CN113717883A - Lactobacillus plantarum FLPL05 capable of promoting body health and longevity and application thereof - Google Patents
Lactobacillus plantarum FLPL05 capable of promoting body health and longevity and application thereof Download PDFInfo
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- CN113717883A CN113717883A CN202110939336.4A CN202110939336A CN113717883A CN 113717883 A CN113717883 A CN 113717883A CN 202110939336 A CN202110939336 A CN 202110939336A CN 113717883 A CN113717883 A CN 113717883A
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Abstract
The invention discloses a lactobacillus plantarum FLPL05(Lactobacillus plantarum) for promoting body health and long life, which is preserved in China center for type culture Collection (Wuhan university, China) in 12 months and 07 days in 2017, and the preservation number is as follows: CCTCC NO: M2017763. The lactobacillus plantarum FLPL05 is separated from feces of the elderly with good health and long life, is gram-positive bacteria, has strong growth capacity, can promote the health and long life of organisms, maintains the integrity of intestinal barriers after the elderly, can reduce the inflammation level after the elderly, and improves the gastrointestinal tract health of the elderly.
Description
Technical Field
The invention belongs to the field of microorganisms, and relates to a novel Lactobacillus plantarum strain for promoting body health and long life and application thereof.
Background
The intestinal flora, as a large 'organ' of a human body, affects the growth and development, nutrient absorption and health state of the human body, even is closely related to metabolic diseases (irritable bowel syndrome, inflammatory bowel disease, cardiovascular diseases, diabetes, obesity and the like), and has close relevance to the health and even longevity of the human body.
Aging of the body, also called aging, is a phenomenon in which the cytoprotective mechanisms of the body, including the organs, are reduced, including the metabolic processes of the tissues and the physiological functions, and these changes affect the overall functions of the body. The function of the organism is gradually reduced, and the function is accompanied by the level changes of a series of genes, cells and epigenetics, such as: decreased stability of the genome, shortened telomere length, altered epigenetics, altered proteome stability, diminished nutrient perception, loss of mitochondrial function, aging of cells, and reduction of stem cells, as well as altered cell-to-cell signaling.
The intestinal tract is affected by external factors and aging of intestinal epithelial cells, shows physiological changes related to age, and plays a key role in the aging process of human beings if the intestinal barrier can normally function. Nobel prize winner-Russian Mei-Nikov states that putrefaction in the intestinal tract can promote life shortening, and microorganisms and toxins produced by metabolism in the intestinal tract can reach the internal circulation system and damage the organism. The body is maintained to be stable depending on the normal function of the intestinal tract, on one hand, the nutrient absorption, electrolyte and water regulation of the body are participated in, and on the other hand, the invasion from the intestinal microorganisms, food antigens and toxins can be resisted. The special function of the gut makes it particularly susceptible to aging-related decay, and therefore dysfunction of the gut barrier function is one of the hallmarks of aging.
The geographical position of the high-field village in Liuyang city of Hunan province is relatively closed, and villagers can not only have long lives, but also generally have no chronic diseases. In contrast, the peripheral area has no life-prolonging phenomenon, and diseases such as cardiovascular diseases, chronic gastroenteritis, cancer and the like occur.
Lactobacillus plantarum is an edible lactic acid bacterium prescribed by the Ministry of health, a gram-positive bacterium that does not produce spores, and a bacterium that is anaerobic or facultative anaerobic, and is often present in a single, paired, or chain form. The optimum pH value is 6.5, the homofermentation and chemoheterotrophic lactic acid bacteria can ferment gluconate or pentose, the most fermentation products are lactic acid, the content of lactic acid is more than 85 percent, and the special lactobacillus can be produced in the propagation process.
The lactobacillus can regulate the balance of gastrointestinal flora, improve immunity, reduce serum cholesterol, lower blood pressure, resist oxidation, inhibit tumor, etc. Bifidobacterium longum BBMN68 is isolated from feces of the elderly with long life in Guangxi Bama, and the viable bacteria liquid has effects of loosening bowel to relieve constipation and enhancing immunity of constipation model mice. The lactobacillus salivarius FDB86 can relieve adverse effects of dimethylhydrazine on intestinal flora, so that the intestinal flora approaches to a normal state, and the intestinal flora change of colon cancer rats is regulated. In addition, aging accumulates inflammatory factors in the body and is prone to chronic inflammation and chronic diseases.
The lactobacillus plantarum is one of edible probiotics issued by the ministry of health of China, and the probiotic functions of lactobacillus plantarum strains from different sources are different. The experiment of the in vitro and in vivo probiotic properties has larger difference. Therefore, it is necessary to find lactobacillus plantarum capable of promoting the health and longevity of the body.
Disclosure of Invention
Based on the above, one of the purposes of the invention is to provide a lactobacillus plantarum capable of promoting the health and longevity of the organism.
Lactobacillus plantarum (Lactobacillus plantarum) FLPL05 with the preservation number of CCTCC NO: M2017763.
The Lactobacillus plantarum FLPL05(Lactobacillus plantarum) of the invention is preserved in the China center for type culture Collection (Wuhan university, China) in 2017, 12 months and 07 days, and has the following numbering: CCTCC NO: M2017763.
The invention also aims to provide application of the Lactobacillus plantarum FLPL05 or a metabolite thereof or an extract thereof in preparation of products for promoting health and longevity of people or animals or delaying senescence.
Another object of the present invention is to provide the use of Lactobacillus plantarum FLPL05, or a metabolite thereof, or an extract thereof, as defined above, for the preparation of a product for repairing or protecting the intestinal barrier of humans or animals.
The invention also aims to provide application of the Lactobacillus plantarum FLPL05 or a metabolite thereof or an extract thereof in preparation of a product for preventing and reducing intestinal inflammation of humans or animals.
It is another object of the present invention to provide a product for promoting a healthy long life in humans or animals, for repairing or protecting the intestinal barrier in humans or animals, and/or for preventing, reducing intestinal inflammation in humans or animals.
A product for promoting the health and longevity of human or animals, repairing or protecting the intestinal barrier of human or animals, and/or preventing or reducing intestinal inflammation of human or animals comprises Lactobacillus plantarum FLPL05 or its metabolite, or its extractive solution, as active ingredient.
The inventors of the present invention isolated and identified a strain of Lactobacillus plantarum FLPL05 (accession number: CCTCC NO: M2017763) that acts to maintain the intestinal barrier in a number of ways, such as: the intestinal villus structural integrity is protected, the expression of intestinal tight junction protein is regulated, the apoptosis is reduced, the survival rate of intestinal epithelial cells is enhanced, intestinal stem cells are regulated and controlled, and the aim of promoting the health of organisms is finally achieved; the strain can obviously prolong the service life of a mouse by 30 percent and reduce inflammation caused by aging, and is a strain with excellent performance. Therefore, the method has high research value and application value.
Drawings
FIG. 1 shows biochemical indicators after the strain intervenes in D-galactose model mice. A: alanine Aminotransferase (ALT), B: aspartate Aminotransferase (AST), C: total Bilirubin (TBIL), D: direct Bilirubin (DBIL)*p<0.05,**p<0.01 compared to model set (D-gal); # p<0.05 compared to normal group (NC).
FIG. 2 the intervention of the strain in the blood sugar and blood lipid levels of D-galactose mice. A: blood Glucose (GLU) B Total Cholesterol (T-CHOL) C: triglyceride (TG) × P <0.05, P <0.01 compared to model group (D-gal); # P <0.05, compared to normal group (NC).
FIG. 3 Effect of strain intervention in D-galactose mice on renal function. A: creatinine (CREA) B: urea (Urea) C: uric Acid (UA)*P<0.05 compared to the D-gal group.
FIG. 4 senescence index of Lactobacillus plantarum intervention in D-galactose model mice; antioxidant index of serum: a: glutathione peroxidase (GSH-PX), B: general assemblySuperoxide dismutase (T-SOD), C: malondialdehyde (MDA) liver antioxidant index: d: GSH-PX, E: T-SOD, F: MDA*P<0.05,**P<0.01 compared to the D-gal group,#P<0.05，##P<0.01 was compared to the NC group.
FIG. 5 transcriptional expression of antioxidant genes in the small intestine of D-galactose mice.
FIG. 6 effect of the strain on transcriptional expression of antioxidant genes in intestinal epithelial cell lines. A: HT-29, B: caco 2.
FIG. 7 Effect of Lactobacillus plantarum FLPL05 on longevity of aging mice.
FIG. 8 the content of Dextran-FITC in the serum of mice after the intervention of Lactobacillus plantarum FLPL05,*P<0.05,**P<0.01 was compared with the aged group (old group).
Fig. 9 mice intestinal H & E staining results (a young mice, B old mice, C old mice gavage FLPL05 group).
Figure 10 serum inflammation levels following dry prognosis with lactobacillus plantarum FLPL 05.
Figure 11 results of TUNEL staining in intestinal tract.
Figure 12 lactobacillus plantarum intervenes in intestinal immunohistochemistry in aged mice. A: results of immunohistochemistry; b: immunohistochemical quantification profile, by: p <0.05, compared to 4M (young) mice; #: p <0.05, compared to 20M (aged) mice.
FIG. 13 effect of Lactobacillus plantarum on interfering with the expression of intestinal gene transcript levels in aged rats; wherein, A: anti-oxidation related genes; b: a senescence-associated gene.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In some embodiments of the present invention, there is provided a strain of Lactobacillus plantarum FLPL05(Lactobacillus plantarum), deposited at the chinese type culture collection (university of wuhan, china) on 12/07 of 2017, No.: m2017763, which has the functions of promoting the health and longevity of human or animals, repairing or protecting the intestinal barrier of human or animals, and/or preventing and reducing the intestinal inflammation of human or animals, and can be prepared into health products, foods or medicines, or microbial agents or feed additives or microecological preparations.
In some of these embodiments, the food product is a product, a vegetable product, a beverage product, and other fermentation-based products.
In some of these embodiments, the food product is a yogurt, a beverage, a granular food product, a powdered food product, a encapsulated food product, or a flaked food product; the health care product is a liquid health care product, a granular health care product, a powdery health care product, a capsule health care product or a sheet health care product; the microecological preparation is a solution, a granule, powder, a capsule or a tablet.
The probiotic of the present invention is preferably administered orally, and can be administered in any amount because of safety of lactobacillus plantarum, and for example, the amount of administration can be set to 10 to 5000, preferably 100 to 5000, billion per 1 day of lactobacillus plantarum.
The present invention will be described in further detail with reference to specific examples.
Example 1: isolation and identification of Lactobacillus plantarum FLPL05
Lactobacillus plantarum FLPL05 is isolated from feces of elderly people with good health and long life. Dissolving 1g of feces in 10mL of 10% peptone solution, dissolving sufficiently, filtering out residues with filter paper, centrifuging the filtrate for 10min at 5000rmp, discarding supernatant, collecting precipitate, and re-suspending with 1mL of 10% peptone water. Inoculating to MRS liquid culture medium at 1%, and culturing overnight. The culture solution is diluted by 10 times of gradient, and the gradient dilution is 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8And 10-91mL of each gradient was applied evenly to an agar plate in MRS broth and incubated anaerobically at 37 ℃ for 48 h. Selecting single colony, streaking and separating on a plate, repeatedly performing purification culture, and observing colony morphology by gram staining method to select gram-positive bacteria. After liquid culture of the separated strain, plate culture is carried out, and single colony is picked by using an inoculating loop as a template to carry out colony PCR. Positive PCR products were sent to the company for sequencing.
The inventor adopts a traditional microorganism separation method to obtain 90 strains of bacteria from 21 intestinal flora of the elderly with long life, which are respectively as follows: 15 strains of Lactobacillus plantarum (Lactobacillus plantarum), 25 strains of Lactobacillus fermentum (L.fermentum), 32 strains of Lactobacillus mucosae (L.mucosae), 16 strains of Lactobacillus salivarius (L.salivarius), 1 strain of Enterococcus hirae (Enterococcus hirae) and 1 strain of Enterococcus faecalis (Enterococcus faecalis). Three strains with excellent performance are screened out through the evaluation of hydrogen peroxide tolerance, gastric juice and intestinal juice simulation and free radical scavenging capacity: lactobacillus plantarum FLPL05, FLPL0302 and FLPL0902, for use in intervention studies in aging model mice. The FLPL05 was identified as Lactobacillus plantarum by whole genome sequencing. And is preserved in China center for type culture Collection in Wuhan, with a preservation number of CCTCC NO: M2017763.
The 16S rRNA base sequence of the isolated bacterial strain was sequenced, wherein the results of FLPL05 were as follows: ACTTGACGTTAATACTGACGCTATTACCTTAACTGGTAGTGATGCTGATATTTCTATTGAAACCACCATTCCAGCAAGTGATGACAACAATACGTTAGTCGTTGAAGATGCCGGTTCAATCGTTTTATGCCCGTTTCTTTAGTGAAATCGTAAAAAAATTACCAGAAGACACCATGACTGTTAACGTGGTCGACGGTTTTCAAACACAAATTACTTCAGGGGCCGCTTCCTTTACAATCAACGGCTTAGACCCGAAACTACCCACACTTACCTGAAATTGATACGACTAATACCATCACGTTAGCTGGGGATGTACTCAAAGAGTTGATTGGTCAGACCGTTATTGCCGTTTCCAATCAGGAAAGTCGACCAATTCTGACGGGTTCATTTTATCTTAGCTAACGGTGAGTTCTTAGCGGTCGCAACCGATTCACACCGCCTAAGTCAACGCCGGATCAAGTTGCCAGAAGCCAATAATGCCAATTATGATGTAATCATTCCTGGTAAATTTGACAGAATTATCTCGGATGATCGGCGACAATAATCCTGACGTGCAGATGCGGTTATCAGAAAACCAAGTCCTATTTGTGCTGGGGAATACGTCCTTCTATTCTCGCTTACTCGAAGGAAATATCCGACACGTCACGGCTGATTCCTAAAGAATCTAATACGACTGTTGAAATTTCAGCACCCGCACTTTCAGCAGCAATTGAACGGGCATCCCTGTTATCCCACGAAAGTCGCAATAATGTGGTTCCTTCTCGTTAACCCAACGGACAAAACGATTACGATTTTCGGTAACTCACCAGATGTCGGGGAAGTAACGGAACAACTACAGCCCACTGATTTGTCAGGTGACGAACTTGAAATTTCATTTAACCCAGACTACATGAAAGAAGCGTTGTTCGTTTGGGCAAGCTATGATCAAAATCTCATTCACGATGGCATTGCGCCCATTCACTTTAGTGCCAACTGAAGAAGGCGAAAACTTTATTCAGTTGATTACACCAGTTCGAACGTTTTAATTAGTAATAAATATACAATTCTAAATAAAAGGCCCGCATTTCACCGTGAAATGCGGGCCTTTTATGACGGCTAAAACTAGCTAAAGATGGGGCAATGAATAGGCAATAGCGATGATGTAACTTTTGAAGTTTACTTATGCATATTATATTAGCTGCTGGTGAATTGTCACGCTAACTATCGATGTTGCCCTACTAAAATCCCACCATCTGATGAACCCCAGTCCTTGCACTCGTTCGCTACAAACGCTCGCTTTCAATATGAATGCTGGCTTTAGCCAATTTTTATAGGTAAAAAACAAGATATTTTATTAAAATCCGCAAAATTAGGGAATTTCAGCAAATTTACCCCTAGGCTAGTATCCCGATATTAAAAAGCCCTAAACGTCTAAAATGGCTCTGAGTGCGGTTATAAATTGAATTTTCATCAAAA (SEQ ID NO.1)
And comparing and analyzing the base sequences, and finding that the base sequences are consistent with the lactobacillus plantarum sequences in a database.
The lactobacillus plantarum FLPL05 strain is anaerobic and non-spore-producing gram-positive bacterium, has short rod-shaped thallus and is milky, transparent, round and smooth colony in MRS agar plate culture medium. Lactobacillus plantarum FLPL05 can grow on MRS broth and is a gram-positive bacterium.
Example 2
In this example and the following examples, Lactobacillus plantarum FLPL0302, FLPL05(CCTCC NO: M2017763), and FLPL0902 were derived from intestinal flora of 21 elderly people with long life.
Female BALB/C mice, 5 weeks old, were raised at 23 + -1 deg.C for 12 h: in 12h light and dark circulation environment, the feed is standard mouse food and purified water, and diet is not limited. After acclimatization, 60 mice were randomly divided into 6 groups of 10 mice each, each group being:
model set (D-gal): injecting 50mg/kg D-galactose into abdominal cavity for 42 days, and intragastrically administering 100 μ L normal saline for 30 days;
FLPL0302 group: injecting 50mg/kg D-galactose into abdominal cavity for 42 days, and re-suspending 100 μ L strain physiological saline for 30 days to obtain 10-concentration bacterial solution8cfu/mL；
FLPL05 group: injecting 50mg/kg D-galactose into abdominal cavity for 42 days, and intragastrically administering 100 μ L physiological saline resuspension of strain for 30 days with bacterial liquid concentration of 108cfu/mL；
FLPL0902 group: injecting 50mg/kg D-galactose into abdominal cavity for 42 days, and intragastrically administering 100 μ L strain physiological saline resuspension for 30 days with bacterial liquid concentration of 108cfu/mL；
Positive control group (Vitamin C): intraperitoneal injection of 50mg/kg D-galactose is carried out for 42 days, and intragastric administration of 100 mu L ascorbic acid (0.50mg/mL) is carried out for 30 days;
control group (NC): the same volume of saline was injected intraperitoneally for 42 days, and 100. mu.L of saline was gavaged for 30 days.
Fasting is carried out for 12 hours after the last gastric lavage, the orbit blood is collected after weighing, the mice are sacrificed, each organ is collected in a sterile manner and weighed respectively, and the organ index is calculated. Standing the collected blood at 4 ℃ for 8h, centrifuging at 4000r/min for 15min, discarding the precipitate, and collecting the supernatant, namely the serum for later use.
Organ index is organ mass (g)/mouse body weight (g) × 100%.
The following experiments were performed.
1) Biochemical index of mouse
Serum was sent to the first subsidiary hospital of Nanchang university to test health indicators such as liver function, kidney function, blood glucose and triglycerides.
2) Serum antioxidant index detection
Collecting supernatant from collected blood, and detecting GSH-PX, T-SOD and MDA contents by using Nanjing construction kit. The kit goods numbers are respectively: GSH-PX (No. A005-1-1), T-SOD (No. A001-1-2), and MDA (No. A003-1-2).
3) Detection of liver antioxidant index
After the liver tissues are ground, the protein concentration of each sample is detected according to a BCA kit, and the contents of GSH-PX, T-SOD and MDA in the liver are detected according to the kit specification. The kit has the product numbers of GSH-PX (No. A005-1-1), T-SOD (No. A001-1-2) and MDA (No. A003-1-2).
4) Small intestine gene transcription level detection
And detecting the change of the transcription level of the antioxidant related gene by using a reverse transcription fluorescence quantitative PCR method.
RNA was extracted from the small intestine tissue in total according to the instructions of the Mini BEST Universal RNA Extraction Kit, and the nucleic acid concentration was determined.
Total RNA was reverse transcribed into cDNA using Takara PrimeScriptTM RT reagent Kit with gDNA Erase Kit according to the instructions, and RT-qPCR detection was performed using this as template.
Reverse transcription:
a system for removing genomic DNA: maintaining at 42 deg.C for 2min
Total RNA amount was less than 1 μ g, 7 μ L was used in this experiment.
b, reverse transcription system: reacting at 37 ℃ for 15min and at 85 ℃ for 5s
Fluorescent quantitative PCR:
a system:
b fluorescent quantitative PCR amplification procedure:
c calculating the expression of the gene, using beta-actin as an internal reference, using 2-ΔΔCTThe relative expression of each gene was calculated by the method, and the information on the gene and the primer is shown in Table 1.
TABLE 1 fluorescent quantitative antioxidant related genes and primer sequences of small intestine
5) Effect of Lactobacillus plantarum on intestinal epithelial cell transcript levels
(1) Caco2 and HT-29 cells were cultured in DMEM complete medium at 37 ℃ with 5% CO2；
(2) Culturing the cells after three passages in a six-hole plate respectively to obtain monolayer cells;
(3) counting the number of cells by using a blood counting plate;
(4) co-incubating the cells and the lactobacillus plantarum for 2 hours in a ratio of 1:1000, and extracting RNA of the cells;
(5) extracting RNA and carrying out reverse transcription, and detecting the gene transcription level of the small intestine by methods such as fluorescence quantification and the like according to the steps of the instruction of a takara kit;
(6) the sequences of the primers used for fluorescence quantification are shown in Table 2.
TABLE 2 fluorescent quantitative primer sequences for cellular antioxidant genes
The experimental results are as follows:
1) body weight and organ index
The results of the body weight and organ index measurements of the mice in the different treatment groups are shown in table 3, and the body weight of the mice was not significantly changed (P <0.05) and only the liver index was significantly increased by 5.69 ± 0.28(P <0.05) after the injection of D-galactose, compared to the normal group. After the lactobacillus plantarum FLPL05 is dried, the body weight is not significantly changed (P <0.05), the liver index is significantly reduced to 4.91 +/-0.23 compared with a model group (P <0.05), the organ index of the kidney is significantly reduced compared with the model group (P <0.05), and the liver index is significantly changed after the ascorbic acid is dried (P < 0.05).
TABLE 3 body weight and organ index for L.plantarum on D-galactose mouse Stem prognosis
The date presented are means±S.D.(n＝10)
Organ index＝weight of organ/body weight×100
ap<0.05,bp<0.01compared with D-gal model mice
2) Biochemical index-liver function
It was investigated whether Lactobacillus plantarum has an effect on the liver function of D-galactose-induced aging mice, and the results are shown in FIG. 1. Compared with the normal group, the glutamic-pyruvic transaminase (P <0.05) and the glutamic-oxalacetic transaminase (P <0.05) in the serum are remarkably increased after D-galactose modeling, the total bilirubin (P >0.05) and the direct bilirubin (P >0.05) have no remarkable change, and the D-galactose has damage effect on the liver function of the mouse. The lactobacillus plantarum FLPL05 and ascorbic acid dry prognosis have similar effects, and the levels of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase in serum are remarkably reduced (P <0.05), which indicates that the lactobacillus plantarum FLPL05 has a protective effect on the liver. The other two strains of Lactobacillus plantarum did not have similar protective effects.
3) Biochemical index-blood sugar and blood fat
The results of investigating whether lactobacillus plantarum has an effect on blood glucose and blood lipid of the D-galactose-induced aging mice are shown in FIG. 2. Compared with the normal group, the blood sugar level of the mice is remarkably reduced (P <0.05) and the triglyceride is remarkably increased (P <0.05) after the intervention of the D-galactose. After three lactobacillus plantarum strains and ascorbic acid are dried, the blood sugar level has no significant difference (P >0.05) compared with the normal group, and has significant improvement (P <0.05) compared with the model group. Total cholesterol levels were not different between the model and normal groups, but increased after lactobacillus plantarum FLPL05 and ascorbic acid intervention. Lactobacillus plantarum FLPL0302 and ascorbic acid dried-up were not different from the normal group, and were significantly reduced compared to the model group (P < 0.001).
4) Biochemical index-renal function
The influence of Lactobacillus plantarum on the renal function of the D-galactose-induced aging mice was investigated, and the results are shown in FIG. 3. Compared with the normal group, D-galactose has no significant effect on creatinine, urea and uric acid of mice, three strains of lactobacillus plantarum and ascorbic acid have no effect on renal function, and only lactobacillus plantarum FLPL0302 can significantly reduce uric acid level (P < 0.05).
5) Influence of strain on biochemical indexes of aging of D-galactose model mouse
By detecting the biochemical aging indexes of the organism, the T-SOD, the GSH-PX and the MDA can evaluate the aging condition. Results as shown in a and D in fig. 4, the activities of serum and liver glutathione peroxidase were both significantly decreased after D-galactose injection (P <0.05) compared to the normal control group, while the activities of serum GSH-PX were significantly increased after the intervention of lactobacillus plantarum FLPL05 and FLPL0302 (P <0.05), but had no significant effect on the liver. As shown in E in FIG. 4, the T-SOD activity of the liver of the model mouse is significantly reduced (P <0.001), the T-SOD activity in the liver is significantly increased by the Lactobacillus plantarum FLPL05 and ascorbic acid (P <0.001), and the T-SOD activity is not significantly different from that of the normal control group. As shown by B in fig. 4, total superoxide dismutase in serum had no significant effect. The content of malondialdehyde in serum and liver of model mice is significantly increased (P <0.01 and P < 0.05).
6) Influence of strain on expression of antioxidant related genes of small intestine of D-galactose model mouse
The expression condition of the small intestine antioxidant gene is detected by a fluorescent quantitative RT-qPCR technology, and the result is shown in figure 5. After the D-galactose is dried, the expression of the antioxidant related genes of the small intestine shows a down-regulation trend. After lactobacillus plantarum FLPL05 and ascorbic acid were pretreated, genes GR, MT1, MT2, GPX1, GPX2 and SOD were all up-regulated, while lactobacillus plantarum FLPL0302 and FLPL0902 did not have significant effects.
7) Effect of strains on expression of Gene transcript levels in intestinal epithelial cells
As shown in a in fig. 6, lactobacillus plantarum FLPL05 significantly up-regulated PRG1, MT1A, MT1M and BAL2 genes of HT-29 cells, and lactobacillus plantarum FLPL0302 up-regulated PRG1, GPX2, MT1A, MT1M and BCL2 genes, as found by fluorescent quantitative RT-qPCR. As shown in B in FIG. 6, the Lactobacillus plantarum FLPL05 also up-regulated the PRG1, GPX2, MT1A, MT1M and BCL2 genes of Caco2 cells, and the Lactobacillus plantarum FLPL0302 had no significant effect on Caco2 cells. Lactobacillus plantarum FLPL0902 had no significant effect on both HT-29 and Caco2 cells.
I.e., lactobacillus plantarum FLPL05, FLPL0302 and FLPL0902, the probiotic function of the strains in vivo was assessed by D-galactose model mice. Lactobacillus plantarum FLPL05 reduced serum alanine Aminotransferase (ALT), glutamate Aminotransferase (AST) and total cholesterol (T-CHOL) levels without affecting body weight and Creatinine (CREA). In vivo damage to the body by free radicals is typically reflected in organ indices and biomarkers, such as: ATL, AST and CREA. ALT and AST are the most important enzymes for judging liver functions, and the lactobacillus plantarum FLPL05 has a protective effect on the liver.
The experiments show that the Lactobacillus plantarum FLPL05 can up-regulate GPX-2, MT1A and MT1M genes of the intestinal tracts of HT-29 and Caco2 cell lines or D-galactose-induced aging model mice. The results show that the lactobacillus plantarum FLPL05 is a potential probiotic for promoting health by enhancing the body antioxidant capacity.
The organism senescence is closely related to the cell senescence, the cell apoptosis is related to various senescence-related pathologies, the cell apoptosis is regulated by a series of genes, the Bcl-2 has the function of inhibiting the cell apoptosis, the GPX-2 also has the functions of resisting inflammation and resisting apoptosis, and the Lactobacillus plantarum FLPL05 can up-regulate the Bcl-2 and GPX-2 genes of the intestinal tract, so that the reduction of the cell apoptosis of the intestinal tract is presumed, and the reduction of the cell apoptosis can be another action mechanism for relieving the senescence of a D-galactose model mouse.
Example 3: strain for prolonging mouse life
Four week old female BALB/c mice were housed in a 23 ± 1 ℃ environment, provided with standard diet and drinking water, and had free diet. Randomized into two groups of 10, experimental (FLPL 05): intragastric administration 100. mu.L Lactobacillus plantarum FLPL05 bacterial liquid (10)8cfu/mouse) to age and die naturally; control group: and (5) perfusing the normal saline with the same volume as the stomach until the mice die naturally. The natural life of two groups of mice are counted respectively and compared and analyzed.
As can be seen from FIG. 7, the Lactobacillus plantarum FLPL05 strain prolonged the lifespan of the mice by 30%.
Example 4: influence of strains on intestinal permeability of aged mice
Older BALB/c mice (20 months old) were randomly divided into two groups, housed in a 23 + -1 deg.C environment, served on standard and potable water, and had free access to the diet. 10 per group, experimental group (FLPL 05): intragastric administration 100. mu.L Lactobacillus plantarum FLPL05 bacterial liquid (10)8cfu/only); control group: and (5) infusing normal saline with the same volume as the stomach. Gastric lavage Dextran-FITC (1) after one monthFluorescein labeling Glucan) And taking serum. The influence of the strain on the intestinal permeability of the aged mice was evaluated by measuring the content of Dextran-FITC in serum.
As can be seen from FIG. 8, the content of Dextran-FITC in the blood of the aged mice is high, which indicates that the intestinal barrier is damaged and the intestinal permeability is reduced after the aged mice are aged, and the lactobacillus plantarum FLPL05 for intragastric administration in the experimental group can obviously reduce the content of Dextran-FITC in the blood and enhance the intestinal permeability of the aged mice.
Example 5: influence of strains on intestinal structures of aged mice
One month after the gavage of the aged mice, the mice were sacrificed and the intestinal tracts were taken for H & E staining, respectively. As can be seen from FIG. 9, the strain has a significant improvement effect on the intestinal structure, and compared with the aged mice, the lactobacillus plantarum FLPL05 has the advantages of dry prognosis, regular intestinal villi and complete crypt.
Example 6: reduction of inflammation levels in aged mice following strain dry prognosis
And collecting mouse serum, and respectively detecting inflammation indexes TNF-alpha and IFN-gamma in the serum according to the steps of an ELISA kit specification.
Referring to FIG. 10, the increase in serum TNF- α levels in young mice (69.64. + -. 6.03pg/mL) was 92.32. + -. 10.25pg/mL in aged mice and the decrease in TNF- α levels in dried Lactobacillus plantarum FLPL05 was 61.63. + -. 11.04 pg/mL.
As shown in FIG. 10, after the Lactobacillus plantarum FLPL05 is dried, the inflammation level can be improved, the TNF-alpha in serum is reduced, and the IFN-gamma is increased.
Example 7
30 female BALB/C mice, 20 months old (20M) and 10 female BALB/C mice, 4 months old (4M), 20 months old female mice were randomly divided into three groups. The experimental design was as follows:
young control group: feeding 10 female mice of 4 months old with skim milk;
control group for the aged: gastric lavage of 10 female mice of 20 months of age;
strain intervention experimental group: each group of 10 20-month-old female mice had a 100. mu.L intragastric lavage of 1X 10 mice9CFU/mL Lactobacillus plantarum FLPL05, another group of gavages 100 μ L1X 109CFU/mL Lactobacillus plantarum FLPL 0302.
After 45 days of gavage, blood was collected by a sterile centrifuge tube after weighing, killed by breaking the neck, and organs were aseptically taken and weighed. Organs were collected in liquid nitrogen and stored at-80 ℃ until use.
A method for collecting serum comprises standing collected blood at 4 deg.C for 8 hr, centrifuging at 10000 Xg for 10min, and collecting supernatant to obtain serum. The serum is reserved for detecting the aging index and the inflammatory factor.
1) Intestinal fluorescent quantitative RT-qPCR
Extracting intestinal RNA, reverse transcription and fluorescence quantification according to conventional method.
2) Intestinal immunohistochemistry
The deparaffinized tissue sections were sent to Wuhan Severer for testing.
3) TUNEL staining of intestinal tract
After paraffin removal of the intestinal sections, the procedure was followed with the instructions of the Roche in situ cell death assay kit, and a photograph of the cell staining was taken with a microscope.
The experimental results are as follows:
1) effect of Lactobacillus plantarum on apoptosis of intestinal cells of mice
The apoptosis condition of intestinal tract cells of mice is detected by tunel staining, and the result is shown in figure 11, compared with the apoptotic cells in the intestinal tracts of old mice, the dry prognosis of the lactobacillus plantarum FLPL0302 and FLPL05 can obviously reduce the apoptotic cells.
2) Effect of Lactobacillus plantarum on mouse Tight protein expression
The claudin has great significance for maintaining the normal function of intestinal barriers, and the expression conditions of the claudin in the intestinal tracts of mice of different treatment groups are detected through immunohistochemistry, so that the results are shown in figure 12, the expression level of ZO-1 in the intestinal tracts of the mice after old age is not significantly changed, the expression level of ZO-1 is increased after lactobacillus plantarum FLPL05 is dried, and the expression level is reduced after lactobacillus plantarum FLPL0302 intervenes. After the old, the expression level of the intestinal occludin is reduced, and the dry prognosis of the lactobacillus plantarum FLPL0302 has no significant improvement effect (P <0.05), while the dry prognosis of the lactobacillus plantarum FLPL05 has no significant difference from the expression level of young mice. The expression level of Claudin-4 is not different in old mice and young mice, while the expression level of Lactobacillus plantarum FLPL05 in intestinal tract is remarkably improved after intervention (P < 0.05).
3) Effect of Lactobacillus plantarum on mouse intestinal transcript levels
The possible action mechanism of the two lactobacillus plantarum strains is evaluated by detecting the transcriptional level change of the intestinal oxidative stress and aging related genes. As shown in FIG. 13, FLPL05 significantly upregulated genes GPx-1, MT-2, CAT and TERT, and down-regulated genes IGF-1 and IGF-1R compared to Lactobacillus plantarum FLPL 0302.
The aging is the gradual decline of organ functions, the capacity of the intestinal tract to absorb nutrition is reduced after aging, and the lactobacillus plantarum FLPL05 also participates in regulating and controlling signal transduction of the intestinal tract and the digestive and endocrine systems. It can be seen that the lactobacillus plantarum FLPL05 of the present invention acts to maintain the intestinal barrier in a number of ways, for example: protecting the structural integrity of intestinal villi, regulating the expression of intestinal tight junction protein, reducing apoptosis, enhancing the survival rate of intestinal epithelial cells, regulating intestinal stem cells and the like, and finally achieving the purpose of promoting the health of organisms.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
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Claims (10)
1. Lactobacillus plantarum (Lactobacillus plantarum) FLPL05 with the preservation number of CCTCC NO: M2017763.
2. Use of Lactobacillus plantarum FLPL05, or a metabolite thereof, or an extract thereof, according to claim 1, for the preparation of a product for promoting good life or delaying aging in humans or animals.
3. Use of Lactobacillus plantarum (FLPL05), or a metabolite thereof, or an extract thereof, according to claim 1 for the preparation of a product for repairing or protecting the intestinal barrier of humans or animals.
4. Use of Lactobacillus plantarum (FLPL05), or a metabolite thereof, or an extract thereof, according to claim 1 for the preparation of a product for preventing, reducing intestinal inflammation in humans or animals.
5. Use according to claim 2 or 3 or 4, wherein the product is a nutraceutical, food or pharmaceutical product or a bacterial preparation or a feed additive.
6. Use according to claim 5, wherein the food products are dairy products, vegetable products, beverage products and other fermented products.
7. A product for promoting the healthy longevity of humans or animals, repairing or protecting the intestinal barrier of humans or animals, and/or preventing, reducing intestinal inflammation of humans or animals, whose active ingredient comprises Lactobacillus plantarum FLPL05, or a metabolite thereof, or an extract thereof, according to claim 1.
8. The product according to claim 7, wherein the product is a nutraceutical, food or pharmaceutical product or a bacterial agent.
9. The product of claim 7, wherein the food product is a dairy product, a vegetable product, a beverage product, and other fermented products.
10. The product of claim 7, wherein the food product is a yogurt, a beverage, a granular food product, a powdered food product, a encapsulated food product, or a tablet; or the health care product is a liquid health care product, a granular health care product, a powdery health care product, a capsule health care product or a sheet health care product; or the medicine is solution, granule, powder, capsule or tablet.
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