Non-genetically modified probiotics with capacity of synthesizing glycans as well as construction method and application of non-genetically modified probiotics
By combining lactic acid bacteria with fusion enzymes of Lactococcus lactic acid lactic acid peptidoglycan hydrolase and fructosacase, lactic acid bacteria synthesize fruit glycans without changing genes, solving the problem of lactic acid bacteria lacking the ability to synthesize fruit glycans. Improves gut health and immune function and avoids genetically modified controversy.
Patent Information
- Application Number
- CN202411409305.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2025-01-10
AI Technical Summary
In the prior art, lactic acid bacteria lack the ability to synthesize fruit glycans, and there is a genetically modified dispute that imparts this ability through genetic modification, and there is a lack of a non-genetically modified solution.
By combining the fusion enzyme of Lactococcus lactica peptidoglycan hydrolase and fructosacase with lactic acid bacteria, the non-gene modification method is used to impart the ability to synthesize fruit glycans. The specific steps include building a recombinant plasmid, transforming E. coli, and purifying fusion. The enzyme is mixed with lactic acid bacteria and undergoes sugar conversion reaction to produce fructans.
It has achieved the synthesis of fructose glycans without changing their genes, which has improved intestinal health and body immune function, avoided controversy caused by genetic modification, and the resulting fructose is of high purity.
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Figure CN119286844A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a non-genetically modified probiotic capable of synthesizing fructan, a construction method thereof and an application thereof. Background Art
[0002] Fructans are a type of water-soluble dietary fiber that can promote the growth of probiotics and has the function of lowering blood lipids and blood sugar. Currently, probiotics represented by lactic acid bacteria do not have the ability to synthesize fructans. The ability to synthesize fructans can be given to them through genetic modification, but genetic modification belongs to the category of transgenics and is controversial in its application. At present, there is a lack of a method to give lactic acid bacteria the ability to synthesize fructans without changing their genes using non-genetic modification. Summary of the invention
[0003] The purpose of the present invention is to provide a non-genetically modified probiotic capable of synthesizing fructan and a construction method and application thereof. The present invention uses a non-genetically modified method to give lactic acid bacteria new functions, thus avoiding the controversy caused by genetic modification.
[0004] The invention provides a non-genetically modified probiotic with the ability to synthesize fructan. The surface of the probiotic is bound with a fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and fructan sucrase; the probiotic comprises lactic acid bacteria.
[0005] Preferably, the lactic acid bacteria include Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus paracasei or Lactobacillus acidophilus.
[0006] Preferably, the levansucrase comprises Bacillus fusogenus levansucrase.
[0007] Preferably, the nucleotide sequence of the gene encoding the Bacillus fusogenus levansucrase is shown as SEQ ID NO.1.
[0008] The present invention also provides a method for constructing the non-genetically modified probiotics described in the above technical solution, comprising the following steps:
[0009] The probiotics are mixed with a fusion enzyme containing peptidoglycan hydrolase and levansucrase from Lactococcus lactis to obtain non-genetically modified probiotics.
[0010] Preferably, the method for preparing the fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase comprises the following steps: constructing the levansucrase gene into a plasmid containing the Lactococcus lactis peptidoglycan hydrolase gene to obtain a recombinant plasmid;
[0011] The recombinant plasmid is transformed into Escherichia coli, cultured, induced and purified to obtain a fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase.
[0012] Preferably, the binding time is 10 to 60 minutes.
[0013] The present invention also provides a method for converting sugar in juice using the non-genetically modified probiotics described in the above technical solution or the non-genetically modified probiotics constructed by the construction method described in the above technical solution, comprising the following steps:
[0014] The non-genetically modified probiotics are mixed with fruit juice to carry out sugar conversion reaction to obtain fructan.
[0015] Preferably, the fruit juice comprises date juice.
[0016] Preferably, the temperature of the sugar conversion reaction is 25 to 40° C.; and the time of the sugar conversion reaction is 6 to 24 hours.
[0017] The present invention provides a non-genetically modified probiotic capable of synthesizing fructan. The non-genetically modified probiotic obtained by the present invention has the ability to synthesize fructan, and the synthesized fructan has a high purity; the non-genetically modified probiotic of the present invention can regulate intestinal health and improve body immunity. The present invention uses a non-genetically modified method to give lactic acid bacteria new functions, avoiding the controversy caused by genetic modification. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] 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.
[0019] Figure 1 This is an SDS-PAGE image of non-genetically modified probiotics constructed from different strains of lactic acid bacteria provided by the present invention;
[0020] Figure 2 This is a full-wavelength scan of fructan obtained after the non-genetically modified probiotics provided by the present invention convert sucrose. DETAILED DESCRIPTION
[0021]
[0022] The present invention also provides a method for constructing the non-genetically modified probiotics described in the above technical solution, comprising the following steps:
[0023] The probiotics are mixed with a fusion enzyme containing a lactococcal peptidoglycan hydrolase and a levansucrase, and the non-genetically modified probiotics are obtained after the combination. In a specific embodiment, the combination time is 10 to 60 minutes. In a specific embodiment, the combination time can be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes. The present invention utilizes the lactococcal peptidoglycan hydrolase to combine the levansucrase with the probiotics more quickly, so the reaction is rapid and time is saved.
[0024] In a specific embodiment, the method for preparing the fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase comprises the following steps: constructing the levansucrase gene into a plasmid containing the Lactococcus lactis peptidoglycan hydrolase gene to obtain a recombinant plasmid;
[0025] The recombinant plasmid is transformed into Escherichia coli, cultured, induced and purified to obtain a fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase.
[0026] The present invention constructs the levansucrase gene into a plasmid containing a lactococcal peptidoglycan hydrolase gene to obtain a recombinant plasmid. In a specific embodiment, the levansucrase includes a brown bacillus levansucrase. In a specific embodiment, the nucleotide sequence of the gene (GenBank ID: ASS72713.1) encoding the brown bacillus levansucrase is shown in SEQ ID NO.1. In a specific embodiment, the primers for amplifying the gene of the brown bacillus levansucrase include a forward primer and a reverse primer. In a specific embodiment, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2: 5'-CGCGGATCCAAAGAAACGGATAAC-3', and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3: 5'-ATAGAATTCTTTATTAACTGTTAG-3'. The present invention does not specifically limit the source of the plasmid containing the peptidoglycan hydrolase gene of Lactococcus lactis, and the plasmid containing the peptidoglycan hydrolase gene of Lactococcus lactis can be constructed by the method reported in the prior art. For example, 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.
[0027] After obtaining the recombinant plasmid, the present invention transforms the recombinant plasmid into Escherichia coli, cultures, induces, and purifies to obtain a fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase. In a specific embodiment, the Escherichia coli includes Escherichia coli BL21 competent cells. In a specific embodiment, the Escherichia coli BL21 competent cells are cultured in LB medium containing kanamycin. In a specific embodiment, IPTG is used to induce the recombinant enzyme. In a specific embodiment, the induction temperature can be 16 to 37°C, specifically 16°C, 25°C or 37°C. After induction, the embodiment of the present invention performs ultrasonic fragmentation, centrifugation, and collects the supernatant to obtain a fusion enzyme (crude enzyme solution) containing Lactococcus lactis peptidoglycan hydrolase and levansucrase.
[0028] The present invention also provides a method for converting sugar in juice using the non-genetically modified probiotics described in the above technical solution or the non-genetically modified probiotics constructed by the construction method described in the above technical solution, comprising the following steps:
[0029] The non-genetically modified probiotics are mixed with fruit juice, and a sugar conversion reaction is performed to obtain fructan. In a specific embodiment, the fruit juice includes jujube juice. In a specific embodiment, the temperature of the sugar conversion reaction is 25 to 40°C, specifically 37°C; the time of the sugar conversion reaction is 6 to 24h, specifically 6h, 12h, 18h or 24h. In a specific embodiment, the lactic acid bacteria include Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus paracasei or Lactobacillus acidophilus. In a specific embodiment, the Lactococcus lactis may be Lactococcus lactis subsp.cremoris NZ9000. In a specific embodiment, the Lactococcus lactis subsp.cremoris NZ9000, GenBank: CP002094.1. In a specific embodiment, the Lactobacillus rhamnosus may be Lactobacillus rhamnosus LGG, the Lactobacillus paracasei may be Lactobacillus paracasei VL8, and the Lactobacillus acidophilus may be Lactobacillus acidophilus CICC 22162. In a specific embodiment, the culture medium for culturing lactic acid bacteria includes MRS culture medium. In a specific embodiment, lactic acid bacteria are statically cultured. In a specific embodiment, the temperature of the static culture can be 37°C, and the time of the static culture can be 12h. After static culture, the present invention collects the bacteria by centrifugation, washes, and obtains the bacteria for subsequent combination. In a specific embodiment, PBS buffer can be used for the washing; the number of washings can be 2 to 3 times. In a specific embodiment, before the combination, the bacteria and the crude enzyme solution are mixed evenly. After the combination, the precipitate is centrifuged. In a specific embodiment, the precipitate is washed; PBS buffer can be used for washing; the number of washings can be 2 to 3 times. After washing, non-genetically modified probiotics are obtained. The non-genetically modified probiotics of the present invention can convert sucrose into fructan. In another specific embodiment, the conversion can be carried out in juice, specifically jujube juice. The present invention does not specifically limit the source of jujube juice, and jujube juice prepared by conventional commercially available jujube juice or conventional juice preparation method can be used.
[0030] To further illustrate the present invention, a non-genetically modified probiotic capable of synthesizing fructans, a construction method thereof and an application thereof 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.
[0031] Example 1
[0032] 1) Construction of pET28a-AcmA-SacB recombinant plasmid.
[0033] According to the Bacillus fusae levansucrase gene (GenBank ID: ASS72713.1), a gene was synthesized and a forward primer and a reverse primer (5'-CGCGGATCCAAAGAAACGGATAAC-3', SEQ ID NO.2) and a reverse primer (5'-ATAGAATTCTTTATTAACTGTTAG-3', SEQ ID NO.3) were designed. The gene of levansucrase SacB was obtained by PCR amplification. The PCR product and plasmid pET28a-AcmA were cut together with restriction endonucleases BamHI and EcoRI, connected and transformed to obtain the recombinant plasmid pET28a-AcmA-SacB.
[0034] 2) Recombinase expression.
[0035] The recombinant plasmid pET28a-AcmA-SacB 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 centrifugation to collect the supernatant to obtain a crude enzyme solution. Lactobacillus lactis subsp.cremoris NZ9000, GenBank: CP002094.1 was used for combination. Then, sugar conversion was performed in jujube juice, and the total polysaccharide content before and after the conversion was determined. The results showed that the total polysaccharide content in jujube increased by 15% at 37°C, 30% at 25°C, and 60% at 16°C.
[0036] Polysaccharide determination method: Centrifuge the converted jujube juice at 8000rpm and 4℃ for 15min, take the supernatant after centrifugation, add three times the volume of 95% ethanol, precipitate at 4℃ for 12h, and centrifuge to get the precipitate. Add 75% ethanol 3 times the original volume, shake and resuspend, centrifuge and take the precipitate to wash once, repeat this operation 2-3 times, and finally get the precipitate. Dry it naturally for 24h, add 10 times the original volume of distilled water, measure the absorbance at 220nm, and bring in the standard curve to get the total polysaccharide content.
[0037] 3) Construction of non-genetically modified probiotics.
[0038] Lactic acid bacteria were cultured using MRS medium (consistent with that used in 2)), statically cultured at 37°C for 12h, the cells were collected by centrifugation, washed 2-3 times with PBS buffer to fully remove the culture medium, and the cells were obtained by centrifugation. The obtained cells were resuspended in the crude enzyme solution obtained in 2), mixed by suction, and the cells and crude enzyme solution were combined for 10min, 30min, and 60min, respectively, and then centrifuged to obtain the precipitate. Resuspended by suction with PBS buffer, the precipitate was obtained by centrifugation, washed once, and this operation was repeated 2-3 times to obtain non-genetically modified probiotics. Non-genetically modified probiotics were used for sugar conversion in jujube juice, and the total polysaccharide content before and after conversion was determined. The results showed that the total polysaccharide content in jujube increased by 40% after 10min of combination, 55% after 30min of combination, and 53% after 60min of combination.
[0039] 4) Influence of bacterial species.
[0040] Different lactic acid bacteria, including Lactobacillus rhamnosus LGG, Lactobacillus paracasei VL8, and Lactobacillus acidophilus CICC22162, were selected to construct non-genetically modified probiotics. The results of SDS-PAGE (mixing the sample with 5*protein loading buffer at a ratio of 4:1 and boiling at 100℃ for 15min to denature the protein sample) are as follows Figure 1 shown. Figure 1 The SDS-PAGE diagram of different strains of lactic acid bacteria and non-genetically modified probiotics constructed from different strains of lactic acid bacteria; wherein, 1: crude enzyme solution obtained after ultrasonic disruption in 2); M: Marker; 2: Lactobacillus paracasei VL8; 3: non-genetically modified Lactobacillus paracasei VL8; 4: Lactobacillus rhamnosus LGG; 5: non-genetically modified Lactobacillus rhamnosus LGG; 6: Lactobacillus acidophilus CICC 22162; 7: non-genetically modified Lactobacillus acidophilus CICC 22162. Obvious bands can be seen at 70KDa in lanes 3, 5, and 7, indicating that the non-genetically modified probiotics were successfully constructed.
[0041] The non-genetically modified probiotics were used to convert sugars in jujube juice, reacted at 37°C for 24 hours, and the total polysaccharide content before and after the conversion was determined. The results showed that the bacteria prepared by Lactobacillus rhamnosus LGG increased the total polysaccharide content in jujube by 20%, the bacteria prepared by Lactobacillus paracasei VL8 increased the total polysaccharide content in jujube by 25%, and the bacteria prepared by Lactobacillus acidophilus CICC 22162 increased the total polysaccharide content in jujube by 120%.
[0042] 4) Non-genetically modified probiotics convert sucrose under different time conditions.
[0043] The non-genetically modified probiotics prepared by Lactobacillus acidophilus CICC 22162 were added to jujube juice for sugar conversion, and the conversion time was 6h, 12h, and 18h, respectively, and the total polysaccharide content before and after the conversion was determined. The results showed that the total polysaccharide content in the jujube increased by 50% after 6h of conversion, 120% after 12h of conversion, and 120% after 18h of conversion.
[0044] The non-genetically modified probiotics prepared by Lactobacillus acidophilus CICC 22162 were added to jujube juice and reacted at 37°C for 18 hours. The obtained jujube juice was centrifuged to obtain the supernatant, and three times the volume of 95% ethanol was added. The mixture was precipitated at 4°C for 12 hours and the precipitate was centrifuged. Three times the original volume of 75% ethanol was added and resuspended by oscillation. The precipitate was centrifuged and washed once. This operation was repeated 2 to 3 times to obtain the fructan precipitate. It was naturally dried for 24 hours, and 10 times the original volume of distilled water was added. The fructan precipitate was obtained by full wavelength scanning at 190-800nm using a quartz cuvette. Figure 2 . Figure 2 This is a full-wavelength scan of the synthesized fructan, which corresponds to the full-wavelength scan result of the fructan obtained after the non-genetically modified probiotics converted sucrose in 4). Figure 2 It was demonstrated that non-genetically modified probiotics have the ability to synthesize fructans, and the synthesized fructans have a high purity.
[0045] The present invention uses a non-genetic modification method to give lactic acid bacteria new functions, thus avoiding the controversy caused by genetic modification.
[0046] 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 non-genetically modified probiotic capable of synthesizing fructan, characterized in that: The probiotics are bound to the surface of a fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase; the probiotics include lactic acid bacteria.
2. The non-genetically modified probiotic according to claim 1, characterized in that The lactic acid bacteria include Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus paracasei or Lactobacillus acidophilus.
3. The non-genetically modified probiotic according to claim 1, characterized in that The levansucrase includes Bacillus fusogenus levansucrase.
4. The non-genetically modified probiotic according to claim 1, characterized in that The nucleotide sequence of the gene encoding the Bacillus fusae levansucrase is shown in SEQ ID NO.
1.
5. The method for constructing the non-genetically modified probiotic according to any one of claims 1 to 4, comprising the following steps: The probiotics are mixed with a fusion enzyme containing peptidoglycan hydrolase and levansucrase from Lactococcus lactis to obtain non-genetically modified probiotics.
6. The construction method according to claim 5, characterized in that: The method for preparing the fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase comprises the following steps: constructing the levansucrase gene into a plasmid containing the Lactococcus lactis peptidoglycan hydrolase gene to obtain a recombinant plasmid; The recombinant plasmid is transformed into Escherichia coli, cultured, induced and purified to obtain a fusion enzyme containing Lactococcus lactis peptidoglycan hydrolase and levansucrase.
7. The construction method according to claim 5, characterized in that: The binding time is 10 to 60 minutes.
8. A method for converting sugar in juice using the non-genetically modified probiotics according to any one of claims 1 to 4 or the non-genetically modified probiotics constructed by the construction method according to any one of claims 5 to 7, characterized in that: The following steps are involved: The non-genetically modified probiotics are mixed with fruit juice to carry out sugar conversion reaction to obtain fructan.
9. The method according to claim 8, characterized in that The fruit juice includes date juice.
10. The method according to claim 8, characterized in that The temperature of the sugar conversion reaction is 25 to 40° C.; the time of the sugar conversion reaction is 6 to 24 hours.