Lactic acid bacteria strains capable of inhibiting multiple drug-resistant food-borne pathogenic bacteria in broad-spectrum manner and application of lactic acid bacteria strains

Abstract

The invention discloses lactic acid bacteria strains capable of inhibiting multiple drug-resistant food-borne pathogenic bacteria in a broad-spectrum manner and an application of the lactic acid bacteria strains. The strains have functions of inhibiting the activity of multiple drug-resistant bacteria and food-borne pathogenic bacteria in a broad spectrum manner, and are named as lactobacillus plantarum GS083, lactobacillus plantarum GS086, lactobacillus plantarum GS090 and lactobacillus fermentum GF110. The lactic acid bacteria strains provided by the invention has a very good bacteriostaticeffect on multiple drug-resistant bacteria and food-borne pathogenic bacteria, and can inhibit gram-negative bacteria and gram-positive bacteria; the strains also have the characteristics of bile salthydrolase activity, acid resistance, bile salt resistance, low drug resistance, no hemolysis, no toxicity gene and the like, enrich the lactobacillus strain resource library, and have wide application prospects in the industries of food, cosmetics, medicines and the like.

Description

technical field

[0001] The invention belongs to the technical field of microbes and relates to lactic acid bacteria, in particular to a lactic acid bacteria strain capable of broad-spectrum inhibiting multidrug-resistant food-borne pathogenic bacteria and an application thereof. Background technique

[0002] The frequent outbreak of food safety problems is mainly due to the growth and reproduction of pathogenic bacteria. Food-borne pathogens can cause human diseases. WHO estimates that there are thousands of cases of food-borne diseases every year in the world, endangering human health. Although, antimicrobial agents, including antibiotics, have saved countless lives and helped people improve their quality of life. Unfortunately, the overuse and misuse of antimicrobials has led to the emergence and mass spread of resistant microorganisms, and antimicrobial resistance has become one of the major threats to human health. Therefore, research on alternatives to antibiotics and...

Examples

Embodiment 1

[0022] Isolation and screening of embodiment 1 lactic acid bacteria

[0023] 1. Preliminary screening of lactic acid bacteria

[0024] Collect homemade kimchi as samples, immerse each sample in 100mL MRS broth medium, enrich and culture at 37°C for 24 hours, and inoculate them in MRS solid medium after 10-fold serial dilution, and culture them anaerobically at 37°C for 48 hours , pick the typical colonies on the plate, separate them by streaking to obtain pure colonies, and carry out microscopic examination, Gram staining and catalase activity test. The purified strains were stored in a -80°C refrigerator.

[0025] A total of 63 suspected strains were screened, among which the results of Gram staining were positive and the results of catalase activity test were negative. The results are shown in Table 1 ("+" is positive, "-" is negative).

[0026] Table 1 Screening results of lactic acid bacteria

[0027]

[0028]

[0029]

[0030] 2. Screening of acid-resistant a...

Embodiment 2

[0061] Identification of embodiment 2 lactic acid bacteria

[0062] (1) API identification

[0063] The strains GS083, GS086, GS090 and GF110 were identified using API50CHL series identification reagent strips, and the identification results are shown in Table 6 ("+" indicates positive, "-" indicates negative, "?" indicates suspicious).

[0064] The identification results of API50CHL series of strains in Table 6

[0065]

[0066]

[0067]

[0068] It can be seen from Table 6 that the bacterial strains GS083, GS086 and GS090 of the present invention are all Lactobacillus plantarum, and the identification rate is 99.9%; the bacterial strain GF110 is Lactobacillus fermentum, and the identification rate is 99.9%

[0069] (2) Identification of the strain by 16S rDNA sequencing

[0070] The DNA of bacterial strains GS083, GS086, GS090 and GS090 was obtained using a genomic DNA extraction kit as a template for PCR amplification, using universal primers (forward primer is 5...

Embodiment 3

[0073] The probiotic property of embodiment 3 lactic acid bacteria

[0074] (1) Simulated gastric juice and pancreatic juice viability test

[0075] Simulated gastric juice: prepared by dissolving 0.35% (w / v) pepsin in phosphate buffered solution (PBS), acidified with 1M HCl to pH=2.0; simulated pancreatic juice: containing 1.1% (w / v) NaHCO 3 , 0.1% (w / v) trypsin solution in PBS, adjusted to pH 8.0 with 1M NaOH.

[0076] After centrifuging the overnight cultured bacteria solution, collect the bacteria, wash twice with PBS, resuspend in the same amount of artificial gastric juice, culture at 37°C, measure the number of viable bacteria by plate counting method at 0h and 3h respectively, and calculate their survival Rate(%). The collected bacteria treated with artificial gastric juice were suspended in the same volume of simulated pancreatic juice, incubated at 37°C for 24h, and the number of viable bacteria was measured by plate counting method at 0h and 24h respectively, and ...

technical field

[0001] The invention belongs to the technical field of microbes and relates to lactic acid bacteria, in particular to a lactic acid bacteria strain capable of broad-spectrum inhibiting multidrug-resistant food-borne pathogenic bacteria and an application thereof. Background technique

[0002] The frequent outbreak of food safety problems is mainly due to the growth and reproduction of pathogenic bacteria. Food-borne pathogens can cause human diseases. WHO estimates that there are thousands of cases of food-borne diseases every year in the world, endangering human health. Although, antimicrobial agents, including antibiotics, have saved countless lives and helped people improve their quality of life. Unfortunately, the overuse and misuse of antimicrobials has led to the emergence and mass spread of resistant microorganisms, and antimicrobial resistance has become one of the major threats to human health. Therefore, research on alternatives to antibiotics and...

Examples

Embodiment 1

[0022] Isolation and screening of embodiment 1 lactic acid bacteria

[0023] 1. Preliminary screening of lactic acid bacteria

[0024] Collect homemade kimchi as samples, immerse each sample in 100mL MRS broth medium, enrich and culture at 37°C for 24 hours, and inoculate them in MRS solid medium after 10-fold serial dilution, and culture them anaerobically at 37°C for 48 hours , pick the typical colonies on the plate, separate them by streaking to obtain pure colonies, and carry out microscopic examination, Gram staining and catalase activity test. The purified strains were stored in a -80°C refrigerator.

[0025] A total of 63 suspected strains were screened, among which the results of Gram staining were positive and the results of catalase activity test were negative. The results are shown in Table 1 ("+" is positive, "-" is negative).

[0026] Table 1 Screening results of lactic acid bacteria

[0027]

[0028]

[0029]

[0030] 2. Screening of acid-resistant a...

Embodiment 2

[0061] Identification of embodiment 2 lactic acid bacteria

[0062] (1) API identification

[0063] The strains GS083, GS086, GS090 and GF110 were identified using API50CHL series identification reagent strips, and the identification results are shown in Table 6 ("+" indicates positive, "-" indicates negative, "?" indicates suspicious).

[0064] The identification results of API50CHL series of strains in Table 6

[0065]

[0066]

[0067]

[0068] It can be seen from Table 6 that the bacterial strains GS083, GS086 and GS090 of the present invention are all Lactobacillus plantarum, and the identification rate is 99.9%; the bacterial strain GF110 is Lactobacillus fermentum, and the identification rate is 99.9%

[0069] (2) Identification of the strain by 16S rDNA sequencing

[0070] The DNA of bacterial strains GS083, GS086, GS090 and GS090 was obtained using a genomic DNA extraction kit as a template for PCR amplification, using universal primers (forward primer is 5...

Embodiment 3

[0073] The probiotic property of embodiment 3 lactic acid bacteria

[0074] (1) Simulated gastric juice and pancreatic juice viability test

[0075] Simulated gastric juice: prepared by dissolving 0.35% (w / v) pepsin in phosphate buffered solution (PBS), acidified with 1M HCl to pH=2.0; simulated pancreatic juice: containing 1.1% (w / v) NaHCO 3 , 0.1% (w / v) trypsin solution in PBS, adjusted to pH 8.0 with 1M NaOH.

[0076] After centrifuging the overnight cultured bacteria solution, collect the bacteria, wash twice with PBS, resuspend in the same amount of artificial gastric juice, culture at 37°C, measure the number of viable bacteria by plate counting method at 0h and 3h respectively, and calculate their survival Rate(%). The collected bacteria treated with artificial gastric juice were suspended in the same volume of simulated pancreatic juice, incubated at 37°C for 24h, and the number of viable bacteria was measured by plate counting method at 0h and 24h respectively, and ...

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