A method for converting sucrose in jujubes into glucan using an immobilized enzyme, and jujube juice rich in glucan and its applications.
By immobilizing glucan sucrase to convert sucrose in jujubes into glucan, the problem of sucrose conversion in jujube juice is solved, and the glucan content in jujube juice is increased. This method is applicable to the field of juice preparation, especially the preparation of jujube juice rich in glucan.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HEBEI AGRICULTURAL UNIV.
- Filing Date
- 2025-01-24
- Publication Date
- 2026-07-31
AI Technical Summary
The lack of an effective method to convert the sucrose in jujubes into biofunctional glucans makes it impossible for people with high blood sugar to eat jujubes, and existing technologies have failed to effectively utilize the sucrose in jujubes.
Immobilized glucan sucrase was mixed with jujube juice, and the immobilized enzyme converted sucrose in jujube into glucan. The glucan sucrase was expressed and immobilized using a bacterial enhancement matrix and a lactococcus lactis peptidoglycan hydrolase expression system. The reaction conditions, such as temperature and time, were optimized, and centrifugation was used to remove the precipitate to obtain jujube juice rich in glucan.
While reducing the sucrose content in jujube juice, the content of prebiotics and dietary fiber glucan was increased, thereby enhancing the health value of jujube juice.
Smart Images
Figure CN119955755B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fruit juice preparation technology, specifically relating to a method for converting sucrose in jujubes into glucan using an immobilized enzyme, and jujube juice rich in glucan and its applications. Background Technology
[0002] Jujubes, as a food with both medicinal and nutritional value, have excellent health benefits. Jujubes contain 15-66% sucrose, which is composed of one molecule of glucose and one molecule of fructose. It is a sugar that is easily and quickly utilized, but its high sugar content makes it unsuitable for people with high blood sugar. Glucan is an extracellular polysaccharide produced by lactic acid bacteria during their growth and metabolism. It possesses a range of biological functions, including anti-inflammatory, anti-tumor, gut microbiota regulation, and immune modulation. Currently, there is a lack of an effective method to convert sucrose into glucan. Summary of the Invention
[0003] The purpose of this invention is to provide a method for converting sucrose in jujubes into glucan using an immobilized enzyme, as well as jujube juice rich in glucan and its applications. The method described in this invention can efficiently convert sucrose into glucan, generating glucan while reducing readily available sugars.
[0004] This invention provides a method for converting sucrose in jujubes into glucan using an immobilized enzyme, comprising the following steps:
[0005] The immobilized glucan sucrase was mixed with jujube juice, and the sugar was converted. After centrifugation, the precipitate was discarded to obtain jujube juice rich in glucan. The nucleotide sequence of the gene encoding the glucan sucrase is shown in SEQ ID NO.1.
[0006] Preferably, the temperature for sugar inversion is 30–35°C, and the time for sugar inversion is 12–20 h.
[0007] Preferably, the immobilized dextran sucrase is obtained by expression and immobilization based on a bacterial enhancement matrix and a lactococcus lactis peptidoglycan hydrolase expression system.
[0008] Preferably, the method for immobilizing dextran sucrase includes the following steps:
[0009] The gene encoding the glucan sucrase was constructed into a plasmid carrying a tag of lactococcus lactis peptidoglycan hydrolase to obtain a recombinant plasmid.
[0010] The recombinant plasmid was transformed into Escherichia coli, cultured, induced, and the bacterial cells were collected. The cells were then sonicated, centrifuged, and the supernatant was collected to obtain crude dextran sucrase enzyme solution.
[0011] The crude dextran sucrase solution was immobilized using a bacterial-enhanced matrix purification method to obtain immobilized dextran sucrase.
[0012] Preferably, the plasmid comprises pET-28a.
[0013] Preferably, the immobilization method includes: mixing the bacterial enhancement matrix with the crude dextran sucrase enzyme solution, centrifuging to collect the precipitate after binding, and washing.
[0014] Preferably, the bonding time is 30 to 60 minutes; the bonding temperature is 0 to 4°C.
[0015] Preferably, the bacteria used to prepare the bacterial enhancement matrix include Lactococcus lactis.
[0016] The present invention also provides jujube juice rich in glucan prepared by the method described in the above technical solution.
[0017] The present invention also provides the application of immobilized dextran sucrase in increasing the dextran content in jujube juice, and the nucleotide sequence of the gene encoding the dextran sucrase is shown in SEQ ID NO.1.
[0018] This invention provides a method for converting sucrose in jujubes into glucan using an immobilized enzyme. This invention utilizes an immobilized enzyme to convert sucrose in jujubes into glucan, which can increase the content of prebiotic / dietary fiber glucan while reducing the sucrose content. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 The image shows the results of agarose gel electrophoresis detection of colony PCR products provided by this invention.
[0021] Figure 2 The SDS-PAGE analysis results of the recombinant dextran sucrase provided by this invention are shown in the figure.
[0022] Figure 3 A graph showing the effect of induction temperature on the expression level of recombinant dextran sucrase, provided by the present invention.
[0023] Figure 4The SDS-PAGE analysis results of the recombinant dextran sucrase provided by this invention are shown in the figure; where lane M: protein marker; lane 1: whole bacteria before induction; lane 2: whole bacteria after induction; lane 3: supernatant after ultrasonic disruption; lane 4: precipitate after ultrasonic disruption; lane 5: BEM (fixation material); lane 6: supernatant with BEM binding; lane 7: precipitate with BEM binding.
[0024] Figure 5 Figure showing the changes of the immobilized dextran sucrase provided by the present invention before and after reaction in sucrose solution and jujube juice;
[0025] Figure 6 This is a full-wavelength scan result diagram of dextran provided by the present invention;
[0026] Figure 7 The diagram shows the optimal temperature results for the immobilized enzyme provided by this invention. Detailed Implementation
[0027] This invention provides a method for converting sucrose in jujubes into glucan using an immobilized enzyme, comprising the following steps:
[0028] The immobilized glucan sucrase was mixed with jujube juice, and the sugar was converted. After centrifugation, the precipitate was discarded to obtain jujube juice rich in glucan. The nucleotide sequence of the gene encoding the glucan sucrase is shown in SEQ ID NO.1.
[0029] In a specific embodiment, the immobilized dextran sucrase is obtained by expression and immobilization based on a bacterial enhancement matrix and a lactococcus lactis peptidoglycan hydrolase expression system.
[0030] In a specific embodiment, the method for immobilizing dextran sucrase includes the following steps: constructing the gene encoding the dextran sucrase into a plasmid carrying a tag of lactococcus lactis peptidoglycan hydrolase to obtain a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli, culturing, inducing, collecting the bacterial cells, sonicating, centrifuging, and collecting the supernatant to obtain a crude dextran sucrase enzyme solution; and immobilizing the obtained crude dextran sucrase enzyme solution using a bacterial enhancement matrix purification method to obtain immobilized dextran sucrase.
[0031] This invention constructs the gene encoding the glucan sucrase into a plasmid carrying a *Lactococcus lactis* peptidoglycan hydrolase tag to obtain a recombinant plasmid. In a specific embodiment, the plasmid includes pET-28a. This invention does not have specific limitations on the source of the plasmid carrying the AcmA tag; conventional methods can be used for construction. Refer to the article "Research on Purification and Immobilization Methods of Recombinant Proteins Based on BEM and AcmA Expression Systems" published by Zhao Fangkun in *Tianjin University* to construct pET28a-AcmA. In a specific embodiment, this invention designs forward primer 5'-CGCCCATGGGAGATAGCACAAACACAGTGAC-3' (SEQ ID NO.2) and reverse primer 5'-ATAGAATTCAGCGACTGAGACAAAGTAAC-3' (SEQ ID NO.3) based on *Leuconostoc mesenteroides* CBA3627 (GenBank: CP042418.1). The glucan sucrase gene is obtained by polymerase chain reaction (PCR). The nucleotide sequence of the gene encoding the dextran sucrase is shown in SEQ ID NO.1. In a specific embodiment, the dextran sucrase gene was ligated and transformed into plasmid pET-28a-AcmA using restriction endonucleases EcoRI and HindIII.
[0032] After obtaining the recombinant plasmid, the present invention transforms the recombinant plasmid into *E. coli*, cultured, induced, and collects the bacterial cells. The cells are then sonicated, centrifuged, and the supernatant is collected to obtain a crude dextran sucrase enzyme solution. In a specific embodiment, the *E. coli* includes *E. coli* BL21. In a specific embodiment, the *E. coli* is *E. coli* BL21 competent cells. In a specific embodiment, IPTG is used for induction expression. In a specific embodiment, the induction temperature can be 16–37°C, or it can be 16°C, 25°C, or 37°C. In a specific embodiment, the induction time can be 24 hours.
[0033] After obtaining the crude dextran sucrase enzyme solution, this invention uses a bacterial enhancement matrix to purify and immobilize the enzyme, thereby obtaining immobilized dextran sucrase. In a specific embodiment, the immobilization method includes: mixing the bacterial enhancement matrix with the crude dextran sucrase enzyme solution, centrifuging to collect the precipitate, and washing. In a specific embodiment, the binding time is 30–60 min, specifically 30 min, 40 min, 50 min, or 60 min. In a specific embodiment, the binding temperature is 0–4 °C, specifically 4 °C. In a specific embodiment, the bacteria used to prepare the bacterial enhancement matrix include *Lactococcus lactis*. In a specific embodiment, the *Lactococcus lactis* is *Lactococcus lactis* subsp. *lactococcus* or *Lactococcus lactis* NZ9000. This invention does not specifically limit the preparation method of the bacterial enhancement matrix; reference can be made to the article "Research on Purification and Immobilization Method of Recombinant Protein Based on BEM and AcmA Expression Systems" published by Zhao Fangkun in *Tianjin University*. In a specific embodiment, *Lactococcus lactis* NZ9000 was cultured overnight, and the bacterial cells were collected by centrifugation, resuspended, boiled, cooled, and then centrifuged again to obtain BEM as the precipitate. In a specific embodiment, the conditions for collecting the bacterial cells by centrifugation were 4000 rpm at room temperature for 20 min. In this invention, the room temperature was 20–30°C. In a specific embodiment, 0.1 M HCl was added for resuspension; specifically, 100 mL of 0.1 M HCl was added for every 100 mL of bacterial cells collected. In a specific embodiment, the boiling time was 30 min, and boiling was used to remove proteins and DNA. In a specific embodiment, after cooling, centrifugation was performed at room temperature at 8000 rpm for 20 min. After obtaining the precipitate, the BEM was thoroughly washed with PBS. The BEM prepared by this invention can be stored for a long time at 4°C.
[0034] In a specific embodiment, the sugar conversion temperature is 30–35°C, specifically 30–35°C. In a specific embodiment, the sugar conversion temperature can be 28°C, 30°C, 32°C, or 35°C. The ideal temperature for dextran sucrase is 30°C; above 30°C, enzyme activity rapidly decreases. The activity of dextran sucrase is relatively high between 20 and 30°C, and still retains 95% activity at 35°C. In a specific embodiment, the sugar conversion time is 12–20 hours. In a specific embodiment, the sugar conversion time can be 12 hours, 14 hours, 16 hours, 18 hours, or 20 hours. Dextran yield growth slows down after 20 hours. Considering economic benefits and other indicators, the optimal sugar production time is 20 hours, with a dextran yield of 191.9 g / L, achieving 77% of the theoretical yield. In a specific embodiment, the enzyme addition during the sugar conversion process is 4000–7000 U / mL, specifically 6000 U / mL.
[0035] This invention also provides jujube juice rich in glucan prepared by the method described in the above technical solution. This invention utilizes immobilized enzymes to convert sucrose in jujubes into glucan, and removes the enzyme by centrifugation after the reaction. The jujube juice of this invention reduces the sucrose content while increasing the content of prebiotic / dietary fiber glucan.
[0036] The present invention also provides the application of immobilized dextran sucrase in increasing the dextran content in jujube juice, and the nucleotide sequence of the gene encoding the dextran sucrase is shown in SEQ ID NO.1.
[0037] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a method for converting sucrose in jujubes into glucan using an immobilized enzyme, as well as jujube juice rich in glucan and its applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.
[0038] The sucrose content was determined by high performance liquid chromatography.
[0039] Conditions: Deionized water was used as the mobile phase, the flow rate was 1 mL / min, a differential refractive index detector was used, the injection volume was 6 μL, the column temperature and detector temperature were both 30 °C, and the chromatographic column was Shodex Asahipak NH2P-504E. The change in sucrose content in jujubes was determined.
[0040] Preparation method of jujube juice
[0041] After washing, pitting, drying, and grinding the Ruoqiang red dates, red date powder was obtained. The red date powder was mixed with deionized water at a ratio of 1g:20mL (w / v), and the red date juice was extracted in a boiling water bath for 4 hours using the hot water extraction method to reduce the volume of the red date juice to half of the original volume. After centrifugation at room temperature (8000×g, 15min), the date residue was discarded to obtain red date juice.
[0042] Example 1
[0043] 1) Selection of glucan sucrase
[0044] The dextran sucrase DsrB synthesized by Leuconostoc mesenteroides CBA3627 (GenBank: CP042418.1) is shown in SEQ ID NO.1.
[0045]
[0046] Recombinant plasmids were constructed (construction method as in Example 2) and expressed at an induction temperature of 16℃. After immobilization using BEM (immobilization method as in Example 3, BEM binding time with crude enzyme solution was 60 min), the plasmids were added to jujube juice for sugar conversion under the following conditions: 30℃, 20 h. The sucrose content before and after conversion was measured. The results showed that the glucan sucrase DsrB could reduce the sucrose content in jujube juice by 67%.
[0047] Example 2
[0048] Construction of pET-28a-AcmA-DsrB recombinant plasmid
[0049] Referring to Zhao Fangkun's article "Study on Purification and Immobilization Methods of Recombinant Proteins Based on BEM and AcmA Expression Systems" published in *Tianjin University*, pET-28a-AcmA was constructed. Forward primer 5'-CGCCCATGGGAGATAGCACAAACACAGTGAC-3' (SEQ ID NO.2) and reverse primer 5'-ATAGAATTCAGCGACTGAGACAAAGTAAC-3' (SEQ ID NO.3) were designed based on *Leuconostoc mesenteroides* CBA3627 (GenBank: CP042418.1). The dextran sucrase gene was amplified by polymerase chain reaction (PCR). The PCR product and plasmid pET-28a-AcmA were cleaved together using restriction endonucleases EcoRI and HindIII, followed by ligation and transformation.
[0050] Figure 1 For the detection of colony PCR products by agarose gel electrophoresis. For example... Figure 1 As shown, lanes 1, 2, 4, 5 and 6 are plasmids that were successfully ligated. Combined with the sequencing results, this indicates that the vector pET-28a-AcmA was successfully ligated to the target gene.
[0051] 3) Recombinant expression of AcmA-tagged dextran sucrase
[0052] The recombinant plasmid pET-28a-AcmA-DsrB was transformed into *E. coli* BL21 competent cells and cultured in LB medium containing 50 mg / L kanamycin. Expression was induced using IPTG, with 37℃, 25℃, and 16℃ selected as optimal induction temperatures. After expression, the cells were collected, sonicated, and the supernatant was collected by centrifugation. The crude dextran sucrase enzyme solution in the supernatant was immobilized using BEM (prepared as in Example 3) (BEM binding time with crude enzyme solution was 60 min). Sugar conversion in jujube juice was then performed (sugar conversion conditions: 30℃, 20 h), and the sucrose content before and after conversion was measured. The results showed that sucrose in jujube decreased by 36% at 37℃, 54% at 25℃, and 67% at 16℃. Compared with the induction temperatures of 37℃ and 25℃, the dextran sucrase enzyme reduced the sucrose content in jujube juice more significantly under the induction condition of 16℃.
[0053] Figure 2 SDS-PAGE analysis of recombinant dextran sucrase. Lane M: protein marker; Lane 1: bacterial culture before induction; Lane 2: bacterial culture after induction; Lane 3: supernatant from sonication; Lane 4: precipitate from sonication. After IPTG induction, the target band was observed in the 150–250 kDa range, demonstrating that IPTG can induce the expression of recombinant dextran sucrase. Comparison of the sonicated supernatant and precipitate showed that recombinant dextran sucrase DsrB is an intracellular soluble protein, and sonication releases the recombinant enzyme.
[0054] Figure 3 To investigate the effect of induction temperature on the expression level of recombinant dextran sucrase, this invention studied the effect of different induction temperatures on protein expression levels, and the results are as follows: Figure 3 As shown, enzyme activity was measured simultaneously with SDS-PAGE. The expression level of the target protein was the highest and the enzyme activity was the highest at an induction temperature of 16℃. Therefore, 16℃ was determined to be the optimal induction temperature.
[0055] Example 3
[0056] Enzyme immobilization
[0057] The crude enzyme solution of dextran sucrase obtained at an induction temperature of 16℃ in Example 2 was purified using the BEM purification and immobilization enzyme technique. The preparation of BEM was based on the article "Research on Purification and Immobilization Method of Recombinant Protein Based on BEM and AcmA Expression System" published by Zhao Fangkun in *Tianjin University*. *Lactococcus lactis* NZ9000 was cultured overnight, and the bacterial cells were collected by centrifugation for 20 min (4000 rpm, room temperature). The cells were resuspended in 0.1 M HCl (100 mL of 0.1 M HCl was added for every 100 mL of collected bacterial cells). The cells were then boiled for 30 min to remove protein and DNA. After cooling, the cells were centrifuged at 8000 rpm for 20 min at room temperature. The resulting precipitate was BEM. BEM was thoroughly washed with PBS to obtain BEM particle precipitate. The prepared BEM was stored at 4℃. BEM is essentially 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.
[0058] An appropriate amount of BEM particle precipitate was bound to crude enzyme solution on ice for 10 min, 30 min, and 60 min. After centrifugation at 12000×g for 10 min at 4℃, the supernatant was discarded. The BEM-bound dextran sucrase precipitate was washed with 50 mM Tris-HCl to obtain immobilized dextran sucrase. The immobilized dextran sucrase was used to perform sugar conversion in jujube juice (sugar conversion conditions: 30℃, 20 h), and the sucrose content before and after conversion was measured. The results showed that after 10 min of binding, the sucrose content in jujube decreased by 30%; after 30 min, it decreased by 50%; and after 60 min, it decreased by 65%.
[0059] Figure 4 SDS-PAGE analysis of recombinant glucan sucrase. Lane M: protein marker; Lane 1: whole cells before induction; Lane 2: whole cells after induction; Lane 3: supernatant after sonication; Lane 4: precipitate after sonication; Lane 5: BEM (fixation material); Lane 6: supernatant with BEM binding; Lane 7: precipitate with BEM binding. Figure 4 It can be seen that the purified recombinant dextran sucrase had very few impurity protein bands compared with the unpurified version, indicating a good purification effect.
[0060] Example 4
[0061] Sucrose in jujube juice is converted into glucan.
[0062] Conversion of sucrose in jujube juice
[0063] The immobilized dextran sucrase obtained in Example 3 under 60 min conditions was added to jujube juice, and the sucrose in the jujube juice was converted at 30°C for 12 h, 14 h, 16 h, 18 h, and 20 h, and at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C, respectively. After the reaction, the immobilized enzyme was removed by centrifugation at 12000×g for 10 min. The sucrose content before and after conversion was measured.
[0064] Immobilized enzymes were used to convert sucrose in jujubes into glucan, and the enzyme was removed by centrifugation after the reaction. This reduced the sucrose content while increasing the content of prebiotic / dietary fiber glucan.
[0065] The results showed that sucrose levels in jujubes decreased by 30% after 12 hours of reaction, by 58% after 16 hours, and by 67% after 20 hours. The growth of dextran production slowed down after 20 hours. Considering economic benefits and other indicators, the optimal sugar production time was determined to be 20 hours, with a dextran yield of 191.9 g / L, achieving 77% of the theoretical yield.
[0066] Figure 5 The images show the changes in immobilized dextran sucrase before and after reaction in sucrose solution and jujube juice. The left image shows the changes in immobilized dextran sucrase before and after reaction in sucrose solution. Figure 5 The left image shows that the sucrose solution became cloudy after the reaction, proving that sucrose was converted into dextran; the right image shows the changes before and after the reaction of immobilized dextran sucrase in jujube juice. Figure 5 As shown in the right figure, the jujube juice became noticeably cloudy after the reaction, indicating that the immobilized glucan sucrase successfully converted the sucrose in the jujube juice into glucan.
[0067] Figure 6 This is a full-wavelength scan result of dextran. After purification, the dextran obtained from the reaction with sucrose solution exhibits a maximum characteristic absorption peak at around 200 nm, which is a characteristic absorption peak of carbohydrates. The absence of a characteristic absorption peak in the 260–280 nm range indicates that the purified dextran does not contain nucleic acids or proteins.
[0068] Figure 7 This figure shows the optimal temperature for immobilized dextran sucrase. The ideal temperature for dextran sucrase is 30℃; above 30℃, enzyme activity decreases rapidly. The activity of dextran sucrase is relatively high between 20 and 30℃, and it still retains 95% activity at 35℃.
[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for converting sucrose in jujubes into glucan using an immobilized enzyme, characterized in that, Includes the following steps: Immobilized glucan sucrase was mixed with jujube juice for sugar conversion, and the precipitate was discarded by centrifugation to obtain jujube juice rich in glucan; the nucleotide sequence of the gene encoding the glucan sucrase is shown in SEQ ID NO.1; the sugar conversion temperature was 30℃; the sugar conversion time was 20h; and the enzyme addition amount during the sugar conversion process was 6000U / mL. The immobilized dextran sucrase was obtained by expression and immobilization based on a bacterial enhancement matrix and a lactococcus lactis peptidoglycan hydrolase expression system. The method for obtaining the immobilized dextran sucrase includes the following steps: The gene encoding the glucan sucrase was constructed into a plasmid carrying a tag of lactococcus lactis peptidoglycan hydrolase to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli, cultured, induced, and the bacterial cells were collected, ultrasonically disrupted, and the supernatant was collected by centrifugation to obtain crude dextran sucrase enzyme solution; the induction temperature was 16℃. The crude dextran sucrase solution was immobilized using a bacterial enhancement matrix purification method to obtain immobilized dextran sucrase. The immobilization method included: mixing the bacterial enhancement matrix with the crude dextran sucrase solution, centrifuging to collect the precipitate after binding, and washing; the binding time was 60 min; and the binding temperature was 4 °C.
2. The method according to claim 1, characterized in that, The plasmid includes pET-28a.
3. The method according to claim 1, characterized in that, The bacteria used to prepare the bacterial enhancement matrix include Lactococcus lactis.
4. Application of immobilized glucan sucrase in increasing glucan content in jujube juice, wherein the nucleotide sequence of the gene encoding the glucan sucrase is shown in SEQ ID NO.1.