Application and methods of Leuconostoc mesenteroides

Abstract

The present invention discloses applications and methods of Leuconostoc mesenteroides, relating to the field of microbial technology. Leuconostoc mesenteroides can be used to prepare phytic acid and improve non-alcoholic fatty liver disease, increased liver weight, elevated blood lipids, elevated blood sugar, elevated serum lipopolysaccharide levels, elevated serum insulin levels, inflammatory responses, and elevated triglyceride levels in the liver caused by a high-fat, high-sugar diet, and inhibit the release of the pro-inflammatory cytokine lipopolysaccharide.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to applications and methods of Leuconostoc mesenteroides. Background Art

[0002] Phytic acid, also known as inositol hexaphosphate, is a phosphorus-containing organic acid compound. Previous studies have shown that phytic acid can form chelate complexes with polyvalent metal ions (such as calcium and magnesium ions), thereby affecting the absorption and utilization of mineral ions, and is considered an antioxidant. However, with in-depth research, it has been found that small molecules of phytic acid do not antagonize the absorption and utilization of minerals and have extremely low toxicity. In recent years, numerous studies have demonstrated that phytic acid has unique physiological activities, such as antioxidant, anti-tumor, immunomodulatory, and weight loss effects, and is a natural nutrient. Currently, phytic acid is widely used in the food industry as an antioxidant, preservative, and stabilizer, such as in oily foods, beverages, and fresh fruits and vegetables.

[0003] Currently, the production of phytic acid still relies on traditional chemical extraction from plant seeds, grains, or fruit shells. This involves crushing the raw materials, then extracting calcium phytate through acid leaching, filtration, and neutralization. Phytic acid is then produced through acidification, filtration, neutralization, ion exchange resin treatment, concentration, and decolorization. This traditional phytic acid production process is cumbersome, time-consuming, and environmentally polluting, limiting its sustainability and environmental friendliness. Microbial fermentation, or biosynthesis, is an emerging technology in recent years. It offers advantages such as mild conditions, minimal environmental pollution, and low raw material costs. It has been widely used in the biosynthesis of various high-value platform compounds, such as lactic acid and D-glucaric acid. This suggests that developing economical, environmentally friendly, and efficient phytic acid biosynthesis pathways is one of the key strategies for addressing the challenges of existing production processes. However, there are currently no reports on the microbial fermentation synthesis of phytic acid. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of Leuconostoc mesenteroides for preparing phytic acid.

[0005] The invention also provides a method for preparing phytic acid.

[0006] According to the first aspect of the present invention, an embodiment of the present invention is the use of Leuconostoc mesenteroides in the preparation of phytic acid.

[0007] According to some embodiments of the present invention, the application is achieved by isolating and purifying phytic acid from the fermentation broth of Leuconostoc mesenteroides.

[0008] According to some embodiments of the present invention, the Leuconostoc mesenteroides includes Leuconostoc mesenteroides with a deposit number of GDMCC No: 61720.

[0009] According to a second embodiment of the present invention, a method for preparing phytic acid is provided by separating and purifying the fermentation broth of Leuconostoc mesenteroides.

[0010] According to some embodiments of the present invention, the Leuconostoc mesenteroides includes Leuconostoc mesenteroides with a deposit number of GDMCC No: 61720.

[0011] Phytic acid can be prepared from fermentation broth through conventional phytic acid separation and purification processes.

[0012] According to some embodiments of the present invention, the purification includes an ion exchange resin method. The ion exchange resin method uses an anion exchange resin to adsorb phytate ions in the solution, and then uses an alkaline eluent to elute to remove anionic impurities such as chloride ions, and then uses a cationic resin to adsorb Na + , Ca 2+ and Mg 2+ Cationic impurities such as PEG are removed to achieve the purpose of purification. The anion resins used include but are not limited to 330, 717, D315, D318, and D301.

[0013] According to some embodiments of the present invention, the method for preparing the fermentation broth includes: inoculating the Leuconostoc mesenteroides into a culture medium, and culturing at 30° C. to 37° C. to obtain the fermentation broth.

[0014] According to some embodiments of the present invention, the fermentation time is 12 h to 96 h, preferably 24 h to 72 h, and more preferably 36 h to 60 h.

[0015] According to some embodiments of the present invention, the inoculation density of Leuconostoc mesenteroides is 10 6 CFU / mL~10 10 CFU / mL. Preferably 10 7 CFU / mL~10 9 CFU / mL.

[0016] According to some embodiments of the present invention, the fermentation temperature is 25°C to 40°C, preferably 35°C to 40°C, and more preferably 34°C to 37°C.

[0017] According to some embodiments of the present invention, the culture medium includes MRS culture medium, Rogosa culture medium, LMM culture medium. It is understood that other well-known culture media or culture solutions for lactic acid bacteria are also applicable.

[0018] According to some embodiments of the present invention, the preparation method further comprises a post-purification treatment, wherein the post-purification treatment comprises at least one of concentration and decolorization.

[0019] According to the third aspect of the present invention, the use of Leuconostoc mesenteroides with the accession number of GDMCC No: 61720 in any one of A1 to A9,

[0020] A1: Preparation of products for the prevention and treatment of non-alcoholic fatty liver disease;

[0021] A2: Preparation of products for preventing and / or reducing weight gain;

[0022] A3: Preparation of a product for preventing and / or reducing liver weight increase;

[0023] A4: Preparation of products for lowering blood lipids;

[0024] A5: Preparation of products for lowering blood sugar;

[0025] A6: Preparation of a product for reducing lipopolysaccharide levels in serum;

[0026] A7: Preparation of a product that reduces serum insulin levels;

[0027] A8: Prepare products that improve inflammatory symptoms;

[0028] A9: Prepare a product for lowering hepatic triglyceride levels.

[0029] According to some embodiments of the present invention, the product is directed to non-alcoholic fatty liver disease, increased liver weight, increased blood lipids, increased blood glucose, increased serum lipopolysaccharide levels, increased serum insulin levels, inflammatory response, and increased triglyceride levels in the liver caused by high fat and / or high sugar intake.

[0030] According to some embodiments of the present invention, the lipid-lowering comprises lowering at least one of the levels of serum total cholesterol, serum total triglycerides, and liver triglycerides.

[0031] According to some embodiments of the present invention, the lowering of blood sugar comprises lowering the level of serum glucose.

[0032] According to some embodiments of the invention, improving inflammatory symptoms comprises reducing the level of interleukin-6 in the liver.

[0033] According to some embodiments of the invention, the product comprises a medicine.

[0034] According to some embodiments of the present invention, the product further comprises a pharmaceutically acceptable excipient.

[0035] According to some embodiments of the present invention, the dosage form of the drug includes at least one of tablets, capsules, granules, and pills.

[0036] The present invention has at least the following beneficial effects:

[0037] Traditional methods for extracting phytic acid are complex, environmentally polluting, and time-consuming. However, Leuconostoc mesenteroides, deposited with GDMCC No. 61720, can biosynthesize high levels of phytic acid through fermentation in an environmentally friendly, economical, and efficient manner. Leuconostoc mesenteroides, deposited with GDMCC No. 61720, can improve non-alcoholic fatty liver disease, increased liver weight, elevated blood lipids, elevated blood glucose, elevated serum lipopolysaccharide levels, elevated serum insulin levels, inflammatory responses, elevated triglyceride levels in the liver, and inhibit the release of the pro-inflammatory cytokine lipopolysaccharide, caused by a high-fat, high-sugar diet.

[0038] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a liquid chromatogram of a fermentation broth of Leuconostoc mesenteroides according to an embodiment of the present invention;

[0040] Figure 2 FIG1 is a schematic diagram of inositol phosphate metabolism of Leuconostoc mesenteroides according to an embodiment of the present invention. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0042] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0043] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to the process, method, product or apparatus.

[0044] In the following examples, Leuconostoc mesenteroides ( Leuconostoc mesenteroides )I1 / 53, deposited on June 11, 2021, at the Guangdong Provincial Microbial Culture Collection Center, Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 61720.

[0045] C57 mice, male, 6–7 weeks old, were provided by Zhuhai Baishitong Biotechnology Co., Ltd.

[0046] Normal mouse feed and high-sugar and high-fat feed (containing 30% sucrose and 35% fat) were provided by Beijing Keao Xieli Feed Co., Ltd.

[0047] Phytic acid was provided by Shanghai MacLean Biochemical Technology Co., Ltd.

[0048] Example 1 (Identification of Phytic Acid in Leuconostoc mesenteroides I1 / 53 Fermentation Broth)

[0049] Active Leuconostoc mesenteroides I1 / 53 was inoculated into 10 mL of MRS medium and cultured at 37°C for 48 h (inoculation size was 1×10 9 CFU); the fermentation broth was then centrifuged at 4°C and 8000 rpm for 10 minutes. 50 µL of the supernatant was added to 750 µL of pre-chilled (4°C) acetonitrile, followed by sonication for 3 minutes and centrifugation at 4°C and 13000 rpm for 15 minutes. 600 µL of the supernatant was used for non-targeted metabolomics analysis using an ultra-high-pressure liquid phase triple quadrupole time-of-flight mass spectrometer. Chromatographic and mass spectral data were collected using Progenesis QI V2.0 software. After a series of quality control procedures, including noise reduction, peak alignment, and zeroing, the high-quality chromatographic and mass spectral data were compared and analyzed with public databases, and accurate identification was achieved based on the secondary mass spectral data.

[0050] The chromatographic column was an ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 μm); the parameters were: column temperature 40°C, mobile phase flow rate 0.35 mL / min, injection volume 2.0 μL. Mobile phase A was pure water, and mobile phase B was acetonitrile. The elution gradient was: 98.0% A at 0 min, 50% A at 6.5 min, and 98% A at 10 min. The total detection time was 12.5 min.

[0051] The mass spectrometer parameters were set as follows: in positive ion mode, M / Z range 50-1200, collision voltage 35 V; desolvation and cone gas were nitrogen, capillary pressure 2.5 kilowatt-volt, cone gas flow rate 40 L / h, and ion source temperature 120°C.

[0052] Identification results such as Figure 1 A total of 1069 metabolites were detected, among which phytic acid had a retention time of 3.6412 and a mass-to-charge ratio of 659.8614. Further quantitative analysis using liquid chromatography and standard analysis revealed a phytic acid content of 36.6 mg / L in the fermentation broth.

[0053] Example 2

[0054] In this example, the whole genome of Leuconostoc mesenteroides I1 / 53 was determined using the PromethlON and NovaSeq6000 platforms, as follows:

[0055] DNA from Leuconostoc mesenteroides I1 / 53 was extracted using the Hipure Bacterial DNA Kit (provided by Shanghai Meiji Biotechnology Co., Ltd.), and the concentration and purity of the DNA were analyzed using Nanodrop 2000 and Qubit 3.0 fluorometers. A DNA library was constructed and sequenced using the SQK-LSK109 kit (provided by Shanghai Meiji Biotechnology Co., Ltd.). The sequencing results were then quality controlled to remove low-quality fragments, and the genome was assembled and structurally analyzed. Functional analysis was also performed using public databases including the protein family database, gene sequence database, homologous sequence database, and KEGG database.

[0056] The analysis results showed that the genome size of L. mesenteroides I1 / 53 was 2,044,872 bps, with a GC content of 37.5%, 2,027 coding DNA sequences (CDS), 16 human RNA operons and 71 transfer RNA genes.

[0057] Functional analysis of the genome of Leuconostoc mesenteroides I1 / 53 revealed a total of 2205 metabolic pathways. Combined analysis with the metabolome revealed that five metabolic pathways were the intersection of the two omics, among which inositol phosphate metabolism was a pathway for the synthesis of phytic acid, such as Figure 2 shown.

[0058] Example 3

[0059] This example evaluated the physiological functions of Leuconostoc mesenteroides I1 / 53 and its metabolite phytic acid through animal experiments on a high-sugar, high-fat, non-alcoholic fatty liver disease mouse model. The steps are as follows:

[0060] Twenty-four C57 mice were randomly divided into four groups: normal group, model group, Leuconostoc mesenteroides I1 / 53 intervention group and phytic acid intervention group, with 6 mice in each group. The mice were gavaged once a day, and the dose of Leuconostoc mesenteroides was 1*10 9CFU / mouse, the phytic acid dosage was 100 mg / kg·bw. During the experiment, the normal group was fed normal mouse feed, and the other three groups were fed high-sugar and high-fat feed. All mice had free access to water for 8 weeks. At the end of the experiment, the mice were fasted for 12 hours and weighed. Serum and liver tissue were obtained after anesthesia. After the liver was weighed, the levels of insulin, glucose, lipopolysaccharide, and cholesterol (including total cholesterol and triglycerides) in serum, and the levels of triglycerides and interleukin-6 in the liver were determined using enzyme-linked immunosorbent assay kits (Wuhan Enzyme Immunity Biotechnology Co., Ltd.).

[0061] The test results are shown in Table 1. Compared with the model group, the body weight, liver tissue, serum insulin, glucose, lipopolysaccharide, total cholesterol, and triglyceride levels of mice in the Leuconostoc mesenteroides and phytic acid intervention groups were significantly reduced. In addition, liver triglyceride and interleukin-6 levels were also significantly reduced in the Leuconostoc mesenteroides and phytic acid intervention groups. This suggests that both Leuconostoc mesenteroides I1 / 53 and its metabolite, phytic acid, have the effect of alleviating non-alcoholic fatty liver disease.

[0062] Table 1 Live Leuconostoc mesenteroides preparation and phytic acid have the effect of alleviating non-alcoholic fatty liver disease

[0063]

[0064] Note: * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0065] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. Use of Leuconostoc mesenteroides in the preparation of phytic acid; the Leuconostoc mesenteroides includes Leuconostoc mesenteroides with a preservation number of GDMCC No: 61720; The phytic acid is obtained by separating and purifying the fermentation broth of the Leuconostoc mesenteroides.

2. A method for preparing phytic acid, characterized in that: The method is obtained by separating and purifying the fermentation liquid of Leuconostoc mesenteroides; the Leuconostoc mesenteroides includes Leuconostoc mesenteroides with a preservation number of GDMCC No: 61720.

3. The preparation method according to claim 2, characterized in that The purification includes ion exchange resin method.

4. The preparation method according to claim 2, characterized in that The method for preparing the fermentation liquid comprises: inoculating the Leuconostoc mesenteroides into a culture medium, and culturing the culture medium at 25° C. to 40° C. to obtain the fermentation liquid.

5. The preparation method according to claim 4, characterized in that The culture time is 12 h to 96 h.

6. The preparation method according to claim 4, characterized in that The inoculation density of the Leuconostoc mesenteroides is 10 6 CFU / mL~10 10 CFU / mL.

7. The preparation method according to claim 4, characterized in that The culture medium includes MRS culture medium, Rogosa culture medium and LMM culture medium.

8. Use of Leuconostoc mesenteroides with a deposit number of GDMCC No: 61720 in any one of items A1 to A9, characterized in that: A1: Preparation of products for the prevention and treatment of non-alcoholic fatty liver disease; A2: Preparation of products for preventing and / or reducing weight gain; A3: Preparation of a product for preventing and / or reducing liver weight increase; A4: Preparation of products for lowering blood lipids; A5: Preparation of products for lowering blood sugar; A6: Preparation of a product for reducing lipopolysaccharide levels in serum; A7: Preparation of a product that reduces serum insulin levels; A8: Prepare products that improve inflammatory symptoms; A9: Preparation of a product for lowering hepatic triglyceride levels; The product is a drug.

9. The use according to claim 8, characterized in that The product targets non-alcoholic fatty liver disease, increased liver weight, elevated blood lipids, elevated blood glucose, elevated serum lipopolysaccharide levels, elevated serum insulin levels, inflammatory responses, or elevated triglyceride levels in the liver caused by high fat and / or high sugar intake.

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