CN110684697A - Lactobacillus fermentum JX306 with antioxidant function and application thereof - Google Patents
Lactobacillus fermentum JX306 with antioxidant function and application thereof Download PDFInfo
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- CN110684697A CN110684697A CN201911107792.1A CN201911107792A CN110684697A CN 110684697 A CN110684697 A CN 110684697A CN 201911107792 A CN201911107792 A CN 201911107792A CN 110684697 A CN110684697 A CN 110684697A
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- lactobacillus fermentum
- lactobacillus
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Abstract
The invention discloses Lactobacillus fermentum JX306 with an antioxidant function and application thereof, belonging to the technical field of bioengineering. The strain has strong DPPH free radical and hydroxyl free radical scavenging ability, high reducing power and strong Fe2+Chelating ability, good lipid peroxidation resistance, good bile salt resistance, gastrointestinal fluid resistance and hydrogen peroxide tolerance; can also obviously increase the activity of glutathione peroxidase, reduce the level of malondialdehyde, obviously up-regulate the levels of five antioxidant enzymes of TR3, Prdx1, Gsr, Gpx1 and nuclear transcription factor Nrf2 and obviously relieve histopathological injury induced by D-gal. Fermentation according to the inventionThe lactobacillus JX306 has great effect on improving the oxidation resistance of food as a fermentation strain, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to lactobacillus fermentum JX306 with an antioxidant function and application thereof.
Background
Oxidative stress is considered as an imbalance between high levels of active oxygen and low activities of antioxidant mechanisms, causing various diseases such as amyotrophic lateral sclerosis, asthma, allergy, diabetes, and further accelerating aging. Antioxidants Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT) and Tertiary Butyl Hydroquinone (TBHQ) in food products have been used to control oxidative stress, and in recent years their safety has been questioned. Lactic acid bacteria (lactobacillus) are important probiotics in the human intestinal tract, and when taken as a dietary supplement or through food (whether fermented or fortified) control the oxidative stress of the body or tissues, providing health benefits.
Lactic Acid Bacteria (LAB) are a general term for a class of bacteria that can ferment carbohydrates to produce large amounts of Lactic acid, are closely related to humans, and widely exist in naturally fermented dairy products, fermented vegetables, and human intestinal tracts. The lactobacillus fermentum is one of lactic acid bacteria, not only improves the nutritive value of the food, but also can be planted in the gastrointestinal tract to play a probiotic role. In the currently published documents and patents, for example, CN107603925A discloses a lactobacillus fermentum inm25 with cholesterol lowering function and its application. CN104430847B discloses a lactobacillus fermentum Lee with health care function for preventing constipation. CN102839135B discloses lactobacillus fermentum for high yield of gamma-aminobutyric acid and application thereof.
However, the patent or patent application of lactobacillus fermentum, which is a traditional fermented vegetable in China as a separation source and has strong free radical scavenging capacity, hydrogen peroxide resistance, good gastrointestinal fluid resistance, oxidation resistance and other performances, is not related at home. The research of lactobacillus fermentum in relieving oxidative stress is still in the initial stage compared to other probiotics, and the key involved molecules in the antioxidant mechanism of lactobacillus fermentum at molecular level are still unknown, which is crucial for how to correctly convert animal model findings into human-related therapies. Therefore, the research on the antioxidation mechanism of the lactobacillus fermentum is of great significance.
Disclosure of Invention
An object of the present invention is to provide an antioxidant Lactobacillus fermentum JX306 strain capable of scavenging free radicals and being resistant to hydrogen peroxide.
It is another object of the present invention to provide the use of said lactobacillus fermentum in fermented food products.
The Lactobacillus fermentum JX306 with antioxidant function provided by the invention is preserved in the China general microbiological Culture collection Center (CGMCC) in 2019, 9 and 17 months, and the addresses of the preservation Center are as follows: the China general microbiological culture Collection center (CGMCC) has a collection number of CGMCC No. 18493.
The invention relates to a Lactobacillus fermentum JX306 strain which is derived from Jiangxiaji old jar pickled vegetables. The Lactobacillus fermentum JX306 strain has obvious advantages in DPPH free radical scavenging capacity and gastrointestinal fluid resistance compared with other Lactobacillus. Can obviously increase the transcription level of antioxidant related genes and effectively inhibit the D-galactose-induced histopathological damage of the kidney, the liver and the skin of an aging mouse. The lactobacillus fermentum of the invention provides a foundation for developing probiotic products.
As a second object of the present invention, there is provided a use of the Lactobacillus fermentum JX306 strain in fermented foods, which are fermented yogurts of lactic acid bacteria, probiotic capsule products of sodium alginate, and probiotic tablets.
Compared with the prior art, the invention has the beneficial effects that:
(1) the lactobacillus fermentum provided by the invention is derived from a lactobacillus strain screened from old jar pickled Chinese cabbage of Jian, Jiangxi, China, is identified As lactobacillus fermentum by utilizing the microbiological characteristics such As morphological characteristics, culture properties, physiological and biochemical characteristics and the like, is derived from traditional fermented vegetables, and belongs to Generally Recognized As Safe (GRAS) strains;
(2) as can be seen from the test results, the Lactobacillus fermentum (Lactobacillus ferm) provided by the present inventionenum) JX306 strain at a cell concentration of 4X 108CFU/mL, DPPH radical clearance of 37.29%, hydroxyl radical clearance of 37.90%, reducing power of 83.95%, Fe2+The chelating capacity is 54.38%, the lipid peroxidation resistance is 28.14%, the sodium alginate liposome can tolerate hydrogen peroxide with the initial concentration of 1.5mM, the sodium alginate liposome can survive for more than 3h in a bile salt solution, the survival rate in artificial gastric juice is 53.05%, and the survival rate in artificial intestinal juice is 42.07%. Can improve the activity of superoxide dismutase and glutathione peroxidase, can remarkably up-regulate the relative expression quantity of 5 antioxidant genes on the molecular level, and can inhibit histopathological damage caused by D-gal.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is colony morphology of Lactobacillus fermentum JX 306;
FIG. 2 shows the bacterial morphology of Lactobacillus fermentum JX 306;
FIG. 3 is a nucleic acid sequence diagram of Lactobacillus fermentum JX 306;
FIG. 4 is a growth curve of Lactobacillus fermentum JX 306;
FIG. 5 is the tolerance of Lactobacillus fermentum JX306 to hydrogen peroxide;
FIG. 6 shows the relative expression level of TR3 gene of Lactobacillus fermentum JX 306;
FIG. 7 shows the relative expression level of Prdx1 gene of Lactobacillus fermentum JX 306;
FIG. 8 shows the relative expression level of Gsr gene in Lactobacillus fermentum JX 306;
FIG. 9 shows the relative expression level of Gpx1 gene of Lactobacillus fermentum JX 306;
FIG. 10 shows the relative expression levels of the Nrf2 gene of Lactobacillus fermentum JX 306;
FIG. 11 is HE staining of different treated kidneys with Lactobacillus fermentum JX 306;
FIG. 12 is HE staining of different treated livers of Lactobacillus fermentum JX 306;
FIG. 13 is HE staining of Lactobacillus fermentum JX306 differently treated skin.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: obtaining and characterizing Lactobacillus fermentum JX306
Separation and purification of Lactobacillus fermentum JX306
The solution of Jiangxi Ji Anlatan pickled vegetable is diluted with sterile saline (0.85%, w/v) in turn, spread on MRS agar plate (containing 0.75% CaCO in MRS liquid culture medium)3And 1.5% agar powder). After static culture for 48h at 37 ℃, separating according to the size, color, shape and edge characteristics of the colony, and repeatedly streaking and purifying on an MRS solid culture medium until a pure single colony appears on the solid culture medium. Continuously passaging the separated lactobacillus for more than 5 times, screening out strains with stable growth state and high growth speed, culturing at 37 ℃ to a stable stage, and storing the strains in MRS liquid culture medium containing 20% glycerol at-80 ℃.
Identification of Lactobacillus fermentum JX306
Characterization of Lactobacillus fermentum JX306 species
The Lactobacillus fermentum JX306 frozen at the temperature of minus 80 ℃ is streaked and inoculated on an MRS solid culture medium, and the mixture is kept still for 48 hours at the temperature of 37 ℃. Observing the characteristics of the size and shape, color, transparency, whether the edges are neat or not, whether the surface is smooth or not, transparency, color of the colony and the like of the colony.
As shown in FIG. 1, the strain JX306 had a diameter of about 0.5 to 2.0mm, a round shape, a uniform edge, a moist and smooth surface, no coloring matter, and a milky colony.
As a result of observing the shape of individual cells under a microscope after gram staining, JX306 was a gram-positive bacterium and the individual cells were rod-shaped as shown in FIG. 2.
2.16 amplification and sequencing of S rDNA sequences
Taking out the frozen Lactobacillus fermentum JX306 single colony at-80 ℃, streaking, extracting the single colony to MRS liquid culture medium, culturing for 18-20h, extracting DNA according to the instruction of a Bateria Genomic DNA Kit (Kangji, China), and using the extracted DNA for the amplification of a 16Sr DNA sequence.
The amplification primer for the 16S rDNA gene sequence is a bacterial universal primer, and the upstream primer is as follows: 27F5 '-AGAGAGTTTGATCCTGGCTCAG-3' downstream primer is: 1492R 5'-GGTTACCTTGTTACGACTT-3'.
The PCR reaction system (50. mu.L) is shown in Table 1
TABLE 1 PCR reaction System (50. mu.L)
The PCR reaction conditions are shown in Table 2
TABLE 2 PCR reaction conditions
Electrophoresis of the amplification product: the PCR amplification product of the DNA was detected by 1.5% agarose gel electrophoresis, as shown in FIG. 3, a specific amplification fragment was obtained at 1500bp observed with a gel imager, the target band was clear and bright, and the recovered product was sequenced by Bejing Ruibo BiotechCo., Ltd after passing the detection.
The nucleotide sequence of 16S rDNA of Lactobacillus fermentum JX306 is sequence 1 in the sequence table.
Lactobacillus fermentum JX306 physiological and biochemical properties and carbohydrate fermentation experiment
The physiological and biochemical characteristics of Lactobacillus fermentum JX306 are measured, the physiological and biochemical characteristics mainly comprise a starch hydrolysis experiment, an esculin hydrolysis experiment, a urease measurement experiment, an arginine ammonia production experiment, a hydrogen sulfide production experiment, a gelatin liquefaction experiment and a peroxidase measurement experiment, and the measurement results are shown in Table 3.
TABLE 3 determination of physiological and biochemical Properties of JX306
| Name of experiment | Results |
| Starch hydrolysis test | - |
| Hydrolysis test of esculin | + |
| Urease assay test | - |
| Arginine ammonia production experiment | + |
| Experiment for production of Hydrogen sulfide | - |
| Experiment of liquefaction of gelatin | - |
| Peroxidase assay test | - |
The results of the fermentation of different carbohydrates by the Lactobacillus fermentum JX306 strain are shown in Table 4.
TABLE 4 fermentation results of JX306 on different carbohydrates
Note: "+": indicating a positive reaction; "-": indicating a negative reaction
Growth Curve determination of the Lactobacillus fermentum JX306 Strain
Lactobacillus fermentum JX306 is inoculated into MRS liquid culture medium with the inoculation amount of 1% (v/v), cultured at 37 ℃, taken out every 2h, adjusted to zero by taking the MRS culture medium as a blank, and the absorbance value is detected at the wavelength of 600 nm. And (3) taking the time as an abscissa and the light absorption value as an ordinate to obtain a growth curve of the strain. The growth curve results are shown in fig. 4. The results show that the lag phase of JX306 is only about 2h, then the growth logarithmic phase begins to enter, and the stationary phase enters about 12 h.
Example 2 in vitro antioxidant indicator assay of Lactobacillus fermentum JX306
Inoculating Lactobacillus fermentum JX306 at 1% (v/v) in 1.0mL MRS liquid culture medium, culturing at 37 deg.C for 24h, centrifuging at 8000 Xg for 10min, washing with sterile water for 2 times, adjusting thallus cell concentration to 4X 108CFU/mL。
DPPH free radical scavenging experiment
Adding 1.0mL of 0.2mmol/L DPPH solution (dissolving DPPH with anhydrous ethanol) into 1.0mL of thallus cells, mixing, reacting at room temperature in the dark for 30min, centrifuging at 8000 Xg for 10min, and collecting supernatant to measure absorbance at 517 nm. The measurement result shows that the DPPH free radical clearance rate of Lactobacillus fermentum JX306 is 37.29%, and the Lactobacillus fermentum JX shows better free radical clearance capacity.
Second, hydroxy radical scavenging ability test
1.0mL of somatic cells, 0.5mL of o-phenanthroline (2.5mmol/L) and 1.0mL of LPBS buffer solution (0.01mol/L, pH7.4), mixing the above solutions, and adding 0.5mL of sulfuric acid (2.5mmol/L) and 0.5mLH2O2(20mmol/L), incubated at 37 ℃ for 1.5h in a thermostated water bath, and the absorbance measured at 536 nm. The measurement result shows that the clearance rate of the hydroxyl radical of Lactobacillus fermentum JX306 is 37.90 percent.
Third, determination of reducing power
0.5mL of the bacterial cells, 1.5mL of PBS (0.1mol/L, pH 6.6), and 1.5mL of potassium ferricyanide solution (1%, w/v). The solution was mixed well and incubated in a 50WC water bath for 20 minutes. Then cooled in an ice bath, 1.5mL of trichloroacetic acid (TCA, 10%, w/v) was added, shaken, centrifuged at 8000 Xg for 10min, and 1.0mL of the supernatant was transferred to a centrifuge containing 1.0mL of sterile physiological saline (0.85%, w/v) and 0.5mL of FeCl3(0.1%, w/v) in a fresh tube. The solution was shaken well, allowed to stand for 10 minutes, and the absorbance at 700nm was measured. The results of the measurement showed that the reduction power of Lactobacillus fermentum JX306 was 83.95%.
Fourth, Fe2+Determination of chelating Capacity
0.5mL of bacterial cells, 0.1mL of ascorbic acid (1%, w/v), 0.1mL of FeSO4(0.4%, w/v) and 1.0mL of sodium hydroxide (0.2mol/L) were mixed and placed in a 37WC water bath for 20 minutes. Then precipitating the protein with 0.5mL of trichloroacetic acid (TCA, 10%, w/v), centrifuging for 10min at 8000 Xg, then uniformly mixing 0.5mL of supernatant with 4.0mL of phenanthroline (0.1%, w/v), carrying out water bath with 37WC for 10min, centrifuging for 10min at 8000 Xg, and measuring the absorbance of the supernatant at 510 nm. The measurement result shows that the Fe of Lactobacillus fermentum JX3062+Chelating energyThe force was 54.38%.
Anti-lipid peroxidation ability
1.0mL of bacterial cells, 0.5mL of LPBS buffer (0.01mol/L, pH7.4), and 0.2mL of FeSO4(0.01%, w/v), 0.02mL of ascorbic acid (0.01%, w/v), 1.0mL of linoleic acid emulsion (20.0mL of linoleic acid emulsion: 0.2mL of Tween 20, 0.1mL of linoleic acid, 19.7mL of sterile water), thermostatting at 37 ℃ for 12 hours, adding 0.2mL of trichloroacetic acid (TCA, 4%, w/v), 2mL of thiobarbituric acid (TBA, 0.8%, w/v) and 0.2mL of butylhydroxytoluene (BHT, 0.4%, w/v), and mixing. Water bath at 100 deg.C for 30min, ice bath cooling, butanol extraction of 2mL, centrifugation at 8000 Xg for 10min, and measuring absorbance of supernatant at 532nm wavelength. The measurement results showed that the lipid peroxidation resistance of Lactobacillus fermentum JX306 was 28.14%.
Example 3 in vitro tolerance assay of Lactobacillus fermentum JX306
Inoculating Lactobacillus fermentum JX306 at 1% (v/v) in 1.0mL MRS liquid culture medium, culturing at 37 deg.C for 24h, centrifuging at 8000 Xg for 10min, washing with sterile water for 2 times, adjusting thallus cell concentration to 4X 108CFU/mL。
Determination of bile salt resistance
Adjusting the cell concentration of the cells to 4X 108CFU/mL, resuspended in 1mL sterile saline solution containing 1mg/mL pancreatin and 0.5% (w/v) bile salts. The cells are incubated for 1h, 2h and 3h at 37 ℃, diluted and coated on an MRS plate, and the survival rate of the strain is measured by a plate counting method after incubation for 48h at 37 ℃. The results of the measurement are shown in Table 5, and it was revealed that the survival rate of the strain showed a decreasing tendency with the lapse of time.
TABLE 5 bile salt resistance of the strains
Ability to withstand gastrointestinal fluids (GI)
Bacterial cells (4X 10)8CFU/mL), suspending in 1mL artificial gastric juice or intestinal juice, culturing thallus cells in artificial gastric juice at 37 deg.C for 3h, and culturing thallus cells in artificial intestinal juiceCulturing at 37 deg.C for 24h, diluting, spreading on MRS plate, incubating at 37 deg.C for 48h, and determining the survival rate of the strain by plate counting method. The results of the measurement are shown in Table 6, and the results show that the strain exhibits better gastrointestinal fluid resistance.
TABLE 6 gastrointestinal fluid (GI) resistance
Third, hydrogen peroxide resistance
Bacterial cells (4X 10)8CFU/mL), with 0mM, 0.4mM, 0.8mM, 1.2mM, 1.5mM of H, respectively2O2Resuspending, standing at 37 deg.C, and measuring the cell concentration at 600nm every 2 h. The results are shown in FIG. 5, and show that H is detected at different concentrations2O2Tolerance, increase H2O2Concentration, prolonged lag phase, indicates H2O2Resulting in oxidative damage, while Lactobacillus fermentum JX306 was still viable at 1.5mM, shown to be responsible for H2O2Good tolerance.
Example 4 mechanism of repair of D-gal-induced oxidative damage by Lactobacillus fermentum JX306
The experimental procedures for the mice were performed strictly in accordance with ethical guidelines of the Shandong university Committee animal welfare agency. 60 male KM mice (20. + -.2 g) were randomly divided into 6 groups of 10 mice each, and the specific grouping was as shown in Table 7. During the experiment, the model group mice had obvious signs of aging. For example, the skin of the mouse gradually shrinks, loses luster, and the elasticity of the skin is weakened. In addition, mice exhibit symptoms such as depression, lethargy, and the like.
TABLE 7 mouse grouping
Note: NC: a normal group; MC: a model group; PC: a positive control group; JFL: a low dose group; JFM, respectively; a medium dose group; JFH: high dose group.
Oxidative stress product detection and analysis
1. Determination of MDA levels in serum, kidney and liver tissues
The specific operation steps of the experiment are operated according to the kit instruction provided by Nanjing institute of built-in bioengineering.
The measurement results are shown in table 8, and the detection results show that the MDA level in the model group is significantly increased compared with that in the normal group, which indicates that the model of the D-gal-induced oxidative damage in mice is successful. Different feeding doses of JX306 can down-regulate the MDA level in serum, kidney and liver to different degrees, and the MDA down-regulated level has a dose-effect relationship with the gavage dose.
TABLE 8 Effect of different treatments on mouse MDA (serum, kidney and liver)
Note: there were differences in the mean values within the different superscript letter lists (P < 0.05). Data are presented as mean ± standard deviation.
2. Determination of GSH-Px Activity in serum, Kidney and liver tissues
The specific operation steps of the experiment are operated according to the kit instruction provided by Nanjing institute of built-in bioengineering.
The measurement results are shown in table 9, and the measurement results show that the GSH-Px levels in the model group are significantly reduced compared to the normal group. Different feeding doses of JX306 can up-regulate the GSH-Px level in serum, kidney and liver to different degrees, and the up-regulated level of the GSH-Px has dose-effect relation with the gavage dose.
TABLE 9 Effect of different treatments on mouse GSH-Px (serum, kidney and liver)
| Group of | Serum (U/mL) | Kidney (U/mgprot) | Liver (U/mgprot) |
| NC | 366.49±3.57b | 413.55±33.43b | 508.94±3.82a |
| MC | 328.84±5.88c | 208.49±10.12d | 304.44±17.95d |
| PC | 390.31±9.27a | 464.91±33.35a | 476.35±21.01ab |
| JFL | 345.02±2.33b | 292.42±8.49c | 380.25±4.74c |
| JFM | 356.49±3.85b | 375.59±4.03b | 442.48±36.31b |
| JFH | 373.26±1.76b | 413.45±7.24b | 451.8±16.78b |
Note: there were differences in the mean values within the different superscript letter lists (P < 0.05). Data are presented as mean ± standard deviation.
3. Determination of superoxide dismutase (SOD) Activity in serum, Kidney and liver tissues
The specific operation steps of the experiment are operated according to the kit instruction provided by Nanjing institute of built-in bioengineering.
The measurement results are shown in table 10, and the measurement results show that the SOD levels were significantly reduced in the model group compared to the normal group. Different feed doses of JX306 were treated with no significant difference.
TABLE 10 Effect of different treatments on mouse superoxide dismutase (serum, kidney and liver)
| Group of | Serum (U/mL) | Kidney (U/mgprot) | Liver (U/mgprot) |
| NC | 300.54±3.41bc | 410.13±12.04ab | 493.67±3.99a |
| MC | 266.13±7.39d | 380.60±8.59c | 458.85±0.05b |
| PC | 293.77±10.40c | 398.33±3.26b | 468.8±10.15b |
| JFL | 284.80±14.88c | 386.73±19.35bc | 459.29±1.65b |
| JFM | 311.4±5.29b | 410.64±14.93ab | 483.40±4.02a |
| JFH | 329.3±4.50a | 427.17±6.29a | 483.13±9.02a |
Note: there were differences in the mean values within the different superscript letter lists (P < 0.05). Data are presented as mean ± standard deviation.
4. Determination of Total antioxidant Capacity (T-AOC) Activity in serum, Kidney and liver tissues
The specific operation steps of the experiment are operated according to the kit instruction provided by Nanjing institute of built-in bioengineering.
The results of the measurements are shown in Table 11, and the results of the measurements show that the levels of T-AOC in the model group are significantly reduced compared to the normal group. Different feeding doses of JX306 can up-regulate the T-AOC level in serum, kidney and liver to different degrees, and the up-regulated level of the T-AOC has dose-effect relationship with the gavage dose.
TABLE 11 Effect of different treatments on the Total antioxidant Capacity of mice (serum, Kidney and liver)
| Group of | Serum (μmol/gprot) | Kidney (mu mol/gprot) | Liver (mu mol/gprot) |
| NC | 917.45±65.48a | 287.33±13.73a | 1130.64±41.60a |
| MC | 613.95±36.52d | 57.34±3.15d | 672.76±37.59c |
| PC | 756.77±47.57bc | 181.26±29.78b | 1035.08±82.90ab |
| JFL | 641.25±71.87cd | 87.80±1.82d | 704.26±21.44c |
| JFM | 772.53±75.61bc | 129.80±11.36c | 1004.62±17.35b |
| JFH | 793.53±45.58ab | 202.27±33.34b | 1057.13±60.79ab |
Note: there were differences in the mean values within the different superscript letter lists (P < 0.05). Data are presented as mean ± standard deviation.
Second, RT-qPCR method is used for detecting the relative expression levels of TR3, Prdx1, Gsr, Gpx1 and Nrf2 genes
Total RNA was extracted from liver tissue by RNAioso Plus following the manufacturer's Takara instructions. By OD260/OD280And agar gel electrophoresis to determine the purity and quality of total RNA. Then, cDNA was synthesized using the PrimeScriptIIFirst Strand cDNAsSynthesis Kit. RT-PCR was performed using 7500Fast Real-Time PCR System and SYBR Green PCR kit. The PCR procedure consisted of denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 5s, and annealing extension at 60 ℃ for 30s for 40 cycles. 2 relative expression levels of 5 antioxidant enzyme genes in liver, such as TR3, Prdx1, Gsr, Gpx1, Nrf2 and the like-ΔΔCTIt is shown that beta-actin is used as an internal reference gene.
The results of measuring the influence of different experimental treatments on the relative expression amounts of TR3, Prdx1, Gsr, Gpx1 and Nrf2 genes in the liver of the oxidative-damaged mouse are shown in FIGS. 6-10, and the results show that the relative expression amounts of the TR3, Prdx1, Gsr, Gpx1 and Nrf2 genes in the liver of the mouse in the model groupThe expression level of the antioxidant enzyme gene is obviously reduced compared with that of the normal group. Different doses of JX306 and antioxidant V compared to model groupCThe treatment can improve the expression level of the antioxidant enzyme genes to different degrees, and the high-dose JX306 can obviously improve the expression level of the antioxidant enzyme genes.
Effects of different experimental treatments on mouse kidney, liver and skin morphology
The kidneys, liver and skin of each group were fixed in 10% omalin buffer and paraffin embedded. The slices were cut to a thickness of 4 to 5 μm, HE-stained, then stained with hematoxylin and eosin, and the degree of lesion of the sections was evaluated using an optical microscope.
1. HE staining of kidney tissue with different treatments
FIG. 11 shows that the normal group of renal tubules and glomeruli are intact in morphological structure. The histological pathological section of the kidney of the model group shows that the glomerulus is seriously injured, the tubulointerstitial lesion, the brush edge is fallen off, and the renal tubular epithelial cells are vacuolated. Adopting Lactobacillus fermentum JX306 with different thallus concentrations and positive antioxidant VCThe mice with the gastric perfusion D-gal induced oxidative damage can reduce the pathological changes of glomeruli and renal tubules in the kidney tissues of the mice to different degrees and present dose-effect relationship.
2. HE staining of liver tissue with different treatments
FIG. 12 shows that the normal group of hepatocytes showed large and round nuclei, distinct nucleoli and intact cytoplasm. The histological pathological section of the kidney of the model group showed necrotic lesions, edematous degeneration and vacuolar degeneration of the liver hepatocytes. Adopting Lactobacillus fermentum JX306 with different thallus concentrations and positive antioxidant VCThe mice with the oxidative damage induced by the gavage D-gal can reduce the pathological change degree in the liver cells of the mice to different degrees and present a dose-effect relationship.
3. HE staining of skin tissue with different treatments
As shown in fig. 13, the model set results were mainly manifested as damage to the hair follicle. Adopting Lactobacillus fermentum JX306 with different thallus concentrations and positive antioxidant VCGavage D-gal induced oxidative injury in mice, noneThe lesion degree of the mouse skin section is reduced to the same degree, and the dose-effect relationship is presented.
Application example 1: lactobacillus acidophilus milk prepared by Lactobacillus fermentum JX306
Firstly, inoculating 2 percent (volume) of the Lactobacillus fermentum JX306 original strain into skim milk sterilized at 110 ℃ for 10min, culturing at 37 ℃ until the strain is curd, and continuously culturing and activating for two generations to be used as a mother starter; inoculating the mother starter to sterilized milk at 37 deg.C for 4-8 hr, and culturing to obtain curd with viable count of about 109CFU/mL, to obtain the working starter, can be added directly to food products or used with commercial starters for symbiotic fermented milks such as Lactobacillus bulgaricus and Streptococcus thermophilus to make fermented milks.
Then, the raw milk is heated and sterilized for 20min at the temperature of 95 ℃, then is rapidly cooled to about 37 ℃, and is inoculated with the Lactobacillus fermentation JX306 working leavening agent according to the inoculation amount of 2-3 percent (volume) to ensure that the concentration of the Lactobacillus fermentation JX306 working leavening agent reaches 106Cooling to 10 ℃ above CFU/mL, transferring to a refrigerator for low-temperature maturation, and obtaining the lactobacillus acidophilus milk containing the lactobacillus fermentum JX306 viable bacteria.
In the embodiment, the raw milk is skim milk; the MRS liquid culture medium is a culture medium for culturing lactic acid bacteria sold by Qingdao Gaokoubo biotechnology limited;
the high-temperature sterilization comprises the following steps: for example, a vertical autoclave of the type LDZX-50KBS sold by Shanghai Shenan medical instruments factory is used.
Application example 2: lactobacillus fermentum JX306 makes sodium alginate based probiotic capsule preparations.
Inoculating 12% (w/v) of activated Lactobacillus fermentum JX306 with inoculum size of 4% into optimized MRS enrichment medium, neutralizing with fed-batch alkaline solution, and culturing with 20% (w/v) Na2CO3Maintaining the pH of the culture medium to be 6.60 constant, carrying out enrichment culture on the thalli for 15h, and then centrifuging and collecting the thalli to obtain wet bacterial sludge. The bacterial sludge and the protective agent are mixed according to the mass ratio of 1:3Mixing uniformly, at which time the thallus cell concentration is 1010CFU/mL。
And then embedding three layers, wherein the first layer of embedding material is soybean protein isolate liquid, the second layer is microporous starch solution, the third layer is sodium alginate solution, and the three layers are required to be uniformly stirred before each layer is embedded. At this time, CaCl was added dropwise to the mixture2Solidifying the solution into microspheres, rinsing the microspheres with sterile water, pre-freezing the microspheres at a low temperature, and freeze-drying the microspheres in vacuum to obtain the Lactobacillus fermentum JX306 microcapsules.
Centrifuging by adopting a centrifuge eppendorf centrifuge 5424R of Shanghai Aijian biological science and technology limited;
the freeze drying is carried out by vacuum freeze drying SCIENTZ-10Z of Ningbo Xinzhi Biotechnology GmbH;
the low-temperature pre-freezing is ultra-low temperature refrigerator DW-HL3985 of Mike Mitsubishi low-temperature science and technology, Inc.;
stirring was carried out by using a Kyosu Jintanda automated Instrument factory CJ 78-1.
Application example 3: preparation of probiotic tablet by using Lactobacillus fermentum JX306
Activating Lactobacillus fermentum JX306, performing amplification culture at 37 deg.C for 18-24 hr, centrifuging, collecting thallus, and spraying bacteria solution containing thallus protectant, wherein the viable count is 1.0 × 109CFU/mL, spray drying to obtain viable bacteria powder.
And then, taking spray-dried mushroom powder as a main material, taking corn starch, dextrin, starch paste powder, mannitol and magnesium stearate as auxiliary materials, taking 5% starch syrup as a binder, uniformly mixing according to a certain proportion, granulating, drying in an oven, and tabletting to obtain a finished product.
The preparation method of the bacteria liquid to be sprayed comprises mixing Arabic gum, soybean protein isolate and maltodextrin at a ratio of 2:2:1, dissolving to obtain 25% solution, and sterilizing at 114 deg.C for 20 min.
The preparation method of the thallus protective agent comprises 16% of trehalose, 6% of glycerol and 0.14% of gelatin.
The strain activation culture is carried out by using a DHP-9162 type electric heating constant temperature culture box of Shanghai constant technology limited;
the oven drying is carried out by using a DHG-9070A type electric heating constant temperature blast drying oven of Shanghai constant technology Limited company;
spray drying was carried out using an experimental spray dryer sold by Shanghai Vondi science and technology, Inc.;
MRS enrichment medium, equipment centrifuge, autoclave were as described above.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
<110> Shandong university of agriculture
<120> a strain of lactobacillus fermentum for relieving oxidative stress
<160>1
<170>PatentIn version 3.5
<210>1
<211>1405
<212>DNA
<213>Lactobacillus fermentum
<400>1
CCTTAGGCGGCTGGCTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCAT 60
GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC 120
GATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAAC 180
GGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGC 240
ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCG 300
GTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGG 360
TTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCW 420
SCACCYSTSWYWKYGKTYCCGAAgGAAACGCCCTATCTCTAGGGtTGGCGCAAGAtGTCA 480
AGACCTGGTAAGGtTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCG 540
GGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTT 600
AATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTA 660
CGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGT 720
CAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCC 780
ACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGC 840
ACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTARAAAAMCSSCYGCACTCTCTT 900
TACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGT 960
AGTTAGCCGTGACTTTCTGGTTAAATACCGTCAACGTATGAACAGTTACTCTCATACGTG 1020
TTCTTCTTTAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCT 1080
CCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGG 1140
CCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTT 1200
GGTAGGCCNTTACCCCACCAACAAGCTAATGCACCGCAGGTCCATCCAGAAGTGATAGCG 1260
AGAAGCCATCTTTTAANCGTTGTTCATGCGAACAACGTTGTTATGCGGTATTAGCATCTG 1320
TTTCCAAATGTTGTCCCCCGCTTCTGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCC 1380
ACTCGTTGGCGACCAAAATCAATCA 1405
Claims (6)
1. The Lactobacillus fermentum JX306 with the antioxidant function is characterized in that the preservation number of the Lactobacillus fermentum JX306 is CGMCC No. 18493.
2. Use of lactobacillus fermentum JX306 according to claim 1 as a strain of fermentum.
3. A starter culture prepared from Lactobacillus fermentum JX306 according to claim 1.
4. A fermented yoghurt product prepared by fermentation with lactobacillus fermentum JX306 according to claim 1.
5. Sodium alginate based probiotic capsule preparation prepared with lactobacillus fermentum JX306 according to claim 1.
6. Probiotic tablets prepared with lactobacillus fermentum JX306 according to claim 1.
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