CN115948281A - Saili yoghurt source lactobacillus plantarum R6-3 and application thereof - Google Patents

Saili yoghurt source lactobacillus plantarum R6-3 and application thereof Download PDF

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CN115948281A
CN115948281A CN202211467765.7A CN202211467765A CN115948281A CN 115948281 A CN115948281 A CN 115948281A CN 202211467765 A CN202211467765 A CN 202211467765A CN 115948281 A CN115948281 A CN 115948281A
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lactobacillus plantarum
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CN115948281B (en
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田洪涛
赵丽娜
张娜
李东尧
李晨
张少刚
许秀媛
柳苏月
王新宇
王晨阳
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Hebei Agricultural University
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Abstract

The invention discloses a cerimic acid milk source lactobacillus plantarum R6-3 and application thereof. The lactobacillus plantarum R6-3 is separated from a traditional fermented food of Sirilmu yogurt in Baicheng county in Aksu region of Xinjiang, has good tolerance and antibacterial property of artificial gastrointestinal fluid and adhesion to Caco-2 cells, and has good in vitro antioxidant capacity; by taking a D-galactose-induced oxidative damage mouse as a model, lactobacillus plantarum R6-3 can cause the SCFAs level in the intestinal tract of the mouse to rise by regulating the intestinal flora of the mouse, and the SCFAs enter the liver to activate an Nrf-2 signal path, so that oxidative stress is relieved; by taking a CUMS-induced depressed mouse as a model, the lactobacillus plantarum R6-3 can improve the SCFAs content in intestinal tracts, improve the immune and oxidative stress state of organisms, relieve monoamine neurotransmitter deficiency and HPA hyperfunction and restore the brain neurotrophic level by regulating the composition and the function of intestinal flora of the mouse, thereby playing a role in preventing depression.

Description

Saili yoghurt source lactobacillus plantarum R6-3 and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus plantarum R6-3 derived from cerimic acid milk and application thereof.
Background
Studies have shown that many metabolic processes in the body and various stimuli from outside the body, such as obesity, high-fat or sugar-rich and processed foods, ultraviolet (UV) radiation, smoking, drinking, chemotherapeutics, hyperthermia, exposure to pesticides or industrial chemicals, even oxygen itself, etc., all cause the body to be in an oxidative stress state, and the excessive oxidative stress induces apoptosis and necrosis, thereby causing various diseases and causing aging. Among them, depression associated with oxidative stress is one of the most common neurological diseases of this age, and is listed by the world health organization as the leading cause of global disability and non-fatal health loss. With the progress of society and the increase of life rhythm of people, various stresses and stresses from life are one of the most easily caused risk factors of depression. The economic cost for depression is expected to double globally by 2030, becoming the first disease burden. Therefore, the prevention, alleviation and treatment of depression has become an urgent global problem to be solved.
However, due to the side effects of current antidepressants, scientists are still looking for new methods of controlling depression. Oxidative stress is one of the causes of depression, and antioxidants may therefore have potential value in the treatment and prevention of depression. Synthetic antioxidants are questioned for safety, and probiotics in natural antioxidants are emerging sources of effective antioxidants for their long-term safe eating tradition and potential multiple health benefits to the body. From a safety point of view, traditional fermented foods with a long history of consumption are a good source for obtaining probiotic resources. The medical level of Baicheng county in Aksu city of Xinjiang falls behind, the geographic environment is severe, the living standard of people is low, but the Xinjiang Aksu county is considered as a world-level 'longevity county', which is probably related to local people habit of eating self-made murmurky xyloacid milk by families, contains abundant microorganism species and has superior quality, is a non-material cultural heritage of Uygur nationality of Xinjiang, and is even known as 'longevity secret formula'. However, the study on the sairita yoghourt is less, and the research is mainly focused on screening specific strains through in vitro tests and application research on the strains.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum R6-3 derived from cerimus acid milk and application thereof.
The method separates a series of lactic acid bacteria strains from the sori milk, carries out preliminary identification by sequencing 16S rDNA, takes the probiotic Lactobacillus rhamnosus GG (ATCC 53103) with the most approved probiotic function and the most thorough probiotic mechanism as a positive control strain (purchased from China general microbiological culture collection center) with the most research at present, screens out a lactic acid bacteria strain with potential probiotic function by taking artificial gastric juice resistance, artificial intestinal juice resistance, bacteriostasis, adhesion capacity and the like as indexes, determines the lactic acid bacteria strain as Lactobacillus plantarum by combining with physiological and biochemical test results, is named as R6-3, and simultaneously evaluates the safety (antibiotic sensitivity, harmful metabolites, hemolysis and the like) and oxidation resistance of the strain by in vitro tests; further evaluating the antioxidant capacity and antioxidant mechanism of the strain in a D-galactose-induced oxidative damage mouse by an in vivo test; and finally, taking a CUMS-induced depressed mouse as an animal model, and checking whether the screened lactobacillus plantarum R6-3 with antioxidant activity has the probiotic effect of preventing depression by regulating intestinal flora on the depressed model mouse.
Lactobacillus plantarum R6-3 with preservation number of CGMCCNO.25884 is provided.
The Lactobacillus plantarum R6-3 is applied to preparation of an antioxidant composition.
The Lactobacillus plantarum R6-3 is applied to preparation of an antidepressant composition.
The Lactobacillus plantarum R6-3 is applied to preparation of a composition for preventing oxidative aging by regulating intestinal flora.
The Lactobacillus plantarum R6-3 is applied to preparation of a composition for preventing depression by adjusting intestinal flora.
A composition comprises the Lactobacillus sakei (Lactobacillus plantarum) R6-3.
A food product comprising said Lactobacillus plantarum R6-3 derived from cericium vulgare.
A food additive comprises Lactobacillus plantarum R6-3 of the species Lactobacillus sakei.
A microbial preparation comprises the Lactobacillus plantarum R6-3 derived from ceric acid milk.
A pharmaceutical composition comprises the Lactobacillus plantarum R6-3 derived from cericium vulgare.
The preservation date of the strain is 2022, 10 months and 09 days, and the preservation number is CGMCC NO.25884. The name of the preservation unit is common microorganism center of China Committee for culture Collection of microorganisms, and the address is No. 3 of West Lu No. 1 of Beijing, facing the sunny district, the institute of microbiology of the Chinese academy of sciences, and the postal code is 100101.
The invention has the beneficial effects that: the Lactobacillus plantarum R6-3 disclosed by the invention has good tolerance to artificial gastric juice and artificial intestinal juice, has broad-spectrum antibacterial activity and high adhesion to Caco-2 cells. In-vitro probiotic function tests show that R6-3 thalli/fermentation supernatant have stronger antioxidant activity. An animal model of D-galactose induced mouse oxidative damage proves that SCFAs in the intestinal tract of a mouse are increased by adjusting the intestinal flora of the mouse through the R6-3 thalli, and the SCFAs enter the liver to activate an Nrf-2 signal channel, so that the oxidative stress of the mouse is relieved. The animal model of CUMS induced mice depression proves that the R6-3 thalli has the function of improving the SCFAs content in intestinal tracts, improving the immune and oxidative stress state of organisms, relieving monoamine neurotransmitter deficiency and HPA hyperfunction and recovering the brain neurotrophic level by adjusting the composition and the function of intestinal flora of mice so as to play a role in preventing depression.
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Figure 1 is a graph of the tolerance of the strains to artificial gastric juice, wherein P <0.001 indicates a significant level of difference in comparison of different strains to LGG compared to different treatment groups.
Fig. 2 is a graph of the tolerance of the strains to artificial intestinal fluid, wherein P <0.001 indicates a significant level of difference in comparison of different strains to LGG compared to different treatment groups.
Fig. 3 is a graph of adherence of strains to Caco-2 cells, where P <0.001 indicates significant levels of difference in comparison between different strains compared to different treatment groups.
FIG. 4 is a diagram showing the colony morphology and cell characteristics of the strain R6-3.
FIG. 5 is a phylogenetic tree based on the 16S rDNA sequence.
FIG. 6 shows R6-3 and LGG at different concentrations of H 2 O 2 Growth profile in culture medium.
FIG. 7 is a graph showing HE staining patterns of mouse livers of different treatment groups, wherein A to E represent a normal group, a model group, an R6-3 group, an LGG group, and a VC group, respectively, and the scale shows 50 μm at a microscope magnification of 400 times.
Figure 8 is a graph of the levels of oxidation indicators in the sera of mice from different treatment groups, wherein P <0.05, P <0.01, and P <0.001 indicate significant differences between the different groups compared in pairs.
Fig. 9 is a graph of the oxidation indicator levels of liver, kidney, brain in mice of different treatment groups, where the upper lower case letters (a-c) represent significant differences in P =0.05 levels compared to different treatment groups.
Fig. 10 is a graph of relative expression of genes related to antioxidant function of liver of mice in different treatment groups, wherein P <0.05, P <0.01, and P <0.001 indicates significant difference between two groups compared with each other.
Figure 11 is a graph of fecal flora composition and differential analysis of mice from different treatment groups, where P <0.05, P <0.01, P <0.001 indicates significant levels of difference between the different groups compared two by two.
Fig. 12 is a graph of correlation analysis, where P <0.05, P <0.01 indicates the level of correlation between the two factors.
Figure 13 is a diagram of an antidepressant animal test protocol.
FIG. 14 is a diagram of a stimulation program schedule for building a CUMS model, wherein water is disabled: the water bottle is removed for 24 hours; fasting: removing the rat food for 24 hours; and (3) cage wetting: spraying 200mL of tap water into the padding for 24 hours; no padding: removing the padding for 24h; the day and night are reversed: adjusting the day and night period, turning on the lamp at night, and covering the mouse cage with black cloth in the day; cold water swimming: swimming the mouse in water at 7 ℃ for 1min; inclined cage: inclining the squirrel cage by 45 degrees for 24 hours; clamping a tail: clamping the part 1cm away from the tail tip with a clamp for 1min; illuminating all over night: turning on the lamp all night at night; and (3) no food intake: the rat food and water bottle were removed for 24h.
FIG. 15 is a graph of body weight change of mice in different treatment groups, where P is compared to P<0.05，**P<0.01，***P<0.001 represents a comparison between the normal group and the model group; a P<0.05， aa P<0.01， aaa P<0.001 represents the comparison between the R6-3 group and the model group; b P<0.05， bb P<0.01， bbb P<0.001 represents the comparison between the drug group and the model group.
Figure 16 is a graph of behavioral indices of mice from different treatment groups, where P <0.05, P <0.01, and P <0.001 indicate significant levels of difference between the different groups compared two by two.
FIG. 17 is a Nicol staining diagram of mouse hippocampus in different treatment groups, wherein A-D respectively represent normal group, model group, R6-3 group, and drug group, and scale represents 400 μm, and microscope magnification is 100 times and 400 times.
Figure 18 is a graph of neuro-biological indicator content for mice in different treatment groups, where P <0.05, P <0.01, and P <0.001 indicate significant levels of difference between the different groups compared two by two.
Figure 19 is a graph of inflammatory factor content in mice from different treatment groups, where P <0.05 and P <0.01 indicate significant levels of difference between the different groups compared in pairs.
Figure 20 is a graph of the levels of oxidation indicators in the sera of mice from different treatment groups, wherein P <0.05, P <0.01, and P <0.001 indicate significant levels of difference between the different groups compared in pairs.
Figure 21 is a graph of oxidation indicator levels of brain tissue of mice in different treatment groups, wherein P <0.05, P <0.01, and P <0.001 indicate significant levels of difference between the different groups compared two by two.
Fig. 22 is a graph of the composition and differential analysis of cecal content flora at KEGG Level3 metabolic pathway in mice of different treatment groups, wherein P <0.05 indicates significant Level of difference compared to the model group.
Figure 23 is a graph of short chain fatty acid content in feces from mice in different treatment groups, where the upper lower case letters (a-c) represent significant differences at the P =0.05 level compared to different treatment groups.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following more detailed description. 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 Serissa acid milk and further characterization
1. Separation, purification and preliminary identification of strains
Gradient dilution is carried out on the senecio yoghourt by using normal saline, proper dilution is selected, MRS and YPD solid culture media are respectively adopted for carrying out coating culture on bacteria and fungi, then bacterial colonies with different forms are selected, and streaking purification is carried out for multiple times until a single bacterial colony is detected by a microscope. And (3) carrying out DNA extraction on the purified strains, respectively carrying out 16S rDNA and ITS sequence amplification sequencing on the bacteria and the fungi, then carrying out homologous sequence comparison, and searching for the strains with high similarity. The strains separated, purified and identified are subjected to de-duplication and numbering, and the activated bacterium liquid is mixed in a glycerol tube with the volume ratio of 20 percent and stored in a refrigerator at the temperature of minus 20 ℃ for later use.
MRS and YPD culture media are respectively utilized to separate and purify bacteria and yeasts from 6 parts of cericid milk, preliminary identification of molecular biology is carried out, 30 strains of bacteria and 5 strains of yeasts are separated and purified in total, and detailed information of the strains is shown in Table 1.
TABLE 1 alignment of the strains
Figure BDA0003957995270000041
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Figure BDA0003957995270000051
2. Preparation of test bacterial solution
Respectively inoculating the preliminarily identified nonpathogenic bacteria and yeast strains into MRS and YPD liquid culture media according to the volume ratio of 1 percent, culturing at 37 ℃ and 30 ℃, activating for 2-3 generations, then respectively culturing for 14h and 24h in the liquid MRS and YPD liquid culture media, centrifuging (4 ℃,8000r/min and 10 min), discarding supernatant, adding sterile physiological saline to wash for 2 times, and then suspending the thalli in 10mL of sterile physiological saline.
3. Determination of strain tolerance to artificial digestive juice
Adding 1.0mL of test bacterial suspension into 9.0mL of artificial gastric juice, mixing uniformly, respectively placing bacteria and yeast in a constant-temperature incubator at 37 ℃ and 30 ℃ for 3h, respectively measuring the viable count of 0h and 3h, and calculating the survival rate of each strain in the artificial gastric juice according to a formula (1):
Figure BDA0003957995270000052
the results are shown in fig. 1, and fig. 1 shows that the strains R1-1, R1-4, R1-5, R1-6, R2-1, R2-6 and R5-2 are completely inhibited from growing by the artificial gastric juice, the survival rate of the strain R4-3 in the artificial gastric juice is very much lower than that of LGG (P < 0.001), the survival rates of the strains R1-3 and R2-3 in the artificial gastric juice are equivalent to that of LGG (P > 0.05), and the survival rates of the other 22 strains in the artificial gastric juice are very much higher than that of LGG (P < 0.001), which indicates that most of the strains screened from the senecio milk have stronger tolerance to the artificial gastric juice, which is caused by the screening of the high-acidity senecio milk for the acid-resistant strains. Therefore, we selected a strain that is stronger than and comparable to LGG in tolerance to artificial gastric juice as a candidate strain for the next step in tolerance to artificial intestinal juice.
4. Determination of artificial intestinal juice resistance of strain
Adding 1.0mL of test bacterial suspension into 9.0mL of artificial intestinal juice, mixing uniformly, respectively placing bacteria and saccharomycetes in a constant-temperature incubator at 37 ℃ and 30 ℃ for 4h, respectively measuring the viable count of 0h and 4h, and calculating the survival rate of each strain in the artificial intestinal juice according to a formula (2):
Figure BDA0003957995270000061
the results are shown in FIG. 2, and it can be seen from FIG. 2 that the survival rates of only the strains R2-5, R4-2 and R6-3 in the artificial intestinal juice are significantly higher than that of LGG (P < 0.001), the survival rates of only the strains R2-2, R3-1, R3-2 and R4-1 in the artificial intestinal juice are completely inhibited from growing, and the survival rates of other strains in the artificial intestinal juice are significantly lower than that of LGG (P < 0.001), which indicates that the intestinal juice resistance of the strain separated from the ceric acid milk is not strong. We select R2-5, R4-2 and R6-3 which have intestinal juice resistance remarkably superior to that of LGG to carry out the next bacteriostatic test.
5. Bacterial inhibition assay
LGG is used as a control strain, and Escherichia coli, staphylococcus aureus, salmonella, shigella flexneri and Listeria monocytogenes are used as pathogen indicator bacteria. Inoculating LGG, R2-5, R4-2 and R6-3 strains in MRS liquid culture medium, culturing at 37 deg.C for 14h, and regulating thallus concentration to (1-2) x 10 by measuring culture solution OD value with MRS liquid culture medium 9 CFU/mL. Culturing pathogen indicator bacteria in LB liquid culture medium at 37 deg.C for 24 hr, washing with sterile physiological saline, resuspending bacteria, and adjusting bacterial suspension concentration to (1-2) x 10 6 And CFU/mL, adding 250 mu L of pathogen indicator bacterial suspension into 25mL of LB agar culture medium in a melting state at 50 ℃, uniformly mixing, pouring the mixture into a flat plate to prepare a bacterial-containing flat plate, punching the solidified flat plate by using a puncher with the diameter of 6mm, adding 100 mu L of bacterial suspension to be detected into the hole, taking sterile MRS as a negative control, placing the flat plate in a constant-temperature incubator at 37 ℃ for culturing for 24 hours, and then measuring the diameter of a bacteriostatic ring.
The results are shown in table 2, and table 2 shows that for e.coli: the diameters of inhibition zones of R4-2, R6-3 and LGG are not obviously different (P is greater than 0.05), and the diameter of the inhibition zone of LGG is obviously greater 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 significantly different (P is more than 0.05), the diameters of the inhibition zones of R4-2 and R6-3 are significantly larger than those of R2-5, and the statistical differences are respectively P <0.01 and P <0.05; for salmonella: the diameters of inhibition zones of R4-2 and R6-3 are not significantly different (P > 0.05), the diameters of inhibition zones of R2-5 and LGG are not significantly different (P > 0.05), but the diameter of the inhibition zone of R4-2 is significantly larger than that of R2-5 (P < 0.01) and LGG (P < 0.01), and the diameter of the inhibition zone of R6-3 is significantly larger than that of R2-5 (P < 0.05) and LGG (P < 0.01); for shigella flexneri: the diameters of inhibition zones of 4 strains of bacteria have no significant difference (P is more than 0.05); for listeria monocytogenes: the zone diameters of R2-5, R4-2 and R6-3 are significantly larger than that of LGG (P < 0.01), and the zone diameters 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 conclusion, the bacteriostatic activity of R6-3 to 5 pathogenic bacteria is better than that of R4-2, and R4-2 is better than that of R2-5 and LGG.
TABLE 2 bacteriostatic Activity of the strains on pathogenic bacteria
Figure BDA0003957995270000071
Note: compared with the different treatment groups, n =3, upper lower case letter ( a -C) represents significant differences at the level of P =0.05, and the upper capital letters (a-C) represent significant differences at the level of P = 0.01.
6. Determination of the adhesion Capacity of the Strain to Caco-2 cells
Inoculating strain LGG, R2-5, R4-2 and R6-3 into MRS liquid culture medium, culturing at 37 deg.C for 14h, washing thallus with sterile normal saline, then re-suspending with MEM culture medium without bovine serum and double antibody, adjusting the concentration of the suspension to (1-2) × 10 8 CFU/mL was used for the next experiment. Caco-2 cells were seeded in 6-well plates at 37 ℃ with 5% CO 2 Culturing in a concentration incubator, replacing culture solution for 1 time in 48h, washing with sterile PBS with pH of 7.2-7.4 for 2-3 times after the cells grow into a monolayer, and washing off dead cells and antibiotics. Then adding 1mL of bovine serum-free and double antibody concentration of (1-2) multiplied by 10 into each hole 8 CFU/mL bacterial suspension, 5% CO at 37 ℃ 2 Incubate for 2h in a concentration incubator. Washed 5 times with sterile PBS pH 7.2-7.4. Adding pancreatin cell with concentration of 0.25% 500 μ L into each well for 3min, adding sterile PBS with pH of 7.2-7.4 500 μ L, and counting viable bacteria. The adhesion rate of the strain to Caco-2 cells was calculated according to the formula (3).
Figure BDA0003957995270000072
The results are shown in FIG. 3, and it can be seen in FIG. 3 that there is no significant difference in the adhesion rates of R6-3 and LGG to Caco-2 cells (P > 0.05), that the adhesion rate of R2-5 to Caco-2 cells is significantly higher than that of R6-3 (P < 0.001) and LGG (P < 0.001), and that the adhesion rate of R4-2 to Caco-2 cells is significantly higher than that of R2-5 (P < 0.001), R6-3 (P < 0.001) and LGG (P < 0.001). I.e., adherence 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 adherence to Caco-2 cells.
Considering comprehensively, the tolerance of the strain to artificial gastric juice and artificial intestinal juice, the bacteriostatic activity to common pathogenic bacteria and the adhesion to Caco-2 cells, the strain R6-3 with stronger artificial gastric juice and bacteriostatic resistance is selected as a target potential probiotic strain for subsequent tests.
7. Identification of strains with potential probiotic properties
The strain R6-3 with potential probiotic characteristics is obtained by screening layer by layer, the strain is subjected to gram staining, the morphology of the strain is observed by using an electron microscope, the strain is coated on an MRS solid culture medium and cultured for 48 hours, and the colony morphology of the strain is observed. Physiological and biochemical identification of R6-3 was carried out according to the instructions for use of the complete biochemical identification tubes for lactic acid bacteria (SHBG 13). Downloading a 16S rDNA sequence of a strain with the highest 16S rDNA sequence homology with R6-3, using MEGA software, utilizing a Neighbor-join statistical method, adopting a Bootstrap method to build a tree, repeating the steps for 1000 times, and calculating the evolutionary distance by using a p-distance method to build a phylogenetic tree.
As a result, as shown in FIG. 4, table 3 and FIG. 5, it can be seen from FIG. 4 that the strain R6-3 is a gram-positive bacterium, and the bacterial body is rod-shaped and nonfilamentous. The R6-3 colony on the MRS solid culture medium is milky white, round and convex, smooth in surface and neat in edge. Table 3 shows that R6-3 can further identify the strain R6-3 as Lactobacillus plantarum by using esculin, maltose, cellobiose, salicin, mannitol, sorbitol, raffinose, sucrose, lactose, inulin and 1% sodium hippurate in comparison with the lactic acid bacteria Classification and Experimental method. FIG. 5 shows that the strain R6-3 and Lactobacillus plantarum are gathered into one branch, and the strain R6-3 is determined to be Lactobacillus plantarum named as Lactobacillus plantarum R6-3 by combining the 16S rDNA sequence comparison result and the physiological and biochemical identification result of the R6-3.
TABLE 3 micro-biochemical identification tube for lactic acid bacteria
Figure BDA0003957995270000081
Note: "+" indicates a positive reaction.
Example 2 in vitro antioxidant Activity assay for R6-3
R6-3 to H 2 O 2 Determination of tolerance ability: LGG as control strain, LGG and Lactobacillus plantarum R6-3 were cultured to initial stable late log phase, and inoculated at a volume ratio of 1% (v/v) to a medium containing 0mmol/L, 0.4mmol/L, 0.8mmol/L, and 1.0mmol/LH 2 O 2 The MRS liquid culture medium is evenly mixed by vortex, cultured for 12h at the constant temperature of 37 ℃, evenly mixed by vortex again, adjusted to zero by a spectrophotometry method, and the cell growth is measured at 600 nm.
The results are shown in FIG. 6, and FIG. 6 shows that H is added to MRS medium 2 O 2 At a concentration of 0.4mmol/L, the growth of Lactobacillus plantarum R6-3 and LGG was not significantly affected (P)>0.05). Adding H into MRS culture medium 2 O 2 At a concentration of 1mmol/L, the growth of Lactobacillus plantarum R6-3 begins to be slowly inhibited: and H 2 O 2 The cell concentration of (2) was not significantly different at a concentration of 0.8mmol/L (P)>0.05 And H) with 2 O 2 The difference in cell concentration was significant when the concentration of (D) was 0.4mmol/L (P)<0.05). LGG in MRS medium is added with H 2 O 2 The concentration of (A) is 0.8mmol/L, the influence is obvious: and H 2 O 2 The difference in cell concentration was very significant when the concentration of (2) was 0.4mmol/L (P)<0.01)，H 2 O 2 The concentration of (2) increased from 0.8mmol/L to 1mmol/L, and the cell concentration decreased again significantly (P)<0.05). The results show that H 2 O 2 Can cause the oxidative damage of the lactobacillus plantarum R6-3 and LGG, and further inhibit the growth of the lactobacillus plantarum R6-3 and LGG. Also shows that the Lactobacillus plantarum R6-3 is H-resistant 2 O 2 The ability to cause oxidative damage is superior to LGG.
Example 3 Lactobacillus plantarum R6-3 relieves oxidative stress in mice by the gut-hepatic axis
1. Design of animal experiments
50 SPF male BALB/c mice 8 weeks old are selected and bred in animal rooms with 22 +/-2 ℃ and 12h alternating illumination/darkness, and the animals are free to drink water and eat. After 1 week of adaptation, the mice were randomly divided into 5 groups (10 mice per group) by body weight, which were designated as a control group, a model group, an R6-3 group, an LGG group, and a VC group, respectively. Mice in 5 groups were weighed 1 time per day, dosed 1 time, injected subcutaneously on the back of the neck 1 time, injected per 10g of mouse weight and gavaged with 0.1mL of liquid. The test period was 8 weeks. The mice in the control group were injected subcutaneously with 200mg/kg D-gal, except for the mice in the control group. The R6-3 group, the LGG group and the VC group of mice respectively gaze lactobacillus plantarum R6-3, LGG bacterial suspension and 50mg/kg VC, and the control group and the model group of mice are gazed with normal saline. LGG group and VC group served as positive strain and positive drug control, respectively.
Feces are directly collected from the anus of the mouse 1 day before the experiment is finished, and each mouse collects two parts, each part is about 0.5g, and the two parts are stored at the temperature of minus 80 ℃ and are used for intestinal flora analysis and SCFAs determination. After the last gastric lavage, the mice are fasted and are not watered for 16h, weighed, blood is taken from eyeballs, and then cervical vertebra is removed for killing. Standing blood at room temperature for 1h, centrifuging (1150g, 10min), and collecting supernatant to obtain serum, and storing at-80 deg.C for antioxidant parameter measurement. The heart, brain, spleen, liver and kidney of each mouse were taken out, weighed and the organ index was calculated according to the formula (8). The liver was cut into 4 portions, 3 of which were immediately frozen in liquid nitrogen and stored at-80 ℃ for antioxidant parameter measurement and real-time quantitative PCR detection of antioxidant gene expression, and 1 portion was fixed in 10% neutral formalin for preparation of histopathological sections. The animal testing method was reviewed and approved by the ethical committee on laboratory animals at the university of agriculture in north and river (2021096).
Figure BDA0003957995270000091
2. HE staining and visualization of liver
Liver tissues of each group of mice were fixed in 10% neutral formalin, then the samples were dehydrated, embedded in normal paraffin, prepared into 5 μm sections, stained with hematoxylin and eosin (H & E) after deparaffinization, observed with an optical microscope and histological images were taken, and read by a pathologist.
The livers of mice from different treatment groups were examined for histopathological changes by HE staining after 8 weeks of the study, 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 mouse in the model group is obviously damaged histologically, and comprises cytoplasm vacuolation, cell swelling, disordered arrangement, irregular shape and deep staining. Compared with the model group, the liver injury of the R6-3, LGG and VC group mice is relieved to different degrees. In particular, the liver structures of the 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 indexes of serum, liver, kidney and brain tissues
The liver, kidney and brain tissue samples were homogenized in 9 times volume of pre-cooled physiological saline for 60s, centrifuged (4 ℃,2010g, 10min), and the supernatant was collected, i.e., the tissue supernatant. The protein concentration of the tissue supernatant was determined using the 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 determined according to the kit instructions.
The effect of Lactobacillus plantarum R6-3 on the oxidation indexes of serum, liver, kidney and brain tissues of D-gal induced oxidative damage mice is shown in FIGS. 8 and 9. As can be seen from fig. 8 and 9, MDA and NO levels in serum, liver, kidney and brain were significantly increased in the model group mice compared to the normal group (P < 0.05). The levels of GSH-PX, SOD, CAT activity and T-AOC in the serum, liver, kidney and brain tissues of the model group of mice are all obviously reduced (P < 0.05) except the level of the serum T-AOC and the renal SOD activity without obvious change (P > 0.05). And the lactobacillus plantarum R6-3, LGG and VC reverse the change of the oxidative stress indexes to a certain extent. Compared with LGG and VC, the Lactobacillus plantarum R6-3 has higher antioxidant capacity and is mainly reflected in the influence 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 the mouse in an oxidative stress state, lactobacillus plantarum R6-3, LGG and VC all relieve the state to a certain extent, and the effect of lactobacillus plantarum R6-3 is better than that of LGG and VC.
4. Real-time quantitative PCR detection of expression of liver Nrf-2 signal channel antioxidation-related gene
Total RNA from liver tissue was extracted using TriQuick Reagent according to the manufacturer's instructions, OD260/OD280 ratio (purity of total RNA) and concentration were measured using an ultraspectrophotometer, and the extracted total RNA was diluted to 1. Mu.g/. Mu.L and then reverse-transcribed to cDNA using PrimeScript RT Reagent Kit with gDNA Eraser Kit. The cDNA is taken as a template, and a Roche LightCycler 96 real-time fluorescent quantitative PCR system and LightCycler 96SW 1.1 software are adopted to carry out real-time fluorescent quantitative PCR analysis. Wherein, the primer, the real-time quantitative PCR reaction system and the reaction program of the antioxidant related genes are respectively shown in tables 4, 5 and 6. Gapdh as internal reference, 2 -△△Ct The relative expression level of the gene is calculated.
The relative expression amount of R6-3 on Nrf-2 antioxidant signal pathway-related genes in liver tissues of mice with D-gal induced oxidative damage is shown in FIG. 10. As can be seen from FIG. 10, the expression levels of mRNA Nrf-2, SOD1, SOD2, CAT, GPx-1, GPx-2, NQO1 and HO-1 in the liver of the model group mice were significantly reduced (P < 0.05) compared with the control group. R6-3, LGG and VC significantly up-regulate the mRNA expression of the genes, and the difference is statistically significant except a few genes (P < 0.05). The result shows that R6-3, LGG and VC activate an Nrf-2 signal channel, and further up-regulate the expression of downstream antioxidant related genes. In comprehensive comparison, R6-3 is superior to LGG and VC in the aspect of up-regulating mRNA expression of genes related to the Nrf-2 signal path.
TABLE 4 fluorescent quantitative PCR primer sequences
Figure BDA0003957995270000101
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Figure BDA0003957995270000111
TABLE 5 fluorescent quantitative PCR reaction System
Figure BDA0003957995270000112
TABLE 6 fluorescent quantitative PCR amplification procedure
Figure BDA0003957995270000113
5. Effect of R6-3 on D-gal-induced oxidative damage of intestinal flora in mice
Randomly selecting 5 samples from each group of the collected mouse feces, placing the samples in a foam box filled with dry ice, and sending the samples to Shanghai Meiji biological medicine science and technology Co., ltd for 16S rRNA amplicon sequencing. Performing quality control on the original sequencing sequence by using Trimmomatic software, and splicing the sequence after the quality control by using FLASH software; OTU clustering (based on 97% similarity) was performed on the sequences using UPARSE software (version 7.0.1090, http:// drive5.Com/UPARSE /), chimera was eliminated using UCHIME software; (ii) performing species class annotation on each sequence using RDP classifier (version 2.11, https:// sourceforce. Net/projects/RDP-classifier), and then comparing (setting comparison threshold 70%) with Silva database (Silva Release138 http:// www. Arb-Silva. De); alpha and beta diversity were analyzed using the Mothur software (version 1.30.1) and the R software (version 3.3.1).
The composition and species differences of the intestinal flora of mice in different treatment groups were analyzed at phylum level and genus level, and the results are shown in fig. 11. The composition and differences of intestinal flora at the phylum level in the mice of the different treatment groups can be seen in fig. 11 (a), and 11 different phyla were identified in the feces of the mice, among which the average relative abundance was highest among the five groups, bacteroidetes (bacteroidata), firmicutes (Firmicutes), proteobacteria (Proteobacteria), actinomyces (actinomycetoma), deironiobacteria (defereribacter), campylobacter (campylobacter), desulfobacteria (desulfobactria) and patescibacter. Fig. 11 (B) shows that the average relative abundances of the model groups bacteroidotita, proteobacteria, deferobacteria and desutrobactera increased from 40.49%, 0.14%, 0.37% and 1.1% to 58.05%, 5.8%, 1.22% and 1.65%, respectively, and conversely, the average relative abundances of firmicites, actinobacteria, campilobacter and patescibacter decreased from 54.06%, 2.03%, 0.80% and 0.84% to 30.69%, 1.79%, 0.52% and 0.24%, respectively, as compared with the normal group. Wherein, the Bacteroidota and Firmicutes with the highest relative abundance have obvious changes, and the statistical differences are P <0.05 and P <0.001 respectively. Lactobacillus plantarum R6-3, LGG and VC reversed the changes in Bacteroides, firmicutes, proteobacteria, camprilobacterota, desufobactrobacteria and Patescibacteria to varying degrees. In addition, the Lactobacillus plantarum R6-3 also improved the increase in the relative abundance of the deferobacteria caused by D-gal injection, while LGG and VC further increased the relative abundance of the deferobacteria in the intestines of D-gal injected mice. Lactobacillus plantarum R6-3, LGG and VC did not contribute to Actinobacteriota changes in the gut of D-gal-injected mice. Furthermore, as can be seen from fig. 11 (C), the ratio of Firmicutes/bacteroidata (F/B) in the intestine of the model group mice was very significantly decreased (P < 0.001) compared to the normal group, but both lactobacillus plantarum R6-3 and LGG could significantly improve this change (P < 0.05). These changes indicate that D-gal injection significantly changes the gut flora of the oxidative stress mice at the phylum level, and that Lactobacillus plantarum R6-3 can ameliorate this change to some extent.
From FIG. 11 (D), the composition and difference of the intestinal flora of mice in different treatment groups at the genus level can be seen, and 137 different genera were identified, among which the first 15 genera with the highest average relative abundance were norak _ f _ Murebacteriaceae, lactobacillus (Lactobacillaceae), alisma (Alisiples), bacteroides (Bacteroides), odorobacter, prevoteriaceae UCG-001 (Prevoteceae _ UCG-001), uncaria (unclassified _ f _ Lachnospiraceae), NK4A136 (Lachnospiraceae _ NK4A136_ group), acinetobacter (Acinetobacter), mucispira, enterobacter (Enunharduces), helicobacter (Helicibacter), spirospiraceae (Lactobacilli), and Clostridium (Clostridium). FIG. 11 (E) shows that the average relative abundance of the model groups Lactobacillus, enterobacter, helicobacter, and norak _ f _ norak _ o _ Clostridia _ UCG-014 was decreased and the average relative abundance of the other genera was increased compared to the normal group. Among them, lactobacillus has a very significant change (P < 0.001). Lactobacillus plantarum R6-3, LGG and VC reversed the changes in norak _ f _ Muricoraceae, lactobacillus, prevotella _ UCG-001, unclassified _ f _ Lachnospiraceae, acinetobacter, helicobacter, norak _ f _ Lachnospiraceae and Desulfovibrio differently. In addition, lactobacillus plantarum R6-3 and VC reversed the changes in Alisipes, and Lactobacillus plantarum R6-3 also reversed the changes in Bacteroides, mucispiralium, and norank _ f _ norank _ o _ Clostridium _ UCG-014. However, L.plantarum R6-3, LGG and VC all had no improving effect on the changes in Odoribacter, lachnospiraceae _ NK4A136_ group and Enterobacter. These changes indicate that the D-gal injection significantly changes the intestinal flora of the oxidative stress mice at the genus level, and the Lactobacillus plantarum R6-3, LGG and VC can improve the changes to some extent.
Comprehensively, D-gal injection changes the distribution and composition of mouse intestinal flora, namely intestinal disorder occurs, lactobacillus plantarum R6-3, LGG and VC can improve the intestinal flora disorder caused by D-gal to a certain extent, the improvement effect is comprehensively compared, and lactobacillus plantarum R6-3 is superior to LGG and VC.
6. Correlation analysis of intestinal microorganisms and liver antioxidant indexes and fecal short-chain fatty acids
The correlation between the species of the intestinal flora on the genus level and the antioxidant index of the liver and the SCFAs content in the feces is respectively detected by the spearman correlation analysis, and the result is shown in figure 12. FIG. 12 (A) shows that the relative abundance of Lactobacillus is significantly positively correlated with both GSH-PX and CAT activities (P < 0.05), the relative abundance of Candidatus-Saccharomyces, colidextracter, norak _ f _ norak _ o _ Clostridia _ UCG-014, norak _ f _ Oscillospiraceae is significantly positively correlated with SOD activities at levels P <0.01, P <0.05, respectively; the relative abundance of Desulfovibrio is in extremely significant negative correlation with the activity of GSH-PX (P < 0.01); the relative abundance of Bacteroides, colidextracter, norak _ f _ Oscillospiraceae was significantly negatively correlated with NO levels (P < 0.05). FIG. 12 (B) shows that the relative abundance of Lactobacillus is significantly positively correlated with propionic acid, isobutyric acid and n-pentanoic acid concentrations (P < 0.05); the relative abundance of the Desulfovibrio is remarkably negative relative to acetic acid and n-butyric acid (P < 0.05); the relative abundance of Mucispirallum is significantly and negatively correlated with acetic acid, propionic acid, n-butyric acid and n-valeric acid, and the correlation levels are P <0.05, P <0.01 and P <0.01 respectively; the relative abundance of norak _ f _ Lachnospiraceae is remarkably and negatively correlated with acetic acid, propionic acid, n-butyric acid and n-valeric acid, and the correlation levels are P <0.05, P <0.01, P <0.05 and P <0.01 respectively; the relative abundance of Prevotellaceae _ UCG-001 was significantly negatively correlated with isobutyric acid (P < 0.05); the relative abundance of Rikenella is obviously negative correlated with n-butyric acid and n-valeric acid (P < 0.05); the relative abundance of unclass _ f _ Lachnospiraceae was significantly negatively correlated with propionic acid, n-pentanoic acid (P < 0.05).
Example 4 Lactobacillus plantarum R6-3 prevention of depression by the gut-brain axis
1. Design of animal experiments
64 male C57BL/6J mice were selected, the breeding conditions were the same as in example 4, and the mice were randomly divided into four groups of 16 mice, each group consisting of a normal group, a model group, an R6-3 group and a drug group, and the weights of the mice were measured weekly, and the specific test schedule is shown in FIG. 13. CUMS is applied to construct a depression mouse model, stimulation of 1 different method is randomly received every day within 8 weeks, the same stimulation cannot be applied for 3 consecutive days, and the animal cannot be expected, and a specific scheme is shown in figure 14. Collecting mouse feces for SCFAs determination; collecting serum after the mice die at the dislocation of cervical vertebra, and using the serum for measuring CORT, oxidation index and inflammatory factor; wherein, 3 mice in each group are randomly selected to take out the whole brain, and the whole brain is fixed in 10% neutral formalin for manufacturing a brain histopathology section; taking out the whole brain tissue and the caecum content of the mouse, immediately quickly freezing by liquid nitrogen, and storing at-80 ℃, wherein the brain tissue is used for measuring monoamine neurotransmitters, BDNF and oxidation indexes, and the caecum content is used for measuring intestinal flora. The animal testing method was reviewed and approved by the ethical committee on laboratory animals of the university of agriculture of north and river (2021095).
2. Effect of R6-3 on CUMS-induced Depression mouse weight
The body weight changes of the mice of the different treatment groups in the 8-week test period are shown in fig. 15, and it can be seen from fig. 15 that the body weight of the mice in the 8-week test period is gradually increased, the body weight of each group in the first two weeks is not different (P > 0.05), the body weight of the mice in the normal group and the body weight of each group in the 3 rd week test period are very significantly different (P < 0.01), the difference level of the normal group and the body weight of the model group in the 4 th to 8 th weeks is P <0.001, the difference level of the R6-3 group and the body weight of the body groups in the 4 th, 5 th, 6 th and 8 th weeks is P <0.01, P <0.05, and the difference level of the R6-3 group and the body weight of the drug group and the body weight of the model group in the 8 th week is P <0.05. It is demonstrated that the CUMS model can slow down the weight gain of mice, while Lactobacillus plantarum R6-3 and fluoxetine can improve the change to different degrees, but cannot recover to normal group level.
3. Behavioural test
(1) Sweet Water Preference Test (SPT)
All mice were fasted for 12h before sucrose preference experiments were performed, and then mice were given equal volumes of two bottles of water: 2% (mass/volume) of sucrose water and tap water, 200 mL/bottle, and after 6h, the positions of the two water bottles were exchanged. After 24h 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)
And (3) sticking a cross bar which is 50cm higher than the ground by using an adhesive tape at a position 1cm away from the tail tip of the mouse to enable the mouse to be in an inverted state, suspending for 6min, struggling and moving the mouse at the beginning, and displaying an desperate state after a period of time when only passive suspension is intermittent and static. After 1min of acclimation, the cumulative immobility time of the mice remaining within 5min was recorded.
(3) Forced swimming Test (Forced Swim Test, FST)
The mice are placed in a swimming bucket with the water depth of 20cm, the diameter of 20cm and the water temperature of 25 +/-1 ℃, and the swimming immobility time is defined as the accumulated time of the mice floating in the water without struggling and only doing slight movement to keep the head floating on the water surface. Forced swimming for 6min, let the mice adapt for 2min first, then record the immobile time of swimming within 4 min.
The effect of R6-3 on sucrose preference, tail suspension immobility time and forced swimming immobility time in CUMS-induced depressed mice is shown in FIG. 16. Sucrose preference test results as shown in fig. 16 (a), the sucrose preference of mice induced by 8-week CUMS treatment was very significantly reduced compared to the normal group (P < 0.001). Compared with the model group, the lactobacillus plantarum R6-3 and fluoxetine both can significantly (P < 0.001) improve the sucrose preference behavior of the mice. But the improvement effects of the two also show very significant difference (P < 0.001), the Lactobacillus plantarum R6-3 can recover the CUMS-induced depression mice to a level which is not significantly different from the normal group (P > 0.05), and the sucrose preference level of the mice after the fluoxetine is improved is still significantly different from the normal group (P < 0.001). The result shows that 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. The results of tail suspension test and forced swimming test are shown in fig. 16 (B) and (C), compared with the normal group, the immobility time of the 8-week CUMS-treated mice in the tail suspension test and the forced swimming test is remarkably increased, and the difference levels are P <0.01 and P <0.001 respectively through statistical analysis. Compared with the model group, the lactobacillus plantarum R6-3 and fluoxetine both can reverse the two despair behaviors of depressed mice remarkably (P < 0.01) and reach the level which has no remarkable difference (P > 0.05) from the normal group. Indicating that the effect of Lactobacillus plantarum R6-3 and fluoxetine is comparable (P > 0.05) in reversing the despair behavior in depressed mice.
In conclusion, the research result shows that compared with the normal group of mice, the model group of mice show that the decrease of the sucrose preference degree and the prolongation of the forced swimming and tail suspension immobility time mean that the mouse is lack of sense of quickness and despair time is increased, and the success of constructing a depression mouse model by using CUMS is shown. Lactobacillus plantarum R6-3 can reverse the behavioral performance of depressed mice to a level that is not significantly different from that of normal mice.
4. Hippocampus Niger staining and Observation
The brain tissues of 3 mice in each group were fixed in 10% neutral formalin, and then sent to Wuhan Severe Biotech Ltd for Nisshi staining, observed by an optical microscope and imaged with histological images, and read by a pathology professional.
After 8 weeks of experiments, the hippocampus of different treatment groups of mice is subjected to Nie's staining to check the histological change of the neuron morphological structure, and the result is shown in figure 17, and as can be seen from figure 17, the neuron cells in the CA1 region of the hippocampus of the normal group of mice are regular in shape, clear in boundary, compact in arrangement, round in nucleus and obvious in nucleolus. The number of the neuron cells of the model group is obviously reduced, the staining is deepened, the shape is irregular and is polygonal, and the nucleus is shrunk, and the changes indicate that the CUMS causes the mouse hippocampal neuron to be damaged, and the cell structural morphology is changed. However, most of the neuronal cells in the R6-3 group and the drug group were very similar to those in the normal group, indicating that Lactobacillus plantarum R6-3 and fluoxetine were effective in restoring the mouse hippocampal tissue damage caused by CUMS.
5. Detection of brain tissue monoamine neurotransmitters, brain derived neurotrophic factor and serum corticosterone
The content of 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE), BDNF, CORT in serum was measured in brain tissue using ELISA (enzyme-linked immunosorbent assay) kits, according to the manufacturer's instructions.
The results are shown in fig. 18, and it can be seen from fig. 18 (a), (B) and (C) that the brain tissue of the CUMS-induced depressed mice has significantly reduced 5-HT, DA and NE contents, with the difference levels of 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 can be improved to different degrees by both lactobacillus plantarum R6-3 and fluoxetine, the levels of improvement of 5-HT by both lactobacillus plantarum R6-3 and fluoxetine are P <0.01, P <0.01 and P <0.05, respectively, the levels of improvement of NE are P <0.001 and P <0.05, respectively, compared with the normal group; as can be seen in fig. 18 (D), the CUMS-induced serum CORT in depressed mice was significantly increased (P < 0.05) compared to the normal group, while lactobacillus plantarum R6-3 could reduce the serum CORT in mice of the depression model group to a level (P > 0.05) that was not significantly different from the normal group, with a very significant effect (P < 0.01). The fluoxetine also has a reducing effect on the serum CORT of the CUMS-induced depressed mice, and the fluoxetine drug group has no significant difference (P > 0.05) from a normal group and a model group, but has a significant difference (P < 0.05) from an R6-3 group; as can be seen in fig. 18 (E), the CUMS-induced BDNF was very significantly decreased in the brain tissue of the depressed mice (P < 0.01) compared to the normal group, while lactobacillus plantarum R6-3 can very significantly (P < 0.001) increase the BDNF in the brain tissue of the mice in the depressed model group to a level that is not significantly different from the normal group (P > 0.05). The fluoxetine also has an increasing effect on BDNF in brain tissues of the CUMS-induced depressed mice, and the fluoxetine drug group has no significant difference (P > 0.05) from a normal group and a model group, but has a very significant difference (P < 0.01) from an R6-3 group.
In conclusion, the lactobacillus plantarum R6-3 has a reversing effect on the reduction of three monoamine neurotransmitters 5-HT, DA and NE and BDNF and the increase of the core hormone CORT of the HPA axis in brain tissues of the CUMS-induced depressed mice, and the lactobacillus plantarum R6-3 can reverse the neurobiological disorder of the CUMS-induced depressed mice.
6. Determination of serum inflammatory factors
The amounts of the inflammatory factors TNF-. Alpha.IL-6, IL-1. Beta., IL-10 in serum were determined by ELISA kits according to the manufacturer's instructions.
As shown in FIG. 19, it can be seen from FIG. 19 that the serum levels of the proinflammatory factors TNF-alpha, IL-6 and IL-1 beta in the CUMS-induced depressed mice are significantly increased (P < 0.05) and the level of the anti-inflammatory factor IL-10 is significantly decreased (P < 0.05) compared with the normal group. Compared with the model group, the Lactobacillus plantarum R6-3 and fluoxetine can reduce the content of TNF-alpha, 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 levels are respectively P <0.05 and P <0.01; the Lactobacillus plantarum R6-3 and fluoxetine can obviously reduce the content of IL-1 beta, and the difference levels are both P <0.05; lactobacillus plantarum R6-3 can increase the IL-10 content very significantly (P < 0.01), and fluoxetine can also increase the IL-10 content, but the difference is not significant (P > 0.05). Compared with the normal group, the R6-3 group and the drug group have no significant difference (P > 0.05), which indicates that the two groups can reverse the states of TNF-alpha, IL-6, IL-1 beta and IL-10 to the normal group. Further, lactobacillus plantarum R6-3 can improve the immune status of CUMS-induced depressed mice.
7. Determination of serum and brain tissue oxygenation indices
The glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities, total antioxidant capacity (T-AOC), and Malondialdehyde (MDA) levels of serum and brain tissue, respectively, were determined using the kit with reference to the manufacturer's instructions.
The results are shown in fig. 20 and 21, and it can be seen from fig. 20 that the CUMS-induced activity of GSH-PX and SOD and T-AOC levels in serum of depressed mice were significantly decreased, and the MDA level was significantly increased, as compared to the normal group, with the difference levels of P <0.01, P <0.05, P <0.001, and P <0.001, respectively. Lactobacillus plantarum R6-3 and fluoxetine can increase GSH-PX activity compared to the model group to a level that is not significantly different from that of the normal group (P > 0.05); the Lactobacillus plantarum R6-3 and fluoxetine can both obviously increase the activity of SOD (P < 0.05), and have no obvious difference with a normal group (P > 0.05); the Lactobacillus plantarum R6-3 and fluoxetine can both increase the level of T-AOC, the difference levels are P <0.05 and P >0.05 respectively, but the difference levels are still significant from the normal group, and the difference levels are P <0.05 and P <0.01 respectively; the Lactobacillus plantarum R6-3 and fluoxetine can reduce the level of MDA, the difference levels are P <0.05 and P <0.001, but the R6-3 group still has significant difference (P < 0.05) from the normal group, and the drug group has no significant difference (P > 0.05) from the normal group; as can be seen in FIG. 21, CUMS-induced significant decrease in GSH-PX and SOD activity, T-AOC levels, and MDA levels were significantly increased in brain tissue of depressed mice compared to the normal group, with differential levels of P <0.01, P <0.05, P <0.001, and P <0.01, respectively. Compared with a model group, the lactobacillus plantarum R6-3 and fluoxetine can obviously improve the GSH-PX activity, the difference level is P <0.05, and the difference level is not obviously different from a normal group (P > 0.05); the Lactobacillus plantarum R6-3 and fluoxetine can obviously increase SOD activity, the difference levels are respectively P <0.01 and P <0.05, and the difference level is not obviously different from that of a normal group (P > 0.05); the Lactobacillus plantarum R6-3 and fluoxetine can obviously increase the T-AOC level, the difference levels are respectively P <0.05 and P <0.001, and the difference level is not obviously different from that of a normal group (P > 0.05); lactobacillus plantarum R6-3 and fluoxetine can significantly reduce MDA level, and the difference levels are P <0.001 and P <0.01 respectively, and have no significant difference (P > 0.05) from the normal group.
In conclusion, CUMS makes the mice under the oxidative stress state, and the lactobacillus plantarum R6-3 can relieve the state to a certain extent and basically reach the normal level.
8. Effect of R6-3 on D-gal-induced oxidative damage of intestinal flora in mice
Randomly selecting 5 samples from each group of the collected mouse cecal contents, placing the samples in a foam box filled with dry ice, and sending the samples to Meiji biological medicine science and technology Co., ltd.
Based on the KEGG database, PICRUSt2 is used for carrying out Level3 Level function prediction analysis on the 16S rRNA sequencing data of different treatment groups of intestinal flora, and the result is shown in FIG. 22. As can be seen from fig. 22: compared with the normal group, the relative abundance of genes of Metabolic pathways (Metabolic pathways) involved in intestinal flora, microbial metabolism in different environments (Microbial metabolism in secondary environment), carbohydrate metabolism (Carbon metabolism), two-component system (Two-component system), glycolysis/Gluconeogenesis (glycogenosis) Metabolic pathways of the mouse in the model group is reduced, but the difference is not significant. The relative abundance of genes involved in amino acid Biosynthesis (Biosynthesis of amino acids), ABC transporters (ABC transporters), ribosomes (ribosomes), quorum sensing (Quorum sensing), starch and sucrose metabolism (Starch and sucrose metabolism), aminoacyl-tRNA Biosynthesis (amino-tRNA Biosynthesis), and Cysteine and methionine metabolism (Cysteine and methionine metabolism) metabolic pathways is enhanced, with ribosomes being the most prominent (P < 0.01). Indicating that the metabolism pathway related to the intestinal flora of the depressed mice induced by CUMS is disordered. Lactobacillus plantarum R6-3 has a reversing effect on genes involved in metabolic pathways, amino acid biosynthesis, ABC transporter, ribosomes, quorum sensing, glycolysis/gluconeogenesis, aminoacyl-tRNA biosynthesis, and cysteine and methionine metabolic pathways, and particularly on genes involved in ribosomal and glycolysis/gluconeogenesis metabolic pathways, the differences were very significant compared to the model group (P < 0.01). Fluoxetine has a regulatory effect on genes involved in the metabolic pathways of microbial metabolism, amino acid biosynthesis, cysteine and methionine metabolism, ribosome and aminoacyl-tRNA biosynthesis in different environments, and particularly ribosome and aminoacyl-tRNA biosynthesis, and compared with a model group, the fluoxetine has a very significant difference (P < 0.01) but is reduced to a level lower than that of a normal group. Therefore, in general, the improvement effect of the lactobacillus plantarum R6-3 is better than that of fluoxetine on the metabolic activity disturbance of the intestinal flora of mice caused by CUMS. Further, it was shown that lactobacillus plantarum R6-3 may mitigate the depressive behavior 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 fecal short-chain fatty acids
Acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid and isovaleric acid were determined in the mouse caecum contents by gas chromatography.
As shown in FIG. 23, it can be seen from FIG. 23 that the content sequence of SCFAs in C57BL/6 mouse feces is: acetic acid, propionic acid, n-butyric acid, isovaleric acid, n-valeric acid and isobutyric acid, and mainly comprises acetic acid, propionic acid and n-butyric acid. Compared with the normal group, CUMS induced a significant decrease in SCFAs in the feces of depressed mice (P < 0.05). Compared with the model group, the lactobacillus plantarum R6-3 can remarkably improve the contents of acetic acid, propionic acid, n-butyric acid and n-valeric acid in the excrement of the mice (P < 0.05) to a level which has no remarkable difference with the normal group (P > 0.05), can also remarkably improve the content of isovaleric acid in the excrement of the mice (P < 0.05), has a remarkable difference with the normal group (P < 0.05) and has no remarkable influence on isobutyric acid in the excrement of the mice (P > 0.05). Fluoxetine can increase the content of acetic acid and propionic acid in mouse feces to a level which is not significantly different from that of the normal group and the model group (P > 0.05), and has no significant influence on other four SCFAs. The experiment shows that the CUMS causes the intestinal SCFAs of the depressed mice to be obviously reduced, the Lactobacillus plantarum R6-3 and the fluoxetine can both play a role in improving to a certain extent, but the effect of the Lactobacillus plantarum R6-3 is better than that of the fluoxetine, which is probably caused by the fact that the Lactobacillus plantarum R6-3 promotes the 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 by using MRS liquid culture medium, culturing for 14h at 37 ℃, then inoculating 3% of inoculum size by volume ratio into a test tube filled with 10mL of sterilized milk, shaking up, and culturing at 37 ℃ until the milk is in a coagulated state. Subculturing to 3 rd time to obtain activated strain, and refrigerating for use.
2. Fermentation preparation of yogurt samples
Redissolving milk powder and water at a ratio of 1: 5, sterilizing at 100 deg.C for 10min, adding 5% white sugar, cooling to 37 deg.C, inoculating 4% Lactobacillus plantarum R6-3, culturing at 37 deg.C until curd (6-8 hr), and transferring to 4 deg.C refrigerator for cold storage.
Example 6 application of Lactobacillus plantarum R6-3 in fermenting fruit and vegetable juice
Respectively taking mango juice, banana juice, purple sweet potato juice, aloe juice, carrot juice, garlic juice and ginger juice, diluting to Brix =12, and sterilizing at 95 ℃ for 5min. Cooling the feed liquid to 30 deg.C, and mixing with 0.002% (1.0 × 10) of Lactobacillus plantarum R6-3 7 CFU/mL) was inoculated into the fermentation base and fermented at 37 ℃. Obtaining the fermented fruit and vegetable juice after 16 hours of fermentation.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. Lactobacillus plantarum (C) (A) of cerimus acid milk sourceLactobacillus plantarum) R6-3, which is characterized in that the preservation number is CGMCC NO.25884.
2. Lactobacillus plantarum (C) of claim 1, derived from a source of cerimus acidLactobacillus plantarum) Application of R6-3 in preparing antioxidant composition.
3. Lactobacillus plantarum of claim 1 (I)Lactobacillus plantarum) The application of R6-3 in preparing antidepressant compositions.
4. Lactobacillus plantarum of claim 1 (I)Lactobacillus plantarum) Application of R6-3 in preparing a composition for preventing oxidative aging by regulating intestinal flora.
5. Lactobacillus plantarum (C) of claim 1, derived from a source of cerimus acidLactobacillus plantarum) The application of R6-3 in preparing a composition for preventing depression by regulating intestinal flora.
6. A composition comprising the Lactobacillus plantarum (C) derived from Sailikurai as defined in claim 1Lactobacillus plantarum）R6-3。
7. A food product comprising the Lactobacillus plantarum (F) (L) derived from a Saili yogurt as defined in claim 1Lactobacillus plantarum）R6-3。
8. A food additive characterized by comprising the Lactobacillus plantarum (C) derived from Sailikura according to claim 1Lactobacillus plantarum）R6-3。
9. A microbial preparation comprising the Lactobacillus plantarum (C) derived from Saili yogurt as defined in claim 1Lactobacillus plantarum）R6-3。
10. A pharmaceutical composition comprising the Lactobacillus plantarum (C) derived from Sailippic acid milk of claim 1Lactobacillus plantarum）R6-3。
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