CN115948281B - Lactobacillus plantarum R6-3 from Saimu yoghurt and application thereof - Google Patents
Lactobacillus plantarum R6-3 from Saimu yoghurt and application thereof Download PDFInfo
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- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a Lactobacillus plantarum R6-3 derived from Saimu yoghurt and application thereof. The lactobacillus plantarum R6-3 is separated from traditional fermented food, namely the siraitia papyrifera in Baicheng county in the Achaku district of Xinjiang, has good tolerance, bacteriostasis and adhesion to Caco-2 cells of artificial gastrointestinal fluid, and has good in-vitro antioxidation capability; by taking a D-galactose-induced oxidative damage mouse as a model, lactobacillus plantarum R6-3 can regulate intestinal flora of the mouse to cause the rise of SCFAs level in the intestinal tract of the mouse, and the SCFAs enter the liver to activate Nrf-2 signal paths so as to relieve oxidative stress; by taking a CUMS-induced depressed mouse as a model, lactobacillus plantarum R6-3 can play a role in preventing depression by regulating the composition and function of intestinal flora of the mouse, thereby improving the content of SCFAs in intestinal tracts, improving the immune and oxidative stress states of organisms, relieving monoamine neurotransmitter deficiency and HPA hyperthyroidism and recovering cerebral neurotrophic level.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum R6-3 of a Saimu yoghurt source and application thereof.
Background
Studies have shown that many metabolic processes in organisms and various stimuli from the outside of the body (e.g. obesity, high fat or diet rich in sugar and processed foods, ultraviolet (UV) radiation, smoking, drinking, chemotherapeutics, hyperthermia, exposure to pesticides or industrial chemicals and even oxygen itself, etc.) lead to the body being in oxidative stress, and that excessive oxidative stress induces apoptosis and necrosis, which in turn causes various diseases and leads to aging. Among them, depression associated with oxidative stress is one of the most common neurological diseases in this age, and is listed by the world health organization as the most dominant factor causing global disability and non-fatal health loss. With the advancement of society, the pace of life of people is accelerated, and various stresses and pressures from life are one of the most likely risk factors to cause depression. The global economic cost for depression is expected to double by 2030, becoming the first disease burden. Thus, the prevention, alleviation and treatment of depression has become a global problem that needs to be addressed urgently.
However, scientists are still looking for new methods of preventing and treating depression due to the side effects of antidepressants. Oxidative stress is one of the causes of depression, and thus antioxidants may have potential value in the treatment and prevention of depression. Synthetic antioxidants are questioned for safety, and probiotics in natural antioxidants are an emerging source of effective antioxidants due to their long-standing safety tradition of eating and the potentially diverse health benefits to the body. From a safety point of view, traditional fermented foods with a long eating history are a good source of obtaining probiotic resources. The medical level of Baicheng county in the Archavey of Xinjiang falls behind, the geographical environment is bad, the living standard of people is low, but the medical level is considered as the world level 'longevity county', which is possibly related to the self-made Sirink yoghurt of the habit of local people for eating the home, the medical level contains abundant microorganism species, the quality is superior, and the medical level is a non-matter cultural heritage of the Uygur of Xinjiang, and even is known as 'longevity secret formula'. However, few studies on siraitia yoghurt are currently conducted, and the research on screening of specific strains and application of the strains by in vitro tests is mainly focused.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum R6-3 of a siraitia yoghourt source and application thereof.
The invention separates a series of lactobacillus strains from the siraitia papyrifera, carries out preliminary identification by sequencing 16S rDNA thereof, takes probiotics Lactobacillus rhamnosus GG (ATCC 53103) with most research and most thorough probiotics as positive control strains (purchased from China general microbiological culture Collection center), screens out a lactobacillus with potential probiotics by taking artificial gastric juice resistance, artificial intestinal juice resistance, bacteriostasis, adhesion capacity and the like as indexes, and then determines the lactobacillus plantarum as R6-3 by combining physiological and biochemical test results, and evaluates the safety (antibiotic sensitivity, harmful metabolites, hemolysis and the like) and the antioxidant capacity of the lactobacillus strain by an in vitro test; further evaluating the antioxidant capacity and the antioxidant mechanism of the strain in the D-galactose induced oxidative damage mice through in vivo tests; finally, using a CUMS induced depressed mouse as an animal model, and checking whether the screened lactobacillus plantarum R6-3 with antioxidant activity has a probiotic effect on the depressed model mouse for preventing depression by regulating intestinal flora.
Lactobacillus plantarum (Lactobacillus plantarum) R6-3 with preservation number of CGMCC NO.25884 is derived from Saururi yogurt.
The application of the Lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the Sirina yoghurt source in preparing an antioxidant composition.
The application of the Lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the Sirina yogurt source in preparing an antidepressant composition.
The application of lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the Sirina yoghurt source in preparing a composition for preventing oxidative aging by regulating intestinal flora.
The application of lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the Sirina yoghurt source in preparing a composition for preventing depression by regulating intestinal flora.
A composition comprising said lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the siraitia yoghurt source.
A food comprising said lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the siraitia yoghurt source.
A food additive comprises Lactobacillus plantarum (Lactobacillus plantarum) R6-3 of Saurus yogurt source.
A microbial preparation, which comprises the lactobacillus plantarum (Lactobacillus plantarum) R6-3 of a Saimu yoghurt source.
A pharmaceutical composition comprising said lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the siraitia yoghurt source.
The strain of the invention has a preservation date of 2022, 10 and 09 days and a preservation number of CGMCC NO.25884. The preservation organization is named as China general microbiological culture Collection center, and the address is the China national academy of sciences microbiological culture Collection center, post code 100101, of the North Star West way 1, 3 rd, the Korean area of Beijing.
The invention has the beneficial effects that: the Lactobacillus plantarum R6-3 with the siraitia papyrifera disclosed by the invention has the advantages of good resistance to artificial gastric juice and artificial intestinal juice, broad-spectrum antibacterial property and higher adhesiveness to Caco-2 cells. In vitro probiotics function test shows that R6-3 thallus/fermentation supernatant has strong antioxidant activity. Animal models of D-galactose induced mice oxidative damage prove that R6-3 thalli can lead to the rise of SCFAs level in the intestinal tract of the mice by regulating the intestinal flora of the mice, and the SCFAs enter the liver to activate Nrf-2 signal paths, thereby relieving the oxidative stress of the mice. The animal model of mouse depression induced by CUMS proves that R6-3 thalli has the functions of preventing depression by regulating the composition and the functions of intestinal flora of the mouse, thereby improving the content of SCFAs in intestinal tracts, improving the immunity and oxidative stress state of organisms, relieving monoamine neurotransmitter deficiency and HPA hyperthyroidism and recovering the cerebral neurotrophic level.
Drawings
Fig. 1 is a graph of strain tolerance to artificial gastric juice, wherein P <0.001 represents significant levels of difference for different strains compared to LGG compared to different treatment groups.
Fig. 2 is a graph of strain tolerance to artificial intestinal fluid, wherein P <0.001 represents significant levels of difference for different strains compared to LGG compared to different treatment groups.
Fig. 3 is a graph of strain adhesion to Caco-2 cells, wherein P <0.001 represents significant levels of difference in pairwise comparisons between different strains compared to different treatment groups.
FIG. 4 is a diagram showing colony morphology and cell characteristics of strain R6-3.
FIG. 5 is a phylogenetic tree based on the 16S rDNA sequence.
FIG. 6 is a graph showing the growth of R6-3 and LGG in medium containing different concentrations of H 2O2.
FIG. 7 is a graph of HE staining of the liver of mice from different treatment groups, wherein A-E represent the normal group, model group, R6-3 group, LGG group, VC group, scale bar 50 μm, magnification of 400 x microscope.
Fig. 8 is a graph of the oxidation index level of serum from mice in different treatment groups, wherein P <0.05, P <0.01, P <0.001 represent the significant level of difference between the pairwise comparisons between the different treatment groups.
Fig. 9 is a graph of oxidation index levels of liver, kidney, brain of mice in different treatment groups, wherein the superscript lower case letters (a-c) represent significant differences in p=0.05 levels compared to the different treatment groups.
Fig. 10 is a graph of relative expression levels of liver antioxidant-associated genes in mice from different treatment groups, wherein P <0.05, P <0.01, P <0.001 represent significant levels of difference between pairwise comparisons between the different treatment groups.
Fig. 11 is a graph of fecal flora composition and differential analysis of mice in different treatment groups, wherein P <0.05, P <0.01, P <0.001 indicate significant levels of differential between pairwise comparisons between different groups.
Fig. 12 is a correlation analysis graph, wherein P <0.05 and P <0.01 represent the correlation level between the two factors.
Fig. 13 is a diagram of an anti-depressant animal test regimen.
FIG. 14 is a stimulus programming diagram for building a CUMS model, wherein water is disabled: removing the water bottle for 24 hours; fasted: removing the mouse grains for 24 hours; wet cage: 200mL of tap water is sprayed into the padding for 24 hours; no padding: removing the padding for 24 hours; day and night reversal: adjusting the day and night period, turning on the lamp at night, and covering the mouse cage by black cloth in daytime; cold water swimming: placing the mice in water at 7 ℃ for swimming for 1min; inclined cage: tilting the squirrel cage by 45 degrees for 24 hours; clamping tail: clamping the tail tip 1cm away by a clamp for 1min; illumination at night: the lamp is turned on at night; water for forbidden food: the mouse grains and water bottles were removed for 24h.
Fig. 15 is a graph showing changes in body weight of mice in different treatment groups, wherein P <0.05, P <0.01, and P <0.001 represent comparisons between normal and model groups; a P<0.05,aa P<0.01,aaa P <0.001 represents a comparison between the R6-3 group and the model group; b P<0.05,bbP<0.01,bbb P <0.001 represents a comparison between the drug group and the model group.
Fig. 16 is a graph of behavioral indicators of mice in different treatment groups, wherein P <0.05, P <0.01, and P <0.001 represent significant levels of difference between pairwise comparisons between different groups.
FIG. 17 is a chart showing Hippocampus Nile staining of mice in different treatment groups, wherein A-D represent a normal group, a model group, an R6-3 group, and a drug group, respectively, with scales of 400 μm, and with microscope magnification of 100 times and 400 times.
Fig. 18 is a graph of the levels of neurobiological index in mice in different treatment groups, wherein P <0.05, P <0.01, P <0.001 indicate significant levels of difference between pairwise comparisons between different treatment groups.
Fig. 19 is a graph of inflammatory factor content in mice from different treatment groups, wherein P <0.05 and P <0.01 represent significant levels of difference between pairwise comparisons between different groups.
Fig. 20 is a graph of the oxidation index level of serum from mice in different treatment groups, wherein P <0.05, P <0.01, P <0.001 represent the significant level of difference between the pairwise comparisons between the different treatment groups.
Fig. 21 is a graph of the oxidation index level of brain tissue of mice in different treatment groups, wherein P <0.05, P <0.01, P <0.001 represent the significant level of difference between the pairwise comparisons between the different treatment groups.
Fig. 22 is a graph of composition and differential analysis of metabolic pathways at KEGG LEVEL levels of cecal content flora in mice from different treatment groups, where P <0.05 represents significant levels of differential compared to the model group.
Fig. 23 is a graph of short chain fatty acid content in the feces of mice from different treatment groups, wherein the superscript lower case letters (a-c) represent significant differences in p=0.05 levels compared to the different treatment groups.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 screening of lactic acid bacteria from siraitia grosvenorii yogurt and further characterization
1. Isolation, purification and preliminary identification of strains
And (3) carrying out gradient dilution on the siraitia papyrifera with normal saline, selecting proper dilutions, respectively adopting MRS and YPD solid culture media to carry out coating culture on bacteria and fungi, then picking out colonies with different forms, and carrying out repeated streak purification until a single colony is detected by a mirror. Extracting DNA from the purified strain, amplifying and sequencing the 16S rDNA and ITS sequences of bacteria and fungi respectively, and then comparing homologous sequences to find the strain with high similarity. The separated, purified and identified strains are subjected to de-duplication and numbering, and the activated bacterial liquid is mixed in a glycerin tube with the volume ratio of 20 percent and is preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
Bacteria and yeasts were isolated and purified from 6 parts of siraitia yoghurt using MRS and YPD media, respectively, and preliminary molecular biology identification was performed, and 30 bacteria, 5 yeasts, and strain detailed information were totally isolated, purified and identified, as shown in table 1.
TABLE 1 alignment of strain sequences
2. Preparation of test bacterial liquid
The non-pathogenic bacteria and yeast strains which are primarily identified are respectively inoculated into MRS and YPD liquid culture media according to the volume ratio of 1 percent, are cultured at 37 ℃ and 30 ℃ for 2-3 generations, are respectively cultured in the liquid MRS and YPD culture media for 14h and 24h, are centrifuged (4 ℃,8000r/min and 10 min), the supernatant is discarded, and are washed for 2 times by adding sterile physiological saline, and then the thalli are suspended in 10mL of sterile physiological saline.
3. Determination of the ability of the Strain to withstand Artificial digestive juice
Adding 1.0mL of the test bacterial suspension into 9.0mL of artificial gastric juice, uniformly mixing, respectively placing bacteria and saccharomycetes in a constant-temperature incubator at 37 ℃ and 30 ℃ for 3 hours, respectively measuring the viable count of 0 hours and 3 hours, and calculating the survival rate of each bacterial strain in the artificial gastric juice according to a formula (1):
As a result, as shown in FIG. 1, strains R1-1, R1-4, R1-5, R1-6, R2-1, R2-6 and R5-2 can be obtained in FIG. 1, the survival rate of the strain R4-3 in the artificial gastric juice is extremely lower than that of LGG (P < 0.001), the survival rates of the strains R1-3, R2-3 and LGG in the artificial gastric juice are equivalent (P > 0.05), the survival rates of other 22 strains in the artificial gastric juice are extremely higher than that of LGG (P < 0.001), and the result shows that most strains screened from the sirius yoghurt have relatively strong tolerance to the artificial gastric juice, which is caused by the screening of the high-acidity sirius yoghurt to acid-resistant strains. Therefore, we selected the strain resistant to artificial gastric juice stronger than and comparable to LGG as the candidate strain resistant to artificial intestinal juice in the next step.
4. Determination of artificial intestinal juice resistance of strain
1.0ML of the test bacterial suspension is added into 9.0mL of artificial intestinal juice, the bacteria and the microzyme are uniformly mixed, the bacteria and the microzyme are respectively placed in a constant temperature incubator at 37 ℃ and 30 ℃ for 4 hours, the viable count of 0 hour and 4 hours is respectively measured, and the survival rate of each bacterial strain in the artificial intestinal juice is calculated according to the formula (2):
As a result, as can be seen from fig. 2, only the survival rate of the strains R2-5, R4-2, R6-3 in the artificial intestinal juice is significantly higher than LGG (P < 0.001), the growth of the strains R2-2, R3-1, R3-2, R4-1 in the artificial intestinal juice is completely inhibited, and the survival rate of the other strains in the artificial intestinal juice is significantly lower than LGG (P < 0.001), indicating that the strain isolated from the siraitia yoghurt has a weak intestinal juice resistance. We select the ability of the intestinal juice resistance to be extremely superior to that of the R2-5, R4-2 and R6-3 of LGG for the next antibacterial test.
5. Bacterial strain bacteriostasis assay
LGG is used as a control strain, and escherichia coli, staphylococcus aureus, salmonella, shigella flexneri and listeria monocytogenes are used as pathogenic indicator bacteria. Strains LGG, R2-5, R4-2 and R6-3 were inoculated into MRS liquid medium, cultured at 37℃for 14 hours, and the cell concentration was adjusted to (1.about.2). Times.10 9 CFU/mL with MRS liquid medium by measuring the OD value of the culture solution. Shaking culture of pathogenic bacteria in LB liquid medium at 37 deg.C for 24 hr, washing with sterile physiological saline, resuspending the bacteria, regulating the concentration of bacterial suspension to (1-2) x 10 6 CFU/mL, adding 250 μL of pathogenic bacteria suspension into 25mL of LB agar medium in molten state at 50 deg.C, mixing, pouring into flat plate to obtain bacteria-containing plate, punching with 6mm puncher after solidification, adding 100 μL of bacterial suspension to be tested in the hole, sterilizing MRS as negative control, culturing in constant temperature incubator at 37 deg.C for 24 hr, and measuring the diameter of bacteria inhibition zone.
The results are shown in Table 2, and Table 2 shows that for E.coli: the diameters of the inhibition zones of R4-2, R6-3 and LGG are not obviously different (P is more than 0.05), and the diameter of the inhibition zone of LGG is obviously larger than that of R2-5 (P is less than 0.05); for staphylococcus aureus: the diameters of the inhibition zones of R4-2, R6-3 and LGG are not obviously different (P is more than 0.05), the diameters of the inhibition zones of R4-2 and R6-3 are obviously larger than R2-5, and the statistical differences are P <0.01 and P <0.05 respectively; for salmonella: the diameters of the inhibition zones of R4-2 and R6-3 are not significantly different (P > 0.05), the diameters of the inhibition zones of R2-5 and LGG are not significantly different (P > 0.05), but the diameters of the inhibition zones of R4-2 are significantly larger than those of R2-5 (P < 0.01) and LGG (P < 0.01), and the diameters of the inhibition zones of R6-3 are significantly larger than those of R2-5 (P < 0.05) and LGG (P < 0.01); for shigella flexneri: the diameter of the inhibition zone of 4 strains of bacteria is not obviously different (P is more than 0.05); for listeria monocytogenes: the diameters of the inhibition zones of R2-5, R4-2 and R6-3 are extremely larger than that of LGG (P < 0.01), and the diameters of the inhibition zones of R2-5 and R4-2 are not significantly different (P > 0.05), but are all significantly smaller than that of R6-3 (P < 0.05). In summary, the bacteriostasis of R6-3 to 5 strains of pathogenic bacteria is superior to R4-2, and R4-2 is superior to R2-5 and LGG.
TABLE 2 bacteriostasis of bacterial strains against pathogenic bacteria
Note that: in comparison to the different treatment groups, n =3, the superscript lowercase (a -C) represents the significant difference at the level p=0.05, and the superscript lowercase (a-C) represents the significant difference at the level p=0.01.
6. Determination of the ability of the Strain to adhere to Caco-2 cells
Strains LGG, R2-5, R4-2 and R6-3 were inoculated in MRS liquid medium, cultured at 37℃for 14 hours, the cells were washed with sterile physiological saline, then resuspended in MEM medium free of bovine serum and diabody, and the concentration of the bacterial suspension was adjusted to (1-2). Times.10 8 CFU/mL for the next experiment. Caco-2 cells are inoculated in a 6-hole plate and cultured in a 5% CO 2 concentration incubator at 37 ℃ for 48 hours, the culture solution is replaced for 1 time, after the cells grow into a single layer, the single layer is washed for 2 to 3 times by sterile PBS with the pH value of 7.2 to 7.4, and dead cells and antibiotics are washed off. Then, 1mL of a bacterial suspension containing no bovine serum and having a double antibody concentration of (1-2). Times.10 8 CFU/mL was added to each well, and incubated in an incubator at 37℃and a concentration of 5% CO 2 for 2 hours. Washed 5 times with sterile PBS at pH 7.2-7.4. After adding 500. Mu.L of 0.25% pancreatin to the wells for 3min, and adding 500. Mu.L of sterile PBS with pH of 7.2-7.4, viable counts were performed. The adhesion rate of the strain to Caco-2 cells was calculated according to formula (3).
As a result, as shown in FIG. 3, FIG. 3 shows that there was no significant difference in the adhesion rates of R6-3 and LGG to Caco-2 cells (P > 0.05), the adhesion rates of R2-5 to Caco-2 cells were significantly higher than those of R6-3 (P < 0.001) and LGG (P < 0.001), and the adhesion rates of R4-2 to Caco-2 cells were significantly higher than those of R2-5 (P < 0.001), R6-3 (P < 0.001) and LGG (P < 0.001). I.e., adhesion to Caco-2 cells, R4-2 is greater than R2-5, R2-5 is greater than R6-3 and LGG, and R6-3 is comparable to LGG adhesion to Caco-2 cells.
Comprehensively considering the tolerance of the strain to artificial gastric juice and artificial intestinal juice, the bacteriostasis to common pathogenic bacteria and the adhesion to Caco-2 cells, we select the strain R6-3 with stronger resistance to artificial gastric juice and bacteriostasis as a target potential probiotic strain for subsequent experiments.
7. Identification of strains with potential probiotic properties
The strain R6-3 is obtained through layer-by-layer screening to have potential probiotics characteristics, the strain is subjected to gram staining, the bacterial morphology is observed by an electron microscope, the strain is coated on an MRS solid culture medium for culturing for 48 hours, and the bacterial colony morphology is observed. The physiological and biochemical identification of R6-3 was performed according to the procedure of the lactobacillus complete biochemical identification tube (SHBG 13). Downloading the 16S rDNA sequence of the strain with the highest homology with the 16S rDNA sequence of R6-3, using MEGA software, utilizing a Neighbor-joining statistical method, adopting a Bootstrap method to build a tree, repeating for 1000 times, and utilizing a p-distance method to calculate the evolutionary distance to build a phylogenetic tree.
The results are shown in FIG. 4, table 3 and FIG. 5, and it can be seen from FIG. 4 that strain R6-3 is a gram-positive bacterium, and the cells are rod-shaped and flagellum-free. R6-3 colony on MRS solid culture medium is milky white, round convex, smooth in surface and neat in edge. Table 3 shows that R6-3 can be further identified as Lactobacillus plantarum by using esculin, maltose, cellobiose, salicin, mannitol, sorbitol, raffinose, sucrose, lactose, inulin and 1% sodium hippurate, as compared with "lactic acid bacteria Classification identification and Experimental methods". FIG. 5 shows that the strain R6-3 and Lactobacillus plantarum are aggregated into one branch, and the strain R6-3 is determined to be lactobacillus plantarum and named Lactobacillus plantarum R-3 by combining the 16S rDNA sequence comparison result and the physiological and biochemical identification result of the R6-3.
TABLE 3 identification results of micro biochemical identification tube for lactic acid bacteria
Note that: "+" indicates a positive reaction.
Example 2 in vitro antioxidant Activity assay of R6-3
Determination of H 2O2 tolerance to R6-3: LGG as a control strain, LGG and Lactobacillus plantarum R6-3 were cultured to the initial stage of log end stabilization, inoculated into MRS liquid medium containing 0mmol/L, 0.4mmol/L, 0.8mmol/L, 1.0mmol/LH 2O2 in a volume ratio of 1% (v/v), vortexed and mixed uniformly, incubated at 37℃for 12 hours, vortexed and mixed uniformly again, and cell growth was measured at 600nm by spectrophotometry with the MRS liquid medium zeroed.
As a result, as shown in FIG. 6, it can be seen in FIG. 6 that the growth of Lactobacillus plantarum R6-3 and LGG was not significantly affected (P > 0.05) when H 2O2 was added to the MRS medium at a concentration of 0.4 mmol/L. At a concentration of 1mmol/L H 2O2 in MRS medium, the growth of Lactobacillus plantarum R6-3 was only slowly inhibited: the cell concentration was not significantly different from that at the concentration of H 2O2 of 0.8mmol/L (P > 0.05), and the cell concentration was significantly different from that at the concentration of H 2O2 of 0.4mmol/L (P < 0.05). While LGG was significantly affected when H 2O2 was added to the MRS medium at a concentration of 0.8 mmol/L: the difference from the cell concentration was very remarkable when the concentration of H 2O2 was 0.4mmol/L (P < 0.01), the concentration of H 2O2 was increased from 0.8mmol/L to 1mmol/L, and the cell concentration was significantly decreased again (P < 0.05). The results demonstrate that H 2O2 is capable of causing oxidative damage to Lactobacillus plantarum R6-3 and LGG, thereby inhibiting their growth. Also, lactobacillus plantarum R6-3 is shown to be superior to LGG in its ability to resist H 2O2 causing oxidative damage.
Example 3 Lactobacillus plantarum R6-3 relieves oxidative stress in mice via the intestinal-hepatic axis
1. Animal test design
50 SPF-class male BALB/c mice of 8 weeks old are selected and fed into an animal house with alternating illumination/darkness at 22+/-2 ℃ for 12 hours, and are free to drink and eat. After 1 week of adaptation, the mice were randomly divided into 5 groups (10 per group) according to body weight, and designated as control group, model group, R6-3 group, LGG group and VC group, respectively. The 5 groups of mice were weighed 1 time a day, dosed 1 time, injected subcutaneously 1 time in the nape of the neck, and 0.1mL of liquid was injected and lavaged per 10g of mice body weight. The test period was 8 weeks. Except for the control mice, 200mg/kg D-gal was injected subcutaneously. The mice in the R6-3 group, the LGG group and the VC group are respectively perfused with the lactobacillus plantarum R6-3, the LGG bacterial suspension and 50mg/kg VC, and the mice in the control group and the model group are perfused with normal saline. LGG and VC groups served as positive strain and positive drug controls, respectively.
Feces were collected directly from the anus of the mice 1 day before the end of the experiment, two mice were collected, each about 0.5g, stored at-80 ℃ for intestinal flora analysis and SCFAs determination. After the last gastric lavage, the mice are fasted and forbidden for 16 hours, weighed, the eyeballs are taken for blood, and then the mice are killed by cervical vertebra removal. Standing blood at room temperature for 1 hr, centrifuging (1150 g,10 min), collecting supernatant, and storing at-80deg.C for antioxidant parameter measurement. The heart, brain, spleen, liver, kidney, and organs of each mouse were taken out, weighed, and organ indexes were calculated according to formula (8). The liver was split into 4 parts, 3 of which were immediately frozen in liquid nitrogen, stored at-80 ℃ for antioxidant parameter determination, real-time quantitative PCR detection of the expression of antioxidant genes, and 1 part fixed in 10% neutral formalin for histopathological section production. The animal testing method is reviewed and approved by the laboratory animal ethics committee of the university of Hebei agriculture (2021096).
2. Liver HE staining and observations
The liver tissue of each group of mice was fixed in 10% neutral formalin, then the samples were dehydrated, paraffin-embedded conventionally, and prepared into 5 μm sections, dewaxed, stained with hematoxylin and eosin (H & E), observed with an optical microscope and photographed into histological images, and read by a pathology professional.
The livers of the mice from the different treatment groups after 8 weeks of the experiment were examined for histopathological changes by HE staining, and the results are shown in FIG. 7. As can be seen from fig. 7, the control mice had normal liver structure, normal cell morphology, uniform staining, and no obvious lesions. The liver of the model group mice was obviously damaged histologically, including cytoplasmatic vacuolation, cell swelling, arrangement disorder, irregular morphology and deep staining. Compared with the model group, the liver injury of the mice in the R6-3, LGG and VC groups is relieved to different degrees. In particular, the liver structures of R6-3 and LGG groups were very similar to those of the control group, indicating that Lactobacillus plantarum R6-3 and LGG were more effective than VC in restoring liver damage caused by D-gal injection.
3. Determination of oxidation index of serum, liver, kidney and brain tissue
Homogenizing liver, kidney and brain tissue samples in 9 times volume of pre-cooled physiological saline for 60s, centrifuging (4deg.C, 2010g,10 min), and collecting supernatant to obtain tissue supernatant. Tissue supernatant protein concentration was determined using BCA protein assay kit. Glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and Catalase (CAT) activities, total antioxidant capacity (T-AOC), and Malondialdehyde (MDA) and Nitric Oxide (NO) levels in serum and tissue supernatants were measured according to the kit instructions.
The effect of Lactobacillus plantarum R6-3 on oxidation index of serum, liver, kidney and brain tissue of D-gal induced oxidative damage mice is shown in FIGS. 8 and 9. As can be seen from figures 8, 9, MDA and NO levels were significantly elevated in the serum, liver, kidney, brain (P < 0.05) in the mice of the model group compared to the normal group. In addition to serum T-AOC levels and kidney SOD activity, there was no significant change (P > 0.05), and GSH-PX, SOD, CAT activity and T-AOC levels were significantly reduced in the serum, liver, kidney and brain tissues of the model group mice (P < 0.05). The Lactobacillus plantarum R6-3, LGG and VC all reverse the changes of the oxidative stress indexes to a certain extent. Comprehensively comparing, lactobacillus plantarum R6-3 shows higher antioxidant capacity than LGG and VC, and mainly shows effects on mouse serum GSH-PX, SOD and T-AOC activity, liver SOD activity, kidney NO level and brain T-AOC level. In conclusion, D-gal makes mice in oxidative stress state, lactobacillus plantarum R6-3, LGG and VC all relieve the state to a certain extent, and Lactobacillus plantarum R6-3 has better effect than LGG and VC.
4. Real-time quantitative PCR detection of expression of liver Nrf-2 signal path oxidation resistance related gene
Total RNA from liver tissue was extracted by TriQuick Reagent according to the manufacturer's instructions, the OD260/OD280 ratio (purity of total RNA was determined) and concentration were measured by an ultra-micro spectrophotometer, and the total RNA that was acceptable for extraction was diluted to 1. Mu.g/. Mu.L and then reverse transcribed to cDNA using PRIMESCRIPTTM RT REAGENT KIT WITH GDNA ERASER kit. The cDNA is used as a template, and a Roche LightCycler-96 real-time fluorescent quantitative PCR system and a LightCycler 96SW 1.1 software are adopted for real-time fluorescent quantitative PCR analysis. Wherein, the primer, real-time quantitative PCR reaction system and reaction procedure of the antioxidant related genes are shown in Table 4, table 5 and Table 6 respectively. The relative expression level of the gene was calculated by 2 -△△Ct method using Gapdh as an internal reference.
The relative expression level of the gene related to the Nrf-2 antioxidant signaling pathway of the liver tissue of the D-gal induced oxidative damage mouse by R6-3 is shown in FIG. 10. As can be seen from FIG. 10, the expression level of Nrf-2, SOD1, SOD2, CAT, GPx-1, GPx-2, NQO1, HO-1mRNA was significantly decreased in the livers of the mice in the model group (P < 0.05) compared with the control group. Whereas R6-3, LGG and VC significantly up-regulated mRNA expression of the above genes, the differences were statistically significant (P < 0.05) except for a few genes. The results indicate that R6-3, LGG and VC activate the Nrf-2 signaling pathway, thereby up-regulating the expression of downstream antioxidant-associated genes. In combination, R6-3 is superior to LGG and VC in up-regulating mRNA expression of genes associated with the Nrf-2 signaling pathway.
TABLE 4 fluorescent quantitative PCR primer sequences
TABLE 5 fluorescent quantitative PCR reaction System
TABLE 6 fluorescent quantitative PCR amplification procedure
5. Effect of R6-3 on D-gal-induced oxidative damage of mice intestinal flora
The collected mouse feces, 5 samples from each group were randomly selected and placed in a foam box filled with dry ice, and sent to Shanghai Meiji Biotechnology Co., ltd for 16S rRNA amplicon sequencing. Performing quality control on the original sequencing sequence by using Trimmomatic software, and then splicing the quality-controlled sequence by using FLASH software; sequences were OTU clustered (based on 97% similarity) using UPARSE software (7.0.1090 version, http:// drive5.Com/uparse /), chimeras were knocked out using UCHIME software; each sequence was annotated for species classification using RDP CLASSIFIER (version 2.11, https:// sourceforge. Net/projects/rdp-classifer /), and then aligned with the Silva database (SILVA RELEASE138 http:// www.arb-Silva. De) (set alignment threshold at 70%); the alpha and beta diversity was analyzed using Mothur software (1.30.1 version) and R software (R3.3.1 version).
The composition and species differences of the intestinal flora of mice from different treatment groups were analyzed at portal and genus level and the results are shown in figure 11. From FIG. 11 (A) it can be seen that the composition and differences in the levels of the gate for the intestinal flora of mice from the different treatment groups, 11 different gates were identified in total in the mouse faeces, with the average relative abundance being highest among the five groups with Bacteroides gate (Bacteroidota), thick-walled bacteria gate (Firmicutes), proteus gate (Proteus), actinomycetes gate (Actinobacteriota), deiron-rod gate (Deferribacterota), currency gate (Campilobacterota), dethiobacilli gate (Desulfobacterota) and Patescibacteria. Fig. 11 (B) shows that the average relative abundance of model groups Bacteroidota, proteobacteria, deferribacterota and Desulfobacterota increased from 40.49%, 0.14%, 0.37% and 1.1% to 58.05%, 5.8%, 1.22% and 1.65%, respectively, compared to the normal group, whereas the average relative abundance of Firmicutes, actinobacteriota, campilobacterota and Patescibacteria decreased from 54.06%, 2.03%, 0.80% and 0.84% to 30.69%, 1.79%, 0.52% and 0.24%, respectively. Wherein, bacteroidota and Firmicutes with the highest relative abundance have significant changes, and the statistical differences are P <0.05 and P <0.001 respectively. Lactobacillus plantarum R6-3, LGG and VC reverse the Bacteroidota, firmicutes, proteobacteria, campilobacterota, desulfobacterota and Patescibacteria changes to varying degrees. In addition, lactobacillus plantarum R6-3 also improved the increase in the relative abundance of Deferribacterota caused by D-gal injection, while LGG and VC further increased the relative abundance of Deferribacterota in the gut of D-gal injected mice. Lactobacillus plantarum R6-3, LGG and VC do not affect changes in Actinobacteriota in the intestinal tract of D-gal injected mice. Furthermore, as can be seen from fig. 11 (C), the ratio Firmicutes/Bacteroidota (F/B) was extremely reduced in the intestinal tract of the model group mice compared to the normal group (P < 0.001), but both lactobacillus plantarum R6-3 and LGG could significantly improve this change (P < 0.05). These changes indicate that D-gal injection significantly alters the intestinal flora of oxidatively stressed mice at the portal level, and that Lactobacillus plantarum R6-3 can ameliorate this change to some extent.
From fig. 11 (D), the composition and differences at the genus level of the intestinal flora of mice of different treatment groups can be seen, and 137 different genera were identified in total, of which the first 15 genera with highest average relative abundance were norank _f_ Muribaculaceae, lactobacillus (Lactobacillus), alismatis (ALISTIPES), bacteroides (bacteriodes), odoribacter, prevoteaceae UCG-001 (Prevotellaceae _ucg-001), lachnospiraceae unclassified genera (unclassified _f_ Lachnospiraceae), lachnospiraceae NK4a136 (Lachnospiraceae _nk4a 136_group), acinetobacter (Acinetobacter), mucispirillum, enterobacter (Enterorhabdus), helicobacter (Helicobacter), lachnum (norank _f_ Lachnospiraceae), vibrio (Desulfovibrio) and clostridium uncleanum (norank _f_ norank _o_ Clostridia _uc014_g-in this order. Fig. 11 (E) shows that model groups Lactobacillus, enterorhabdus, helicobacter and norank _f_ norank _o_ Clostridia _ucg-014 have reduced average relative abundance compared to the normal group and other genera have increased average relative abundance. Among them, lactobacillus has very significant changes (P < 0.001). Lactobacillus plantarum R6-3, LGG and VC reverse the norank_f_Muribaculaceae、Lactobacillus、Prevotellaceae_UCG-001、unclassified_f_Lachnospiraceae、Acinetobacter、Helicobacter、norank_f_Lachnospiraceae and Desulfovibrio changes differently. In addition, lactobacillus plantarum R6-3 and VC reverse ALISTIPES changes, lactobacillus plantarum R6-3 also reverses Bacteroides, mucispirillum and norank _f_ norank _o_ Clostridia _UCG-014 changes. However, lactobacillus plantarum R6-3, LGG and VC all had no improving effect on the change of Odoribacter, lachnospiraceae _NK4A136_group and Enterorhabdus. These changes indicate that D-gal injection significantly alters the intestinal flora of oxidatively stressed mice at the genus level, and that Lactobacillus plantarum R6-3, LGG and VC can improve this change to some extent.
By combining the above, the D-gal injection changes the distribution and composition of the intestinal flora of the mice, namely the intestinal tract is disturbed, and the lactobacillus plantarum R6-3, the LGG and the VC can improve the intestinal flora disturbance caused by the D-gal to a certain extent, and the comprehensive comparison of the improvement effect is superior to the LGG and the VC of the lactobacillus plantarum R6-3.
6. Correlation analysis of intestinal microorganisms and liver oxidation resistance indexes and fecal short-chain fatty acids
The correlation between species of intestinal flora at genus level and liver antioxidant index and SCFAs content in feces were detected by spearman correlation analysis, and the results are shown in FIG. 12. Fig. 12 (a) shows that the relative abundance of Lactobacillus correlated significantly with both GSH-PX and CAT activity (P<0.05),Candidatus_Saccharimonas、Colidextribacter、norank_f_norank_o_Clostridia_UCG-014、norank_f_Oscillospiraceae correlated significantly with SOD activity at correlation levels P <0.01, P <0.05, respectively; the relative abundance of Desulfovibrio is very significantly inversely correlated with GSH-PX activity (P < 0.01); the relative abundance of Bacteroides, colidextribacter, norank _f __ Oscillospiraceae was significantly inversely correlated with NO levels (P < 0.05). Figure 12 (B) shows that the relative abundance of Lactobacillus is significantly positively correlated with propionic acid, isobutyric acid and n-valeric acid concentrations (P < 0.05); the relative abundance of Desulfovibrio was significantly inversely correlated with acetic acid, n-butyric acid (P < 0.05); the relative abundance of Mucispirillum is significantly inversely correlated with acetic acid, propionic acid, n-butyric acid, n-valeric acid, at correlation levels of P <0.05, P <0.01, respectively; the relative abundance of norank _f_ Lachnospiraceae is significantly inversely correlated with acetic acid, propionic acid, n-butyric acid, and n-valeric acid, with correlation levels of P <0.05, P <0.01, P <0.05, and P <0.01, respectively; the relative abundance of Prevotellaceae _ucg-001 is significantly inversely correlated with isobutyric acid (P < 0.05); the relative abundance of RIKENELLA is significantly inversely related to n-butyric acid, n-valeric acid (P < 0.05); the relative abundance of unclass _f_ Lachnospiraceae was significantly inversely related to propionic acid, n-valeric acid (P < 0.05).
EXAMPLE 4 Lactobacillus plantarum R6-3 prevention of depression by the intestinal-brain axis
1. Animal test design
The feeding conditions of 64 male C57BL/6J mice were selected and randomly divided into four groups of 16 mice each, namely a normal group, a model group, an R6-3 group and a drug group, and the weights of the mice were measured every week, and the specific experimental arrangement is shown in FIG. 13. The CUMS was performed to construct a depressed mouse model, 1 different method of stimulation was randomly received daily for 8 weeks, and the same stimulation could not be applied for 3 consecutive days, ensuring that the animals could not be expected, and the specific scheme is shown in FIG. 14. Collecting mouse feces for determination of SCFAs; after the cervical dislocation of the mice is killed, serum is collected and used for measuring CORT, oxidation indexes and inflammatory factors; wherein 3 mice are randomly selected from each group, and the whole brain is taken out and fixed in 10% neutral formalin for preparing brain histopathological sections; taking out the whole brain tissue of the mouse and the cecum content, immediately freezing with liquid nitrogen, storing at-80 ℃, and determining monoamine neurotransmitter, BDNF and oxidation indexes of the brain tissue, wherein the cecum content is used for determining intestinal flora. The animal testing method is reviewed and approved by the laboratory animal ethics committee of the university of Hebei agriculture (2021095).
2. Influence of R6-3 on the body weight of CUMS-induced depression mice
The change of the weight of the mice in the different treatment groups in the 8-week test period is shown in fig. 15, it can be seen from fig. 15 that the weight of the mice in the 8-week test period is gradually increased, the weight of each group is not different (P > 0.05) in the first two weeks, the normal group and the model group are extremely significantly different (P < 0.01) from the 3 rd week, the difference level of the normal group and the model group in the 4-8 weeks is P <0.001, the difference level of the R6-3 th group and the model group in the 4, 5, 6 and 8 weeks is P <0.01, P <0.05, and the difference level of the drug group and the model group is P <0.05 in the 7 th and 8 weeks. It was demonstrated that the CUMS model slowed the weight gain in mice, while Lactobacillus plantarum R6-3 and fluoxetine improved this change to varying degrees, but failed to return to normal group levels.
3. Behavioural test
(1) Sugar water preference test (sugar PREFERENCE TEST, SPT)
All mice were fasted for 12h before conducting sucrose preference experiments and then given equal volumes of two bottles of water: 2% (mass/volume) sucrose water and tap water, 200 mL/bottle, 6h later the positions of the two water bottles were exchanged. After 24 hours the consumption of water and sucrose solution in the flask was weighed and the sucrose preference calculated. Sucrose preference = (volume sucrose consumed/total volume liquid consumed) ×100%
(2) Tail suspension test (Tail Suspension Test TST)
The mouse is stuck on a cross rod which is 50cm higher than the ground by using an adhesive tape at the position 1cm away from the tip of the tail of the mouse, so that the mouse is in an inverted state and is suspended for 6min, the mouse can struggle and move just before beginning, and only passive suspension intermittent rest can appear after a period of time, namely a hopeless state is displayed. After 1min of acclimation, the mice were recorded for a cumulative period of immobility remaining for 5min.
(3) Forced swimming test (Forced Swim Test FST)
The mice were placed in a swimming bucket with a water depth of 20cm, a diameter of 20cm and a water temperature of 25+ -1deg.C, and swimming immobility time was defined as the time the mice were floating in the water without struggling, and only slight movements were made to maintain the head floating above the water surface, the accumulated time. Forced swimming for 6min, adapting the mice for 2min, and recording swimming immobility time within 4 min.
Influence of R6-3 on sucrose preference, tail suspension immobility time and forced swimming immobility time of CUMS-induced depressed mice, the results are shown in FIG. 16. Sucrose preference test results as shown in fig. 16 (a), sucrose preference was very significantly reduced in 8 week CUMS treated induced mice compared to normal group (P < 0.001). Both lactobacillus plantarum R6-3 and fluoxetine can significantly (P < 0.001) improve sucrose preference behavior in mice compared to the model group. However, the improvement effect of the two compounds also shows extremely significant difference (P < 0.001), and lactobacillus plantarum R6-3 can restore the level of CUMS induced depression mice to the level which is not significantly different from the normal group (P > 0.05), and the sucrose preference level of the mice after fluoxetine improvement still has extremely significant difference from the normal group (P < 0.001). The effect of the lactobacillus plantarum R6-3 is better than that of the antidepressant fluoxetine in improving the sucrose preference behavior of depressed mice. Results of tail suspension test and forced swimming test as shown in fig. 16 (B) and (C), compared with the normal group, the immobility time of 8-week CUMS-treated induced mice in tail suspension test and forced swimming test was extremely significantly increased, and the difference levels were P <0.01 and P <0.001, respectively, through statistical analysis. Both lactobacillus plantarum R6-3 and fluoxetine were able to reverse both of these despair behaviors very significantly (P < 0.01) in depressed mice compared to the model group and reached levels that were not significantly different (P > 0.05) from the normal group. It was demonstrated that Lactobacillus plantarum R6-3 and fluoxetine were quite effective (P > 0.05) in reversing the destinating behavior in depressed mice.
In summary, the results of this study showed that model mice exhibited reduced sucrose preference, forced swimming and prolonged tail suspension time compared to normal mice, meaning that mice had a lack of pleasure and increased hopeless time, indicating success in constructing a model of depressed mice using CUMS. Lactobacillus plantarum R6-3 can reverse behavior of depressed mice to a level that is not significantly different from normal mice.
4. Hippocampus Nishi staining and observation
Each group was fixed with 3 mouse brain tissues in 10% neutral formalin, then sent to the wuhan seville biosciences, inc, for nikose staining, light microscopy and histological image taking, and read by pathology professionals.
After 8 weeks of experiment, the sea horse of different treatment groups of mice is dyed by Nishi to check the histological change of the morphological structure of the neurons, the result is shown in figure 17, and the result is shown in figure 17, the shape rule, the limit, the compact arrangement, the nuclear circle and the obvious nucleolus of the CA1 region of the sea horse of the normal group of mice are shown. The number of the neurons of the model group is obviously reduced, the staining is deepened, the shape is irregular, the polygon is formed, the nucleus is shrunken, and the changes indicate that the CUMS causes the damage to the hippocampal neurons of the mice, and the morphology of the cell structure is changed. However, most of the neuronal cells in R6-3 and drug groups were very similar to the normal group, indicating that Lactobacillus plantarum R6-3 and fluoxetine were effective in restoring CUMS-induced damage to the hippocampal tissue of mice.
5. Detection of brain tissue monoamine neurotransmitters, brain-derived neurotrophic factors and serum corticosterone
Referring to the manufacturer's instructions, ELISA (enzyme-linked immunosorbent assay) kits were used to detect the levels of 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE), BDNF, CORT in serum in brain tissue.
As shown in fig. 18, as can be seen from fig. 18 (a), (B) and (C), compared with the normal group, the levels of 5-HT, DA and NE in brain tissues of the mice with CUMS-induced depression were significantly reduced, the difference levels were P <0.05, P <0.01 and P <0.05, respectively, the levels of monoamine neurotransmitters in the brains of the mice in the model group could be improved to different extents by lactobacillus plantarum R6-3 and fluoxetine, the levels of improvement of 5-HT by lactobacillus plantarum R6-3 and fluoxetine were P <0.01, the levels of improvement of DA were P <0.01 and P <0.05, respectively, and the levels of improvement of NE were P <0.001 and P <0.05, respectively; fig. 18 (D) shows that, compared to the normal group, the cot in serum was significantly elevated in the mouse serum from CUMS induced depression (P < 0.05), while lactobacillus plantarum R6-3 could significantly (P < 0.01) lower the cot in serum from mice from the depression model group to levels not significantly different from the normal group (P > 0.05). The fluoxetine also has a reduction effect on CORT in serum of a mouse with depression induced by CUMS, and the fluoxetine drug group has no obvious difference (P > 0.05) from the normal group and the model group, but has obvious difference (P < 0.05) from the R6-3 group; fig. 18 (E) shows that CUMS induced a very significant decrease in BDNF in brain tissue of depressed mice (P < 0.01) compared to the normal group, whereas lactobacillus plantarum R6-3 could significantly (P < 0.001) raise BDNF in brain tissue of mice of the depression model group to a level (P > 0.05) that is not significantly different from the normal group. Fluoxetine also has an increasing effect on BDNF in brain tissues of mice induced by CUMS, and the fluoxetine drug group has no significant difference (P > 0.05) from the normal group and the model group, but has extremely significant difference (P < 0.01) from the R6-3 group.
In conclusion, the lactobacillus plantarum R6-3 has a reverse effect on the reduction of 5-HT, DA and NE monoamine neurotransmitters and BDNF and the increase of HPA axis core hormone CORT in brain tissues of mice with the depression induced by CUMS, which indicates that the lactobacillus plantarum R6-3 can reverse the neurobiological disorder of the mice with the depression induced by CUMS.
6. Determination of serum inflammatory factors
Referring to the manufacturer's instructions, ELISA kits were used to determine the levels of inflammatory factors TNF-alpha, IL-6, IL-1β, IL-10 in serum.
As a result, as shown in fig. 19, it can be seen from fig. 19 that the levels of pro-inflammatory factors TNF- α, IL-6, IL-1β were significantly increased (P < 0.05) and the levels of anti-inflammatory factor IL-10 were significantly decreased (P < 0.05) in serum of the mouse with CUMS induced depression compared to the normal group. Lactobacillus plantarum R6-3 and fluoxetine can reduce TNF- α content compared to the model group, but the difference is not significant (P > 0.05); the lactobacillus plantarum R6-3 and fluoxetine can obviously reduce the content of IL-6, and the difference level is P <0.05 and P <0.01 respectively; the lactobacillus plantarum R6-3 and fluoxetine can obviously reduce the content of IL-1 beta, and the difference level is P <0.05; lactobacillus plantarum R6-3 can significantly increase the IL-10 content (P < 0.01), fluoxetine can also increase the IL-10 content, but the difference is not significant (P > 0.05). The R6-3 group and the drug group were not significantly different (P > 0.05) compared to the normal group, respectively, indicating that both could reverse TNF- α, IL-6, IL-1 β, IL-10 to the normal group status. Further, it was shown that Lactobacillus plantarum R6-3 may improve the immune status of CUMS-induced depressed mice.
7. Determination of serum and brain tissue Oxidation index
Referring to the manufacturer's instructions, the serum and brain tissue glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities, total antioxidant capacity (T-AOC), and Malondialdehyde (MDA) levels were measured separately using a kit.
As shown in fig. 20 and 21, it can be seen from fig. 20 that GSH-PX and SOD activities and T-AOC levels in serum of mice with CUMS induced depression were significantly reduced, and MDA levels were significantly increased, compared to normal groups, with difference levels of P <0.01, P <0.05, P <0.001, and P <0.001, respectively. Lactobacillus plantarum R6-3 and fluoxetine can elevate GSH-PX activity to levels (P > 0.05) that are not significantly different from the normal group, as compared to the model group; both lactobacillus plantarum R6-3 and fluoxetine can significantly increase the activity of SOD (P < 0.05), and have no significant difference from the normal group (P > 0.05); both lactobacillus plantarum R6-3 and fluoxetine can raise the level of T-AOC, the difference level is P <0.05 and P >0.05 respectively, but the difference level is P <0.05 and P <0.01 respectively; both lactobacillus plantarum R6-3 and fluoxetine can reduce the MDA level, the difference level is P <0.05 and P <0.001, but the R6-3 group is still significantly different from the normal group (P < 0.05), and the drug group is not significantly different from the normal group (P > 0.05); as can be seen from fig. 21, the levels of GSH-PX and SOD activity and T-AOC in brain tissue of the CUMS-induced depressed mice were significantly reduced, and the levels of MDA were significantly increased, compared to the normal group, with the difference levels of P <0.01, P <0.05, P <0.001, and P <0.01, respectively. Compared with the model group, the lactobacillus plantarum R6-3 and fluoxetine can significantly increase GSH-PX activity, the difference level is P <0.05, and the difference is not significantly different from the normal group (P > 0.05); lactobacillus plantarum R6-3 and fluoxetine can obviously raise SOD activity, the difference level is P <0.01 and P <0.05 respectively, and no obvious difference (P > 0.05) is generated from the normal group; lactobacillus plantarum R6-3 and fluoxetine can significantly raise the level of T-AOC, the difference level is P <0.05 and P <0.001 respectively, and no significant difference (P > 0.05) from the normal group exists; lactobacillus plantarum R6-3 and fluoxetine can significantly reduce MDA levels, the difference levels are P <0.001 and P <0.01 respectively, and no significant difference (P > 0.05) exists from the normal group.
In summary, CUMS places the mice in oxidative stress, and Lactobacillus plantarum R6-3 can relieve this state to some extent, and can reach normal levels basically.
8. Effect of R6-3 on D-gal-induced oxidative damage of mice intestinal flora
The harvested mouse cecal contents, 5 samples per group, were placed in foam boxes filled with dry ice at random, and mailed to Shanghai Meiji Biotechnology Co., ltd for 16S rRNA amplicon sequencing.
Based on the KEGG database, a Level3 functional predictive analysis was performed on the intestinal flora 16S rRNA sequencing data of different treatment groups using PICRUSt, and the results are shown in fig. 22. As can be seen from fig. 22: the relative abundance of metabolic pathway genes involved in the intestinal flora of mice in the model group (Metabolic pathways), microbial metabolism in different environments (Microbial metabolism IN DIVERSE environments), carbohydrate metabolism (Carbon metabolism), two-component system (Two-component system), glycolysis/gluconeogenesis (Glycolysis/Gluconeogenesis) was reduced but the differences were not significant compared to the normal group. Whereas the relative abundance of metabolic pathway genes involved in amino acid biosynthesis (Biosynthesis of amino acids), ABC transporter (ABC transporters), ribosomes (riboname), quorum sensing (Quorum sensing), starch and sucrose metabolism (STARCH AND sucrose metabolism), aminoacyl-tRNA biosynthesis (Aminoacyl-tRNA biosynthesis), cysteine and methionine metabolism (CYSTEINE AND methionine metabolism) was enhanced, especially with the most pronounced ribosomes (P < 0.01). Demonstrating that the metabolic pathway related to the intestinal flora of the mice with depression induced by CUMS is disturbed. Lactobacillus plantarum R6-3 has a reverse effect on genes involved in metabolic pathways, amino acid biosynthesis, ABC transport vectors, ribosomes, quorum sensing, glycolysis/gluconeogenesis, aminoacyl-tRNA biosynthesis, cysteine and methionine metabolic pathways, and in particular has a very significant difference compared to the model group (P < 0.01). Fluoxetine has a regulating effect on genes involved in microbial metabolism, amino acid biosynthesis, cysteine and methionine metabolism, ribosome, aminoacyl-tRNA biosynthetic metabolic pathways in different environments, especially ribosome, aminoacyl-tRNA biosynthesis, and the difference is very significant (P < 0.01) compared with the model group, but is reduced to a lower level than the normal group. Thus, overall, the improvement effect of Lactobacillus plantarum R6-3 was superior to fluoxetine for CUMS-induced metabolic activity disturbance in the mouse intestinal flora. It was further shown that lactobacillus plantarum R6-3 might be able to alleviate the depressive behaviour of mice caused by CUMS by balancing the metabolic activity of the intestinal flora.
9. Effect of R6-3 on D-gal-induced oxidative damage of mouse faeces short chain fatty acids
Acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid and isovaleric acid were measured in the cecal content of mice using gas chromatography.
As shown in FIG. 23, the content sequence of SCFAs in feces of C57BL/6 mice is shown in FIG. 23: acetic acid > propionic acid > n-butyric acid > isovaleric acid > n-valeric acid > isobutyric acid, and mainly consists of acetic acid, propionic acid and n-butyric acid. Compared to the normal group, CUMS induced a significant decrease in SCFAs in the stool of depressed mice (P < 0.05). Compared with a model group, the lactobacillus plantarum R6-3 can obviously improve the contents of acetic acid, propionic acid, n-butyric acid and n-valeric acid in the mouse excrement (P < 0.05) to a level (P > 0.05) which is not obviously different from that in the normal group, can also obviously improve the content of isovaleric acid in the mouse excrement (P < 0.05), but is obviously different from that in the normal group (P < 0.05), and has no obvious influence on the isobutyric acid in the mouse excrement (P > 0.05). Fluoxetine can increase the acetic acid and propionic acid content in the mouse feces to a level (P > 0.05) which is not significantly different from that of the normal group and the model group, and has no significant effect on the other four SCFAs. It is shown that CUMS causes obvious reduction of SCFAs in depressed mice intestinal tracts, and both the lactobacillus plantarum R6-3 and fluoxetine can play a role in improving to a certain extent, but the effect of the lactobacillus plantarum R6-3 is better than that of fluoxetine, which is probably caused by the fact that the lactobacillus plantarum R6-3 promotes proliferation of SCFAs producing bacteria in intestinal flora.
Example 5 application of Lactobacillus plantarum R6-3 in yogurt
1. Strain activation and starter preparation
Activating lactobacillus plantarum R6-3,37 ℃ with MRS liquid culture medium, culturing for 14h, inoculating the lactobacillus plantarum R6-3,37% inoculum size with the volume ratio of 3% into a test tube filled with 10mL sterilized milk, shaking uniformly, and culturing at 37 ℃ until the milk becomes coagulated. And (3) subculturing to obtain activated strain, and refrigerating for later use.
2. Preparation of yoghurt sample by fermentation
Re-dissolving the milk powder and water at a ratio of 1:5, sterilizing at 100deg.C for 10min, adding 5% white granulated sugar, cooling to 37deg.C, inoculating 4% Lactobacillus plantarum R6-3, culturing at 37deg.C until curd is coagulated (6-8 h), and refrigerating in a refrigerator at 4deg.C.
Example 6 application of Lactobacillus plantarum R6-3 in fermented fruit and vegetable juice
Respectively taking mango juice, banana juice, purple potato juice, aloe juice, carrot juice, garlic juice and ginger juice, diluting to Brix=12, and sterilizing at 95deg.C for 5min. After the feed liquid was cooled to 30℃the Lactobacillus plantarum R6-3 was inoculated into the fermentation base in an amount of 0.002% (1.0X10 7 CFU/mL) and fermented at 37 ℃. And (5) obtaining the fermented fruit and vegetable juice after 16 hours of fermentation.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (9)
1. Lactobacillus plantarum (Lactobacillus plantarum) R6-3 with a Saimu yoghurt source is characterized in that the preservation number is CGMCC No.25884.
2. Use of lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1 for the preparation of an antioxidant composition.
3. Use of lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1 for the preparation of an antidepressant pharmaceutical composition.
4. Use of lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1 for the preparation of a pharmaceutical composition for the prevention of depression by modulating the intestinal flora.
5. A composition comprising lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1.
6. A food product comprising lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1.
7. A food additive comprising lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1.
8. A microbial preparation comprising lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1.
9. A pharmaceutical composition comprising lactobacillus plantarum (Lactobacillus plantarum) R6-3 of the cerimum yoghurt source of claim 1.
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