AU2020101589A4

AU2020101589A4 - A Lactobacillus Brevis ZJ401 with antioxidant activity and its application

Info

Publication number: AU2020101589A4
Application number: AU2020101589A
Authority: AU
Inventors: Jundun Huang; Haotian Liu; Lequan Qiu; Jinshi Wu; Tingzhou Zhang
Original assignee: Zhejiang Kefeng Biotechnology Co Ltd
Current assignee: Zhejiang Kefeng Biotechnology Co ltd; Zhejiang University of Technology ZJUT
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-10-15
Anticipated expiration: 2028-07-31

Abstract

The invention provides a Lactobacillus Brevis ZJ401 with antioxidant activity and its application. The strain is preserved in China Center for Type Culture Collection with address Wuhan University, Wuhan, China, zip code 430072, preservation number CCTCC M 2017666, and preservation date: November 7, 2017. It can significantly increase the activities of SOD and CAT in A549 cells, significantly reduce the content of LDH and ROS in cell supernatant, and significantly increase the expression of Nrf2 and y - GCS genes in Caco-2 cells. The Lactobacillus brevis ZJ401 was prepared into powder and then made into antioxidant food or health products, which can be eaten or drunk directly. It is easy to use and can enhance the immunity of the body. The Lactobacillus brevis ZJ401 has important research significance and application prospects for the development of live microorganismal agent and functional fermented dairy products. -1/6 e aFigure1

Description

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e aFigure1

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

A Lactobacillus Brevis ZJ401 with antioxidant activity and its application

The invention is described in the following statement:-

A Lactobacillus Brevis ZJ401 With Antioxidant Activity And Its Application

TECHNICAL FIELD

[0001] The present invention relates to a strain of Lactobacillus breris ZJ401 having the

activity of anti-oxidative stress damage of cells in vivo and in vitro, and the application

thereof.

BACKGROUND

[0002] In 1956, British scholar Harmna first put forward the theory of free radical

senescence, which hypothesized that the change of aging was caused by free radical

reaction. In 1990, professor Sohal, the American authority of aging research, pointed out

the defects of the theory of free radical aging, and put forward the concept of oxidative

stress for the first time. Oxidative stress is the process of oxidative damage caused by the

accumulation of free radicals in the body, which results in the disorder of oxidation system

and oxidation resistance system caused by the production of free radicals increased or the

ability of scavenging free radicals decreased. Oxidative stress is not only closely related to

senility, but also to the occurrence of tumor, asthma, depression and various chronic

inflammatory diseases. In addition, oxidative stress also participates in that occurrence and

development of various pathophysiological processes of cardiovascular disease, causing a

variety of cardiovascular diseases such as atherosclerosis, heart failure, hypertension and

myocardial injury.

[0003] The body's own antioxidant capacity is limited, so the intake of exogenous

antioxidants has become an important way to alleviate oxidative stress. Lactic acid bacteria

is a kind of probiotic which is widely used by human beings which is often used in food fermentation industry and microecological preparation. It is well known that lactic acid bacteria has the functions of regulating intestinal flora and improving human immunity. It has been found that some lactic acid bacteria can scavenge active oxygen free radicals such as DPPH and hydroxyl free radicals to relieve oxidative stress. Therefore, functional lactic acid bacteria can be used as natural food additives to alleviate oxidative stress and the occurrence of some diseases. It is of great significance and market value to screen lactic acid bacteria which have better function of relieving oxidative stress. In that invention, lactic acid bacteria capable of relieve oxidative stress is screened from the traditional self made ferment food through in vitro oxidation resistance experiment, so as to lay a foundation and theoretical basis for the development of lactic acid bacteria food starter with the function of relieving oxidative stress.

SUMMARY

[0004] The object of the present invention is to provide a new strain Lactobacillus brevis

ZJ401, which has in vivo and in vitro cell antioxidant stress damaging activity.

[0005] In order to achieve that purpose of the invention, the technical scheme adopted by

the invention is as follow:

[0006] The present invention provides a new strain, Lactobacillus brevis ZJ401, deposited

in the Chinese Type Culture Collection, on November 7, 2017, with accession No. CCTCC

No.of M2017666, address of Wuhan, China. Wuhan University, and zip code of 430072.

In that invention, the pure culture with the antioxidant function is obtained from the pickled

vegetable through preliminary screening and re-screening, and is subject to morphological

identification, physiological and biochemical test identification and 16S rDNA sequence analysis. The strain was identified as Lactobacillus breris and named as Lactobacillus breris

ZJ401.

[0007] The invention also relates to the use of said Lactobacillus brevis ZJ401 in the

preparation of cellular antioxidants, which is human lung cancer cells A549 or human colon

cancer cells Caco-2. The cellular antioxidants is able to increase the SOD and CAT activity

in the cells, decrease the contents of LDH and ROS in the cell supernatant, and increase

the expression of Nrf2, SOD and HO-i genes.

[0008] The cell antioxidant is the wet thalli obtained by fermentation culture of

Lactobacillus brevis ZJ401 or the supernatant after ultrasonic crushing of the wet thallus,

and is prepared according to the following method:

[0009] (1) Lactobacillus brevis ZJ401 is inoculated into MRS solid medium, and cultured

at 37C for 24 hour to obtain slant mycelium; MRS solid medium comprises

[0010] peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 10.0 g / L, diammonium

citrate 2.0 g / L, sodium acetate 5.0 g / 1, magnesium sulfate 0.58 g / 1, manganese sulfate

0.25 g / L, diammonium hydrogen phosphate 2.0 g /L, glucose 20.0 g / L, tween-80 1 mL

/ L and agar 18 g / L. The solvent was deionized water with pH 5.0;

[0011] (2) Inoculating the slant cell to MRS liquid medium, culturing at 37C for 16 hours

to obtain seed liquid, and the MRS solid medium is the one removes agar;

[0012] (3) Inoculating the seed solution with the inoculation amount of 3% of the volume

concentration into the MRS liquid medium, and carrying out fermentation culture at 37 'C

to obtain the fermentation liquid containing the wet thalli, that is, the complete cell

suspension in that concentration of 109 cfu / kg. 5ml of the above-mentioned complete cell

suspension was broken by ultrasonic wave with the power of 400 W, working for 5 s, stopping for 5 s, 60 times. After ultrasonic breaking, the bacterial suspension was centrifugedat4 °C, 10000 r/ min for 10 min, and 4 ml of supernatant was collected, which was the cell-free extract.

[0013] The present invention also provides an application of Lactobacillus brevis ZJ401 in

the preparation of an antioxidant feed, wherein the content of the antioxidant feed is above

109 cfu / kg, preferably 109 cfu / kg.

[0014] The invention also provides the application of the Lactobacillus brevis ZJ401 in

preparing the antioxidant food or the health care product, wherein the content of the L.

brevis zJ401 is more than 109 cfu / kg.

[0015] The beneficial effect of the invention is mainly reflected in that the invention

obtains a strain of Lactobacillus brevis ZJ401 with antioxidant activity, which not only has

high antioxidant activity but also has good acid and bile salt resistant characteristics. The

DPPH and hydroxyl radical scavenging rates of ZJ401 cell-free extracts were 25.68% and

23.72% respectively, and the reductive activity was equivalent to L-cysteine 104.67 p

mol/L. ZJ401 fermentation broth and cell-free extract could significantly increase the

activities of SOD and CAT in A549 cells, and significantly decrease the contents of LDH

and ROS in supernatant of A549 cells. In that Caco-2 oxidative stress model, the total

antioxidant capacity in the culture supernatant was significantly increased, and the

expression of Nrf2 and y -GCS genes in Caco-2 cell was significantly increased by the

whole cell suspension of ZJ401. In that invention, the lactobacillus brevis ZJ401 is

prepared into a bacterial powder, and then is made into an anti-oxidation food or a health

care product for organism, which is directly eaten or taken for drink, is convenient to use,

and can enhance the immunity of the organism. The Lactobacillus brevis ZJ401 has important research significance and application prospect for the development of microecological living bacteria preparation and functional fermented dairy products.

BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 is the colony morphology of strain ZJ401 on MRS solid medium;

[0017] FIG. 2 is a gram-stained microscopic image of strain ZJ401 (the red rod strain is

the negative control, and the purple rod is strain ZJ 401);

[0018] FIG. 3 is a growth curve of strain ZJ401;

[0019] FIG. 4 is the evolutionary tree of strain ZJ401;

[0020] FIG. 5 shows the effect of strain ZJ401 on ROS production; A is the blank control

group; B is the oxidative stress control group, C-FS is the experimental group; D-CFE is

the experimental group, E-VC is the positive control group.

[0021] FIG. 6 shows the effect of strain ZJ401 on the expression of oxidative stress-related

genes Nrf2 (a), SOD (b), HO-i (c), y-GCS (d) mRNA in Caco-2 cells; CFE: Cell-free

extract; IC: Intact cell suspension; The a- e indicate the significance analysis between

different groups in the same gene, and the same letter indicates no significance, and

different letters indicate significance (p < 0.05).

DESCRIPTION OF THE INVENTION

[0022] In the following, the present invention will be further described with reference to

specific examples, but the scope of the invention is not limited thereto:

[0023] Example 1 Screening and Identification of Lactobacillus brevis ZJ401

[0024] 1.The screening of strain ZJ401

[0025] (1) Strain primary screen is carried out by collecting pickle soup juice prepared by

a traditional fermentation method by using a disposable sterile syringe. Diluting the sterile collect sample with physiological saline in a gradient way. 100 ~ 200 p L of the diluted sample was uniformly coated on the MRS solid medium with pH 5.0, and then was cultured upside down for 24 ~ 48 hours after being dried in a biochemical incubator at 37 °C for minutes. Observe the colony shape of single colony on the plate, and select the colony conforming to the shape of lactic acid bacteria (the colony is round, medium in size, convex, slightly white, moist and neat at the edges). The isolated and purified colonies were subjected to Gram stain and contact enzyme tests. The strain with Gram positive stain and negative contact enzyme test could be preliminarily identified as lactic acid bacteria.

[0026] (2)Tolerance screening: The two times activated strains in 3% volume were

inoculated in MRS liquid medium with pH 2.0, 3.0, 4.0, 5.0, 6.0 respectively and inoculated

in MRS liquid medium with the mass fraction of pig bile salt 0, 0.3%, 0.5% respectively,

and inoculated in MRS liquid medium with H202 concentration 0.4, 0.7, 1.0 mmol / L

respectively. After incubation at 37 °C for 16 h, the strain with the best tolerance was

selected by measuring the absorbance value at 600 nm. In this way, strain ZJ401 that could

maintain its activity in the environment of pH 3.0, 0.5% of pig bile salt and 1.0 mmol / L

H202 can be obtained.

[0027] (3)Sample treatment: Strain ZJ401 was inoculated in MRS liquid medium, cultured

at 37 °C for 16 hours, centrifuged at 4 °C for 10 min at 4000 r / min. And the the

supernatant was discarded and bacteria was collected. The cells were washed with PBS

buffer with pH 7.2 for 3 times and suspended again, and the number of cells was adjusted

to 1 X 109 cfu / mL as complete cell suspension. The complete cell suspensions were

ultrasonically disrupted at a power of 400 W for 5 s, and then stop for 5 s and repeat for 60 times. After ultrasonic disruption, the suspension was centrifuged at 4 °C for 10,000 r/ min for 10 min, and the supernatant was collected as cell-free extract.

[0028] (4) The anti-oxidation activity detection and screen: The anti-oxidation activity

detection and screen are carried out on the sample of the whole cell suspension and the

cell-free extract, and the DPPH clearance rate, the hydroxyl free radical clearance ratio, the

lipid peroxidation resistance, the chelating ferrous ion ability and the reducing activity are

detected; The activities of SOD, GSH-Px and NADH oxidase were detected in cell-free

extracts. In the ZJ401 whole cell suspension and the cell-free extract, that percentage of

DPPH scavenge was 19.33% and 25.68% respectively, the percentage of hydroxyl radical

scavenging was 19.82% and 23.72% respectively, and the anti-lipid peroxidation was

12.11% and 8.62% respectively. The capacity of chelating ferrous ion was 12.61% and

15.43% respectively, and the reducing activity was equivalent to 97.00 p mol / L and

104.67 P mol / L L-cysteine. The activity of SOD, GSH PX and NADH oxidase of ZJ401

was 7.068 U / mgprot, 156.73 U / Mg prot and 0.0615 U / Mg prot respectively.

[0029] MRS liquid culture medium: Peptone 10.0 g / L, beef extract 10.Og /1, yeast extract

10.0 g/ L, diammonium citrate 2.0 g /L, sodium acetate 5.0 g / I, magnesium sulfate 0.58 g

/ L. Manganese sulfate 0.25 g / L, diammonium hydrogen phosphate 2.0 g / L. Glucose

20.0 g / 1, Tween-80 1 mL / L. The solvent was deionized water with pH 5.0.

[0030] In that MRS solid medium, 18 g / L agar was added to the MRS liquid medium.

[0031] 2. Identification of the strain ZJ401

[0032] (1) Identification of colony morphology: Strain ZJ401 was inoculated on MRS solid

medium and cultured at 37 °C for 24 hours, and the single colony was slightly white,

convex and smooth and moist round (as shown in Fig. 1). Microscopic examination showed that strain ZJ401 was positive for leatheroid, with a straight or slightly curved short rod shape, sometimes singly, sometimes in pairs or chains (as shown in FIG. 2). The strain

ZJ401 entered the logarithmic phase after 6 h, and the growth of the bacteria entered the

stationary phase after 18 h. The logarithmic growth phase of the strain ZJ401 was 6-18 h

and the pH of the fermentation broth decreased from 6.0 to 4.4 (as shown in FIG. 3).

[0033] (2) Physiological and biochemical identification: Strain ZJ401 was inoculated on

MRS solid medium, cultured at 37 °C for 24 hours, and then transferred into API 50CHL

medium, and two McGregor suspensions were prepared by using the method of Michaelis

turbidimetric control. Connect the bacterial suspension to API50CH reagent strip, and

incubate anaerobically at 37 °C for 24-48h. The physiological and biochemical results

showed that the strain ZJ401 was Lactobacillus brevis at 24h and 48h (see Table 1 for the

specific physiological and biochemistry results).

[0034] Table 1 Physiological and biochemical results of strain ZJ401

Tube Reaction result Tube Reaction result numbe economic index 24h 48h number economic index 24h 48h r 0 Blank control - - 25 Horse leaf spirit - 1 glycerin - - 26 Salicin - 2 Erythritol - - 27 D-cellobiose - 3 D-arabinose - - 28 D-maltose + +

4 L-arabinose + + 29 D-lactose - 5 D-ribose + + 30 D-Melibiose - +

6 D-xylose + + 31 D-sucrose - 7 L-xylose - - 32 D-trehalose - 8 Adong sugar - - 33 Inulin - 9 B-methyl-D-xyloside - - 34 D-melezitose - 10 D-galactose - + 35 D-Raffinose - 11 D-glucose + + 36 starch - 12 D-fructose + + 37 Glycogen - 13 D-Mannose - - 38 Xylitol - 14 L-sorbose - - 39 D-gentiobiose - 15 L-rhamnose - - 40 D-Trone - 16 Fumarol - - 41 D-lyxose - 17 Inositol - - 42 D-tagatose - -

18 Mannitol - - 43 D-Rock Sugar - 19 Sorbitol - - 44 L-Rock Sugar - 20 A-methyl-D-mannoside - - 45 D-arabinitol - 21 A-methyl-D-glucoside - + 46 L-arabinitol - 22 N-acetylglucosamine - + 47 Gluconate +

+ 23 Amygdalin - - 48 2-keto-gluconate - 24 Arbutin - - 49 5-keto-gluconate +

+

[0035] (3)Molecular identification: The DNA of strain ZJ401 was amplified by PCR, and

a 1455bp DNA fragment (shown in SEQ ID NO. 1) was amplified with 16S rDNA

universal primer. The sequences were aligned by BLAST on NCBI. The result showed that

the strain with the highest homology of ZJ401 sequence was Lactobacillus brevis

(similarity 99%, sequence number NR116238.1). The genetic distance of the strain ZJ401

and its closely related strains was calculated by selecting the typical strain with high

homology from above NCBI. The system development tree was constructed using MAGE

5.0 software (as shown in Figure 4). Based on the results of morphology, 16S rDNA

sequence comparison and physiological and biochemical analysis, the strain ZJ401 was

identified to be Lactobacillus brevis named ZJ401which was deposited in the Chinese

Typical Culture Collection with collection No. of CCTCC No: M 2017666, and date of

collection is Nov. 7, 2017, address is Wuhan University, Wuhan, China, 430072.

[0036] Example 2 Lactobacillus brevis ZJ401 fermentation broth

[0037] (1) Lactobacillus brevis ZJ401 was inoculated into MRS solid medium and cultured

at 37 °C for 24 hours to obtain Slant thallus.

[0038] (2) Inoculating the Slant thallus to MRS liquid medium and culturing at 37 °C for

16 hours to obtain the seed liquid.

[0039] (3) Inoculate the seed solution with the inoculation amount of 3% by volume

concentration into the MRS liquid medium, and carry out fermentation culture at 37 °C to

obtain the fermentation liquid, which is the complete cell suspension with the concentration

of 109 cfu / kg. 5 ml of the above-mentioned complete cell suspension was broken by

ultrasonic wave with power of 400 W, working for 5 s, stopping for 5 s, 60 times. After

ultrasonic crushing, the bacterial suspension was centrifuged at 4 °C,10000R/min for10

min, and 4 ml of supernatant was collected, which was the cell-free extract.

[0040] Example 3 Protective Effect of Lactobacillus brevis ZJ401 on Oxidative Stress

Injury of A549 Cells in Vitro

[0041] Preparation of PM2.5 solution: A sampling filter membrane (Whatman PM2.5,

purchased from Orfurson) was cut to a size of lcm X 1cm, immersed in ultrapure water,

allowed to stand at room temperature for 30 min, and subjected to ultrasound for 30 min.

Three times were repeated to elute PM2.5 particles. The obtained filtrate was filtered with

6 layers of gauze, and the filtered filtrate was frozen at -80°C to be a lyophilized powder.

In application, sterile PBS is added to configure into high-concentration mother liquor, and

then diluted into PM2.5 solution with different concentration.

[0042] PM2.5 solution was used as oxidative stress for A549 cells. A549 cells were plated

at 1 x 104 cells per well (i.e. at a concentration of 1 x 105, 100 L per well) in a 96-well

plate containing 1640 basal medium with 10% calf serum by volume + 1% celadon by

volume (100 U / ml), cultured at 37 °C for 24h, and suck off the culture medium after the

cell attaching to the wall. After washing with PBS, divide it into groups according to Table

2, add PM2.5 to the final concentration of 100kg / mL. The whole cell suspension (IC) of

Lactobacillus brevis ZJ401 prepared in Example 2 was added in an amount of 0.5 ml, the

cell-free extract (CFE) in an addition amount of 0.5ml, Vc in a final concentration of 20 g

/ mL, and PBS in an equal amount is a blank control. Culture them at 37 °C for 24 h. The

fermentation broth and the cell-free extract of ZJ401 significantly increased the activities

of SOD and CAT in the cells, significantly reduced the contents of LDH and ROS in the

supernatant of cells. ZJ401 can alleviate the oxidative stress injury of A549 cells caused by

PM2.5 (as shown in Table 3 and FIG. 5).

Table 2. Experimental grouping

Blank control Oxidative Positive control Group group stress group group test group

Additives PBS PM2.5 PM2.5+ Vc PM2.5+IC PM2.5+CFE

Table 3. Effect of Lactobacillus brevis ZJ401 on SOD, CAT and LDH of A549 cells Blank control Oxidative Positive test group Group group stress group control group PM2.5+IC PM2.5+CFE SOD(U 1 8 .5 8 +1.4 1 ab 15.38+1.13a 22.56+1.81c 23.61+0.48c 2 0 . 8 3 +1. 4 4be mgprot) Cat(U/ 25.65+2.15ab 20.87+0.97a 23.83+1.51a 3 0 .9 9 +2. 30 b 2 5 . 1 2 +1. 4 2 ab mgprot) LDH(U/ 9 6 . 7 6 +4. 2 5b 1 4 2 . 1 2 +7. 8 5 ' 119.55+5.84c 55.18+3.95a 67.83+0.46a gprot) Notes: 1. In the table, a ~ d indicates the significance analysis of different groups under the same antioxidant enzyme index, and the same letter indicates no significance, and different letters indicate significance (p < 0.05). 2. CFE: Cell-free extract; IC: Complete cell suspension

[0043] Example 4 Protective Effect of Lactobacillus brevis ZJ401 on Oxidative Stress

Injury of Caco-2 Cells in Vitro

[0044] H202 was used as an oxidative stress for Caco-2 cells. A549 cells were plated in

96-well plates in DMEM basal medium containing 20% fetal bovine serum by volume and

1% celestine by volume (100 U / mL) at 1 X 104 cells per well (i.e. 1 X 105

concentration, 100 p L per well) and cultured at 37 °C for 24 hours. Suck off the culture

medium after cell adhering to the wall. After washing with PBS, A549 cell were divided

into groups according to Table 4, in which H202 is added with a final concentration of 100

p mol / L, the whole cell suspension (IC) of Lactobacillus brevis ZJ401 prepared in

Example 2 was added in an amount of 0.5 ml, and the cell-free extract (CFE) was in an

addition amount of 0.5ml. The complete cell suspension and cell-free extract of ZJ401

significantly increased the total antioxidant capacity (T-AOC) in the cells (as shown in

Table 5).

[0045] Table 4. Experimental grouping

Blank control Oxidative Positive control group stress group group Additives PBS H20 2 H 2 0 2 +Vc H202+1C H 202+CFE

Table 5 Effect of Lactobacillus brevis ZJ401 on antioxidant activity of Caco-2 Blank control Oxidative Positive test group Group group stress group control group H 2 0 2 +IC H 20 2+CFE SOD (U/ 5. 84 +0. 3 5b 6.90+0.64ac 6 .2 5 +0. 4 9bc 6 .4 7 +0. 7 1ab 7.26+0.59a mgprot) GSH (

8.56+1. 2 6ab 7 .8 7 +0.13ab 7.37+1. 3 0 b 9.35i0.44a 8.61+0. 3ab mg/mgprot) Pod (U / 1.43±0.15a 1.41+0.09a 1.49±0.37a 1.78±0.82a 2.08+0.70a mgprot) LDH 70.10 ± 2.27° 77.00+3.67a 72 .18i 2 .14be 5 7 .9 0 + 3 .9 0 ' 75.04 ± 4.90ab (U/gprot) T-AOC 12. 3 3 +1. 14 d 1 0 . 4 9 +1. 3 2d 14 . 0 2 +1. 2 4be 16.88+1.12a 1 5 . 7 8 +0. 9 1 ab (U/mgprot) CAT 8.25±0.603a 7 .8 9 +0.58ab 6 .9 9 +0. 84 b 8.24+0.60a 7 .4 6 +0. 4 0ab (U/mgprot) Notes: 1. In the table, a ~ d indicates the significance analysis of different groups under the same antioxidant enzyme index, and the same letter indicates no significance, and different letters indicate significance (p < 0.05). 2. CFE: Cell-free extract; IC: Complete cell suspension

[0046] Example 5 Effect of Lactobacillus brevis ZJ401 on mRNA expression of Nrf2,

SOD, HO-1, y -GCS in intestinal epithelial Caco-2 cells in vitro

[0047] Caco-2 cells were purchased from ATCC. Remove the frozen cells from the -80 C

refrigerator or liquid nitrogen tank, and quickly put them into a 37 C water bath with

occasional shaking to melt the cells. Add 5 mL of complete cell culture medium and thawed

cell suspension to the centrifuge tube. After centrifugation at 1000 r / min for 3 min, the

supernatant was discarded, and the cells suspended in complete medium were added, and

the cells were thoroughly blown with a pipetting gun. The cell suspension was transferred

to a cell culture dish, placed in an incubator with a temperature of 37C, and a carbon dioxide concentration of 5%, and the culture medium was changed once every 2 days.

Intestinal epithelial Caco-2 cells were cultured in vitro, stimulated with different doses (2

X 105, 2 X 106 and 2 X 107 CFU) of Lactobacillus brevis ZJ401 respectively for 8

hours, and total RNA of the stimulated cells was extracted and reverse transcribed into

cDNA. The changes of Nrf2, SOD, HO-i and y -GCS mRNA expression in intestinal

epithelial cells were detected by FQ-PCR, and PBS solution was used as a blank control,

as shown in FIG. 6. The results showed that the gene expressions of Nrf2, SOD and HO-i

in the oxidative stress group was significantly higher than that in the control group (p <

0.05), indicating that Caco-2 cells produced oxidative stress under the stimulation of H202.

The intracellular signal pathway of Keap1-Nrf2-ARE is activated and the downstream

antioxidant enzymes such as SOD and HO-i are activated.

[0048] Example 6 Effect of the addition of Lactobacillus brevis ZJ401 to the grains

on the anti-oxidation function of mice

[0049] Lactobacillus brevis ZJ401 was added to the diet of 48-day-old mice to make the

number of viable bacteria reach 109 cfu / kg. After 21 days of continuous feeding, T-SOD

and MDA in serum were detected. ZJ401 could significantly increase the activity of total

superoxide dismutase (T-SOD) in serum and intestinal mucosa of 21 days' mice (p < 0.05),

and decrease the content of malondialdehyde (MDA) of serum of 21 day mice (P < 0.01).

On the 21st day, it makes the content of MDA in intestinal mucosa has the decreasing trend

(p = 0.057), and can significantly increase the serum T-SOD activity and total antioxidant

capacity (T-AOC) on the 42nd day (p < 0.01).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. Lactobacillus breris ZJ401 is deposited in China Center for Type Culture Collection with

deposited number CCTCC No: M 2017666, and date November 7, 2017, and the address is

Wuhan University, Wuhan, China with zip code 430072.

2. An application of Lactobacillus brevis ZJ401 in the preparation of cell antioxidant in claim

1, wherein the cell antioxidant is the fermentation liquid obtained by fermentation of

Lactobacillus brevis ZJ401 or the supernatant of the fermentation broth after ultrasonic

crushing.

3. The application according to claim 2 wherein the cell is a human lung cancer cell A549 or a

human colon cancer cell Caco-2.

4. The application according to claim 2 wherein the cell antioxidant is a preparation for

improving the activity of SOD or CAT in cells.

5. The application according to claim 2 wherein the cell antioxidant is a preparation for

reducing the content of LDH or ROS in the cell.

6. The application according to claim 2 is characterized in that the cell antioxidant is a

preparation for increasing the gene expression of Nrf2, SOD or HO-1 in cells.

7. Lactobacillus brevis ZJ401 in claim 1 applied to the preparation of antioxidant feed.

8. An application of Lactobacillus brevis ZJ401 according to claim 7 wherein the content of

Lactobacillus brevis ZJ401 in the antioxidant feed is more than 109 cfu / kg.

9. Lactobacillus brevis ZJ401 in claim 1 applied to preparation of antioxidant food or health

care products.

10. An application of Lactobacillus brevis ZJ401 according to claim 9 wherein the content of

Lactobacillus brevis ZJ401 in the antioxidant food or health product is more than 109 cfu / kg.

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Figure 1

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Figure 2

-3/6- 31 Jul 2020

3.5 5.8 菌体干重（mg/mL） 3.0 pH 5.6 2020101589

2.5 5.4 mg/mL）

2.0 5.2

5.0

pH 菌体干重（

1.5 Cell dry weight

4.8 1.0 4.6

0.5 4.4

0.0 4.2 0 2 4 6 8 10 12 14 16 18 20 22 24 时间（h）

Figure 3

-4/6-

Figure 4

-5/6-

Figure 5 d b

Figure 6 -6/6-

Relative expression Relative expression

阳性对照 c a

c CFE组 d

IC组 e

空白对照氧化应激组 a

b 1.5

1.2

0.9

0.6

0.3

0.0 HO-1 mRNA相对表达量 Relative expression Relative expression 31 Jul 2020 2020101589