CN113308416B - Lactobacillus plantarum capable of inhibiting kidney stone formation and application thereof - Google Patents
Lactobacillus plantarum capable of inhibiting kidney stone formation and application thereof Download PDFInfo
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- CN113308416B CN113308416B CN202110820572.4A CN202110820572A CN113308416B CN 113308416 B CN113308416 B CN 113308416B CN 202110820572 A CN202110820572 A CN 202110820572A CN 113308416 B CN113308416 B CN 113308416B
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- lactobacillus plantarum
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Abstract
The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus plantarum (A) capable of inhibiting kidney stone formationL.plantarum) J-15 and applications thereof. The lactobacillus plantarum J-15 provided by the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No.22140, preservation date: 2021, 4 and 6. The strain is separated from feces of a healthy person in urban province of Sichuan province, grows well on an MRS agar culture medium, has certain tolerance capacity to acid and bile salt, is sensitive to various antibiotics, has certain oxalate degradation capacity, has strong kidney crystallization inhibition capacity, has strong carbohydrate metabolism capacity, membrane transport capacity and environmental adaptability as shown by whole genome sequencing, is applied to the field of functional foods and clinical kidney stone prevention and adjuvant therapy, has actual production value, and has very important significance on human health.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum capable of inhibiting kidney stone formation, and particularly relates to the field of research and development of functional lactic acid bacteria and products thereof.
Technical Field
Kidney stones are one of the common urinary system diseases worldwide, can cause pain, urinary tract infection, chronic kidney diseases and even loss of kidney function, and the prevalence rate thereof is increasing worldwide year by year. Calcium oxalate calculi are the most prominent type of calculi, accounting for about 80%. Only a few renal calcium oxalate calculi are caused by primary hyperoxaluria, and the cause of most renal calcium oxalate calculi patients remains unclear. Conventional treatment methods include drug therapy, external shock wave lithotripsy, ureteroscopy and the like, and although these treatment methods can successfully remove most kidney stones, the postoperative recurrence rate is still high, the recurrence rate after 5 years is as high as 53%, and the patients are greatly burdened with economy and physiology. The main prevention means at present are the traditional means of increasing water drinking amount and limiting the intake of stone-causing components, but the effect is not obvious. Therefore, the search for new methods for treating and/or preventing renal calcium oxalate calculi is urgent.
Mammals lack the enzyme for oxalic acid bioconversion. Its metabolism of oxalic acid depends mainly on its limited absorption, excretion and microbial degradation, and oxalic acid is degraded by microorganisms in the gastrointestinal tract to reduce oxalic acid in the blood, and thus is considered to be an important factor for reducing the risk of kidney disease. Previous studies have focused on a few oxalate-degrading bacteria, such as Lactobacillus (B)Lactobacillus) Bifidobacterium (b)Bifidobacterium) And Acidobacterium (A), (B)Oxalobacter Formiges). Sadaf et al found that oxalate bacteria in intestinal microorganisms can degrade oxalate to formate by means of oxalyl-CoA decarboxylase and formyl-CoA transferase; the study by Hatch et al indicated that the oxaloacetic acid excretion was reduced in hyperoxaloacetic rats after the administration of oxalic acid bacteria. In recent years, several reports have emerged on the correlation between intestinal microorganisms and kidney stones: 16S rRNA gene sequencing is carried out on feces of kidney stone patients and healthy people by a research team of Guangxi medical university, and the research team discovers that species abundance and diversity of intestinal flora of the kidney stone patients are remarkably reduced, the structure of the intestinal flora of the kidney stone patients is obviously different from that of the intestinal flora of the healthy people, and the abundance of certain proinflammatory bacteria is remarkably increased. The Italy Andrea ticinesi team analyzes the composition and function of intestinal flora of idiopathic calcium calculus patients (SF) by using a metagenome method, identifies 5 groups related to SF and urooxalic acid, comprises a plurality of degradable oxalic acid groups which are not found before, finds that the total content of flora genes related to oxalic acid degradation and taxa containing the genes in SF feces is reduced, is negatively related to UOE, and indicates that the intestinal-renal axis is possibly involved in the formation of kidney stones.
Probiotics show great potential in the treatment of urinary system related diseases. It has been reported by Lieske et al that the ingestion of probiotics controls oxidative stress and systemic inflammation in patients with chronic kidney disease. The study by Camphieri et al showed that the administration of lactic acid bacteria also reduced the urooxalic acid levels in patients with kidney stones, suggesting that supplementation with probiotic bacteria containing lactic acid bacteria may be a potential strategy for treating kidney stones.
Lactic Acid Bacteria (LAB) are a general term for a class of spore-free, gram-positive bacteria that utilize fermentable carbohydrates to produce large amounts of Lactic acid. Lactic acid bacteria, a group of probiotics, are considered highly safe food-grade microorganisms by the U.S. Food and Drug Administration (FDA). The lactobacillus plantarum is a member of lactobacillus families, is widely existed in fermented foods, and has various probiotic functions of regulating intestinal flora balance, reducing cholesterol level, cardiovascular disease incidence and the like. At present, the lactobacillus plantarum has the capacity of degrading oxalic acid proved by in vitro experiments, and a plurality of recombinant lactobacillus plantarum have the capacity of inhibiting calculus formation proved by animal experiments. In the research of Sasikumar and the like, the lactobacillus plantarum recombinant strains WCFS1OxdC and NC8OxdC can degrade 70-77% of oxalic acid in vitro, the ratio of oxalic acid, calcium, uric acid, creatinine, serum uric acid and urea nitrogen/creatinine in rat urine is obviously reduced, the oxalic acid content in kidney homogenate is obviously reduced, and the kidney crystallization is very little. The recombinant strain can reduce the excretion of oxalic acid in urine and the deposition of oxalic acid crystals by increasing the degradation of oxalic acid in intestinal tracts, but the genetically modified recombinant bacteria have uncertain safety when being applied to clinical treatment and probiotic edible products in the future and face the limitation of relevant regulations. In addition, there are few other natural lactic acid bacteria which have been confirmed to have the ability to reduce calculus formation by animal experiments, and Kwak et al found thatLactobacillus casei HY2743 andLactobacillus casei the combined treatment of HY7201 has reduced urinary oxalate excretion and reduced renal crystal of rat, but the physiological characteristics of the two strains are not reported in detail, and the action mechanisms are not clearAnd genomic sequence information is not queried in NCBI. In conclusion, the application of the lactobacillus plantarum which is separated from a human body and is not genetically modified in the aspect of reducing the formation of the renal calcium oxalate calculus is rarely reported.
There is increasing evidence that intestinal microbial metabolism plays a role in kidney stone formation and that lactic acid bacteria can serve as a potential therapeutic strategy for the prevention of urinary stones. Therefore, the lactobacillus with high kidney stone inhibition capability is screened, the kidney stone formation inhibition capability in vivo is evaluated, and the lactobacillus has great potential when being applied to the field of functional foods and the adjuvant therapy of clinical treatment, and has very important significance on human health.
It is known that oxalic acid is an important factor for forming calcium oxalate calculus, and oxalic acid in human body is mainly classified into exogenous oxalic acid and endogenous oxalic acid. Endogenous oxalic acid constitutes 85-90% of blood oxalic acid, and 5-10% of oxalic acid in blood is secreted to an intestinal cavity; 90-98% of the exogenous oxalic acid is combined with calcium ions in the intestinal tract to form calcium oxalate which is discharged out of the body along with the feces or degraded by microorganisms in the intestinal tract. It is seen that intestinal tract and intestinal microorganisms play an important role in oxalate catabolism. The application is based on the latest results obtained from the previous research subjects on calcium oxalate kidney stones and intestinal microorganisms.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum (A) with the capability of inhibiting kidney stone formationLactiplantibacillus plantarum) J-15 and applications thereof.
The invention also aims to: providing a lactobacillus plantarum capable of inhibiting kidney stone formationLactiplantibacillus plantarum) J-15 gene combination.
In order to achieve the purpose, the invention adopts the following technical scheme:
lactobacillus plantarum J-15 was deposited in CGMCC (CGMCC, accession No. 3, ministry of microbiology 1, ministry of China academy of sciences, japan, and Japan, as CGMCC No. 4, 6, 2021, 4 months and 6 days), and the number of the deposit was CGMCC No.22140.
The lactobacillus plantarum J-15 is obtained by separating excrement of a healthy person in urban province, sichuan province. The main factors considered in the category of sample collection include the age-matched persons who have not used antibiotics or immunosuppressants and have no inflammatory bowel disease, no irritable bowel syndrome, no digestive tract infection, no digestive system tumor, no intestinal surgery or diarrhea or constipation within three months before sampling the stool.
The invention obtains and separates the Lactobacillus plantarum J-15 from the excrement and urine and has the following concrete steps: temporarily storing the feces sample in a refrigerator at 4 deg.C, taking out about 2g of the middle part with sterile cotton swab during experiment, placing in a tube filled with 18ml of sterile PBS prepared in advance, adding sterile glass beads, shaking on a shaking table at 37 deg.C and 180 rpm for 10 min, mixing, and diluting to 10 deg.C -4 . Choose 10 -2 ，10 -3 ，10 -4 Three dilutions, 100 μ L of each dilution was pipetted and plated on GAM medium, repeated three times. Then, the cells were individually placed in 37 ℃ facultative anaerobic incubators and incubated for about 3 days. Then selecting different bacteria according to the morphological characteristics of the bacterial colony, purifying the bacteria on a GAM culture medium for multiple times, transferring the bacteria to a liquid culture medium for amplification culture, extracting DNA, sending the bacteria to a company for sequencing after PCR amplification, and carrying out strain comparison and identification by NCBI. Preserving the seeds by adopting glycerol with the final concentration of 30 percent, and preserving at the temperature of minus 80 ℃. The method introduces a step of predicting the separation culture medium in the early stage on the basis of the traditional separation process, is not only limited to the use of a general culture medium for wide separation and blind screening of bacteria, but also purposefully provides a basis for predicting the culture medium formula of a target microorganism by combining a bioinformatics method. The culture medium of the closely related species with high similarity is deduced according to the phylogenetic similarity of 16S rDNA and the species of the known culture medium. The method greatly improves the probability and success rate of separating the target bacteria.
The lactobacillus plantarum J-15 disclosed by the invention has the advantages of relatively high growth speed on an MRS agar culture medium, relatively large bacterial colony, obvious morphological characteristics, milky white and opaque bacterial colony, smooth surface, neat edge and negative catalyst, and the gram stain is purple and rod-shaped when the bacterial colony is subjected to microscopic examination.
The lactobacillus plantarum J-15 disclosed by the invention is proved to have certain tolerance to acid and bile salt through acid-resistant and bile salt-resistant tests.
The lactobacillus plantarum J-15 has certain degradation capacity on sodium oxalate as shown in oxalate degradation experiments.
The lactobacillus plantarum J-15 is sensitive to most antibiotics and has certain tolerance capability to individual antibiotics.
Animal experiments show that the lactobacillus plantarum J-15 can reduce the urooxalic acid and urinary calcium levels of kidney stones and rats, relieve the pathological conditions of kidneys and reduce the formation of kidney crystals.
The lactobacillus plantarum J-15 is subjected to PCR amplification by using a bacterial universal primer 16S rDNA 27F/1492R to obtain a target gene SEQUENCE consisting of 1465 bp base pairs by splicing, wherein the target gene SEQUENCE is shown as SEQUENCE LISTING. Inputting the gene sequence obtained by sequencing into an EZBioCloud database for comparison, and comparing the gene sequence with a standard strain in a bacterial 16S rRNA gene databaseLactiplantibacillus plantarum The ATCC 14917 similarity reaches 100%, and the strain can be preliminarily identified to be lactobacillus plantarum (Lactobacillus plantarum) (C)L. plantarum). Then, all genes and related regulatory information in the J-15 genome are detected by a whole genome sequencing method.
Compared with the prior art, the invention has the innovativeness that:
1. the lactobacillus plantarum provided by the inventionL. plantarum J-15 is obtained by screening by combining bioinformatics with a traditional culture method. Because the microorganism separation and screening has unpredictability and uncertainty, the same microorganism cannot be separated from the same sample in different batches of experiments, and the target genus can not be cultured even if a selective culture medium and a specific temperature condition are used; even if the target bacteria is separated, the microbial characteristics of the same genus and species are different due to the species and strain specificity, the microorganisms of the same genus and species are often found, the different strains show larger biological characteristics and functional differences, and the strains are artificially selected and purified according to the morphological characteristics of the strainsIn the process, randomness is often high, and strains with the same morphological characteristics can be different types of bacteria, so that the result of the whole bacteria classification process is unpredictable. Therefore, the invention is upgraded and optimized by combining a new technical method on the basis of the traditional separation culture method, firstly carries out high-throughput 16S amplicon sequencing on the collected feces samples of healthy people and kidney stone patients in the early stage, compares and differentially analyzes the bacterial types and abundances of the feces samples of healthy people and kidney stone patients, and finds out the key bacterial types possibly influencing the formation of kidney stones. The KNMODO (Known Media Database) website was then used to predict the medium recipe for culturing the bacteria based on the 16S rDNA sequence of the concerned bacteria. The culture medium formulas are applied to the separation culture of human fecal bacteria, and the lactobacillus plantarum J-15 with the kidney stone inhibiting effect is obtained by artificially selectively selecting colonies growing on a solid culture medium and purifying the colonies. The method is different from the traditional microorganism isolation culture, realizes the culture medium formula with basis for predicting the target microorganism, organically links the microbial diversity data based on 16S rDNA with the isolation culture of the target strain, and greatly accelerates the process from ecological research to microbial resource mining. The method can organically link the microbial diversity data based on 16S rDNA with the separation culture of a target strain, and greatly accelerate the process from ecological research to microbial resource mining.
2. The lactobacillus plantarum provided by the inventionL. plantarum J-15, separating and screening a sample of the excrement of the healthy person, belonging to the human lactobacillus plantarum, being not genetically modified, coming from a primary micro-ecological environment to be applied, being safe, reliable and more easily adaptable to the human environment. Because the strain is derived from the human body and the host is a healthy person, the strain has higher safety, is not easy to cause rejection and other adverse reactions when being applied to the auxiliary treatment of clinical kidney stone patients in the later period, and is suitable for human bodiesThe intestinal tract of the body has better adaptability and is easier to fix. And the safety of the strains from other sources needs to be further evaluated firstly, and only animal experiments are possibly insufficient, and the animal and human are slightly different, so that even if no harm is found in the animal experiments, no danger can be completely guaranteed to be applied to the human body, and more risks are caused.
3. At present, no whole genome sequencing report of lactobacillus plantarum with an inhibiting effect on kidney stone formation is found.L. plantarumJ-15 was isolated from feces by the above procedure and the sequence was obtained by whole genome sequencing. The strain can be subsequently prepared into different forms such as a lactic acid bacteria agent or a capsule and the like through spray drying and embedding technologies, is applied to patients and is applied to the clinical auxiliary treatment of calcium oxalate kidney stones.
Compared with the prior art, the invention has the advantages and benefits that:
1. the lactobacillus plantarum provided by the inventionL. plantarum J-15 grows well on MRS agar medium, has certain tolerance to acid and bile salt, and has certain tolerance to antibiotics. Has better survival ability in severe environment and is easy to colonize in intestinal tract.
2. The lactobacillus plantarum provided by the inventionL. plantarum J-15 was derived from a different sample than the two other species of lactic acid bacteria, y104 and y96, which were both isolated from human feces, but had a different gene sequence. Compared with other two strains, the strain with stronger oxalic acid degrading capability can be selected for the early-stage experiment of the inventor to be applied to the later animal experiment, and the possibility that the inventor finds that the strain has the effect of inhibiting the formation of the kidney stone in vivo is increased. In vitro oxalate degradation tests carried out by lactic acid bacteria separated from human feces, stronger oxalate degradation capability is shown, in culture media without other carbon sources except sodium weedicide, the in vitro sodium oxalate degradation rate reaches 3.51%, and the in vitro oxalate degradation capability has stronger application potential, namely the in vitro oxalate degradation capability shows that the in vitro oxalate degradation capability has higher possibility and success rate when the in vitro oxalate degradation capability is applied to the following animal tests. As previously mentioned, the oxalate degradation rate in vitro tests is not directly indicative of the inhibitory effect on kidney stones, and has been shown to beThe capability of degrading oxalate is reduced.
3. The lactobacillus plantarum provided by the inventionL. plantarum J-15, animal experiments show that the strain can reduce the formation of rat kidney crystals, reduce the level of oxalic acid in the urine of a rat with a kidney calcium oxalate calculus model, and reduce the content of Ca in the urine. Therefore, the method has great development potential and has very important significance on human health.
4. The lactobacillus plantarum provided by the inventionL. plantarum J-15 is not only capable of degrading a certain amount of oxalate in vitro, but also capable of effectively reducing the formation of renal crystals in a rat body. Therefore, the preparation method can be applied to the field of functional foods and has practical application value in clinical adjuvant therapy. According to the HE staining and sectioning results, (it was indeed observed, see FIG. 2), the J-15 perfused rats had a significant reduction in renal crystallization in vivo, and the examination results showed a reduction in Ca content in the urine of the rats. On the other hand, the effect of J-15 in inhibiting the formation of renal calcium oxalate stones was confirmed by the fact that accumulation of oxalate, which can combine with Ca to form calcium oxalate, can lead to renal calculi of calcium oxalate type, and that the amount of Ca ions excreted with urine increases after the amount of free oxalate that can form calcium oxalate with Ca in vivo decreases.
5. The lactobacillus plantarum provided by the inventionL. plantarum J-15 has better carbohydrate metabolism, membrane transport and adaptive capacity to environment as shown by whole genome sequencing, and besides in vitro and in vivo experiments, the lactobacillus plantarum is disclosed from the gene levelL. plantarum J-15 has unique genetic characteristics and potential biological functions. Through whole genome sequencing, the gene and related regulation and control information of the strain can be obtained, and a molecular biology basis is provided for researching the specific biological characteristics of the strain; and subsequently, by comparing genome analysis, theoretical guidance can be provided for researching functional difference and evolutionary relationship in and among strains. Different strains have different genes, which cause them to exhibit different functions, and when a strain exhibits a certain better function, the cause can be found by reverse focusing on the genes. As can be seen from the whole genome sequencing result of J-15, the protein has better carbohydrate metabolism, membrane transport and environmental protectionAdaptability, which is the reason that oxalate can survive in oligotrophic culture medium and be utilized, and the reason that oxalate can pass through the strong acid and high bile salt environment in vivo and can be successfully colonized in intestinal tract.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum J-15 of the present invention on MRS agar medium.
FIG. 2 is a morphogram (X5) of the kidney of each group of rats subjected to HE staining under a light microscope in example 5 of the present invention, in which CK is a control group, M is a model group, and A is a treatment group).
FIG. 3 is an ion chromatogram for detecting oxalate concentration in example 3 of the present invention.
FIG. 4 is a circle diagram of the CGVIEW genome in example 6 of the present invention.
FIG. 5 is a statistical histogram of GO classification in example 6 of the present invention.
Fig. 6 is a statistical histogram of COG classification in embodiment 6 of the present invention.
FIG. 7 is a KEGG Classification statistics histogram in example 6 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples. The scope of the present invention is not limited to the following embodiments, and any modifications, equivalents, improvements, etc. made within the principle of the present invention should be included in the scope of the present invention.
The methods used in the following examples are conventional methods unless otherwise specified, and the percentages referred to in the medium are mass to volume ratios.
Example 1
Separation, screening and molecular biological identification of lactobacillus plantarum J-15
1. Material preparation
The human fecal sample is provided by a healthy person in Sichuan province;
the general primer pair is synthesized by the company of biological engineering (Shanghai) and has the following 27F/1492R sequence:
27F：AGAGTTTGATCMTGGCTCAG
1492R：TACGGYTACCTTGTTACGACTT
MRS liquid medium: 20.0g of glucose, 10.0g of peptone, 10.0g of beef extract, 6.0g of yeast powder, 1.0mL of Tween 80, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1L of pure water and pH 6.2 +/-0.2 (1.6-1.8% of agar is added as a solid culture medium).
2. Detailed description of the invention
And (4) a healthy human fecal sample meeting the conditions. 1.0g of the sample is weighed and quickly and fully stirred in a centrifuge tube filled with 9mL of sterile water, so that the sample is completely dispersed. And (3) sucking the suspension, diluting by 10 times in a gradient manner, respectively coating the suspension on MRS solid culture media, placing the MRS solid culture media in a facultative anaerobic incubator for culturing for 48 hours at 37 ℃, observing by naked eyes, picking single colonies with different shapes and sizes, and performing plate streaking and purification for 3 times. Extracting strain DNA by using a Tiangen bacteria genome DNA extraction kit, amplifying 16S rRNA by using a bacteria universal primer 27F/1492R, comparing the measured 16S rRNA sequence with NCBI BLAST, and comparing the sequence with that in EzBioCloudLactiplantibacillus plantarumThe similarity rate of the standard strain ATCC 14917 reaches 100 percent, and the strain is preliminarily identified to be one strainLactiplantibacillus plantarumI.e. lactobacillus plantarum. The purified strain was stored in glycerol at a final concentration of 30% (v/v) and stored frozen at-80 ℃ until use.
The lactobacillus plantarum J-15 is streaked on an MRS agar culture medium, inverted culture is carried out for 48 hours in a facultative anaerobic environment at 37 ℃, and then the colony morphology of the strain is observed, as shown in figure 1, the colony is milky white, the surface is smooth, the edge is neat, the colony is opaque, and the catalyst is negative.
Example 2
Detection of main physiological characteristics of lactobacillus plantarum J-15
PBS buffer: 0.20g of monopotassium phosphate, 1.15g of disodium hydrogen phosphate, 8.0g of sodium chloride and 0.2g of potassium chloride, 800mL of pure water is added, stirring and dissolving are carried out, concentrated hydrochloric acid is adjusted to the corresponding pH value, and the volume is adjusted to 1L.
1. Acid resistance test: the strain with the third generation of activationL. plantarum The J-15 is respectively inoculated into PBS buffer solution with pH of 4.0, 3.0 and 2.0 according to the inoculation amount of 1 percent, and is statically cultured for 3 hours at 37 ℃, and the viable count of the lactobacillus is measured by a gradient dilution plate method.
2. Bile salt resistance test: inoculating the activated third generation lactobacillus in an amount of 1% into MRS liquid culture medium containing 0.1%, 0.2% and 0.3% of bovine bile salt, standing and culturing at 37 deg.C for 5 hr, and measuring viable count of lactobacillus by gradient dilution plate method.
The acid and bile salt resistance results are shown in table 1. As can be seen from Table 1, lactobacillus plantarum J-15 has certain tolerance to acid and bile salt, can survive under the conditions of pH 4.0 and 0.1% of bile salt concentration, has survival rates of 53.60% and 3.5%, respectively, and has viable counts gradually decreasing with decreasing pH and increasing bile salt concentration, and survival rates of 11.40% and 0.14% under the conditions of pH2.0 and 0.3% of bile salt concentration.
The premise that the lactic acid bacteria can live and colonize in human intestinal tracts on the premise that the lactic acid bacteria can live and colonize in human intestinal tracts, whether the lactic acid bacteria can live and colonize in the human intestinal tracts is mainly related to the acid and bile salt resistance of the lactic acid bacteria, and whether the lactic acid bacteria can tolerate the high acidity of the stomach and the high bile salt concentration of the duodenum, so the acid and bile salt resistance is an important index for screening the probiotic lactic acid bacteria.
Example 3
To ascertain the oxalate-degrading properties of J-15, J-15 was tested for its ability to degrade sodium oxalate in vitro. Two other lactic acid bacteria y96 and y104 isolated from human feces were tested together for comparison.
Oxalate degradation test: j-15 was inoculated into a self-modified TM liquid medium containing 10mmol/L sodium oxalate at a concentration, cultured at 37 ℃ for 9 days, and the oxalate ion concentrations in the medium at day 0 and day 9 were measured by Ion Chromatography (IC) to calculate the decomposition utilization rate of oxalate, and the ion chromatogram of the oxalate concentration is shown in FIG. 3.
TM liquid medium: dipotassium hydrogen phosphate 2.0g, sodium dihydrogen phosphate 2.0g, ammonium sulfate 2.0g, magnesium chloride 0.58g, manganese sulfate 0.25g, pure water 1L
Degradation rate (%) = (1-A) 1 /A 0 ）×100%
In the formula: a. The 0 Is the initial sodium oxalate concentration after inoculation of the lactic acid bacteria; a. The 1 The concentration of sodium oxalate after 9 days of culture was used
The degradation rate of the lactobacillus plantarum J-15 on oxalate in a culture medium without other carbon sources except sodium weedicide is 3.51 percent, which is superior to that of other two strains of lactic acid bacteria, and through comparison (see table 2), the lactobacillus plantarum J-15 has the best oxalate degradation effect in three tested strains, which shows that the J-15 can utilize oxalate to carry out self-growth metabolism without other carbon sources and has stronger oxalate degradation capability.
Example 4
Drug resistance experiments to evaluate the in vitro safety of the J-15 potential probiotic strain, the antibiotic susceptibility of the strain was determined.
The test adopts a filter paper method to research the tolerance of lactobacillus plantarum J-15 to 7 antibiotics such as tetracycline, ampicillin, sulfamethoxazole, rifampicin, erythromycin, nalidixic acid, chloramphenicol and the like, the activated J-15 is fully and uniformly mixed, 100 mu L of bacterial liquid is taken to be put on an MRS solid culture medium, a daub bar is used for evenly coating and airing, a drug sensitive strip is pasted on the MRS solid culture medium coated with the bacterial liquid by a tweezer and is put in a constant temperature incubator at 37 ℃ for culturing for 24 hours, the diameter of a bacteriostatic circle is measured, the test is repeated three times, and the average value of the three times is taken as the final result. Results were judged according to the American society for Clinical and Laboratory Standards Institute (CLSI) Standards. The results of the antibiotic susceptibility test of Lactobacillus plantarum J-15 are shown in Table 3.
Test results show that J-15 has certain drug resistance to ampicillin and nalidixic acid, is moderately sensitive to tetracycline and rifampicin, and is sensitive to sulfamethoxazole, erythromycin and chloramphenicol.
Example 5 animal model test
1. Laboratory animals and groups
40 male SPF grade Sprague Dawley (SD) rats 6-7 weeks old weighing 210-230g. All rat basal diet adapted fed 7d, SD rats and basal diet were purchased from Duoduo Biotechnology Ltd. Randomized into 3 groups (see table 4): control group (CK), model group (M), treatment group (A), 6 rats per group, gavage for 28 days, 0.5 ml/rat/day, according to different modes (grouping and feeding mode see Table 3). After the experiment, pentobarbital is used for carrying out intraperitoneal injection anesthesia, and the anesthesia dosage is 30mg pentobarbital/kg rat. Blood was collected from the abdominal aorta, and the kidneys, cecum contents, urine, etc. were dissected and collected.
2. Content of the experiment
(1) Kidney section preparation and Observation
When the rats are dissected, the appearance of the kidneys of each group of rats is observed by naked eyes and photographed; the kidney is washed by sterile PBS and then is fixed in 4 percent paraformaldehyde solution, after dehydration, paraffin embedding and HE staining after slicing, the observation result of a common optical microscope is divided into (+ + +), (+ + -) -and (-) according to the existence, the quantity, the polymerization degree and the position of the crystal according to a five-grade classification method of a rapid calcium oxalate animal model, wherein the (-) grade is stone-forming negative, the (+/-), (+ +) and (+) grade are stone-forming positive, and the statistical result is shown in Table 5.
FIG. 2 is a cross-sectional view of a portion of the kidney tissue under microscope at 5 Xmagnification, showing that except for the control group, the kidneys of rats in other groups all had different degrees of calculus formation, showing significant tubular dilation and different degrees of deposition of crystals in the medulla, cortex or papilla. And comparing the renal medullary calculus conditions of the calculus model group and the experiment group (treatment group), wherein a large amount of crystals are distributed in the renal medullary calculus of the calculus group (model group), and the experiment group A has fewer crystals.
3) Detection of urine indicators
The specific detection indexes of urine are as follows: ca, mg, K, P, na, creatinine, uric acid, oxalic acid and citric acid.
The results in Table 6 show that the content of oxalic acid in urine in CK group is the lowest, the M group and CK group have significant difference,P <0.05, the content of oxalic acid in group A is greatly reduced compared with that in group M, which is close to that in group A, and therefore, the effect of lactobacillus plantarum J-15 in reducing the oxalic acid in urine is better. The urine detection result of rats shows that no significant difference exists among K, na, P, mg, creatinine, uric acid and citric acid groups, while the urine Ca group shows very significant difference,P <0.01, the difference between the CK group and the A group is very large, and the obvious difference exists, so that the content of Ca in urine is greatly reduced by J-15. Because lactobacillus plantarum J-15 degrades part of oxalate in vivo, free oxalate capable of forming calcium oxalate with Ca is reduced, and Ca ions are greatly discharged along with urine.
The experimental results show that the lactobacillus plantarum J-15 provided by the invention can effectively reduce the formation of renal crystals of a rat with renal calcium oxalate calculus, relieve renal tissue lesion, and has the potential of developing into probiotic functional food and being applied to clinical adjuvant therapy.
Example 6 Whole genome sequencing
1. Genome composition
The invention adopts the currently most widely used second-generation sequencing platform Illumina Hiseq multiplied by 10 platform to construct a fragment with an insertion fragment of about 400bp for a DNA sample qualified by J-15 quality inspection, performs PE150 (pair-end) sequencing, reads the length of 150bp by single-ended sequencing, outputs original sequencing data which is not less than 100 multiplied by the coverage depth of a genome, performs quality control and assembly on the sequenced sequence, and finally assembles to obtain 69 genomes scaffold, the total base length is 3310895bp, and the average GC content of all bases in the genome is 44.36%. Then, the gene prediction was carried out on the assembly sequence, and it was found that the gene factor of J-15 coding gene was 3243, the total length was 2741751bp, the gene accounted for 82.81% of the genome, and the genome circle is shown in FIG. 4.
2. Genome annotation
By functional annotation of the predicted encoded genes compared to several databases such as COG, GO and KEGG, 2380 genes with GO annotation were found to account for 72.47% of all genes, with 1192 genes related to Cellular composition (Cellular Component), 1889 genes related to Molecular Function (Molecular Function), 1668 genes related to Biological Process (Biological Process), the most genes related to Integral Component of membrane (Molecular Component), and the annotation classification statistics are shown in fig. 5.
COG annotation found a total of 2589 genes in J-15 with COG functional classification of 78.84% of the total gene factor divided into 4 major classes, 20 different classes, with the largest proportion of genes of unknown function, among those of known function, the largest genes involved in Carbohydrate transport and metabolism (Carbohydrate transport and metabolism), followed by Transcription (Transcription) and transport and metabolism of Amino acids (Amino acid transport and metabolism), see fig. 6 for COG classification statistics.
The annotated gene factor of KEGG is 1489, mainly genes involved in metabolism (metabolism), wherein the maximum number of genes involved in Carbohydrate metabolism (Carbohydrate metabolism) is 225, the second is genes involved in membrane transport (membrane transport), 178, and the above results show that J-15 has strong Carbohydrate utilization capacity and membrane transport capacity.
3. Metabolic system analysis
Analysis of the metabolic system revealed that carbohydrate-active enzymes were annotated to 5 different major classes (see table 7 for details, where the relative proportion of Glycoside hydrolase (Glycoside Hydrolases) genes was maximal and 48 genes were involved, accounting for about 44.44%.
The secondary metabolite synthesis gene cluster of J-15 was predicted to obtain 2 gene clusters, which were located in Scaffold2 and Scaffold42 (see Table 8 for details), wherein 25 genes involved in the synthesis of terpene compounds and 2 genes involved in the synthesis of bacteriocin. Bacteriocins are small molecular weight bioactive antimicrobial peptides that are synthesized by bacteria in ribosomes and released extracellularly to kill or inhibit prokaryotic growth, and help J-15 to form a microflora preponderance in the gut.
The results are of great significance for the subsequent deep CAZyme research, the revealing of the metabolic mechanism of J-15 carbohydrate and the understanding of the synthetic gene cluster of natural products on the J-15 genome.
4. Two-component regulation and control system
The two-component regulation system can enable bacteria to sense, respond and adapt to wide environment, pressure source and growth conditions, the typical two-component regulation system mainly comprises sensing protein and regulatory protein, the two-component regulation system of J-15 is predicted by using hmmer3, analysis shows that 30 genes which are annotated to the regulatory protein exist in the J-15, the maximum number of the genes which are annotated to Response regulation (Response _ reg) protein exists, 14 genes exist, and the genes are Response regulator receiver domains (Response regulator receiver domains), and the genes belong to 5 different structural domains; there are 12 genes annotated with a sensory protein, and at most 9 genes annotated with the Histidine protein kinase (HATPase _ c-Histine kinase-, DNA gyrase B-, and HSP90-like ATPase) domains, which belong to 4 different domains. In conclusion, the J-15 diversified bi-component system is beneficial to regulating and controlling various physiological and biochemical processes to adapt to the change of the external environment, and is possibly beneficial to improving the tolerance capability of the bi-component system to gastric juice, the low pH environment and the high bile salt concentration environment of intestinal tracts and the adhesion capability of the bi-component system to the intestinal tracts.
SEQUENCE LISTING
<110> Sichuan university
<120> lactobacillus plantarum capable of inhibiting kidney stone formation and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1480
<212> DNA
<213>Lactobacillus plantarum (A)Lactiplantibacillus plantarum）
<400> 1
tccagagatt tgcaccttgt cacttggcgg ctggtttcta aaaggttacc ccaccgactt 60
tgggtgttac aaactctcat ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc 120
accgcggcat gctgatccgc gattactagc gattccgact tcatgtaggc gagttgcagc 180
ctacaatccg aactgagaat ggctttaaga gattagctta ctctcgcgag ttcgcaactc 240
gttgtaccat ccattgtagc acgtgtgtag cccaggtcat aaggggcatg atgatttgac 300
gtcatcccca ccttcctccg gtttgtcacc ggcagtctca ccagagtgcc caacttaatg 360
ctggcaactg ataataaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac 420
gagctgacga caaccatgca ccacctgtat ccatgtcccc gaagggaacg tctaatctct 480
tagatttgca tagtatgtca agacctggta aggttcttcg cgtagcttcg aattaaacca 540
catgctccac cgcttgtgcg ggcccccgtc aattcctttg agtttcagcc ttgcggccgt 600
actccccagg cggaatgctt aatgcgttag ctgcagcact gaagggcgga aaccctccaa 660
cacttagcat tcatcgttta cggtatggac taccagggta tctaatcctg tttgctaccc 720
atactttcga gcctcagcgt cagttacaga ccagacagcc gccttcgcca ctggtgttct 780
tccatatatc tacgcatttc accgctacac atggagttcc actgtcctct tctgcactca 840
agtttcccag tttccgatgc acttcttcgg ttgagccgaa ggctttcaca tcagacttaa 900
aaaaccgcct gcgctcgctt tacgcccaat aaatccggac aacgcttgcc acctacgtat 960
taccgcggct gctggcacgt agttagccgt ggctttctgg ttaaataccg tcaatacctg 1020
aacagttact ctcagatatg ttcttcttta acaacagagt tttacgagcc gaaacccttc 1080
ttcactcacg cggcgttgct ccatcagact ttcgtccatt gtggaagatt ccctactgct 1140
gcctcccgta ggagtttggg ccgtgtctca gtcccaatgt ggccgattac cctctcaggt 1200
cggctacgta tcattgccat ggtgagccgt taccccacca tctagctaat acgccgcggg 1260
accatccaaa agtgatagcc gaagccatct ttcaagctcg gaccatgcgg tccaagttgt 1320
tatgcggtat tagcatctgt ttccaggtgt tatcccccgc ttctgggcag gtttcccacg 1380
tgttactcac cagttcgcca ctcactcaaa tgtaaatcat gatgcaagca ccaatcaata 1440
ccagagttcg tcgacttgca ttatagctgc cgcacttccc 1480
Claims (2)
1. Lactobacillus plantarum (A) capable of inhibiting kidney stone formationLactiplantibacillus plantarum) J-15, deposited in China general microbiological culture Collection center on 6 th 4 th 2021 with the preservation number of CGMCC No.22140.
2. The use of the Lactobacillus plantarum J-15 having a accession number CGMCC No.22140, as defined in claim 1, for the preparation of a medicament having kidney stone formation-inhibiting ability.
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| CN103013893A (en) * | 2013-01-21 | 2013-04-03 | 黑龙江八一农垦大学 | Lactobacillus plantarum CCL67 and application of same |
| CN107227278A (en) * | 2017-07-17 | 2017-10-03 | 浙江鸣食品股份有限公司 | A kind of Lactobacillus plantarum A11 and its application |
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| US9283252B2 (en) * | 2008-09-30 | 2016-03-15 | Meiji Co., Ltd. | Lactic acid bacterium having high oxalic acid decomposition ability |
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| CN103013893A (en) * | 2013-01-21 | 2013-04-03 | 黑龙江八一农垦大学 | Lactobacillus plantarum CCL67 and application of same |
| CN107227278A (en) * | 2017-07-17 | 2017-10-03 | 浙江鸣食品股份有限公司 | A kind of Lactobacillus plantarum A11 and its application |
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