CN114099555B - Lactobacillus plantarum lipoteichoic acid and application thereof in inhibiting amyloid aggregation - Google Patents
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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Abstract
The invention provides lactobacillus plantarum lipoteichoic acid and application thereof in inhibiting amyloid aggregation. The main chain of the lipoteichoic acid of the lactobacillus plantarum contains glycerol, and the side chain contains a large number of N-acetylglucosamine and alanine modifications. The lipoteichoic acid can effectively inhibit aggregation of beta-amyloid 1-42 (Abeta 42), is an Abeta 42 potential aggregation inhibitor, and can be applied to developing medicines, health products or functional foods for preventing and/or improving Alzheimer's disease.
Description
Technical Field
The invention belongs to the technical field of medicines, health-care products or foods, and particularly relates to separation and extraction, structural identification and application of novel lactobacillus plantarum lipoteichoic acid in inhibiting amyloid aggregation, in particular to application of lactobacillus plantarum MA2 serving as a beta-amyloid 1-42 (Abeta 42) aggregation inhibitor in preparing medicines, health-care products and functional foods for preventing and/or improving Alzheimer's disease.
Background
Aggregation and misfolding of amyloid proteins in cells and tissues to form toxic intermediates and amyloid fibers can lead to various biological dysfunctions. These aggregated intermediates and fibers are presumed to be associated with some neurodegenerative diseases and other disorders, such as Alzheimer's Disease (AD), parkinson's Disease (PD), type II diabetes (diabetes type II), and the like. Numerous studies have demonstrated that the more toxic fractions are oligomers or fibril intermediates, and the development of highly potent inhibitors of amyloid aggregation or functional foods against amyloid aggregation is one of the effective means of alleviating and treating these diseases. Aggregation and deposition of beta-amyloid 1-42 (aβ42) are closely related to the development of AD, and thus inhibition of aβ42 aggregation and deaggregation of aβ42 fibers have become effective pathways for development of anti-AD drugs.
Probiotics are a large class of microorganisms beneficial to the human body, including lactic acid bacteria, bifidobacteria, etc., which exert a probiotic effect by regulating intestinal microecological balance. Several studies have shown that ingestion of probiotics increases the adhesion of the intestinal flora, effectively improves intestinal micro-ecology, activates the immune system to reduce inflammation, promotes release of neurotransmitters and other beneficial metabolites, and thus improves AD and other neurodegenerative diseases. After the AD model rats ingest mixed probiotics consisting of 4 bacteria such as lactobacillus acidophilus and bifidobacterium lactis, the spatial memory capacity and the oxidative stress of the AD model rats are obviously improved. Clinical trials also evaluated the effect of a number of probiotics consisting of lactobacillus and bifidobacteria on learning and memory capacity of AD patients, and the results indicated that the minimum mental state of the tested patients was significantly improved. Thus, it is expected that a microecological therapy for improving intestinal flora by probiotics is an effective way to prevent and improve AD. The existing known probiotics for preventing and treating AD are lack of species, the complexity of intestinal flora and metabolites thereof increases the research difficulty, and the action mechanism for improving AD by utilizing intestinal microecology is not clear, which are key scientific problems for preventing and relieving AD research by using probiotics.
In recent years, the concept of a metagen has been attracting attention, and the metagen is a generic term for a probiotic metabolite component and a bacterial cell obtained by processing a probiotic. Research proves that the immunoregulation capability of some metazoan is better than that of the original viable bacteria, and the metazoan still has high physiological activity even though being treated by high temperature, ultrasonic or gastrointestinal digestive juice. Wherein teichoic acid is a representative component of metazoan, and is a key for determining the acid, alkali and heat tolerance of metazoan. Teichoic acid is a specific component of the cell wall of gram-positive bacteria, and is divided into a main chain and a side chain, wherein the main chain is generally formed by polymerizing 16-40 phosphoric acid-glycerol or phosphoric acid-ribitol, and the side chain is generally modified by hydroxyl, N-acetylglucosamine or D-alanine and the like. Teichoic acids can be classified into lipoteichoic acid (Lipoteichoic acid, LTA) and teichoic acid (Wallteichoic acid, WTA), which are anchored to the cell membrane and peptidoglycan of the cell wall, respectively. Wherein, gram positive bacterial lipoteichoic acid plays an important role in physiological functions, and mainly comprises the following aspects: (1) determining bacterial cell colonization; (2) Maintaining the balance of anions and cations in the cell walls of the bacteria, so that the bacteria are prevented from being damaged by antibacterial peptides, antibiotics and the like; (3) Recognizes and interacts with host cell-associated receptors, specifically recognizes and activates animal pattern recognition receptors and elicits corresponding immune response.
Lactobacillus plantarum MA2 is a novel lactobacillus strain separated from the subject group, and early researches show that the strain has better acid resistance, cholate resistance and antioxidation activity and has a remarkable function of improving the cognitive dysfunction of AD mice. Meanwhile, the thallus treated by high temperature or ultrasonic has good AD improving effect, so the invention firstly separates and extracts the lactobacillus plantarum MA2 lipoteichoic acid (lpTLA), identifies the structure of the lactobacillus plantarum MA2 lipoteichoic acid (lpTLA), explores the effect of the lactobacillus plantarum MA2 lipoteichoic acid in inhibiting Abeta 42 aggregation, and has important theoretical significance and application value for researching that the lipoteichoic acid improves neurodegenerative diseases and developing and preventing and/or improving AD medicines, health care products or functional foods.
Disclosure of Invention
The invention provides separation, extraction and structural identification of novel lactobacillus plantarum lipoteichoic acid and application thereof in inhibiting amyloid aggregation, and application thereof in preparing medicines, health-care products or functional foods for preventing and/or improving AD.
Further, the lactobacillus plantarum lipoteichoic acid is lactobacillus plantarum MA2 lipoteichoic acid.
Further, the lactobacillus plantarum MA2 lipoteichoic acid is separated, extracted and structurally identified.
Further, the use of lactobacillus plantarum MA2 lipoteichoic acid in the manufacture of a medicament, a health product or a functional food for preventing and/or ameliorating a disease characterized by the aggregate deposition of aβ42.
Further, the disease is Alzheimer's disease.
Further, the lactobacillus plantarum MA2 lipoteichoic acid is present in an aqueous dispersion.
Further, the Lactobacillus plantarum MA2 lipoteichoic acid is present in an aqueous dispersion having a concentration of 0.25,0.50,1.00,2.00 mg/mL.
The lipoteichoic acid of the lactobacillus plantarum is lipoteichoic acid which is firstly separated, purified and subjected to structural identification, and also has the following advantages in the aspect of developing an Abeta 42 aggregation inhibitor, wherein the lipoteichoic acid is the function of inhibiting Abeta 42 aggregation for the first time:
the application of the lactobacillus plantarum MA2 lipoteichoic acid in inhibiting Abeta 42 aggregation is derived from lactobacillus plantarum with a probiotic effect on human bodies, and the safety is high; the lpLTA side chain obtained by the invention has higher content of N-acetylglucosamine, alanine and fatty acid groups, has important effect on physiological functions, can effectively inhibit Abeta 42 aggregation, can be used as a potential prodrug or food functional factor, is used for developing new drugs, health care products or functional foods, and is used for preventing and/or improving diseases characterized by Abeta 42 aggregation and the abnormal conception thereof.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is an infrared spectrum of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA) in example 2 of the present invention.
FIG. 2 is a nuclear magnetic resonance hydrogen diagram of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA) in example 2 of the present invention
FIG. 3 is a fluorescence plot of the co-culture of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA) and Abeta 42 at various concentrations in example 3 of the present invention.
FIG. 4 is a scanning Atomic Force Microscope (AFM) scan of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA) and Abeta 42 co-culture according to example 4 of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The embodiment of the invention provides separation and extraction of novel lactobacillus plantarum lipoteichoic acid, structural identification and application thereof in inhibiting amyloid aggregation.
In the embodiment of the invention, the lactobacillus plantarum is lactobacillus plantarum MA2 separated and screened in the laboratory, and is derived from the Kefir grains of Tibetan farmhouses, and the lactobacillus plantarum is preserved in the China general microbiological culture Collection center (CGMCC) 3005 in 2009. Amyloid is beta-amyloid 1-42 (aβ42), consisting of 42 amino acids, with a molecular weight of about 4.5kDa, comprising a highly hydrophobic domain, and aggregation and deposition of this amyloid in nerve cells is closely related to the occurrence and progression of AD. The invention provides a new thought for developing Abeta 42 aggregation inhibitor and researching probiotics and metaplasia improving AD.
The embodiment of the invention provides application of lactobacillus plantarum lipoteichoic acid serving as an Abeta 42 aggregation inhibitor in preparing medicines, health-care products or functional foods for preventing and/or improving AD. The invention fully utilizes the characteristics of lactobacillus plantarum MA2 lipoteichoic acid as a prebiotic product after probiotics, has high safety, good patient adaptability and the like, can be widely applied to the development of medicines, health care products or functional foods, and further promotes the research progress of AD
Preferably, the Lactobacillus plantarum MA2 lipoteichoic acid is present in an aqueous dispersion. In other words, lactobacillus plantarum MA2 lipoteichoic acid can be taken in the form of an aqueous dispersion system, such as injection, soft capsule, beverage, oral liquid and the like.
Preferably, the Lactobacillus plantarum MA2 lipoteichoic acid is present in an aqueous dispersion having a concentration of 0.25-2.00 mg/mL. Specific concentrations were 0.25,0.50,1.00 and 2.00mg/mL.
The isolation and extraction of novel lactobacillus plantarum lipoteichoic acid, structural identification and application thereof in inhibiting amyloid aggregation are further described below with reference to specific examples.
Example 1: isolation, extraction and purification of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA)
(1) Inoculating lactobacillus plantarum MA2 in a biosafety cabinet, and culturing to an exponential growth phase under corresponding conditions;
(2) Taking bacterial liquid, centrifuging at 7000rpm at 4 ℃ for 20min in a centrifugal cup, collecting bacterial cells, and weighing the wet weight of the bacterial cells;
(3) Resuspending wet bacteria in 0.1M sodium citrate buffer solution with pH of 4.7 according to the ratio of 1:1 (w/v), repeatedly freezing and thawing for 3 times, crushing the bacteria by using an ultrasonic crusher, wherein the amplitude is 30%, opening for 1s, closing for 1s, and the total working time is 30min;
(4) Adding equal volume of n-butanol into the mixed bacterial suspension, fully mixing, stirring for 30min on a magnetic stirrer, centrifuging at 4 ℃ and 7000rpm for 30min, and gently collecting a lower water phase;
(5) Performing rotary evaporation and dialysis on the water phase, wherein the cut-off molecular weight of a dialysis bag is 2000Da;
(6) Preparation of a2 x 20cm octyl-Sepharose column, equilibrated with 0.1M sodium acetate buffer (pH 4.7) containing 15% n-propanol;
(7) Mixing the lipoteichoic acid crude extract obtained in the step (5) with 0.1M sodium acetate buffer (pH 4.7) in a ratio of 1:5 (v/v), diluting, and passing through a column;
(8) Washing with 0.1M sodium acetate buffer (pH 4.7) of 15% n-propanol to wash out unbound impurities;
(9) The column was eluted with a gradient of 0.1M sodium acetate buffer (pH 4.7) containing 20%, 35%, 45% n-propanol, and the eluate was collected in a 10mL centrifuge tube;
(10) Detecting inorganic phosphorus in the eluent, and collecting the eluent with higher phosphorus content for rotary evaporation and dialysis;
(11) Preparation of DEAE-FastFlow (1.5 x 10 cm) anion chromatography column secondary purification of lipoteichoic acid, equilibrated column with 0.1M sodium acetate buffer (pH 4.7) containing 30% n-propanol;
(12) Pretreating and loading the dialyzed sample in the step (10), performing gradient elution by using 0-1M sodium chloride solution (prepared by 0.1M sodium acetate buffer), and collecting eluent in a 10 mL/tube;
(13) Detecting inorganic phosphorus in the eluent collected in the step (12), and collecting the eluent with higher phosphorus content for rotary evaporation and dialysis;
(14) Freezing the dialysate obtained in the step (13) for 4 hours at the temperature of minus 80 ℃ in advance, then placing the dialysate into a freeze dryer, and obtaining lactobacillus plantarum MA2 lipoteichoic acid solid after sublimation and drying, and freezing and preserving the lactobacillus plantarum MA2 lipoteichoic acid solid.
Example 2: structural identification of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA)
(1) Infrared spectroscopic analysis
Weighing a proper amount of lpLTA powder, grinding with potassium bromide according to a mass ratio of 1:100, tabletting the mixed powder, and analyzing the functional group of lpLTA by using an infrared spectrometer, wherein the result is shown in figure 1.
Referring to FIG. 1, band 3419.95cm -1 Characterization of the-NH group, band 2929.31cm -1 Characterization of the-CH 2 、-CH 3 Radicals, band 1651.56cm -1 Characterization of the-C=O group, band 1547.32cm -1 Characterization of the-C=H group, band 1379.09cm -1 Characterization of the-P=O group, band 1231.18cm -1 Characterization of sugar residue groups, band 1038.81cm -1 Characterization of the-C-O group. The result is basically consistent with the characteristic functional group of the general structure of lipoteichoic acid, and the infrared spectrum result shows that lpLTA contains the phosphoglyceride structure, the amide, the sugar residue and the like.
(2) Nuclear magnetic resonance analysis
An appropriate amount of lpLTA powder was weighed, fully dissolved with deuterated reagent, and the H spectrum of the sample was measured on a 400MHz nmr, and the results are shown in fig. 2.
Referring to fig. 2, lplta has distinct absorption peaks at σ= 5.096,4.182,3.807,3.511,1.983,1.762,1.177 and 0.784, and in combination with standard chemical shifts, 5.096ppm is-COO vibration, 4.182 and 3.807ppm is glycerol and glycosyl vibration, 3.511ppm is N-acetyl vibration, 1.983,1.762,1.177 and 0.784ppm is N-acetylglucosamine, alanine and fatty acid vibration. As can be seen, the lpLTA backbone contains glycerol and the side chains contain N-acetylglucosamine, alanine and fatty acid modifications. Meanwhile, compared with the reported nuclear magnetic resonance result of teichoic acid (Wu et al, 2021,Journal ofBiological Chemistry,296:100384; patent application CN 104161776A-lipoteichoic acid from clostridium butyricum and the application thereof in regulating the immune response of livestock and poultry), the nuclear magnetic resonance result in FIG. 2 shows that the characteristic peak of the group with chemical shift between 0.5 and 2.0ppm is rich, and the vibration signal is strong, which indicates that the contents of N-acetylglucosamine, alanine and fatty acid groups on the lpLTA side chain are high, and the nuclear magnetic resonance result plays an important role in physiological functions.
Example 3: detection of aggregation inhibition of Abeta 42 by Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA) at different concentrations by Thioflavine T (ThT) method
A solution of Abeta 42 with a final concentration of 25 mu M is prepared, 0.25,0.50,1.00 and 2.00mg/mL of lpLTA are added, 25 mu M of ThT dye is added, the mixture is fully and uniformly mixed and placed in an enzyme-labeled instrument, the mixture is continuously cultured at 37 ℃ and fluorescence of a sample is detected every 1h, the detection condition is that the excitation light wavelength is 440nm, the emission light wavelength is 480nm, and the result of the ThT fluorescence is shown in figure 3.
Referring to FIG. 3, the fluorescence intensity of Abeta 42 itself gradually increased over time, reaching a plateau after 24h, and the fluorescence intensity of Abeta 42 solution also gradually increased over time after 0.25mg/mL of lpTLA was added, but reaching a plateau after 12h, with a maximum fluorescence intensity of about 60% of the maximum fluorescence value of the blank, indicating that Abeta 42 aggregation was inhibited by lpTLA. After addition of 0.50,1.00,2.00mg/mL of lpLTA, the fluorescence intensity of the Abeta 42 solution was hardly changed, indicating that the aggregation of Abeta 42 was completely inhibited by high concentration of lpLTA. In summary, lpTLA has a good inhibition effect on Abeta 42 aggregation, and Abeta 42 aggregation can be completely inhibited at a concentration of 0.50 mg/mL.
Example 4: the influence of Lactobacillus plantarum MA2 lipoteichoic acid (lpLTA) on Abeta 42 aggregation to form fibers is observed by using an atomic force microscope
The result of adding lpLTA at a final concentration of 0.50mg/mL to 25. Mu. M A. Beta.42 solution, culturing at 37℃for 48 hours, and then detecting the aggregation of Aβ42 in the presence or absence of lpLTA to form a fiber by atomic force microscopy is shown in FIG. 4,
Referring to FIG. 4, Aβ42 aggregates into distinct fiber morphology after 48h of culture, and is between about 200-1200nm in length. And after 0.50mg/mL lpLTA is added, the aggregation of Abeta 42 is inhibited, no obvious long fibers appear, and further the lpLTA can effectively inhibit the aggregation of Abeta 42, thus the inhibitor is a potential Abeta 42 aggregation inhibitor.
The application of the patent application CN 112386614A-lactobacillus plantarum MA2 in the aspect of preparing medicines or foods for preventing or improving AD is application of the lactobacillus plantarum MA2, and the invention further extracts and identifies lipoteichoic acid of the lactobacillus plantarum MA2 and explores the application of the lipoteichoic acid in the aspect of inhibiting the aggregation of AD pathogenic protein Abeta 42, and has the following advantages: (1) More deeply and specifically demonstrates the effect of the lactobacillus plantarum MA2 on relieving AD, and provides theoretical support for researching the action mechanism of the lactobacillus plantarum MA 2; (2) 0.5mg/mL lpLTA lipoteichoic acid can completely inhibit aggregation of Abeta 42, and the lactobacillus plantarum MA2 has no function; (3) The lpLTA lipid phosphorus wall has stable acidic property, easy storage and low energy consumption in the preparation process; (4) Can be accurately added into functional foods, beverages, health products and other products in various forms quantitatively, and is also helpful for developing AD-resistant medicaments.
A great deal of researches show that the aggregation of the Abeta 42 is a key factor for the occurrence and the development of the AD, the inhibition of the aggregation and the deposition of the Abeta 42, and the development of new anti-AD medicines based on the Abeta 42 are hot spots in the current medical field, and are one of effective ways for preventing and treating the AD. The lipoteichoic acid of the lactobacillus plantarum MA2 is obtained through ultrasonic crushing, hydrophobic gel filtration chromatography, ion exchange chromatography separation and extraction, the structure of the lipoteichoic acid is primarily analyzed through infrared spectrum and nuclear magnetic resonance, and the application of the lipoteichoic acid in inhibiting the aggregation of amyloid A beta 42 is provided and verified by using ThT fluorescent staining and atomic force microscope analysis. The present invention has been described by way of example of implementation, and it will be apparent to those skilled in the relevant art that modifications and variations can be made in the methods herein to practice the technology of the invention without departing from the spirit or scope of the invention. It should be understood that the above description is only illustrative of the preferred embodiments of the present invention and should not be taken as limiting the invention, but rather, all similar substitutes and modifications apparent to those skilled in the art are deemed to be included within the spirit, scope and content of the invention.
Claims (4)
1. Use of lactobacillus plantarum lipoteichoic acid for the preparation of a medicament for the prevention and/or amelioration of a disease characterized by the aggregate deposition of beta-amyloid 1-42 (aβ42), characterized in that: the disease is Alzheimer disease, the main chain of lipoteichoic acid of the lactobacillus plantarum contains glycerol, the side chain of the lipoteichoic acid contains N-acetylglucosamine and alanine modification, the lactobacillus plantarum is used for preparation, lactobacillus plantarum MA2 is separated and screened for the laboratory, the lactobacillus plantarum MA2 is derived from Tibetan farmhouse Kefir grains and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC3005; the preparation process comprises the steps of separation, extraction and purification, and comprises the following specific steps:
(1) Inoculating lactobacillus plantarum MA2 into MRS liquid culture medium, and culturing in a 37 ℃ incubator for 16-20h;
(2) Taking the bacterial liquid in the step (1), centrifuging the bacterial liquid in a centrifugal cup at 4 ℃ and 7000rpm for 20min, collecting bacterial cells, and weighing the wet weight of the bacterial cells;
(3) Re-suspending wet bacteria in 0.1M sodium citrate buffer solution with pH of 4.7 according to the ratio of 1:1 (w/v), repeatedly freezing and thawing for 3 times, and crushing the bacteria by using an ultrasonic crusher to obtain mixed bacterial suspension, wherein the amplitude is 30%, the opening is 1s, the closing is 1s, and the total working time is 30min;
(4) Adding equal volume of n-butanol into the mixed bacterial suspension obtained in the step (3), fully mixing, stirring for 30min on a magnetic stirrer, centrifuging at 7000rpm for 30min at 4 ℃, and gently collecting a lower water phase;
(5) Performing rotary evaporation and dialysis on the water phase, and intercepting lipoteichoic acid with the molecular weight of 2000Da by a dialysis bag to obtain a lipoteichoic acid crude extract;
(6) Preparing a2 x 20cm octyl-Sepharose gel column, equilibrated with 0.1M sodium acetate buffer at pH 4.7 containing 15% n-propanol;
(7) Mixing and diluting the lipoteichoic acid crude extract obtained in the step (5) with 0.1M sodium acetate buffer solution with the pH of 4.7 according to the volume ratio of 1:5, and passing through a column;
(8) Washing with 0.1M sodium acetate buffer of pH 4.7 of 15% n-propanol to remove unbound impurities;
(9) The column was eluted with a gradient of 0.1M sodium acetate buffer pH 4.7 containing 20%, 35%, 45% n-propanol, and the eluate was collected in a 10mL centrifuge tube;
(10) Detecting inorganic phosphorus in the eluent, and collecting the eluent with higher phosphorus content for rotary evaporation and dialysis;
(11) Preparation of DEAE-FastFlow 1.5 x 10cm anion chromatography column secondary purification of lipoteichoic acid, equilibrated column with 0.1M sodium acetate buffer at pH 4.7 containing 30% n-propanol;
(12) Pretreating the eluent after dialysis in (10) and loading the eluent on a column, performing gradient elution by using 0-1M sodium chloride solution, and collecting the eluent by using a 10 mL/tube; the sodium chloride solution is prepared by 0.1M sodium acetate buffer solution;
(13) Detecting inorganic phosphorus in the eluent collected in the step (12), and collecting the eluent with higher phosphorus content for rotary evaporation and dialysis;
(14) Freezing the dialysate obtained in the step (13) for 4 hours at the temperature of minus 80 ℃ in advance, then placing the dialysate into a freeze dryer, and obtaining lactobacillus plantarum MA2 lipoteichoic acid solid after sublimation and drying, and freezing and preserving the lactobacillus plantarum MA2 lipoteichoic acid solid.
2. The use according to claim 1, characterized in that: the lactobacillus plantarum MA2 lipoteichoic acid exists in an aqueous dispersion system.
3. The use according to claim 1, characterized in that: the lactobacillus plantarum MA2 lipoteichoic acid is present in an aqueous dispersion having a concentration of 0.25,0.50,1.00,2.00 mg/mL.
4. The use according to claim 3, wherein said lactic acid producing agent in the form of an aqueous dispersion is formulated as an injection, injectable solution, soft capsule or oral liquid.
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