain immobilized levansucrase.

5. The preparation method according to claim 4, characterized in that: The plasmids include pET-28a.

6. The preparation method according to claim 4, characterized in that: The immobilization method comprises: mixing the bacterial enhancement matrix with the crude levansucrase enzyme solution, centrifuging to obtain a precipitate after combining, and washing.

7. The preparation method according to claim 6, characterized in that: The combining time is 10 to 60 minutes; the combining temperature is 0 to 4°C.

8. The preparation method according to claim 6, characterized in that: The bacteria used in the preparation of the bacteria-enhanced matrix include Lactococcus lactis.

9. Jujube juice rich in fructan prepared by the preparation method according to any one of claims 1 to 8.

10. Application of immobilized levansucrase in increasing the levan content in jujube juice, the nucleotide sequence of the gene encoding the levansucrase is shown in SEQ ID NO.1.

Images