Recombinant lactic acid bacteria preparation for promoting body to synthesize melatonin and construction method of recombinant lactic acid bacteria preparation
By introducing AANAT and HIOMT genes into lactic acid bacteria, recombinant lactic acid bacteria were solved, and the problem of low bioavailability of oral melatonin supplements was achieved to safely and effectively improve serum melatonin levels.
Patent Information
- Application Number
- CN202510722411.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-29
AI Technical Summary
In the prior art, oral melatonin supplements have low bioavailability and have side effects, making it difficult to safely and effectively increase the endogenous melatonin level in the human body.
Through genetic engineering technology, the melatonin synthetase AANAT and HIOMT genes were introduced into Lactococcus lactis to construct recombinant lactic acid bacteria, so that they secrete melatonin synthetase in the intestine, promote the synthesis of intestinal cells and absorb into the blood circulation.
It has achieved safe and effective improvement of the serum melatonin level, avoided the side effects of traditional supplements, and has broad application prospects.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant lactic acid bacterium capable of increasing serum melatonin levels and a method for constructing the same. Background Art
[0002] Melatonin, a hormone secreted by the pineal gland, is involved in regulating circadian rhythms, sleep, and immune function. However, with aging and other factors, endogenous melatonin levels in the human body decrease significantly, affecting sleep quality and overall health. Oral melatonin supplements are commonly used to increase melatonin levels, but these methods carry risks such as low bioavailability and side effects. Therefore, developing a method that can safely and effectively increase endogenous melatonin levels is of great significance. Summary of the Invention
[0003] The present invention aims to provide a recombinant lactic acid bacterium capable of increasing serum melatonin levels and a method for its construction. The recombinant lactic acid bacterium, through genetic engineering techniques, introduces the melatonin synthase AANAT and HIOMT genes into Lactococcus lactis. This recombinant lactic acid bacterium is capable of secreting melatonin synthase in the intestine, promoting melatonin synthesis in intestinal cells, and thereby increasing serum melatonin levels. The recombinant lactic acid bacteria of this invention lack resistance selection markers, are highly safe, and are suitable for oral administration. They can survive in the intestine and effectively secrete melatonin synthase. Melatonin synthesized in the intestine is absorbed into the bloodstream through the intestinal epithelium. Compared with existing technologies, this invention provides a safe and effective method for increasing endogenous melatonin levels, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0004] Figure 1 This is the vector map for inserting the AANAT-T2A-HIOMT gene sequence; Figure 2 To identify AANAT and HIOMT proteins expressed by recombinant lactic acid bacteria; Figure 3 To detect the melatonin content in the serum of mice fed with recombinant lactic acid bacteria. DETAILED DESCRIPTION
[0005] The first aspect of the present invention provides a method for constructing a lactic acid bacteria preparation expressing AANAT and HIOMT Recombinant strains were constructed and screened using a non-resistance selection marker technique. Based on information published in the NCBI database, T2A-linked AANAT and HIOMT gene fragments (AANAT-T2A-HIOMT) were designed and synthesized. These fragments were assembled into the pNZ8149 vector, which contains a lactic acid promoter. The recombinant vector was then transduced into Lactococcus lactis NZ3900 via electroporation. Recombinant lactic acid bacteria were screened in Elliker selection medium, and the plasmids from the recombinant lactic acid bacteria were extracted and identified. Figure 1
[0006] The second aspect of the present invention provides an identification experiment of recombinant lactic acid bacteria expressing and secreting AANAT and HIOMT proteins The recombinant lactic acid bacteria preparation provided by the present invention was induced to express AANAT and HIOMT proteins using the food-grade inducer Nisin. After screening the induction concentration, induction temperature, and induction time, the final Nisin induction dose was (50-100) ng / mL, the induction temperature was 30°C, and the induction time was 2.5 h-4 h. Western blotting was used to determine the expression of the melatonin synthase AANAT-HIOMT protein. Figure 2 As shown, the recombinant lactic acid bacteria preparation provided by the present invention can express and secrete AANAT and HIOMT proteins.
[0007] The third aspect of the present invention provides an evaluation of the effect of expressing a recombinant lactic acid bacteria preparation on promoting melatonin synthesis in mice Eighteen 6-8 week old Kunming white mice were selected and divided into three groups. The first group was the NC group. The mice were fed with an equal amount of normal saline by gavage. The second group was the low-dose group. The mice were gavaged with a recombinant lactic acid bacteria preparation at a dose of 108 CFU / 50g body weight / day. The third group was the high-dose group. The mice were gavaged with a recombinant lactic acid bacteria preparation at a dose of 5×108 CFU / 50g / day. After 14 consecutive days of gavage, peripheral blood was collected and serum was separated. The melatonin content in the serum was detected by ELISA. Figure 3 As shown, the melatonin content in the serum of mice administered with low or high doses of recombinant lactic acid bacteria preparation was significantly higher than that in the control group.
[0008] application The recombinant lactic acid bacteria of the present invention can be prepared into an oral preparation, which enters the intestine through oral administration, colonizes in the intestine and secretes melatonin synthase, promotes intestinal cells to synthesize melatonin and increases serum melatonin levels.
[0009] advantage The recombinant lactic acid bacteria of the present invention are safe, have no resistance screening markers, are suitable for oral administration, can stably express melatonin synthase in the intestine, significantly increase serum melatonin levels, and have wide application value.
Claims
1. The present invention provides a lactic acid bacteria preparation that can effectively promote the body to synthesize melatonin, characterized in that: The recombinant lactic acid bacteria preparation introduces melatonin synthase AANAT and HIOMT genes into the lactic acid bacteria through genetic engineering technology, so that the melatonin synthase can be secreted.
2. The recombinant lactic acid bacteria according to claim 1, characterized in that The recombinant lactic acid bacteria is an orally administrable strain that can survive in the intestine and secrete melatonin synthase.
3. The recombinant lactic acid bacteria according to claim 1 or 2, characterized in that The lactic acid bacteria is Lactococcus lactis, has no resistance screening marker, and is a food-grade strain.
4. The method for constructing a recombinant lactic acid bacterium according to any one of claims 1 to 3, wherein: The following steps are involved: (1) The gene sequences of melatonin synthases AANAT and HIOMT were designed and synthesized, and linked with a T2A element (AANA-T2A-HIOMT); (2) AANA-T2A-HIOMT was integrated into the vector pNZ8149 controlled by a lactic acid bacteria promoter using genetic engineering techniques; (3) The recombinant vector was transduced into Lactococcus lactis (NZ3900) by click transformation and the recombinant lactic acid bacteria were screened using Elliker screening plates; (4) The protein expression effect and effectiveness of the recombinant lactic acid bacteria were verified.
5. The AANAT and HIOMT sequences according to claims 1-4 are conserved sequences and are applicable to multiple species, including humans, mice, dogs, cats, chickens, cattle, sheep, etc.
6. Use of the recombinant lactic acid bacteria according to any one of claims 1 to 3 for increasing serum melatonin levels, characterized in that: The recombinant lactic acid bacteria enter the intestine through oral administration, secrete melatonin synthase, promote intestinal cells to synthesize melatonin, and thus increase the concentration of melatonin in serum.
7. The use of the recombinant lactic acid bacteria according to claim 6 in improving serum melatonin levels, wherein the dosage is 10 10 CFU / kg body weight / day.