Method for converting sucrose in jujube into glucan by immobilized enzyme, jujube juice rich in glucan and application of jujube juice

Abstract

The invention provides a method for converting cane sugar in jujubes into glucan through immobilized enzyme, jujube juice rich in glucan and application, and particularly belongs to the technical field of fruit juice preparation. The method comprises the following steps: mixing immobilized dextran sucrase with jujube juice, carrying out sugar conversion, centrifuging, and discarding precipitate, so as to obtain jujube juice rich in dextran; the nucleotide sequence of the gene for coding the glucan sucrase is as shown in SEQ ID NO. 1. The method provided by the invention can efficiently convert cane sugar into glucan, and glucan is produced while sugar easy to utilize is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit juice preparation, and particularly relates to a method for converting sucrose in jujube into glucan by using an immobilized enzyme, jujube juice rich in glucan and its application. 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. Excessive sugar content makes it inedible for people with high blood sugar. Glucan is an extracellular polysaccharide produced by lactic acid bacteria during their growth and metabolism. It has a series of biological functions such as anti-inflammatory, anti-tumor, regulation of intestinal flora, and immune regulation. At present, there is a lack of a method that can effectively convert sucrose into glucan. Summary of the invention

[0003] The purpose of the present invention is to provide a method for converting sucrose in jujube into glucan by using an immobilized enzyme, and jujube juice rich in glucan and its application. The method of the present invention can efficiently convert sucrose into glucan, and produce glucan while reducing readily available sugar.

[0004] The present invention provides a method for converting sucrose in jujube into glucan by using an immobilized enzyme, comprising the following steps:

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

[0006] Preferably, the temperature of the sugar conversion is 30-35°C; and the time of the sugar conversion is 12-20 hours.

[0007] Preferably, the immobilized dextran sucrase is expressed and immobilized based on a bacterial enhanced matrix and a Lactococcus lactis peptidoglycan hydrolase expression system.

[0008] Preferably, the method for immobilizing dextran sucrase comprises the following steps:

[0009] constructing the gene encoding the dextran sucrase 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 enzyme solution of glucansucrase;

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

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

[0013] Preferably, the immobilization method comprises: mixing the bacterial enhancement matrix with the crude dextran sucrase enzyme solution, centrifuging to obtain a precipitate after combining, 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 in preparing the bacteria-enhanced matrix include Lactococcus lactis.

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

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

[0018] The present invention provides a method for converting sucrose in jujube into glucan by using an immobilized enzyme. The present invention uses an immobilized enzyme to convert sucrose in jujube into glucan, which can reduce the sucrose content while increasing the content of prebiotic / dietary fiber glucan. 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 A graph showing the results of agarose gel electrophoresis for detecting colony PCR products provided by the present invention;

[0021] Figure 2 This is a diagram showing the results of SDS-PAGE analysis of the recombinant glucansucrase provided by the present invention;

[0022] Figure 3 A result diagram showing the effect of the induction temperature on the expression amount of the recombinant glucan sucrase provided by the present invention;

[0023] Figure 4The SDS-PAGE analysis result diagram of the recombinant glucansucrase provided by the present invention; wherein, 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 (fixed material); lane 6: supernatant combined with BEM; lane 7: precipitate combined with BEM;

[0024] Figure 5 The diagram is a graph showing the changes of the immobilized dextran sucrase provided by the present invention before and after the reaction in sucrose solution and jujube juice;

[0025] Figure 6 This is a graph showing the full wavelength scanning results of the dextran provided by the present invention;

[0026] Figure 7 This is a graph showing the optimum temperature results for the immobilized enzyme provided by the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a method for converting sucrose in jujube into glucan by using an immobilized enzyme, comprising the following steps:

[0028] The immobilized dextran sucrase is mixed with jujube juice, sugar conversion is performed, and the precipitate is discarded by centrifugation to obtain jujube juice rich in dextran; the nucleotide sequence of the gene encoding the dextran sucrase is shown in SEQ ID NO.1.

[0029] In a specific embodiment, the immobilized dextran sucrase is expressed and immobilized based on a bacterial enhanced matrix and a Lactococcus lactis peptidoglycan hydrolase expression system.

[0030] In a specific embodiment, the method for immobilizing glucan sucrase comprises the following steps: constructing the gene encoding the glucan sucrase into a plasmid with a Lactococcus lactis peptidoglycan hydrolase tag 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 glucan sucrase enzyme solution; and immobilizing the obtained crude glucan sucrase enzyme solution using a bacterial enhanced matrix purification method for immobilized enzymes to obtain immobilized glucan sucrase.

[0031] The present invention constructs the gene encoding the glucan sucrase into a plasmid with a Lactococcus lactis peptidoglycan hydrolase 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 using a conventional method. 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-AcmA. In a specific embodiment, the present invention designs a forward primer 5'-CGCCCATGGGAGATAGCACAAACACAGTGAC-3' (SEQ ID NO.2) and a reverse primer 5'-ATAGAATTCAGCGACTGAGACAAAGTAAC-3' (SEQ ID NO.3) based on Leuconostoc lemon CBA3627 (GenBank: CP042418.1). The glucan sucrase gene is obtained by polymerase chain reaction (PCR) amplification. The nucleotide sequence of the gene encoding the glucansucrase is shown in SEQ ID NO. 1. In a specific embodiment, the glucansucrase gene is ligated and transformed with the plasmid pET-28a-AcmA using restriction endonucleases EcoRI and HindIII.

[0032] After obtaining the recombinant plasmid, the present invention transforms the recombinant plasmid into Escherichia coli, cultures, induces, collects the cells, ultrasonically breaks, centrifuges and takes the supernatant to obtain a crude enzyme solution of glucansucrase. 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, IPTG is used to induce expression. In a specific embodiment, the induction temperature can be 16 to 37°C, or 16°C, 25°C or 37°C. In a specific embodiment, the induction time can be 24h.

[0033] After obtaining the crude enzyme solution of dextran sucrase, the present invention uses a method for purifying immobilized enzymes using a bacterial enhanced matrix to immobilize the obtained crude enzyme solution of dextran sucrase to obtain immobilized dextran sucrase. In a specific embodiment, the immobilization method comprises: mixing the bacterial enhanced matrix with the crude enzyme solution of dextran sucrase, centrifuging to obtain a precipitate after combining, and washing. In a specific embodiment, the combining time is 30 to 60 minutes, specifically 30 minutes, 40 minutes, 50 minutes or 60 minutes. In a specific embodiment, the combining temperature is 0 to 4 degrees Celsius, specifically 4 degrees Celsius. 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 lactis subspecies lactis, Lactococcus lactis NZ9000. The present invention does not specifically limit the preparation method of the bacterial enhanced matrix, and reference can be made to the article "Research 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 present invention cultured Lactococcus lactis NZ9000 overnight, collected the cells by centrifugation, resuspended and boiled, and centrifuged after cooling to obtain a precipitate of BEM. In a specific embodiment, the conditions for centrifugal collection of the cells are 4000rpm and centrifuged at room temperature for 20min. In the present invention, the room temperature is 20-30°C. In a specific embodiment, 0.1M HCl is added for the resuspension; specifically, 100mL 0.1M HCl is added to each 100mL of bacterial liquid collected. In a specific embodiment, the boiling time is 30min, and boiling is used to remove protein and DNA. In a specific embodiment, centrifugation is performed at room temperature after cooling, and the centrifugation condition is 8000rpm for 20min. After obtaining the precipitate, the present invention uses PBS to fully wash the BEM. The BEM prepared by the present invention can be stored in a 4°C refrigerator for a long time.

[0034] In a specific embodiment, the temperature of the sugar conversion is 30-35°C, specifically 30-35°C. In a specific embodiment, the temperature of the sugar conversion can be 28°C, 30°C, 32°C or 35°C. The ideal temperature of glucan sucrase is 30°C. After the temperature exceeds 30°C, the enzyme activity decreases rapidly. The activity of glucan sucrase is relatively high between 20 and 30°C, and it still has 95% activity at 35°C. In a specific embodiment, the time of the sugar conversion is 12-20h. In a specific embodiment, the time of the sugar conversion can be 12h, 14h, 16h, 18h or 20h. The growth of glucan yield slows down at 20h. Considering indicators such as economic benefits, it is concluded that the optimal sugar production time is 20h, the glucan yield is 191.9g / L, and the theoretical yield reaches 77%. In a specific embodiment, during the sugar conversion process, the enzyme addition amount is 4000-7000U / mL, specifically 6000U / mL.

[0035] The present invention also provides a jujube juice rich in glucan prepared by the method described in the above technical solution. The present invention uses an immobilized enzyme to convert sucrose in jujube into glucan, and removes the enzyme by centrifugation after the reaction. The jujube juice of the present invention reduces the sucrose content while increasing the content of prebiotic / dietary fiber glucan.

[0036] The present invention also provides the use of immobilized glucan sucrase in increasing the glucan content in jujube juice. The nucleotide sequence of the gene encoding the glucan sucrase is shown in SEQ ID NO.1.

[0037] To further illustrate the present invention, a method for converting sucrose in jujube into glucan using an immobilized enzyme, jujube juice rich in glucan and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] The sucrose content was determined by HPLC.

[0039] 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.

[0040] Preparation method of jujube juice

[0041] 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.

[0042] Example 1

[0043] 1) Selection of dextran sucrase

[0044] The dextran sucrase DsrB was synthesized according to Leuconostoc citrinum CBA3627 (GenBank: CP042418.1), and the nucleotide sequence of the dextran sucrase DsrB is shown in SEQ ID NO.1:

[0045]

[0046] The recombinant plasmid was constructed (the construction method was the same as in Example 2) for expression, the induction temperature was 16°C, and after being immobilized by BEM (the immobilization method was the same as in Example 3, and the BEM and crude enzyme solution were combined for 60 minutes), it was added to jujube juice for sugar conversion, and the sugar conversion conditions were: 30°C, 20 hours. The sucrose content before and after the conversion was measured. The measurement 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] With reference 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, pET-28a-AcmA was constructed. According to Leuconostoc citrinum CBA3627 (GenBank: CP042418.1), forward primer 5'-CGCCCATGGGAGATAGCACAAACACAGTGAC-3' (SEQ ID NO.2) and reverse primer 5'-ATAGAATTCAGCGACTGAGACAAAGTAAC-3' (SEQ ID NO.3) were designed. The glucansucrase gene was obtained by polymerase chain reaction (PCR) amplification. The PCR product and plasmid pET-28a-AcmA were sheared with restriction endonucleases EcoRI and HindIII, and connected and transformed.

[0050] Figure 1 Agarose gel electrophoresis was used to detect the PCR products of colonies. Figure 1 As shown, lanes 1, 2, 4, 5 and 6 are successfully connected plasmids. Combined with the sequencing results, it shows that the vector pET-28a-AcmA is successfully connected to the target gene.

[0051] 3) Recombinant expression of AcmA-tagged dextran sucrase

[0052] The recombinant plasmid pET-28a-AcmA-DsrB was transformed into Escherichia coli BL21 competent cells and cultured in LB medium containing 50 mg / L kanamycin. IPTG was used for induction expression. 37°C, 25°C and 16°C were selected as induction temperatures for optimization. After the expression, the cells were collected for ultrasonic crushing and supernatant was collected by centrifugation. The crude enzyme solution of glucansucrase in the supernatant was immobilized by BEM (the preparation method is the same as that in Example 3) (the combination time of BEM and crude enzyme solution is 60min) and then sugar conversion in jujube juice was performed (sugar conversion conditions are: 30°C, 20h), and the sucrose content before and after conversion was determined. The results showed that the sucrose in jujube was reduced by 36% at 37°C, 54% at 25°C, and 67% at 16°C. Compared with the two induction temperatures of 37°C and 25°C, under the induction condition of 16°C, glucansucrase can reduce more sucrose content in jujube juice.

[0053] Figure 2 This is the SDS-PAGE analysis of recombinant glucansucrase. Lane M: protein marker; Lane 1: bacterial solution before induction; Lane 2: bacterial solution after induction; Lane 3: supernatant after ultrasonic disruption; Lane 4: precipitate after ultrasonic disruption. After IPTG induction, there are target bands in the range of 150-250KDa, proving that IPTG can induce the expression of recombinant glucansucrase; by comparing the supernatant after ultrasonic disruption and the precipitate after ultrasonic disruption, it is concluded that the recombinant glucansucrase DsrB is an intracellular soluble protein, and the recombinant enzyme can be released after ultrasonic disruption.

[0054] Figure 3 The effect of induction temperature on the expression of recombinant glucan sucrase. The present invention uses different induction temperatures to affect the protein expression, and the results are as follows Figure 3 As shown, while SDS-PAGE was used for determination, the enzyme activity was also used for determination. When the induction temperature was 16°C, the expression level of the target protein was the largest and the enzyme activity was the highest, so 16°C was determined to be the optimal induction temperature.

[0055] Example 3

[0056] Enzyme immobilization

[0057] The crude enzyme solution of the glucansucrase obtained at an induction temperature of 16°C in Example 2 was purified using the BEM technology for purifying immobilized enzymes. The preparation of BEM refers 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. Lactococcus lactis NZ9000 was cultured overnight, and the cells were collected by centrifugation for 20 minutes (4000rpm, room temperature), and 0.1MHCl was added to resuspend the cells (100mL 0.1MHCl was added to each 100mL of the collected cells), and then the cells were boiled for 30 minutes to remove protein and DNA. After cooling, the cells were centrifuged at 8000rpm for 20 minutes at room temperature. The precipitate obtained was BEM, and the BEM was fully washed with PBS to obtain a BEM particle precipitate. The prepared BEM was stored in a 4°C refrigerator. The essence of BEM is peptidoglycan. 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] After combining an appropriate amount of BEM granular precipitate with crude enzyme solution on ice for 10min, 30min, and 60min, centrifuge at 12000×g for 10min at 4℃ to remove the supernatant, and wash the precipitate of dextran sucrase after BEM combination with 50mMTris-HCl to obtain immobilized dextran sucrase, and use the immobilized dextran sucrase to convert sugar in jujube juice (sugar conversion conditions: 30℃, 20h), and determine the sucrose content before and after conversion. The results showed that the sucrose content in jujube decreased by 30% after combining for 10min, 50% after combining for 30min, and 65% after combining for 60min.

[0059] Figure 4 This is the SDS-PAGE analysis of recombinant dextran sucrase. 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 (fixed material); lane 6: supernatant bound to BEM; lane 7: precipitate bound to BEM. Figure 4 It can be seen that compared with the recombinant dextran sucrase before purification, there are fewer impurity protein bands after purification, and a good purification effect is obtained.

[0060] Example 4

[0061] Sucrose-to-dextran in jujube juice

[0062] Transformation of Sucrose in Jujube Juice

[0063] The immobilized dextran sucrase obtained in Example 3 under the conditions of 60 min was added to the jujube juice, and the sucrose in the jujube juice was converted at 30° C. The reaction time was 12 h, 14 h, 16 h, 18 h, and 20 h, and the reaction temperature was 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., and 60° C. After the reaction was completed, the immobilized enzyme was removed by centrifugation at 12000×g for 10 min. The sucrose content before and after the conversion was measured.

[0064] Immobilized enzymes are used to convert sucrose in dates into glucan, and the enzyme is removed by centrifugation after the reaction. This reduces the sucrose content while increasing the content of prebiotic / dietary fiber glucan.

[0065] The results showed that the sucrose content in the jujube decreased by 30% after 12 hours of reaction, 58% after 16 hours of reaction, and 67% after 20 hours of reaction. The growth of glucan production slowed down after 20 hours. Considering the economic benefits and other indicators, the best sugar production time was 20 hours, the glucan production was 191.9g / L, and the theoretical production reached 77%.

[0066] Figure 5 The left figure shows the changes of immobilized dextran sucrase before and after the reaction in sucrose solution and jujube juice. The right figure shows the changes of immobilized dextran sucrase before and after the reaction in sucrose solution. Figure 5 The left figure shows that the sucrose solution becomes turbid after the reaction, proving that sucrose is converted into dextran; the right figure shows the changes before and after the immobilized dextran sucrase reacts in jujube juice. Figure 5 As can be seen from the right figure, the jujube juice becomes obviously turbid after the reaction, and the immobilized dextran sucrase successfully converts the sucrose in the jujube juice into dextran.

[0067] Figure 6 This is the result of full wavelength scanning of dextran. After purification, the dextran obtained after the reaction of sucrose solution has the largest characteristic absorption peak at a wavelength of about 200nm, which is the characteristic absorption peak of carbohydrates, and there is no characteristic absorption peak in the range of 260-280nm, indicating that the purified dextran does not contain nucleic acids and proteins.

[0068] Figure 7 The results of the optimal temperature of immobilized dextran sucrase are shown in Figure 2. The ideal temperature of dextran sucrase is 30°C. When the temperature exceeds 30°C, the enzyme activity decreases rapidly. The activity of dextran sucrase is relatively high between 20 and 30°C, and it still has 95% activity at 35°C.

[0069] 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 converting sucrose in jujube into glucan using an immobilized enzyme, characterized in that: The following steps are involved: The immobilized dextran sucrase is mixed with jujube juice, sugar conversion is performed, and the precipitate is discarded by centrifugation to obtain jujube juice rich in dextran; the nucleotide sequence of the gene encoding the dextran sucrase is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The temperature of the sugar conversion is 30-35°C; the time of the sugar conversion is 12-20h.

3. The method according to claim 1, characterized in that The immobilized dextran sucrase is expressed and immobilized based on a bacterial enhanced matrix and a lactococcus lactis peptidoglycan hydrolase expression system.

4. The method according to claim 1 or 3, characterized in that: The method for immobilizing dextran sucrase comprises the following steps: constructing the gene encoding the dextran sucrase 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, and centrifuged to obtain the supernatant to obtain a crude enzyme solution of glucansucrase; The obtained crude dextran sucrase enzyme solution is immobilized by using a bacteria-enhanced matrix purification method to obtain immobilized dextran sucrase.

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

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

7. The method according to claim 6, characterized in that The combining time is 30 to 60 minutes; the combining temperature is 0 to 4°C.

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

9. The jujube juice rich in glucan prepared by the method according to any one of claims 1 to 8.

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

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