A kind of Lactococcus lactis lactic acid subspecies capable of regulating colon tumor signaling pathway and its use

A technology of Lactococcus lactis and Lactic acid subsp. is applied in the field of microorganisms, which can solve the problems of inability to be used in food, unclear regulation of tumor-related signaling pathways, etc., and achieves alleviation of inflammation, good resistance to gastric acid and bile salts, and improvement of integrity. Effect

Active Publication Date: 2020-08-11
JIANGNAN UNIV YANGZHOU FOOD BIOTECH INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] CN104523761A discloses a genetically engineered strain of Lactococcus lactis capable of inducibly expressing interleukin 12, which can inhibit the growth of intestinal tumors, but this bacterium is not obtained from nature and can only be used for medicine and not for food
The above three patented strains are clear and have clear functions, but the animal model involved uses colon cancer cell lines subcutaneously bearing tumors, which is quite different from the tumor environment in the intestine, and the role in the regulation of tumor-related signaling pathways is not clear

Method used

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  • A kind of Lactococcus lactis lactic acid subspecies capable of regulating colon tumor signaling pathway and its use
  • A kind of Lactococcus lactis lactic acid subspecies capable of regulating colon tumor signaling pathway and its use
  • A kind of Lactococcus lactis lactic acid subspecies capable of regulating colon tumor signaling pathway and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Lactococcus lactis subspecies VM5 has good tolerance to simulated gastrointestinal fluid

[0066] Streak-inoculate the cryopreserved Lactococcus lactis subsp. lactic acid VM5 in the solid medium of MRS medium, culture it aerobically at 37°C for 48 hours, and subculture it in MRS medium for 2 to 3 times, then take Lactococcus lactis Lactic acid subspecies VM5 culture fluid was collected by centrifugation at 8000×g for 5 min, resuspended in (1:1) artificial simulated gastric juice of pH 2.5 (MRS medium containing 1% pepsin, pH=2.5), and then mixed in Anaerobic culture was carried out at 37°C. Samples were taken at the beginning (0h), 1h, 2h and 3h respectively, and MRS agar medium was poured for culture to count plate colonies, determine the number of viable bacteria and calculate their survival rate. The survival rate is the ratio of the number of viable bacteria in the culture medium to the number of viable bacteria at 0h, expressed in %.

[0067] Take the c...

Embodiment 2

[0072] Example 2: Regulation of Lactococcus lactis subsp. lactic acid VM5 on Notch1, VEGFR2 and colon cancer-related signaling pathways in HT-29 cells

[0073] Use RPMI1640 cell culture medium without antibiotics to resuspend the bacteria sludge of Lactococcus lactis subsp. lactis VM5 (or control Lactococcus lactis subsp. lactis LM35, LGG, Escherichia coli E.coli) washed twice with PBS, and adjust the bacteria Bulk density to about 2×10 8 CFU / mL; add VM5 (or LM35, LGG, E.coli) suspension into the 6-well plate cultured with HT-29 according to the amount of 2mL bacterial suspension per well (cell fusion degree reaches 95%), blank group Add 2 mL of antibiotic-free cell culture medium and place at 37 °C with 5% CO 2 Incubate in a cell culture incubator for 2 hours; wash the cells 3 times with PBS, add 1 mL TRIzol to each well (6-well plate), let stand at room temperature for 5 minutes, repeatedly pipette with an enzyme-free pipette tip, and then transfer to 1.5 mL EP without enzy...

Embodiment 3

[0078] Example 3: Lactococcus lactis subspecies VM5 has no acute toxic side effects on mice

[0079] Lactococcus lactis subspecies VM5 was resuspended in 2% (w / v) sucrose solution, and the cell density was 4.0×10 9 CFU / mL. Take 10 healthy male BALB / c mice with a body weight of about 25 g, give the suspension of this concentration orally once a day, observe for a week, and record the death and body weight.

[0080] The results of these tests are listed in Table 4. These results indicated that feeding concentrations of 4.0×10 9 CFU / mL of Lactococcus lactis subsp. lactic acid VM5 had no significant impact on the mice, no significant change in body weight, and no death. The appearance of the mice had no obvious pathological symptoms.

[0081] Table 4 Changes in body weight and death conditions of mice

[0082] time (days) 1 2 3 4 5 6 7 weight (g) 25.6±1.4 25.7±1.5 25.7±1.9 25.8±2.3 25.8±1.6 25.9±1.6 26.0±1.5 Circumstances of death - - - -...

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Abstract

The invention discloses a Lactococcus lactis subsp. lactis capable of significantly inhibiting the occurrence of colorectal cancer and an application thereof, belonging to the technical field of microorganisms. Lactococcus lactis subsp. lactic acid VM5 provided by the present invention has a preservation number of GDMCC No.60256, can tolerate gastric acid and bile salts, and can significantly reduce the inflammatory response in the colorectal region of colorectal cancer model mice. The expression of Notch1 signaling pathway and VEGFR2 molecules in tissues can reduce the number of tumors in the colon and rectum of model mice. In addition, Lactococcus lactis subsp. lactic acid VM5 can also improve the structure of intestinal flora and the level of short-chain fatty acids in the intestine. The Lactococcus lactis lactic acid subspecies VM5 is used to prepare fermented food for inhibiting the occurrence of colorectal cancer, and has very broad application prospects.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a Lactococcus lactis subsp. lactis capable of significantly inhibiting the occurrence of colorectal cancer by regulating Notch1 and VEGFR2 signaling pathways and its use. Background technique [0002] Colorectal cancer is a malignant gastrointestinal tumor. As the morbidity and mortality of colorectal cancer continue to rise in my country, its threat to the health of our people is gradually deepening. At present, the mainstream methods for the clinical treatment of colorectal cancer all have relatively large side effects, which greatly affect the quality of life of patients. As a safe edible food, probiotics have been gradually confirmed by researches to alleviate and treat various diseases, such as promoting digestion and absorption, enhancing immunity, preventing reproductive system infections, alleviating allergic reactions, preventing and inhibiting Tumors, preventio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A23C9/12A23L11/00A23C11/10A23L19/20A23L33/00A23L33/135A61K35/744A61P35/00C12R1/01A23L11/50A23L11/60
Inventor 陈卫王刚赵建新张灏
Owner JIANGNAN UNIV YANGZHOU FOOD BIOTECH INST
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