CN112300962B - Lactobacillus plantarum with specific molecular target and antioxidation function and application thereof - Google Patents
Lactobacillus plantarum with specific molecular target and antioxidation function and application thereof Download PDFInfo
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- CN112300962B CN112300962B CN202011184917.3A CN202011184917A CN112300962B CN 112300962 B CN112300962 B CN 112300962B CN 202011184917 A CN202011184917 A CN 202011184917A CN 112300962 B CN112300962 B CN 112300962B
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses a lactobacillus with antioxidant activity and application thereof, wherein the lactobacillus is lactobacillus plantarum (Lactobacillus plantarum) which is preserved in the Guangdong province microorganism strain collection with the preservation number of GDMCC NO 61123; the lactobacillus can be used for preparing microbial preparations, expands the variety of lactobacillus strains, and has wide application prospects in the fields of foods, cosmetics, medicines and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum with a specific molecular target and an antioxidant function and application thereof.
Background
Oxidative stress and injury based on free radicals and reactive oxygen species and intervention strategies are research hotspots in the fields of life sciences, medicine and health and nutrition sciences today. Oxidative stress and injury have become the most common targets and research models for drug discovery and functional food development as important physiological processes for the development and progression of systemic and metabolic diseases. Dietary intervention schemes based on nutritional regulation are an effective control strategy for oxidative stress related diseases, and have attracted considerable attention from researchers. Around redox balance, oxidation stress is taken as an action target point, antioxidants and oxidation stress regulators with different sources are developed, and the method has extremely important research significance and theoretical value on scavenging oxygen free radicals, maintaining normal redox balance of an organism, protecting cells and tissues from damage and further intervening in the occurrence and development of diseases related to the oxidation stress.
Lactic acid bacteria are a class of food grade microorganisms with a long recognized safety (GRAS) history of safe consumption. Lactic acid bacteria have become the main ingredients of related products such as foods and medicines due to their specific physiological functions and properties suitable for biological processing. Many in vivo and in vitro experimental studies have shown that lactic acid bacteria cells and cell-free extracts have antioxidant-like activity in vitro and that ingestion of lactic acid bacteria in vivo has significant regulatory effects on oxidative stress of the body or cells. At present, the research of the intervention of lactic acid bacteria on oxidative stress can be mainly divided into three layers: (1) Development and antioxidation capability evaluation of antioxidation lactic acid bacteria based on in vitro antioxidation activity; (2) Analysis and evaluation of antioxidant lactic acid bacteria based on a cell model, an in vitro tissue model and an animal model; (3) Evaluation of the activity of lactic acid bacteria in alleviating oxidative stress based on human and clinical experiments.
Lactobacillus plantarum (Lactobacillus plantarum) is one of the most common lactic acid bacteria, widely distributed in the environment, food, human and animal hosts. A large number of research results on the probiotic functional characteristics of lactic acid bacteria show that a plurality of lactic acid bacteria have antioxidant functions, but the antioxidant capacities of different strains are different, so that the screening of lactic acid bacteria with high antioxidant functions is an important research content in the fields of food microorganisms, medicine, food science, health care and the like at present. In addition to the study of the antioxidant activity of lactic acid bacteria strains themselves, some studies have now found that the use of lactic acid bacteria having antioxidant function can improve the antioxidant function of fermentation products. For example, the fermentation of soybean milk by using lactobacillus plantarum B1-6, such as Xiao, etc., shows that the total phenol content, the antioxidant activity and the DNA damage protection capability of the fermented soybean milk are improved to different degrees; wu and other researches show that after the echeveria glabra is fermented by lactobacillus plantarum (Lactobacillus plantarum) BCRC10357, the total antioxidant capacity, the superoxide factor scavenging capacity and the DPPH free radical scavenging capacity are improved, and the total phenol content, the flavone content and the gallic acid content are improved. Marazza et al fermented soy milk with Lactobacillus rhamnosus (Lactobacillus rhamnosus) CRLPSl and fed mice with diabetes, found that this soy milk significantly reduced the blood glucose level, total cholesterol level, triacylglycerol level and increased the antioxidant enzyme level in mice. Marazza et al fermented soymilk with Lactobacillus rhamnosus (Lactobacillus rhamnosus) CRLP81, has increased total antioxidant capacity and can inhibit oxidative damage of DNA compared to unfermented soymilk. After fermentation of kelp by means of Lactobacillus brevis (Lactobacillus brevis) BJ20 by Kang et al, the levels of gamma-glutamyl transpeptidase and malondialdehyde in serum of volunteers consuming fermented kelp are reduced and the activities of superoxide dismutase (SOD) and Catalase (CAT) in serum are increased as compared to volunteers consuming no fermented kelp. Xu Yin and the like utilize streptococcus thermophilus (Streptococcus thermophilus) grx9 to ferment soybean milk, compared with the in-vitro antioxidant capacity of unfermented soybean milk, the in-vitro antioxidant capacity of the fermented soybean milk is remarkably improved, alanine Aminotransferase (ALT) and aspartic acid Aminotransferase (AST) in the serum of a mouse with acute alcohol liver injury model can be remarkably reduced, and the activities of superoxide dismutase (SOD) and glutathione-peroxidase (GSH-Px) in liver tissues of the mouse can be improved, so that the Malondialdehyde (MDA) content in the liver tissues of the mouse can be reduced.
Compared with other functional researches and developments, the related lactobacillus has not formed systematic researches on the regulation effect of organism oxidative stress, aiming at oxidative stress and injury caused by different factors, the screening, excavation and evaluation researches of the lactobacillus with the effect of relieving oxidative stress are developed, the original lactobacillus strain with the capability of relieving oxidative stress is still to be screened and researched, and at present, the lactobacillus strain with the effect of remarkably relieving oxidative stress in China is not more, so that functional lactobacillus, especially antioxidant lactobacillus, still need to be further developed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the lactobacillus with oxidation resistance and the application thereof, and the lactobacillus has good strain safety.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a lactic acid bacterium, designated lactobacillus plantarum (Lactobacillus plantarum) 124, deposited at the cantonese microbiological strain collection center, 8/7/2020, at the address: the China Guangzhou City first China No. 100 Guangdong province microbiological institute, deposit number is GDMCC NO 61123.
According to the invention, a plurality of potentially beneficial bacterial groups (Akkermansia, lactobacillus) are found in the century old group through bioinformatics analysis of the 16S amplicon and metagenome for the first time, and the abundance of the bacterial groups gradually increases with the age. Such probiotics are directionally separated under the guidance of metagenome big data, and then target bacteria with excellent in-vitro antioxidation capability are screened by utilizing in-vitro antioxidation activity evaluation indexes (DPPH free radical scavenging capability, reduction activity, hydroxyl free radical scavenging capability, lipid peroxidation resisting capability, superoxide anion scavenging capability and ferrous ion chelating capability) of whole cells of the probiotics, and finally lactobacillus plantarum with excellent antioxidation activity is screened in-vitro and named as lactobacillus plantarum (Lactobacillus plantarum) 124. The test example I shows that the safety evaluation experiment shows that the kit has no hemolytic, no drug resistance gene and no toxic gene. Through a live thallus feeding D-galactose induced oxidative damage mouse experiment, the levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and Malondialdehyde (MDA) in the liver and kidney of the mouse and morphological analysis of the liver, kidney and small intestine tissues of the mouse are detected, and finally, the lactobacillus plantarum (Lactobacillus plantarum) 124 can regulate the GSH-Px and SOD levels in the liver and kidney tissues to different degrees, and can reduce the pathological changes of cells in the liver cells and kidney tissues of the mouse and relieve the pathological changes of intestinal villus arrangement irregularity and the like of the small intestine tissues of the mouse, and finally, the strain is verified to have good antioxidant capacity through an in vivo mouse experiment.
The invention separates and identifies the collected 247 biological samples of the life-long family of 'world life-long country-Chinese abaca', dilutes the samples to prepare bacterial liquid, inoculates the bacterial liquid into a solid or liquid culture medium for culture, observes the morphology, the size and the luster of the cultured bacterial colony, then adopts a flat plate streaking method to separate and purify the bacterial colony to obtain a single bacterial colony, inoculates the single bacterial colony into the solid or liquid culture medium for culture, and then carries out 16S rDNA molecular biological identification and PCR amplification.
In a second aspect, the invention provides the use of the lactic acid bacteria described above for the preparation of a microbial preparation having antioxidant activity.
In a third aspect, the present invention provides the use of the lactic acid bacteria as described above as an antioxidant in the preparation of a food, pharmaceutical or cosmetic product.
In a fourth aspect, the invention provides a microbial preparation with antioxidant activity, comprising the lactic acid bacteria described above and identifying specific molecular targets of said lactic acid bacteria.
As a preferred embodiment of the microbial preparation, the nucleic acid sequence of lactobacillus plantarum (Lactobacillus plantarum) 124 is shown in SEQ ID NO. 1; wherein the specific molecular target nucleic acid sequence is shown as SEQ ID NO.2.
As a preferred embodiment of the microbial preparation according to the invention, the microbial preparation further comprises adjuvants and/or carriers.
As a preferred embodiment of the microbial preparation according to the present invention, the adjuvant comprises corn flour, inulin, whey powder, glucose, sucrose, fructo-oligosaccharides or galacto-oligosaccharides, and the carrier comprises magnetic microspheres, wheat bran, diatomaceous earth or talc.
In the technical scheme of the invention, the microbial preparation further comprises auxiliary materials and/or carriers to improve the performance of the microbial preparation, wherein the auxiliary materials and the carriers are not limited to the substances, but can be other substances conventional in the field.
In a fifth aspect, the present invention provides the use of a microbial preparation as described above for the preparation of a food, pharmaceutical or cosmetic product.
As a preferred embodiment of the application of the microbial preparation of the present invention, the microbial preparation is a food starter.
As a preferred embodiment of the use of the microbial preparation according to the invention, the microbial preparation is used as an antioxidant in food, pharmaceutical or cosmetic products.
The invention provides the local antioxidant lactobacillus from the longevity countryside-Chinese abaca, which has clear sources and definite antioxidant functions of scavenging free radicals, active oxygen and the like in vitro. The in vitro antioxidant index (DPPH, hydroxyl radical removal and lipid peroxidation resistance) is higher than that of lactobacillus bulgaricus (Lactobacillus bulgaricus) used for lactobacillus products at home and abroad, the hydroxyl radical removal, ferrous ion chelation and lipid peroxidation resistance are higher than that of antioxidant Vc, and the DPPH, reduction activity, hydroxyl radical removal, ferrous ion chelation, superoxide anion removal and lipid peroxidation resistance indexes are equal to and higher than those of the standard strain Lactobacillus rhamnosus GG ATCC 53103; the in vivo antioxidation indexes (GSH-Px and SOD) are stronger than those of the domestic Xinjiang yoghurt isolate Lactobacillus plantarum KSFY, and the MDA index is better than that of the foreign natural fermentation product isolate Lactobacillus plantarum AR501, namely the in vivo action of relieving oxidative stress is obvious; the safety evaluation experiment shows that the characteristics of no hemolysis, no drug resistance gene, no toxicity gene and the like, and the method makes up the lack of lactobacillus strains with outstanding effect of relieving oxidative stress in the related industrial fields of lactobacillus biological processing in China at present. The lactobacillus strain resource library is enriched, and the lactobacillus strain resource library has wide application prospect in the fields of foods, cosmetics, medicines and the like.
In summary, the invention has the following beneficial effects:
the invention firstly obtains a plurality of potentially beneficial bacterial groups in the century old group through 16S amplicon and metagenome bioinformatics big data analysis, directionally separates the probiotics under the guidance of metagenome big data, then screens a lactobacillus plantarum with good antioxidant activity by using in vitro antioxidant activity evaluation index, is named as lactobacillus plantarum (Lactobacillus plantarum) 124, has good safety, has better antioxidant effects of scavenging free radicals, active oxygen and the like outside antibodies than standard strains, and expands the variety range of lactobacillus.
Drawings
FIG. 1 is a graph showing the results of the bioinformatics analysis of 16S amplicons in example 1, at the annotation level of species;
FIG. 2 is a graph showing the results of the 16S amplicon bioinformatics analysis of the distribution of some potentially beneficial bacterial groups in the centella aged population of example 1;
FIG. 3 is a graph of the results of the metagenomic bioinformatics analysis of example 1 annotated with functions in the egNOG database;
FIG. 4 is a graph showing the results of in vitro antioxidant screening index analysis of Lactobacillus plantarum (Lactobacillus plantarum) 124 in example 3;
FIG. 5 is a graph showing the results of the levels of GSH-Px, SOD, MDA in liver and kidney tissue of mice in test example II;
FIG. 6 is a graph showing the results of morphological observation of liver, kidney and small intestine of mice in test example III;
FIG. 7 is a diagram i of detection of a molecular target specific for Lactobacillus plantarum (Lactobacillus plantarum) 124 in test example four;
FIG. 8 is a graph ii of detection of a molecular target specific for Lactobacillus plantarum (Lactobacillus plantarum) 124 in test example four;
FIG. 9 is a diagram iii of a molecular target specific for Lactobacillus plantarum (Lactobacillus plantarum) 124 in test example four.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
EXAMPLE 1 preservation information of lactic acid bacteria
The invention provides a lactobacillus, which is named as lactobacillus plantarum (Lactobacillus plantarum) 124 and is preserved in the microorganism strain preservation center of Guangdong province in 8/7/2020, address: the China Guangzhou City first China No. 100 Guangdong province microbiological institute, deposit number is GDMCC NO 61123.
Example 2, sample collection, sample 16S amplicon and metagenomic bioinformatics analysis
1) Collecting biological samples of characteristic crowd in longevity areas:
all collected individuals in the invention come from a 'long-life village-Chinese abaca' queue (the city abaca county of plum, guangdong province), all 247 subjects are strictly grouped, and no serious diseases exist. Subjects were divided into six age groups: group Y20 (0-20 years old), group Y40 (21-40 years old), group Y60 (41-60 years old), group Y80 (61-80 years old), group Y100 (81-100 years old) and group Y120 (100-120 years old). The exclusion criteria for the subjects included the following: (i) History of chronic disease (diabetes, hypertension) and (ii) use of antimicrobial drugs (antibiotic or antifungal therapy) in the last two months. Fecal samples freshly collected from each subject were immediately frozen at-20 ℃ and transported to the laboratory with an ice bag for storage in-80 ℃ refrigerator until use. Sample information is as follows in table 1:
table 1 demographic and clinical characteristics of different groups
2) 16S amplicon bioinformatics analysis
Performing a 16S amplicon bioinformatics analysis from the biological sample collected in step 1), comprising the steps of:
s1 sequencing data processing: the V4 and V5 hypervariable regions of the 16S rRNA genes are sequenced based on an IonS5TMXL sequencing platform, and a small fragment library is constructed by a Single-ended sequencing (Single-End) method to carry out Single-ended sequencing. Cut is sheared and filtered through Cutadapts (V1.9.1, http:// cutadapts.readthes/en/stable /), 85,170 Reads are measured for each sample on average, 80,024 pieces of effective data (Clean Reads) are obtained through quality control average (https:// gitub. Com/torognes/vsearch /), and the quality control effective rate is 94.01%.
S2 OTU clustering and species annotation: sequences were clustered to OTUs (Operational Taxonomic Units) with 97% Identity (Identity) using the Upparse software (Upparse v7.0.1001, http:// www.drive5.com/Uparse /), yielding 3,436 OTUs in total, and then species annotation analyses (thresholding 0.8-1) were performed on the OTUs sequences using the Mothur method with the SSUrRNA database of SILVA132 (http:// www.arb-SILVA. De /). In the annotated results, the proportions annotated to the kingdom, phyllum, class, order, family, genus levels were 100.00%,98.14%,97.12%,95.55%,90.75%,62.43%, respectively. And finally, carrying out homogenization treatment on the data of each sample, and carrying out homogenization treatment by taking the minimum data amount in the sample as a standard. The species annotation is shown in figure 1.
S3 LEfSe analysis: using LEfSe software, the LDA Score was set to a screening value of 3.Metastats analysis uses R software to make inter-group permutation test under each classification level (Phylum, class, order, family, genus, species) to obtain a p value, then uses Benjamini and Hochberg False Discovery Rate method to correct the p value to obtain a q value, uses R software to make inter-group T_test test and plot, finally makes two-by-two wilcox test on the above-mentioned difference species, the difference significance is represented by diamond color, and the difference species is shown by heat map, as shown in figure 2.
As can be taken from fig. 1, the abundance of species Methanobacteriaceae, akkermansiaceae, muribaculaceae, ruminococcaceae, christensenellaceae, lactobacillaceae, etc. is higher in the group of Y120 centenarian at the family level; lachnospiraceae, burkholderiaceae, bifidobacteriaceae species are less abundant. Some potentially beneficial bacterial populations Akkermansiaceae, lactobacillaceae, christensenellaceae were found in the Y120 centella aged group with increasing abundance with age.
As can be taken from fig. 2, the Y120 centella old age group has a differential species distribution with other age groups, mainly Synergistetes, euryarchaeota, verrucomicrobia, lentisphaerae at the portal level. There is mainly Akkermansiaceae, muribaculaceae, erysipelotrichaceae, rikenellaceae, methanobacteriaceae, synergistaceae, christensenellaceae at the department level. The main levels are Akkermansia, methanobrevibacter, alistipes, lactobacillus, parabacteroides, butyrivibrio, barnesiella. Among these differential species, some potentially beneficial bacterial populations Akkermansia, lactobacillus, christensenellaceae were noted that gradually increased in abundance with age, eventually being highest in the Y120 centenarian group. It is currently accepted that Akkermansia, lactobacillus, christensenellaceae, which is related to health, is a beneficial microorganism for humans, so it can be inferred initially that these bacteria are likely to be positively correlated with longevity. However, these probiotic strains were positively or negatively correlated with longevity, and we will culture and characterize the relevant strains in conjunction with in vivo/in vitro experiments for further validation.
3) Metagenomic bioinformatics analysis
Performing a metagenomic bioinformatic analysis from the biological sample collected in step 1), the steps being as follows:
s1, sequencing all samples by using an Illumina NovaSeq sequencing platform to obtain 2,858,406.53Mbp of original Data (Raw Data) (the average Data volume is 11,619.54 Mbp), obtaining 2,854,730.06Mbp of effective Data (clear Data) (the average Data volume is 11,604.59 Mbp) by quality control, and obtaining 45,538,807,910 bp of Scaftigs after single sample assembly and mixed assembly. Gene prediction was performed on each sample and the results of the mixed assembly using MetaGeneMark software to obtain a total of 53,937,694 Open Reading Frames (ORFs) (average 219,259), and after redundancy removal, a total of 5,364,988 ORFs were obtained, with a total length of 3,953.51Mbp.
S2 common function database annotation (e-value < = 10-5) was performed on non-redundant gene sets using diameter software, 3,264,476 (60.85%) ORFs were aligned to KEGG database, 3,204,116 (59.72%) ORFs were aligned to eggNOG database. As shown in fig. 3.
As can be seen from FIG. 3, there are higher methyltransferases, oxydoreducase oxidoreductases, acetyltransferase acetyltransferases, dehydrogenase dehydrogenases, RNA Polymerase in the egNOG database cluster.leve2 level Y120 century old group. The intestinal microbiota of the centenarian reveals the possible mechanisms of flora life extension: intestinal microbiota makes longevity population have higher metabolism and stronger exogenous biodegradability, wherein methyltransferase, oxidoreductase, acetyltransferase, dehydrogenase and RNA polymerase have abnormal functions, and can possibly improve in vivo antioxidant capacity and promote metabolism and prolong life. The correlation mechanism requires further verification.
Example 3 isolation and identification of lactic acid bacteria
Based on 247 biological samples of the life-prolonging family of 'world longevity county-Chinese abaca' collected in example 2, the separation and identification of lactic acid bacteria are carried out as follows:
s1, taking 1-3g of fecal sample, placing the fecal sample in a 15mL centrifuge tube filled with 10mLTPY broth, fully shaking the sample on a vortex homogenizer to uniformly disperse the sample, and culturing the sample at 37 ℃ for 24 hours under anaerobic conditions to perform enrichment treatment.
S2 0.5ml of fecal sample suspension was aspirated from it with a pipette and poured into a 4.5ml tube of sterile PBS (0.9%), and purged three times to allow adequate mixing.
S3A pipette is used to aspirate 0.5ml from this tube into another tube containing 4.5ml of sterile PBS (0.9%) and so on to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Bacterial solutions of various dilutions.
S4, sucking 0.2mL of bacterial liquid with each dilution, respectively dripping the bacterial liquid on a TPY solid culture medium plate, uniformly coating a coating rod, culturing the bacterial liquid in the TPY solid culture medium for 48 hours at 37 ℃ under anaerobic conditions.
S5, observing the morphology, the size and the gloss of each colony on a comparison plate, selecting different colonies, and separating and purifying by a plate streaking method. Streaking on TPY solid culture medium by plate streaking method, culturing for 48 hr at 37deg.C, separating and purifying for 2 times. And after the last streaking, if the sizes and the shapes of the colonies on the plate are consistent, the purification is successful.
S6, picking single colony again, inoculating on TPY liquid culture medium, and culturing for 24 hours at 37 ℃ under anaerobic condition.
S7, sucking 0.5mL of amplified bacterial liquid, adding the bacterial liquid into a 1.5mL centrifuge tube containing 0.5mL of glycerol (60% (v/v)), and preserving at-80 ℃ or-20 ℃.
S8 16S rDNA molecular biology identification: total DNA extraction, using 16SrDNA universal primer, forward primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (see SEQ ID NO. 3), reverse primer 1492R:5'-GGTTACCTTGTTACGACTT-3' (see SEQ ID NO. 4) was amplified.
PCR reaction System (25. Mu.L): 1 mu L of DNA template, 1 mu L of 10 mu mol/L forward and reverse primer, 12.5 mu L of 2X Taq PCR MasterMix II premix, and 9.5 mu L to 25 mu L of deionized water. PCR amplification reaction procedure: 95 ℃ for 5min;94℃1min,58℃1min,72℃1.5min for 30 cycles; and at 72℃for 10min.
Inputting the obtained 16S rDNA gene sequence data of the strain into a GenBank nucleic acid database, comparing and analyzing the sequence in the GenBank database by utilizing an online BLAST program, determining classification status of the strain, finally separating 303 strains of lactic acid bacteria, and removing the weight of the strain to obtain 185 strains of lactic acid bacteria.
Example 4 in vitro screening of lactic acid bacteria
The lactobacillus obtained by separation and identification in example 3 is subjected to in vitro antioxidation screening, and the method comprises the following steps:
1) Preparation of sterile physiological saline bacterial suspension, fermentation supernatant, cell-free extract and thallus broken body
Inoculating single colony of lactobacillus with excellent growth state obtained by the separation into 1.5mL of TPY liquid culture medium, culturing at 37 ℃ for 18-24h, taking the culture solution as an inoculating solution, inoculating into 50mL of TPY liquid culture medium according to 2% of inoculating amount, and standing and culturing for 18-24h to obtain the culture solution of the strain. Centrifuging at 4deg.C for 10min at 10000r/min, collecting supernatant, namely fermentation supernatant, and collecting 10-15mL with 15mL centrifuge tube; the cells were collected by a 15mL centrifuge tube, washed twice with sterile physiological saline, resuspended in sterile physiological saline, and the cell density was adjusted to 1.01.+ -. 0.05×10 9 CFU/mL，OD 600 =1.0±0.1, which is the bacterial suspension; at least taking a centrifuge tube with a volume of 3mL to 15mL, performing ultrasonic crushing (750W, 5s for crushing, 5s for intermittence, 7.5min for working time and 15min for total time) in an ice bath, centrifuging at a temperature of 4 ℃ for 25min at 10000r/min, and collecting the supernatant to obtain a cell-free extract; collecting the precipitate, and adding physiological saline with corresponding volume to obtain thallus crushed body;
2) The in vitro antioxidant activity of lactic acid bacteria is determined as follows:
determination of the ability of S1 lactic acid bacteria to scavenge 1, 1-diphenyl-2-trinitrophenylhydrazine free radical (DPPH)
The measurement of the ability of live cells of lactic acid bacteria to scavenge free radicals (DPPH. Cndot.) is carried out as follows: taking 0.5mL of physiological saline suspension of lactobacillus to be detected, adding 0.5mL of DPPH solution (DPPH is prepared by dissolving with absolute ethyl alcohol, and the final concentration is 0.2 mmol/L), uniformly mixing, then placing the mixture at room temperature for shading reaction for 30min, centrifuging the mixture at the rotating speed of 10000r/min for 10min, taking a supernatant part, and measuring the absorbance of the sample at the wavelength of 517 nm.
Clearance (%) = (a) Control -A Sample of )/A Control x100
Determination of S2 lactic acid bacterium isolate reduction Activity (Reducing activity)
The method for measuring the reduction activity of the lactobacillus living cell suspension comprises the following specific operation steps: adding 0.25mL of physiological saline suspension of lactobacillus into 0.25mL of PBS solution (0.2 mol/L, pH 6.6), adding 0.25mL (1% m/v) of potassium ferricyanide solution, placing the mixture in water bath at 50deg.C for heat preservation for 20min, rapidly cooling to room temperature in ice water, adding 0.25mL of trichloroacetic acid (TCA, 10%) into the mixture, fully mixing, centrifuging at 10000r/min for 10min to remove precipitate such as protein, collecting 0.25mL of supernatant, and simultaneously adding 0.05mL of FeCl 3 (0.1%, m/v), sufficiently shaking to uniformly mix the materials, standing and incubating for 10min, and measuring the absorbance value of the reaction system at the wavelength of 700 nm. As a result, L-cysteine was used as a standard to express the reducing power of lactic acid bacteria. Standard solutions of L-cysteine (0-400 mu mol/L) with different concentrations are prepared respectively, and the standard curves are prepared by measuring according to the steps.
S3 determination of lipid peroxidation resistance of lactic acid bacteria
In the study, linoleic acid is selected as the research of the peroxidation of unsaturated fatty acid lipid. Thiobarbituric acid (TBA) method was used to measure lipid peroxidation. Preparation of linoleic acid emulsion: 0.1mL of linoleic acid, 0.2mL of Tween 20 was added to 19.7mL of deionized water. 0.5mL phosphate buffer (0.02 mol/L, pH 7.4), 1mL linoleic acid emulsion, 0.2mL FeSO 4 (0.01%, m/v), 0.02mL of ascorbate (0.01%, m/v) and 0.4mL of a physiological saline suspension of lactic acid bacteria were thoroughly mixed, incubated at 37℃for 1.5h,2mL of the above reaction, 0.2mL of trichloroacetic acid (TCA, 4%, m/v) was added, 2mL of thiobarbituric acid (TBA, 0.8%, m/v), 0.2mL of di-tert-butyl-p-cresol (BHT, 0.4%, m/v) was added. Mixing the above materials, standing at 00deg.C for 10min, and cooling on ice. At 10000 r-Centrifuging at min speed for 10min, collecting supernatant, and measuring absorbance of the extractive solution at 532 nm.
Inhibition of linoleic acid peroxidation (%) = (1-a) Sample of /A Blank space )x100
Determination of the ability of S4 lactic acid bacteria to scavenge hydroxyl radicals (. OH)
The Fenton reaction system is used for measuring the scavenging capacity of bacterial liquid to ferrous ion catalytic hydrogen peroxide to generate OH to measure the scavenging capacity of bacterial cells to scavenge hydroxyl free radicals, and the specific operation is as follows: 0.25mL of bright green (0.435 mmol/L), 0.5mL of ferrous sulfate (0.5 mmol/L), 0.4mL of hydrogen peroxide (3.0%, w/v) and lmL of lactobacillus physiological saline suspension, and the above materials were mixed uniformly and then subjected to water bath at 37 ℃ for 30min. Centrifugation was carried out at 10000r/min for 10min, and the supernatant was taken and absorbance was measured at a wavelength of 525nm, and the blank group was distilled water instead of the sample but contained hydrogen peroxide.
Hydroxyl radical clearance (%) = (a) Sample of -A Control )/(A Blank space -A Control )x100
S5 lactic acid bacteria scavenge superoxide anions (. O) 2- ) Capacity determination
1mL Tris-HCl (150 mmol/L, pH=8.2), 1mL diethylenetriamine pentaacetic acid (3 mmol/L), 1mL pyrogallol (1.2 mmol/L) and 0.5mL sample to be tested were added, and the total reaction volume was 3.5mL. The absorbance was measured at 325nm in a water bath at 25℃for 10min. Vitamin C solutions with different mass concentrations (0-0.40 mg/mL) are used as positive control, the scavenging rate of superoxide anions is measured by the same method, and the scavenging rate of the superoxide anions of the sample to be measured is converted into Vc equivalent.
Superoxide anion clearance (%) = [1- (A3-A2)/(A1) ] x100
A1 is sample-free and contains pyrogallol;
a2 is a sample containing no pyrogallol;
a3 is sample-containing pyrogallol
Determination of Fe2+ sequestration Capacity of S6 lactic acid bacteria
0.1mL of ascorbic acid (mass fraction 1%), 0.1mL of ferrous sulfate (FeSO 4 mass fraction 0.4%), 0.5mL of lactic acid bacteria are added into 1mL of mixed solution of sodium hydroxide (NaOH concentration is 0.2 mol/L), water bath is carried out for 20min at 37 ℃ of the mixed solution, 0.2mL of trichloroacetic acid (TCA, 4%, m/v) precipitates protein, 8000g of the protein is centrifuged for 5min at 4 ℃,0.2mL of supernatant is taken, 2mL of o-phenanthroline (mass fraction 0.1%) is added for reaction for 10min, the absorbance is measured at 510nm, and Phosphate Buffer Solution (PBS) or distilled water is used as blank control.
Fe 2+ Chelation rate (%) = (a) Control -A Sample of )/A Control x100
S7, screening strains with antioxidant activity in vitro through lactobacillus suspension to finally obtain lactobacillus plantarum with good antioxidant activity, namely lactobacillus plantarum (Lactobacillus plantarum) 124, wherein the in vitro antioxidant index result is shown in figure 4. Lactobacillus plantarum (Lactobacillus plantarum) 124 strain 16S rDNA gene sequence is shown in SEQ ID NO.1.
Test example I, evaluation of safety of lactic acid bacteria
(1) Hemolysis test: respectively picking the freshly cultured lactobacillus plantarum (Lactobacillus plantarum) 124 and the standard control strain LGG ATCC 53103, and carrying out single colony sterile underline inoculation on a blood plate containing 7% sheep blood (v/v), wherein staphylococcus aureus is used as a control, and the staphylococcus aureus is beta-type; culturing at 37deg.C for 48 hr to observe hemolytic circle, wherein there is no obvious change around colony, and its hemolytic group is gamma-type; the beta-type transparent annular hemolysis ring is arranged around the colony, and the hemolysis phenomenon of staphylococcus aureus is referred; the colonies were alpha-type in their surroundings in brown or dark green.
The results showed no significant change around the colony of Lactobacillus plantarum (Lactobacillus plantarum) 124 and the standard control strain LGG ATCC 53103, and that the hemolytic type was gamma-type, i.e., lactobacillus plantarum (Lactobacillus plantarum) 124 was non-hemolytic.
(2) Resistance gene alignment: the whole genome of Lactobacillus plantarum (Lactobacillus plantarum) 124 was sequenced and aligned using the resistance gene database (Card, http:// arpc ard. Mcmaster. Ca) with no coverage of 95% or more and no resistance gene with identification of 90% or more, i.e., lactobacillus plantarum (Lactobacillus plantarum) 124 no resistance gene.
(3) Drug sensitivity test: lactobacillus plantarum (Lactobacillus plantarum) 124 was tested for sensitivity to antibiotics using a paper sheet diffusion method. Seven of seven antibiotics are selected as antibiotics for test according to the anaerobic drug sensitivity test method in Clinical and Laboratory Standards Institute (CLSI) standard procedure, and comprise beta-lactam ampicillin, penicillin amoxicillin, cephalosporin cefoperazone, aminoglycoside gentamicin, tetracycline, phenylpropanoid chloramphenicol, carbapenem, and the results are shown in Table 2 (note: R is drug resistance, S is sensitive)
TABLE 2 lactic acid bacteria antibiotic sensitivity
| Numbering device | Antibiotic class | Antibiotic name | Lactobacillus plantarum 124 |
| 1 | Beta-lactams | Ampicillin (Amoxicillin) | S |
| 2 | Penicillin | Amoxicillin | S |
| 3 | Cephalosporium species | Cefoperazone | S |
| 4 | Aminoglycosides | Gentamicin | S |
| 5 | Tetracyclines | Tetracycline | S |
| 6 | Phenylpropanols | Chloramphenicol | S |
| 7 | Carbapenems | Imipenem | S |
As can be seen from Table 2, lactobacillus plantarum (Lactobacillus plantarum) 124 was sensitive to the antibiotics ampicillin, amoxicillin, cefoperazone, gentamicin, tetracycline, chloramphenicol and imipenem, which are consistent with the alignment of the whole genome in the resistance gene database, i.e., lactobacillus plantarum (Lactobacillus plantarum) 124 was free of resistance genes.
(4) Virulence gene alignment: the whole genome of the lactobacillus plantarum (Lactobacillus plantarum) 124 is sequenced, and the virulence genes of the lactobacillus plantarum (Lactobacillus plantarum) 124 are compared by utilizing a virulence gene database (VFDB, http:// www.mgc.ac.cn/VFs/main.htm), wherein the comparison result has no virulence genes with coverage rate more than or equal to 95 percent and identification rate more than or equal to 90 percent.
Test example two, animal experiment and measurement of oxidoreductase Activity
In vivo mouse experiments were performed according to the antioxidant active lactobacillus plantarum (Lactobacillus plantarum) 124 screened in example 4, as follows:
s1, experimental design of mice: 1. normal group: subcutaneous injection of physiological saline (200 mg/kg/day), and lavage of 0.2mL physiological saline per day; 2. model group: d-galactose (200 mg/kg/day) was subcutaneously injected, and 0.2mL of physiological saline was infused daily; 3. strain a treatment group: d-galactose (200 mg/kg/day) was subcutaneously injected, and 0.2mL was fed daily at a concentration of 1.01.+ -. 0.05xl0 9 CFU/mL A bacterial liquid; 4. strain B treatment group: d-galactose (200 mg/kg/day) was subcutaneously injected, and 0.2mL was fed daily at a concentration of 1.01.+ -. 0.05xl0 9 CFU/mL B bacterial liquid; 5. strain a plus strain B treatment group: d-galactose (200 mg/kg/day) was subcutaneously injected, and 0.1mL was fed daily at a concentration of 1.01.+ -. 0.05xl0 9 CFU/mL A bacterial liquid and 0.1mL concentration of 1.01+ -0.05 xl0 9 CFU/mL B bacterial liquid; 6. positive drug Vc group: d-galactose was injected subcutaneously (200 mg/kg/day) and antioxidant Vc was fed daily (200 mg/kg/day). The feeding period was 9 weeks. Strain a: lactobacillus plantarum 124, 124; strain B: akkermansia muciniphila strain ATCC BAA-835. D-galactose (200 mg/kg/day): subcutaneously injecting D-galactose at 200mg/kg bw.d;
s2, preparing liver and kidney tissue homogenate for determination and analysis of a kit of oxidative stress markers such as glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA) and the like, and the determination result is shown in figure 5. ( Early-stage bioinformatics analysis of the 16S amplicon and metagenome revealed that some potentially beneficial bacterial groups (Akkermansia, lactobacillus, christensenellace) were found in the century old group, with increasing abundance with age; literature reports that Akkermansia delays longevity in mice with premature senility, so that the selection of isolate Lactobacillus plantarum 124 and standard Akkermansia muciniphila strain ATCC BAA-835 together were used in a mouse experiment )
As can be seen in FIG. 5, the same doses Lactobacillus plantarum and Akkermansia both up-regulate GSH-Px (FIGS. 5a, d) and SOD levels (FIGS. 5c, f) in liver and kidney tissues to varying degrees, down-regulate MDA levels (FIGS. 5b, e) in liver and kidney tissues. The independent feeding of Lactobacillus plantarum and Akkermansia can improve the activity of GSH-Px and SOD in the kidney and liver of the mice and reduce the MDA content in the kidney; the simultaneous feeding of Lactobacillus plantarum and Akkermansia has a greater capacity to increase GSH-Px and SOD activity and reduce MDA levels in the kidneys than each alone. The mice experiments prove that both strains have certain antioxidant capacity, and the total Lactobacillus plantarum 124,124 is stronger than Akkermansia.
Test example III, liver, kidney and small intestine histomorphology observations
In vivo mouse liver, kidney and small intestine histomorphometric verification was performed according to the antioxidant active lactobacillus plantarum (Lactobacillus plantarum) 124 screened in example 4.
The effect of mouse liver, kidney and small intestine tissue morphology was analyzed using HE staining technique and the results are shown in fig. 6.
As can be seen from fig. 6, liver cells of the liver model group were moderately watery denatured (++), loose in cytoplasms, accompanied by moderate steatosis, filled with large and small lipid droplets, compared to those of the normal control group. The kidney model group showed moderate water sample denaturation (++) of the tubular epithelium compared to the normal control group, and showed no interstitial vascular congestion and inflammatory cell infiltration. The intestinal tissue of the small intestine model group is irregular in intestinal villus arrangement, and intestinal mucosa and submucosa are edematous and shed compared with the normal control group. From the above results, it can be seen that long-term D-galactose intake can lead to lesions in liver, kidney cell morphology and small intestine tissue, indicating that the D-galactose-induced oxidative damage model was successful. Compared to the model group, when mice were fed with Lactobacillus plantarum and Akkermansia, the extent of lesions of cells in liver cells (fig. 6 g) and kidney tissues (fig. 6 h) of mice was reduced, and the extent of lesions such as irregular arrangement of intestinal villi in small intestine tissues of mice was reduced (fig. 6 i).
Test example four detection of Lactobacillus plantarum (Lactobacillus plantarum) 124 specific molecular target
The unique gene sequence of the lactobacillus plantarum (Lactobacillus plantarum) 124 strain is mainly obtained according to the result of the genome analysis of the lactobacillus plantarum. Genomic sequences of 535 representative lactobacillus plantarum (containing lactobacillus plantarum (Lactobacillus plantarum) 124) were co-selected for flood genomic analysis. The genome is analyzed by adopting an MP method in prokaryotic genome automation analysis software (Pan-Genomics Analysis Pipeline, PGAP), and analysis results are processed through a local Perl script to obtain core gene and non-core gene information of all strains.
The specific gene protein sequence of Lactobacillus plantarum (Lactobacillus plantarum) 124 strain was extracted and aligned back to the Lactobacillus plantarum protein repertoire and NCBI non-redundant protein database (NR) by local Blast, respectively. The sequence of the known lactobacillus plantarum protein can be removed and compared, and the rest is a special gene fragment of lactobacillus plantarum (Lactobacillus plantarum) 124 strain, wherein the nucleotide sequence of the gene fragment is shown as SEQ ID NO.2. The specific genes were tested for their specificity by PCR amplification in Lactobacillus plantarum (Lactobacillus plantarum) 124 strain, other Lactobacillus plantarum strains, and other Lactobacillus (total 227 representative strains) microorganisms. The results are shown in fig. 7 and 8.
A step of detecting a lactobacillus plantarum (Lactobacillus plantarum) 124 specific molecular target:
primer design: specific PCR amplification primer sets (including forward primers and reverse primers) were designed based on the gene fragment sequence SEQ ID NO.2 specific to Lactobacillus plantarum (Lactobacillus plantarum) 124 strain, and the primer set sequences are shown in Table 3 below.
TABLE 3 specific PCR primer sets
PCR system: 25. Mu.L of the reaction system.
PCR amplification procedure:
and (3) taking PCR amplified products to carry out gel electrophoresis, and observing whether the position of the primer group corresponding to the size of the products has single amplified band only in lactobacillus plantarum (Lactobacillus plantarum) 124. If the target is present, the specificity of the molecular target is high; if other strains appear in the same position in a band, it is indicated that the molecular target is not specific.
The gel results of the PCR amplified products are shown in figures 7-9, M is DL2000 DNA standard marker, 1-46 are other lactobacillus plantarum strains, 47-190 are non-target lactobacillus strains, 191-227 are non-lactobacillus strains, + is target positive lactobacillus plantarum (Lactobacillus plantarum) 124 group, C is negative control group, and the template of the control group is an aqueous solution without genome.
The strains used and the detection results are shown in Table 4 below; in the table, "+" in the test result column indicates positive, and "-" indicates negative.
TABLE 4 detection results of Lactobacillus plantarum 124 specific targets of the invention
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As can be seen from FIGS. 7-9 and Table 4, the primer set showed only Lactobacillus plantarum (Lactobacillus plantarum) 124 strain showing specific amplified bands, none of Lactobacillus plantarum (Lactobacillus plantarum) 124 strain had specific bands, indicating that sequence SEQ ID NO.2 is a specific molecular target of Lactobacillus plantarum (Lactobacillus plantarum) 124 strain in the present invention.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Guangdong province microbiological institute (microbiological analysis and detection center, guangdong province), guangdong CycloKai Biotech Co., ltd
<120> lactic acid bacterium and use thereof
<130> 2020.9.24
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1412
<212> DNA
<213> Synthesis
<400> Lactobacillus plantarum (Lactobacillus plantarum) 124
aggttacccc accgactttg ggtgttacaa actctcatgg tgtgacgggc ggtgtgtaca 60
aggcccggga acgtattcac cgcggcatgc tgatccgcga ttactagcga ttccgacttc 120
atgtaggcga gttgcagcct acaatccgaa ctgagaatgg ctttaagaga ttagcttact 180
ctcgcgagtt cgcaactcgt tgtaccatcc attgtagcac gtgtgtagcc caggtcataa 240
ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg cagtctcacc 300
agagtgccca acttaatgct ggcaactgat aataagggtt gcgctcgttg cgggacttaa 360
cccaacatct cacgacacga gctgacgaca accatgcacc acctgtatcc atgtccccga 420
agggaacgtc taatctctta gatttgcata gtatgtcaag acctggtaag gttcttcgcg 480
tagcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag 540
tttcagcctt gcggccgtac tccccaggcg gaatgcttaa tgcgttagct gcagcactga 600
agggcggaaa ccctccaaca cttagcattc atcgtttacg gtatggacta ccagggtatc 660
taatcctgtt tgctacccat actttcgagc ctcagcgtca gttacagacc agacagccgc 720
cttcgccact ggtgttcttc catatatcta cgcatttcac cgctacacat ggagttccac 780
tgtcctcttc tgcactcaag tttcccagtt tccgatgcac ttcttcggtt gagccgaagg 840
ctttcacatc agacttaaaa aaccgcctgc gctcgcttta cgcccaataa atccggacaa 900
cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg ctttctggtt 960
aaataccgtc aatacctgaa cagttactct cagatatgtt cttctttaac aacagagttt 1020
tacgagccga aacccttctt cactcacgcg gcgttgctcc atcagacttt cgtccattgt 1080
ggaagattcc ctactgctgc ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg 1140
ccgattaccc tctcaggtcg gctacgtatc attgccatgg tgagccgtta ccccaccatc 1200
tagctaatac gccgcgggac catccaaaag tgatagccga agccatcttt caagctcgga 1260
ccatgcggtc caagttgtta tgcggtatta gcatctgttt ccaggtgtta tcccccgctt 1320
ctgggcaggt ttcccacgtg ttactcacca gttcgccact cactcaaatg taaatcatga 1380
tgcaagcacc aatcaatacc agagttcgtt cg 1412
<210> 2
<211> 1707
<212> DNA
<213> Synthesis
<400> specific molecular targets
atggctaaaa agattattgt ggttggtggc gttgctggtg gtgcttccgt agcagctcgt 60
gcaaggcggt tagatgaaaa ggccgaagtt gtgatgtttg aaaaggggcc caacgtgtcc 120
ttctcaaact gcgccttacc ttatcactta tctggaacca ttccggatgc tgacagcatt 180
gtcctcatgg atcccccaca gtttaaagcg caatataaca ttgatgccgt tgttaaccat 240
gaagtgactg ggattgatcc caaaaagcaa accgttaccg tgaaaaacgt cttggatgat 300
accgaagaga gcgttgctta cgacgaatta gttctatcac caggtgctga accaatccgg 360
ccaaaatcaa ttgcgggtgt tgatggtgaa aacgtcttta ctatgcgtaa cgttgtggac 420
atcaagaaga ttaaatccta cttggacgaa aaaaaggagg tcgaaaccgt ttccgttatc 480
ggtggtggct ttattgggat tgaaactgcc gaaaacctta tccagggtgg ttacaaagtt 540
aacttgattg aaggcatgga tcacgtgctg ggaacgattg attatgatat ggcccagatc 600
attcaaaaaa cgatgctgga tcacggaatc aacctgttaa ccagtgaaac ggtcactaag 660
attacacctg atagcgttaa attggcgtcc ggaaagagtt taccaagcca ggccgttgtt 720
ctgtccgtgg gtgttttacc ggacacccgg ttggcaaccc aagtaggtgc tgaaatcggt 780
aaaacgggtg gtattaaagt ggatcaaggg tatcaaacca cggtgccgca tatctacgcc 840
gttggggatg cggtggaagt tcaaaaccgg attacccgca agccagctaa gttaaactta 900
gccttcccag ctcaaattga agcacgaatg gccgtggacc acatgtatgg tcgccccatc 960
caaaaccgtg gtgttatcgg ctcacaatgt attccgattt ttgaaatgaa cgtggcttcc 1020
actggtttaa ccgaagcaga agcgaaggaa aacgacattg actaccgcgc cgttaccatc 1080
atcccgaagg ataaggtcgc tttgatgcca cacgctaagc cgctctactt taagttagtt 1140
tttgcttatc caagtggtga aattctgggt gctcaagcca tcggtgaaag ctccgtggac 1200
aagcaggtcg acgttatcgc cactgaaatt accaatggcg gctacgtgga ggacctagaa 1260
gcactggaac tgtgctacca gccaatgttt agtactgcta agaatgccgt taacatggcc 1320
ggtttagtgg catccaacgt gttaaacaat gaattcaagc aagtcagtgt gacggaagtt 1380
cgtgatctgg ttgaaaaggg tgccgatatt atcgatgtcc gtgaaaagtt tgaatatgac 1440
gaaggtcacg tgaaaacagc caagaacatc ccaatgagtg aattccgtga tcgtttggat 1500
gaaattccaa cggacgagcc agtttatatc cattgtctga gcggccaacg gagttacaac 1560
gtggtccgag cactggtcaa cctgggctac accaacgttt ataacattgc aggatcctat 1620
ctggatattt ccgaatatga atactatacg gatacggtta cgggtcgcga cccaattgtg 1680
acgaaatacc ggtttgactt gttataa 1707
<210> 3
<211> 20
<212> DNA
<213> Synthesis
<400> Forward primer 27F
agagtttgat cctggctcag 20
<210> 4
<211> 19
<212> DNA
<213> Synthesis
<400> reverse primer 1492R
ggttaccttg ttacgactt 19
Claims (9)
1. Lactobacillus plantarumLactobacillus plantarum) 124, which is deposited with the Guangdong province microorganism strain collection center under the accession number GDMCC NO 61123;
the lactobacillus plantarum is [ ]Lactobacillus plantarum) 124 comprises the nucleotide sequence shown as SEQ ID NO.2.
2. Primers for detecting specific molecular targets in preparation of lactobacillus plantarumLactobacillus plantarum) 124, wherein the specific molecular target is a nucleotide sequence shown as SEQ ID NO. 2;
the lactobacillus plantarum is [ ]Lactobacillus plantarum) 124 is deposited in the Guangdong province microorganism strain collection center with the deposit number of GDMCC NO. 61123;
primers for detecting a specific molecular target shown as SEQ ID NO.2 comprise a forward primer and a reverse primer;
the nucleotide sequence of the forward primer is GGCCGAAGTTGTGATGTTTGA;
the nucleotide sequence of the reverse primer was AGTCAAACCGGTATTTCGTCA.
3. The lactobacillus plantarum of claim 1Lactobacillus plantarum) 124 in the preparation of a microbial preparation having antioxidant activity.
4. The lactobacillus plantarum of claim 1Lactobacillus plantarum) 124 as an antioxidant in the preparation of food, pharmaceutical or cosmetic products.
5. A microbial preparation having antioxidant activity, comprising the Lactobacillus plantarum strain according to claim 1Lactobacillus plantarum) 124, said lactobacillus plantarum [ ]Lactobacillus plantarum) 124 is deposited in the Guangdong province microorganism strain collection center with the deposit number of GDMCC NO. 61123; the lactobacillus plantarum is [ ]Lactobacillus plantarum) 124 contains a specific molecular target as shown in nucleotide sequence SEQ ID NO.2.
6. The microbial preparation having antioxidant activity according to claim 5, wherein the microbial preparation further comprises an auxiliary material and/or a carrier.
7. The microbial preparation having antioxidant activity according to claim 6, wherein the adjuvant comprises corn flour, inulin, whey powder, glucose, sucrose, fructo-oligosaccharides or galacto-oligosaccharides, and the carrier comprises magnetic microspheres, wheat bran, diatomaceous earth or talc.
8. Use of a microbial preparation according to any one of claims 5 to 7 for the preparation of a food, pharmaceutical or cosmetic product.
9. The use according to claim 8, wherein the microbial preparation is a food starter.
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| WO2019154823A1 (en) * | 2018-02-06 | 2019-08-15 | European Molecular Biology Laboratory | In-vitro model of the human gut microbiome and uses thereof in the analysis of the impact of xenobiotics |
| CN110272846A (en) * | 2019-07-08 | 2019-09-24 | 江苏省农业科学院 | Lactobacillus plantarum and its application with ABTS free radical and PTIO free radical scavenging activity |
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