CN116836879B

CN116836879B - Lactobacillus fermentum with colon cancer cell growth inhibition effect and application thereof

Info

Publication number: CN116836879B
Application number: CN202310899300.7A
Authority: CN
Inventors: 岳碧松; 尚可; 李琰; 邹方东
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-07-21
Filing date: 2023-07-21
Publication date: 2024-01-23
Anticipated expiration: 2043-07-21
Also published as: CN116836879A

Abstract

The invention discloses a lactobacillus fermentum with a colon cancer cell growth inhibition effect and application thereof, wherein the lactobacillus fermentum has stronger adhesion capability to human colon cancer cells, and fermentation supernatant and colon cancer cells HCT-116 after co-culture show stronger cancer cell growth inhibition activity and cancer cell apoptosis promotion capability, and can greatly enhance the toxicity to cancer cells when being used together with anticancer drugs; the strain has good safety, does not generate hemolysis phenomenon on Columbia blood plates, is sensitive to various common antibiotics, does not carry antibiotic resistance genes, and has better tolerance to artificial stomach and intestinal juice; meanwhile, the strain has strong acetic acid and butyric acid production capability, and fermentation liquor has strong inhibition capability and strong antioxidation effect on escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi b, shigella dysenteriae and the like. Therefore, the strain has obvious probiotic effect and can be applied to the field of functional foods for human and animals.

Description

Lactobacillus fermentum with colon cancer cell growth inhibition effect and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus fermentum with a strong inhibition effect on colon cancer cells and various intestinal pathogenic bacteria.

Background

Lactobacillus fermentum (Lactobacillus fermentum) belongs to the genus Lactobacillus. Research shows that the strain can produce lactic acid and other short chain fatty acids, reduce pH value in intestinal tract, increase the number of beneficial bacteria in intestinal tract, inhibit growth of harmful bacteria, maintain acid-base balance in intestinal tract, and promote intestinal tract health. The strain can promote immunoglobulin production, increase leukocyte quantity, and improve disease resistance of organism. Lactobacillus fermentum can decompose complex carbohydrates, proteins and fats in food, and promote digestion and absorption of food. In addition, the lactobacillus fermentum can promote intestinal peristalsis, increase the volume and the moisture of excrement and relieve the symptoms of dyspepsia such as constipation and the like.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides lactobacillus fermentum with the effect of inhibiting the growth of colon cancer cells and application thereof, and the lactobacillus fermentum has the effects of inhibiting the growth of colon cancer cells, inhibiting bacteria, resisting inflammation, producing acid, reducing blood sugar and improving the adaptability of human intestinal environment.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a strain of lactobacillus fermentum (Lactobacillus fermentans) GS1108 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 27395 in the year 2023 and the month 5 and the day 22.

The lactobacillus fermentum GS1108 is applied to the preparation of anti-colon cancer drugs or anti-colon cancer auxiliary drugs.

Application of lactobacillus fermentum GS1108 in inhibiting escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi B or shigella dysenteriae for non-disease treatment purpose.

The application of the lactobacillus fermentum GS1108 in preparing medicaments for treating diseases caused by escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi B or shigella dysenteriae.

Use of lactobacillus fermentum GS1108 as described above in functional food for modulating the intestinal flora of humans or animals.

An anti-colon cancer drug or an anti-colon cancer adjuvant drug comprising lactobacillus fermentum GS1108 as described above.

An antioxidant drug comprising lactobacillus fermentum GS1108 as described above.

A functional food comprising lactobacillus fermentum GS1108 as described above.

Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 in combination with 5-fluorouracil or oxaliplatin for the preparation of a medicament for treating colon cancer.

The lactobacillus fermentum GS1108 is obtained by separating and screening from Guizhou fermented food. Lactobacillus fermentum GS1108 grows well on MRS agar medium, and the colony is milky white, smooth in surface, neat in edge, opaque, and has a purple and rod-shaped gram stain by microscopic examination of the bacterial form. The total genome DNA of the bacterial strain is amplified by PCR by adopting 27F/1492R of bacterial universal primer 16S rRNA as a template, the gene sequence obtained by sequencing is input into NCBI database for comparison, the similarity rate of the gene sequence with a standard strain Lactobacillus fermentans strain TMPC F335 in GenBank reaches 98.53%, and the strain can be initially identified as lactobacillus fermentum (Lactobacillus fermentans).

The lactobacillus fermentum GS1108 strain has good safety, does not generate hemolysis phenomenon on a Columbia blood plate, is sensitive to various common antibiotics, and does not carry antibiotic resistance genes;

the lactobacillus fermentum GS1108 strain has strong intestinal tract adaptability and better tolerance to artificial stomach and intestinal juice;

the lactobacillus fermentum GS1108 strain has obvious probiotics performance, has stronger adhesion capability to human colon cancer cells, has stronger cancer cell activity inhibition capability after the fermentation supernatant and colon cancer cells HCT-116 are co-cultivated, has stronger inhibition capability to escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi B, shigella dysenteriae and the like, can generate various short-chain fatty acids such as acetic acid, butyric acid and the like, and has stronger antioxidation effect.

The beneficial effects of the invention are as follows:

1. the lactobacillus fermentum GS1108 provided by the invention is separated and screened from Guizhou pickled Chinese cabbage, grows well on an MRS agar medium, and has good tolerance to acid and bile salts.

2. The lactobacillus fermentum GS1108 provided by the invention has good adhesion capability to human colon cancer cells and has stronger cancer cell activity inhibition capability.

3. The lactobacillus fermentum GS1108 provided by the invention has strong bacteriostasis to common intestinal pathogenic bacteria such as escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi B, shigella dysenteriae and the like.

4. The lactobacillus fermentum GS1108 provided by the invention can produce various short-chain fatty acids such as acetic acid, butyric acid and the like, and has strong antioxidation.

Preservation information:

lactobacillus fermentum (Lactobacillus fermentans) GS1108 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 27395, which is No.1 and No. 3 in North Xili road, korean, beijing, 5 months, 22 days in 2023

Drawings

FIG. 1 is a diagram showing the phylogenetic relationship between Lactobacillus fermentum GS1108 and other strains according to the present invention;

FIG. 2 shows the inhibition of colon cancer cell growth at various concentrations of fermentation supernatant of Lactobacillus fermentum GS 1108; in the figure, A is control, B is fermentation supernatant (1%), C is fermentation supernatant (1%) +cisplatin (DDP) 20. Mu.M, D is fermentation supernatant (1%) +oxaliplatin (Oxaliptin) 20. Mu.M), E is fermentation supernatant (1%) +5-fluorouracil (5 fu) 20. Mu.M.

FIG. 3 is a GC-MS detection ion flow diagram of short chain fatty acid of fermentation supernatant of lactobacillus fermentum GS1108.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.

Thus, the following detailed description of the embodiments of the invention, as provided, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.

It is noted that relational terms such as "first" and "second", and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises an element.

The features and capabilities of the present invention are described in further detail below with reference to the examples and figures.

Example 1 isolation, screening and molecular biological identification of lactobacillus fermentum GS 1108:

1. material preparation

The strain is derived from Guizhou fermented food;

the 16S rRNA universal primers 27F and 1492R were synthesized by biological engineering (Shanghai) Inc., and the sequences were as follows:

27F：5-AGAGTTTGATCMTGGCTCAG-3，

1492R：5-GGTTACCTTGTTACGACTT-3

formulation of MRS broth (per liter): 10.0g of casein enzyme digest, 10.0g of beef extract powder, 4.0g of yeast extract powder, 2.0g of tri-ammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 20.0g of glucose, tween-80 and a final pH of about 5.7.

Formulation of MRS agar Medium (per liter): 10.0g of peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 1.0mL of tween 80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 15.0g of agar and a final pH of about 6.2.

2. Detailed description of the preferred embodiments

1mL of the sample is taken in 20mLMRS broth, fully and uniformly mixed by shaking, and placed in a shaking table at a constant temperature of 37 ℃ for 24 hours. 10-fold gradient dilution is adopted, the culture is spread and inoculated on MRS agar culture medium, single colony is picked after 24h culture at 37 ℃, and single colony purification is carried out continuously for 3 times. The purified strain is inoculated into 600 mu L of MRS broth culture medium, shake-cultured for 18h at 37 ℃, 400 mu L of 50% (V/V) sterile glycerol is added, and the strain is frozen in an ultralow temperature refrigerator at-80 ℃ for standby. After the purified strain is amplified and cultivated by MRS broth, the strain DNA is extracted by adopting a Tiangen bacterial genome DNA extraction kit, the 16S rRNA amplification is finished by adopting a colony PCR technology, the sequencing of PCR products is finished by a biological engineering (Shanghai) limited company, the nucleotide sequence is shown as SEQ ID No.1, the sequence is compared with BLAST in NCBI, the similarity of one strain with a standard strain Lactobacillusfermentans strain TMPC F335 reaches 98.53%, the strain can be initially identified as lactobacillus fermentum (Lactobacillus fermentans), the strain is named as GS1108, and the phylogenetic relationship between the strain and other strains is shown as figure 1. Lactobacillus fermentum GS1108 was gram stained using the kit method, and the stained bacterial morphology was observed and recorded under a microscope. The lactobacillus fermentum GS1108 grows well on an MRS agar culture medium, the colony shape is milky white, round convex, smooth in edge and smooth in surface, and after dyeing, the thallus is rod-shaped and purple in microscopic examination, so that the bacterial colony meets the morphological characteristics of the lactobacillus fermentum. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 22 days of 5 months of 2023, and the preservation number is 27395.

Example 2 growth-inhibiting and apoptosis-promoting effects of Lactobacillus fermentum GS1108 fermentation broth on colon cancer cells

2.1 adhesion of Lactobacillus fermentum GS1108 to human colon cancer cells

The lactobacillus fermentum GS1108 frozen strain is inoculated into MRS broth culture medium after resuscitating and culturing, and is cultured at the constant temperature of 37 ℃ for 24 hours. After the cultivation, the mixture is centrifuged for 10min at-4 ℃ and 5000r/min, and washed with sterile PBS buffer solution for a plurality of times. Adjusting the concentration of the bacterial suspension to 1X 10 ⁶ CFU/mL for later use. Resuscitates human colon cancer cells HT-29, inoculates them into six well cell culture dishes, adds DMEM complete medium and places them in 5% CO at 37 deg.C ₂ Medium culture, and medium culture is replaced once in two days. When the cell attachment state reached 80%, digestion was performed using 0.25% pancreatin-EDTA, and subcultured. After the completion of the culture, the cells were counted by a cell counting plate and the cell concentration was adjusted to 5X 10 ⁶ And each mL. 1mL of the cell suspension was added to one of the culture wells of a six-well cell culture dish and placed in an incubator for culture. Cells in the plates were grown to a monolayer, DMEM medium was discarded and each well was rinsed 3 times with sterile PBS. 1mL of the prepared bacterial suspension is added into a cell hole, the cell culture plate is slightly shaken, a small amount of bacterial liquid in the hole is sucked for plate counting, and the result is taken as the initial viable bacterial count in the bacterial suspension. The cell plates were incubated at 37℃for 2h, the medium was discarded and washed 3 times with sterile PBS buffer. The cells were digested with 0.7mL of 0.25% trypsin-EDTA for 10min, and after the cells were completely detached, the digestion was terminated by adding 0.3mL of DMEM culture solution, and the culture solution after the end of the adhesion experiment was collected for plate counting, and the result was used as the number of adhesion viable bacteria. Adhesion rate (%) = lactic acid at end periodNumber of bacteria/initial lactic acid bacteria inoculation number X100%

The results are shown in Table 1. As a result, it was found that the adhesion rate of Lactobacillus fermentum GS1108 to human colon cancer cell HCT116 was 35.41%.

TABLE 1 adhesion Rate of Lactobacillus fermentum GS1108 to human colon cancer cells HCT (%)

2.2 growth inhibition of colon cancer cells by Lactobacillus fermentum GS1108 fermentation supernatant

Preparation of fermentation supernatant of lactobacillus fermentum GS 1108: resuscitating lactobacillus fermentum GS1108, inoculating into MRS broth, and culturing at 37deg.C for 24 hr to obtain bacterial suspension with concentration of 1×10 ⁹ CFU/mL. Centrifuging at-4deg.C and 5000r/min for 10min after culturing, collecting fermentation supernatant, and filtering with bacterial filter membrane.

Recovery of cancer cells: pre-heated DMEM medium containing 10% fbs and 1% diabody was prepared in 15mL centrifuge tubes, placed on a sterile operating table, and frozen human colon cancer cells (HCT 116) were removed from-80 ℃ refrigerator or liquid nitrogen in a 37 ℃ water bath for rapid thawing. After thawing the cells, they were transferred on a sterile operating table to a prepared 15mL centrifuge tube containing the medium, centrifuged at 1000r/min for 3min to resuspend the cells, aspirated into a 6cm cell culture dish, and incubated in 5% CO ₂ After 24h incubation at 37℃in the incubator, fresh medium was changed.

Cell passage: and when the cell growth state is good and the density reaches about 80 percent, carrying out passage. After the medium and sterile PBS were preheated in a 37℃water bath, old medium in the cell culture dish was aspirated off on a sterile operating table, cells were washed with preheated PBS to remove dead cells and residual medium, cells were digested with 0.25% pancreatin, pancreatin was aspirated off with a pipette after the cells became round, the digestion was stopped, and cells were collected in a sterile centrifuge tube. The supernatant was aspirated off by centrifugation at 1000r/min for 3min, and the cells were resuspended in fresh medium and then aspirated into fresh cell culture dishes and placed in a cell incubator for culture.

Cytotoxicity experiment: the influence of fermentation supernatant of lactobacillus fermentum GS1108 on the growth of colon cancer cells is determined by using a CCK-8 kit (Cell Counting Kit-8 cell counting reagent), wherein the kit uses tetrazolium salt WST-8 with high water solubility, the tetrazolium salt WST-8 can be reduced by some dehydrogenases in mitochondria to generate yellow formazan under the condition of existence of an electronic coupling reagent, the amount of formazan generated by the dehydrogenases and the number of living cells are in a direct linear relation, the more and faster the proliferation of cancer cells, the darker the color, the more toxic to the cancer cells, the lighter the color, the high sensitivity of the method and the simplicity and convenience in operation are realized. HCT116 cells in the logarithmic growth phase were evenly spread into 96-well plates after digestion and centrifugation, and the count ensured 2000 cells per well. Culturing for 12 hours, adding 1% of prepared probiotic fermentation liquor after cell adhesion is stable, and simultaneously adding three anticancer drugs respectively: pentafluorouracil (5 fu), oxaliplatin (Oxaliptin) and cisplatin (DDP) were each 20 μm in final concentration, 5 replicates were set for each treatment, and after 48 hours of incubation, CCK-8 kit detection was performed, and incubation was continued in a cell incubator for 1 hour, and absorbance was measured with a microplate reader at 450 nm. Meanwhile, no fermentation broth and anticancer drug control were set.

The measurement results are shown in Table 2. The fermentation supernatant of 1% lactobacillus fermentum GS1108 shows obvious inhibition effect on the growth of human colon cancer cells, and the colon cancer cell density after 48 hours of culture is 73.87% of that of a control. The effect was most pronounced when 1% of the fermentation supernatant was used in combination with the anticancer drug 5-fluorouracil (5 fu, 20. Mu.M) or Oxaliplatin (20. Mu.M), and the colon cancer cell densities after 48h of culture were 37.86% and 47.95% of the control, respectively. It was demonstrated that lactobacillus casei GS1108 fermentation supernatant alone or in combination with anticancer drugs had significant inhibitory effect on human colon cancer cell growth (figure 2).

TABLE 2 influence of GS1108 fermentation supernatants on colon cancer cell growth in combination with several anticancer drugs

Pro-apoptotic effects of 2.3GS1108 fermentation supernatants on human colon cancer cells

2.3.1 materials and methods

Cell culture: HCT116 cells in the logarithmic growth phase were evenly spread into 96-well plates after digestion and centrifugation, and the count ensured 2000 cells per well. After 12 hours of culture, GS0605 fermentation supernatant is added for 1% after cell attachment is stabilized, 5 replicates are set for each treatment, and protein is extracted after 48 hours of culture.

Protein extraction: after washing the cultured cells twice with PBS, all remaining PBS on the cell surface was aspirated as much as possible. Cells were placed in an ice bath, RIPA lysate containing the protease inhibitor Cocktail (protease inhibitor diluted 1:100) was added, and the whole cells were scraped into a 1.5ml sterile centrifuge tube and placed in an ice bath for 30min. Centrifuging at 12000r/min and 4 ℃ for 10min, and collecting supernatant into a new 1.5ml sterile centrifuge tube to obtain the extracted protein sample. The protein sample can be directly subjected to protein quantitative analysis or stored in a refrigerator at-80 ℃ for standby. For the protein sample after quantitative analysis, protein buffer solution can be added, and the protein sample is placed in a metal bath and boiled at 98 ℃ for 10min to denature the protein, and the protein sample is stored in a refrigerator at-20 ℃ for standby after instantaneous centrifugation.

Protein quantification: and (5) carrying out quantitative analysis by using the Biyundin protein quantitative kit. Protein samples were diluted 20-fold and added to labeled 96-well plates. Preparing BCA working solution according to the ratio of BCA reagent A to BCA reagent B of 50:1, fully and uniformly mixing, adding 200 μl of BCA working solution into each hole, shaking and uniformly mixing for 30s, covering each sample hole, and placing in a constant temperature incubator at 37 ℃ for 30min. The absorbance at 562nm was measured, the protein concentration of each sample was read out in a linear range of the standard curve based on the corrected absorbance for each protein sample, and the amount of protein in the original sample was calculated based on the sample volume and dilution.

2.3.2 experimental results

(1) Effect of GS1108 fermentation supernatant on cell adhesion factor beta-catenin

Cancer cell spread is characterized by disordered interactions between cells and cell adhesion. Beta-catenin (beta-catenin) is a multifunctional protein, is an important cell adhesion molecule, participates in cell growth and repair, and plays an important role in the processes of tumorigenesis and metastasis. The postoperative recurrence rate of colorectal cancer is high, the survival time of patients is short, and mutations of Wnt/beta-catenin signal channels in almost all colorectal cancers finally lead to accumulation of beta-catenin. Therefore, the content of β -catenin can be used as an auxiliary factor for clinically judging the development and prognosis of colorectal cancer, and nuclear accumulation of β -catenin can be a malignant-related marker for invasion, metastasis, poor prognosis and the like of colorectal cancer. Our experimental results show that after adding 1% of GS1108 fermentation supernatant to colon cancer cell (HCT 116) culture solution and culturing for 48 hours, the content of beta-catenin is greatly reduced, which is only 36.94% of the control without fermentation solution. At the same time, the content of phosphorylated beta-catenin (P-beta-catenin) is also obviously reduced, and the protein content is only 50.04 percent of the control. Phosphorylated β -catenin (P- β -catenin) separates from the adhesion complex and migrates into the cytoplasm, is degraded or translocated to the nucleus, triggering Wnt pathway activation. The Wnt/beta-catenin signal channel is a multifunctional channel, and the disorder of beta-catenin signal occurs in various cancers, and the Wnt/beta-catenin signal channel is involved in the regulation and control of canceration of cancers such as colorectal cancer, prostate cancer, breast cancer, malignant hematopathy and the like. Our results demonstrate that GS1108 fermentation supernatant can reduce the expression of cell adhesion factor β -catenin, thereby reducing adhesion and spread of cancer cells.

TABLE 3 influence of GS1108 fermentation supernatant on colon cancer cell beta-catenin content (gray value)

Protein of interest/reference protein	Control	GS1108
			Beta-catenin/gapdh	1.6243	0.6061
Phosphorylated beta-catenin (P-beta-catenin)/gapdh	1.2770	0.6398

(2) Effect of GS1108 fermentation supernatant on apoptosis-inhibiting Gene protein content

Myeloid leukemia 1 (Mcl-1) is one of the anti-apoptotic bcl2 family members, and overexpression or amplification of Mcl1 is common in many cancer types and is therefore considered one of the most relevant oncoproteins. Our study shows that adding 1% of GS1108 fermentation supernatant to the culture medium can reduce the total MCL-1 protein content of colon cancer cells by 88.2% (Table 4), which shows that the fermentation broth has pro-cancer cell apoptosis activity. The research shows that mRNA and protein level expression of mcl1 gene subtype 1 in colon tumor tissue and corresponding side tissue is higher than that of normal group, and mcl1 subtype 1 and subtype 2 are positively correlated to malignant degree of colon cancer tumor, and can be used as one of effective diagnostic indexes for clinical evaluation of malignant degree of colon cancer.

The B cell lymphoma/leukemia-2 gene (bcl-2) also has obvious effect of inhibiting apoptosis, can inhibit cell death caused by various cytotoxins, is closely related to various cancers such as colon cancer, ovarian cancer and the like, and can enhance the resistance of cells to most cytotoxins through the overexpression of the gene. Our experiments show that the addition of 1% of GS1108 fermentation supernatant to the cell culture medium can reduce the total amount of Bcl-2 protein in colon cancer cells by 48.6% (Table 4), indicating that the fermentation supernatant has pro-apoptotic activity.

TABLE 4 influence of GS1108 fermentation supernatant on apoptosis inhibitor Gene protein content of colon cancer cells (Gray scale value)

(3) Effect of GS1108 fermentation supernatant on apoptosis factor protein content

The P62 protein is an oncogene protein, and a target gene for transcriptional regulation is involved in the processes of regulation of cell cycle, autophagy, cell proliferation, apoptosis, immortalization and the like, and plays an important role in the occurrence of a plurality of tumors. The P62 gene can help the human body to better recognize and resist exogenous antigens, thereby reducing the harm caused by infection. The P62 protein can also exert a cancer suppressing effect by promoting selective autophagy to prevent the accumulation of genotoxic and oncogenic mutations. When the P62 gene binds to an antigen, it triggers antigen-specific T cell and B cell functions. T cells secrete specific Cytokine to kill foreign pathogens, while B cells secrete specific antibodies to inhibit pathogen replication. Thus, the P62 gene can bind to an antigen, promote an immune response, and help the human body resist the foreign antigen, thereby reducing injury caused by viral infection or bacterial infection. Our experiments showed that the addition of 1% of the GS1108 fermentation supernatant to the cell culture medium greatly increased the total P62 protein of colon cancer cells, approximately 2.64 times that of the control (table 5), demonstrating the pro-apoptotic activity of the GS1108 fermentation supernatant.

Poly ADP-ribose polymerase, a poly a-p, is a DNA repair enzyme, and is a cleavage substrate for caspases (caspases) that are the core members of apoptosis, and thus plays an important role in DNA damage repair and apoptosis. PARP cleavage is considered an important indicator of apoptosis and is also commonly considered an indicator of Caspase 3 activation. PARP plays an important role in colon cancer growth and metastasis. Our results showed that the addition of 1% of the GS1108 fermentation supernatant to the medium increased the total poly-apyrase C-Parp content of colon cancer cells by 63% over the control (table 5), demonstrating the pro-apoptotic activity of the GS1108 fermentation supernatant.

TABLE 5 influence of GS1108 fermentation supernatant on the pro-apoptotic factor proteins of colon cancer cells (gray scale value)

Protein of interest/reference protein	Control	GS1108
			P62/gapdh	0.4673	1.2355
Poly (apyrase) C-Parp/gapdh	1.0479	1.7091

In conclusion, the lactobacillus fermentum GS1108 strain has good adhesion to colon cancer cells, the fermentation supernatant has obvious effect of inhibiting the growth of colon cancer cells, the content of beta-catenin related to adhesion diffusion can be reduced, the content of MCL-1 and Bcl-2 proteins representing apoptosis inhibition genes can be reduced, the content of pro-apoptosis factors, namely, the clear-parp of poly-adenosine diphosphate and the P62 protein representing autophagy can be increased, and the effects of inhibiting the growth of colon cancer cells and promoting apoptosis of the lactobacillus fermentum GS1108 are proved from multiple aspects, so that the lactobacillus fermentum GS1108 strain has wide application prospect.

Example 3 adaptation and safety of Lactobacillus fermentum GS1108 to the human intestinal tract

3.1 simulation of gastric and intestinal fluid tolerance

Simulated gastric and intestinal fluids were purchased from Shanghai leaf Biotechnology Inc. The artificial gastric juice simulated liquid comprises dilute hydrochloric acid, pepsin and sodium chloride, and the final pH is 2.5; the artificial intestinal juice simulation solution comprises potassium dihydrogen phosphate and trypsin, and has a final pH of 6.8. Resuscitating lactobacillus fermentum GS1108 of strain to be detected on MRS agar plate, activating for 3 generations, and regulating initial concentration of bacterial liquid to 1×10 ⁶ CFU/mL. 1mL of lactobacillus fermentum GS1108 bacterial liquid of the strain to be detected is inoculated into simulated gastric fluid and cultured for 3 hours at 37 ℃. After the completion of the culture, 20. Mu.L of the bacterial liquid was spread on an MRS agar plate medium, 3 replicates were set, and the culture was performed at 37℃for 24 hours, and the surface of the MRS plate after the culture was observed for colony growth. Similarly, 1mL of lactobacillus fermentum GS1108 bacteria liquid of the strain to be detected is inoculated into simulated intestinal juice, the simulated intestinal juice is cultured for 6 hours at 37 ℃, then a flat plate is coated, the culture is carried out for 24 hours at 37 ℃, and the growth condition of bacterial colonies is checked.

The simulated gastric fluid tolerance test result shows that the lactobacillus fermentum GS1108 can still grow normal colony on the MRS agar plate after being treated in the simulated gastric fluid for 3 hours; the simulated intestinal juice tolerance test result shows that the lactobacillus fermentum GS1108 can still grow normal colony on the MRS agar plate after being tolerant in the simulated intestinal juice for 6 hours; the lactobacillus fermentum GS1108 has stronger acid resistance and cholate resistance.

3.2 Lactobacillus fermentum GS1108 Strain hemolytic and antibiotic resistance

After resuscitating and activating the lactobacillus fermentum GS1108 strain for 3 generations, streaking and inoculating the strain on a Columbia blood plate, culturing the strain at 37 ℃ for 24 hours, and observing whether hemolytic rings appear around the colony to be detected, wherein the hemolytic staphylococci are used as positive control. The susceptibility of the strain to common large antibiotics was tested by a paper sheet agar diffusion method. Resuscitating lactobacillus fermentum GS1108 strain, activating for 3 passages, and regulating bacterial liquid concentration to 1×10 ⁶ CFU/mL, uniformly smearing bacterial liquid on the surface of MRS culture medium flat plate with sterile cotton swab, placing drug sensitive paper sheets after room temperature for 10min, culturing at 37deg.C for 24h, measuring the diameter of antibacterial circle around each drug sensitive paper sheet with vernier caliper, repeating each antibiotic 3 times, and determining drug sensitivity of strain by reference to American clinical laboratory standards Committee (NCCLS) standard, and measuring the results by sensitivity (S), mediator (I) and drug resistance (R) tableShown.

The results of the haemolytic experiments showed that no haemolytic loops occurred around the colonies, which were more sensitive to the 5 antibiotics tested, demonstrating the safety of the lactobacillus fermentum GS1108 strain (table 4).

TABLE 6 sensitivity of Lactobacillus fermentum GS1108 Strain to 5 antibiotics

Numbering device	Tetracycline	Ampicillin (Amoxicillin)	Gentamicin	Clarithromycin	Chloramphenicol
						GS1108	S	S	S	S	I

Example 4 probiotic Properties of Lactobacillus fermentum GS1108 Strain

4.1 short chain fatty acid production Capacity

Preparation of fermentation liquor: after the lactobacillus fermentum GS1108 preservation strain is activated and cultured for 24 hours, 4 mu L of bacterial liquid is sucked and added into 4mL of broth culture medium, and the broth culture medium is cultured for 24 hours at 37 ℃ for standby.

Detection of short-chain fatty acids: the detection instrument was a gas chromatograph-mass spectrometer (GCMS-QP 2010 Plus) from Shimadzu corporation, and the chromatographic column was a Rtx-5 fused silica capillary column (30 m. Times.0.25 mm. Times.0.25 μm) from RESTEK (Rasteck) corporation, USA. The GC temperature program was maintained at an initial temperature of 40℃for 5min, 5℃to 150℃per minute, 10℃to 280℃per minute, and 2min. The carrier gas is high purity helium (purity > 99.999%), flow rate: 1.0mL/min. MS conditions: the ionization mode is EI; the temperature is 200 ℃, the interface temperature is 220 ℃, and the mass scanning range m/z is 33-500. Taking 4mL of fermentation liquor, adding 10 mu L of 2-ethylbutyric acid internal standard solution with the concentration of 200 mu g/mL, sampling 1 mu L of sample in a mode of 1:3 of a split flow mode, setting the solvent delay time to 0.1min, and setting the temperature of a sample inlet to 270 ℃. The concentrations of 5 short chain fatty acids (acetic acid, n-butyric acid, isobutyric acid, isovaleric acid, 2-methylbutyric acid) were calculated by the internal standard method and the measurement results are shown in Table 7.

TABLE 7 Lactobacillus fermentum GS1108 fermentation broth short chain fatty acid content (ug/mL)

Strain number	Strain name	Acetic acid	Isopentanoic acid	2-methylbutyric acid
					GS1108	Lactobacillus fermentum	44.397	0.425	13.12

GC-MS detects the content of 5 short chain fatty acids, and the acetic acid content in the fermentation liquid of the lactobacillus fermentum GS1108 is found to be the highest, namely 44.397 mug/mL, and the fermentation liquid further comprises 2-methylbutyric acid and isovaleric acid, namely 13.12 mug/mL and 0.425 mug/mL respectively. Recent studies have found that acetic acid not only increases IgA production in the colon (immunoglobulin A is the most abundant immunoglobulin in mammals, a component that maintains mucosal surface homeostasis), but also alters IgA's ability to bind to specific microorganisms, including E.coli. Thus, acetic acid produced by intestinal microorganisms is thought to have the effect of regulating IgA production to maintain mucosal homeostasis. Butyric and isovaleric acids also have very important physiological functions.

4.2 bacteriostatic Activity against common intestinal pathogens

Escherichia coli (Escherichia coli CMCCB 44102), pseudomonas aeruginosa (Pseudomonasaeruginosa CMCCB 10104), staphylococcus aureus (Staphylococcus aureus CMCCB 50094), salmonella typhimurium (Salmonella typhimurium ATCC 14028), salmonella paratyphi B (Salmonellaparatyphi B CMCCB 50094) and Shigella dysenteriae (Shigella dysenteriae CMCCB 51105) were inoculated respectively to nutrient agar medium, recovered and activated 3 times. Sucking proper amount of trypticase liquid culture medium into a centrifuge tube, inoculating activated pathogenic bacteria into the broth culture medium, and regulating bacterial liquid concentration to 1×10 ⁸ CFU/mL. 1mL of the mixture of the pathogenic bacteria and the broth is sucked up and added into 500mL of nutrient agar culture medium which is not solidified temporarily after sterilization (the temperature is cooled to about 40 ℃), and the mixture is fully mixed and split-packed into culture dishes according to the amount of 20mL per dish. After the culture medium is cooled and solidified, a puncher with the diameter of 6mm is used for punching holes on a flat plate, so that a pathogenic bacteria agar plate is manufactured, each plate corresponds to one strain of bacteria, and three holes are formed as repetition. Resuscitating and activating the strain to be detected, and regulating the concentration of the cultured bacterial liquid to be 1 multiplied by 10 ⁸ CFU/mL. And (3) sucking 50 mu L of bacteria liquid to be detected, adding the bacteria liquid to the hole of the pathogenic bacteria agar plate, and culturing for 24 hours at 37 ℃. After incubation, the diameter of the zone of inhibition around the perforation point was measured using a vernier caliper and recorded. The above experimental procedure was performed simultaneously with the strain to be tested using the standard strain LGG as a control strain.

The fermentation broth has strong inhibitory activity on the growth of pathogenic bacteria such as escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi b, shigella dysenteriae and the like, and has basically equivalent effect to LGG standard strains (table 8). The microbial inoculum prepared from lactobacillus fermentum GS1108 strain can be used for preventing and controlling the pathogenic bacteria.

TABLE 8 evaluation of antibacterial Activity of Lactobacillus fermentum GS1108 Strain (diameter: mm)

4.3 antioxidant Activity of Lactobacillus fermentum GS1108 fermentation broth

Antioxidants are any substance that is effective in inhibiting the oxidation reaction of free radicals at low concentrations to counteract oxidative attack of the free radicals on human cells. The mechanism of action of the compound can be directly acting on free radicals or indirectly consuming substances which are easy to generate free radicals, so that further reaction is prevented, and the more the organism has strong oxidation resistance, the more healthy the organism is and the longer the life is. More and more studies have shown that antioxidant is an important step in preventing aging, because free radicals or oxidants break down cells and tissues, affect metabolic functions, and cause different health problems. If it is capable of eliminating excessive oxidative free radicals, it can be prevented from many diseases caused by free radicals and related to aging. Such as common cancers, arteriosclerosis, diabetes, cataracts, cardiovascular diseases, senile dementia, arthritis, etc., which are all considered to be related to free radicals.

Preparation of fermentation liquor: resuscitating and culturing lactobacillus fermentum GS1108 frozen strain for 24 hours, inoculating the strain into MRS liquid culture medium according to 2% of inoculum size, culturing for 24 hours at 37 ℃, centrifuging the bacterial liquid for 15 minutes at 4 ℃ and 4000r/min, and filtering the supernatant by a 0.22 mu m filter membrane to obtain fermentation supernatant (CFS) for freezing and storing.

Reagents and instrumentation: the total antioxidant capacity (T-AOC) detection kit (A015-1-2), the hydroxyl radical assay kit (A018-1-1), the DPPH radical scavenging capacity kit (A153-1-1) and the superoxide anion radical inhibition and generation assay kit (colorimetric method A052-1-1) are all produced by Nanjing's established bioengineering research. An ultraviolet visible spectrophotometer (UV 752N type), manufactured by Shanghai you family instruments and meters limited; the antioxidant capacity of the fermentation broth was measured by Chengdu Biotechnology Co.

Total antioxidant capacity (Total antioxidant capacity, T-AOC) refers to the total antioxidant level of the various antioxidant substances make up. The research on antioxidation can effectively overcome the harm brought by the antioxidation, so that the enterprises of the antioxidation healthcare products and cosmetics are one of main research and development directions and one of the most important functional requirements of the market. Many antioxidant substances can reduce Fe3+ into Fe2+, and the Fe+ can form a firm complex with phenanthrene substances, and the oxidation resistance of the Fe+ can be measured by colorimetry. Absorbance was measured using a wavelength 520nm,1cm optical path, double distilled water zeroed. The specific steps are carried out according to the instruction of a total antioxidant capacity (T-AOC) detection kit. The measurement results of the total antioxidant capacity of the fermentation broth of lactobacillus fermentum GS1108 are shown in Table 9, and the total antioxidant capacity of the fermentation broth of Pediococcus pentosaceus TE0307 is 39.10+/-0.65U/mL, which indicates that the fermentation broth of GS1108 has better antioxidant capacity.

Ability to inhibit hydroxyl radicals: one of the hydroxyl radical active oxygen has extremely strong electron-obtaining ability, namely, has extremely strong oxidizing ability. Hydroxyl radicals kill red blood cells and degrade DNA, cell membranes and polysaccharide compounds. Fenton (Fenton) reaction is the most common chemical reaction generating hydroxyl radicals, H ₂ O ₂ The amount of (2) is proportional to the amount of hydroxyl radicals generated by the Fenton reaction, and after the electron acceptor is given, the color is developed by the Griess reagent to form a red substance, and the color is proportional to the amount of the hydroxyl radicals. The absorbance was measured at 550nm, exactly as per the instructions. The calculation formula is as follows:

ability to inhibit hydroxyl radicals (U/mL) = (a) _Control -A _Measurement )/(A _{Standard of} -A _{Blank space} )×C _{Standard of} ×(1/V _Sample )×N

In the formula, the standard concentration is 8.824mmol/L; v sample: sampling amount, 0.2mL; n: dilution fold before sample testing.

The measurement results are shown in Table 8, and the fermentation liquid of the lactobacillus fermentum GS1108 has stronger capability of inhibiting hydroxyl radicals, namely 3150.54 +/-111.27U/mL.

DPPH radical scavenging ability: DPPH is also called 1, 1-diphenyl-2-trinitrophenylhydrazine, and is a very stable free radical of nitrogen center. Since DPPH free radical has single electron, there is a strong absorption at 517nm, its alcohol solution is purple, when free radical scavenger exists, its absorption gradually disappears due to pairing with its single electron, the light color is presented, therefore, the DPPH scavenging ability in the sample can be quantitatively analyzed. According to the instruction of the kit, one standard substance powder is dissolved by adding 2mL of absolute methanol to obtain 0.5mg/mL (Trolox) standard application liquid, and then the standard substance powder is diluted into 5 mug/mL, 10 mug/mL, 15 mug/mL, 20 mug/mL and 25 mug/mL respectively by using absolute methanol, the light path with the wavelength of 517nm and 1cm is zeroed by using absolute ethanol, and the absorbance of each tube is measured to prepare a standard curve.

DPPH radical clearance (%) = (1- (assay-a control)/(a blank) ×100%

The DPPH radical scavenging capacity of the samples was expressed as the amount corresponding to the antioxidant Trolox calculated from the standard curve. Fermentation broth samples DPPH free radical scavenging capacity (μg Trolox/mL) =substituted into the standard curve to give a concentration corresponding to Trolox x dilution factor. The DPPH radical scavenging ability measurement results of the fermentation broth of the lactobacillus fermentum GS1108 are shown in Table 9, and the DPPH radical scavenging ability 177.20 + -1.42 mug Trolox/mL of the fermentation broth of the lactobacillus fermentum GS1108 shows that the fermentation broth of the lactobacillus fermentum GS1108 has better DPPH radical scavenging ability.

Resistance to superoxide anions: the superoxide anion free radical is used as a free radical generated in the metabolic process of organisms, and can attack biological macromolecules such as lipid, protein, nucleic acid, polyunsaturated fatty acid and the like, so that the biological macromolecules are crosslinked or broken, the cell structure and the function are damaged, and the superoxide anion free radical has a close relationship with the aging and pathological changes of the organisms. The experiment simulates a xanthine and xanthine oxidase reaction system in an organism to generate superoxide anion free radicals, an electron transfer substance and a Gress's color developing agent are added to enable the reaction system to be purple red, a spectrophotometer is used for measuring the absorbance of the reaction system, when a tested sample contains a superoxide anion free radical inhibitor, the absorbance of a measuring tube is lower than that of a control tube in colorimetric process, and the inhibition capacity of a tested object to the superoxide anion free radicals can be calculated by taking vitamin C as a standard. When in measurement, a 1cm optical path cuvette is used, double distilled water is used for zeroing, and color comparison is carried out at the wavelength of 550 nm.

Superoxide anion resistance (U/L) = (a) _Control -A _Measurement )/(A _Control -A _{Standard of} )×C _{Standard of} ×1000×N

In the reaction system, the change value of the superoxide anion radical inhibited by the reaction of each liter of fermentation broth at 37 ℃ for 40 minutes corresponds to 1mg of the superoxide anion radical inhibited by vitamin C, and the change value is one activity unit. As shown in Table 8 below, the measurement results of the anti-superoxide anion capacity of the fermentation broth of Lactobacillus fermentum GS1108 show that the inhibition capacity of the fermentation broth of Lactobacillus fermentum GS1108 against superoxide anion radicals is 785.47 + -7.56 (U/L), which indicates that the inhibition capacity of the fermentation broth of Lactobacillus fermentum GS1108 against superoxide anion radicals is stronger.

TABLE 9 antioxidant capacity of Lactobacillus fermentum GS1108 fermentation broth

In conclusion, the GS1108 fermentation liquor has strong antioxidant capacity and potential application value in the field of functional foods, including being used for improving the health level of human bodies.

While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims

1. A strain of lactobacillus fermentum (Lactobacillus fermentans) GS1108 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 27395 in the year 2023 and the month 5 and the day 22.

2. Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 according to claim 1 for the manufacture of an anti-colon cancer medicament or an anti-colon cancer adjuvant.

3. Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 of claim 1 for inhibiting escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi b or shigella dysenteriae for non-disease treatment purposes.

4. Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 according to claim 1 for the manufacture of a medicament for the treatment of a disease caused by escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi b or shigella dysenteriae.

5. Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 according to claim 1 for the preparation of a medicament with antioxidant function.

6. Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 according to claim 1 in a functional food for modulating the intestinal flora of a human or animal.

7. An anti-colon cancer drug or an anti-colon cancer adjuvant comprising lactobacillus fermentum (Lactobacillus fermentans) GS1108 of claim 1.

8. An antioxidant drug comprising lactobacillus fermentum (Lactobacillus fermentans) GS1108 of claim 1.

9. A functional food comprising lactobacillus fermentum (Lactobacillus fermentans) GS1108 of claim 1.

10. Use of lactobacillus fermentum (Lactobacillus fermentans) GS1108 in combination with 5-fluorouracil or oxaliplatin according to claim 1 for the preparation of a medicament for the treatment of colon cancer.