CN116463264B - Lactobacillus plantarum with colon cancer cell growth inhibition effect and application thereof - Google Patents
Lactobacillus plantarum with colon cancer cell growth inhibition effect and application thereof Download PDFInfo
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- CN116463264B CN116463264B CN202310440651.1A CN202310440651A CN116463264B CN 116463264 B CN116463264 B CN 116463264B CN 202310440651 A CN202310440651 A CN 202310440651A CN 116463264 B CN116463264 B CN 116463264B
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Abstract
The invention discloses a lactobacillus plantarum with a colon cancer cell growth inhibition effect and application thereof, wherein 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, has better tolerance to acid, bile salts, artificial gastric juice and artificial intestinal juice, has stronger adhesion capability to human colon cancer cells, has stronger cancer cell activity inhibition capability after fermentation supernatant and colon cancer cell HCT-116 are co-cultured, has stronger inhibition capability to escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli and the like, can generate various short-chain fatty acids such as acetic acid, butyric acid and the like, and has stronger inhibition effect on alpha-glucosidase and stronger antioxidation. Therefore, the strain has obvious probiotic effect and can be applied to the field of functional foods for human and animals.
Description
Technical Field
The invention belongs to the technical field of microorganisms, in particular to lactobacillus plantarum with a strong inhibition effect on colon cancer cells and various intestinal pathogens, and particularly relates to the field of functional lactobacillus and product production and development thereof.
Background
Lactobacillus plantarum (Lactobacillus plantarum) is widely found in a variety of environments in plants, fermented foods, human and animal intestines, and is known for its ability to ferment sugars to lactic acid and health benefits. In particular, lactobacillus plantarum has been studied to demonstrate the ability to improve intestinal health by regulating intestinal flora balance and reducing inflammation. It has also found potential application in food preservation and fermentation and in the production of probiotics and other functional foods.
Lactobacillus plantarum can regulate intestinal flora balance by inhibiting the growth of harmful bacteria and increasing the number of beneficial bacteria; the health condition of the intestinal canal can be improved by reducing the inflammation of the intestinal canal, promoting the health of the intestinal mucosa, enhancing the immune function and the like; can reduce cholesterol level in human body, and can be used for preventing cardiovascular diseases. Therefore, the lactobacillus plantarum can bring various benefits to human health, and has great development and application values. However, the current lactobacillus plantarum does not have the effect of treating colon cancer, so that the development of the lactobacillus plantarum with the effect of inhibiting the growth of colon cancer cells has great value.
The pickled Chinese cabbage is rich in various nutritional ingredients including vitamin C, dietary fiber, lactobacillus and the like, has the effects of regulating intestinal flora, promoting appetite, helping digestion and the like, and is beneficial to enhancing human immunity and maintaining intestinal health. Therefore, the pickled Chinese cabbage is a traditional special food with rich nutrition and delicious taste, and is deeply favored by people. According to research and analysis, the probiotics in Guizhou pickled Chinese cabbage are rich, especially lactobacillus plantarum is rich, and conditions are created for separating and screening lactobacillus plantarum with good comprehensive probiotics.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides lactobacillus plantarum with a colon cancer cell growth inhibition effect and application thereof, and the lactobacillus plantarum has the effects of colon cancer cell growth inhibition, bacteriostasis and anti-inflammatory effects, acid production and blood glucose reduction and improvement of human intestinal environment adaptability.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
lactobacillus plantarum GZ1806 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 20 months in 2023, and is named as Lactobacillus plantarum (Lactobacillus plantarum) in the North Celu No. 1, no. 3 of the Korean area of Beijing, with a preservation number of CGMCC No. 26590.
The lactobacillus plantarum GZ1806 is applied to preparation of anti-colon cancer drugs or anti-colon cancer auxiliary drugs.
The use of lactobacillus plantarum GZ1806 as described above for inhibiting escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli, listeria monocytogenes, staphylococcus hemolyticus, pasteurella multocida or yersinia enterocolitica for non-disease therapeutic purposes.
Application of lactobacillus plantarum GZ1806 in preparing medicines for treating diseases caused by escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli, listeria monocytogenes, staphylococcus hemolyticus, pasteurella multocida or yersinia enterocolitica.
The lactobacillus plantarum GZ1806 is applied to the preparation of the hypoglycemic or antioxidant drugs.
The application of the lactobacillus plantarum GZ1806 in functional food for regulating intestinal flora of human beings or animals.
An anti-colon cancer drug or an anti-colon cancer auxiliary drug, comprising the lactobacillus plantarum GZ1806.
A hypoglycemic agent comprises Lactobacillus plantarum GZ1806.
An antioxidant drug comprises Lactobacillus plantarum GZ1806.
A functional food comprises the Lactobacillus plantarum GZ1806.
The lactobacillus plantarum GZ1806 is obtained by separating and screening acid and hot pickled peppers collected from Fengshui town in Li Ping county, guizhou. Lactobacillus plantarum GZ1806 grows well on MRS agar culture medium, and the colony is milky white, smooth in surface, neat in edge, opaque, and is subjected to microscopic examination to form of the bacterium, and gram staining is purple and rod-shaped. The target gene sequence consisting of 1000 base pairs (bp) is obtained by PCR amplification by using the bacterial universal primer 16S rRNA 27F/1492R as a template, and the sequence is as follows: GCGCAGTGCGGGGTGCTATAATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGAATGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTATTTCGAATCTACGCGAAGAACCTTAACAGATCTTGACATTCTATGCAAT
The gene sequence obtained by sequencing is input into NCBI database for comparison, the similarity rate of the gene sequence with the standard strain Lactobacillus plantarumstrain 103B3 in GenBank reaches 98.80%, and the strain can be initially identified as lactobacillus plantarum (Lactobacillus plantarum).
The lactobacillus plantarum GZ1806 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 plantarum GZ1806 strain has strong intestinal tract adaptability and better tolerance to acid, bile salts, artificial gastric juice and artificial intestinal juice;
the lactobacillus plantarum GZ1806 strain has obvious probiotics performance, has stronger adhesion capability to human colon cancer cells, has stronger cancer cell activity inhibition capability after fermentation supernatant and colon cancer cells HCT-116 are co-cultivated, has stronger inhibition capability to escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli and the like, can generate various short-chain fatty acids such as acetic acid, butyric acid and the like, has stronger inhibition effect to alpha-glucosidase and stronger antioxidation effect.
The beneficial effects of the invention are as follows:
1. the lactobacillus plantarum GZ1806 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 plantarum GZ1806 provided by the invention has good adhesion capability to human colon cancer cells and has stronger cancer cell activity inhibition capability.
3. The lactobacillus plantarum GZ1806 provided by the invention has stronger antibacterial capability on common intestinal pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli, listeria monocytogenes, staphylococcus hemolyticus, pasteurella multocida, yersinia enterocolitica and the like.
4. The lactobacillus plantarum GZ1806 provided by the invention can generate various short-chain fatty acids such as acetic acid, butyric acid and the like, has a strong inhibition effect on alpha-glucosidase and has a strong antioxidation effect.
Drawings
FIG. 1 is a diagram showing the phylogenetic relationship between Lactobacillus plantarum GZ1806 and other strains;
FIG. 2 shows colony morphology (A) and gram staining (B) of Lactobacillus plantarum GZ1806 strain of the present invention on MRS agar medium;
FIG. 3 is a standard graph of MTT assay for cellular activity;
FIG. 4 shows the inhibition of colon cancer cell growth at different concentrations of lactobacillus plantarum GZ1806 fermentation supernatant of the present invention;
FIG. 5 shows the hemolytic activity of Lactobacillus plantarum GZ1806 strain according to the present invention;
FIG. 6 is a GC-MS detection ion flow diagram of short chain fatty acid of lactobacillus plantarum GZ1806 fermentation supernatant of the invention.
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 plantarum GZ 1806:
1. material preparation
The sour and hot pickled peppers are obtained from Fengshen in Li Ping county of Guizhou;
the 16S rDNA universal primers 27F and 1492R universal primers 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
2g of the fermented food sample is taken in 20mM RS 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 biological engineering (Shanghai) limited company, the sequences are compared with BLAST in NCBI to obtain 181 lactobacillus plantarum, one strain has 99.60 percent of similarity with a standard strain Lactobacillus plantarumstrain 103B3, the strain can be initially identified as lactobacillus plantarum (Lactobacillus plantarum), the strain is named GZ1806, and the phylogenetic relationship between the strain and other strains is shown as figure 1. The Lactobacillus plantarum GZ1806 is subjected to gram staining by a kit method, and the bacterial morphology after staining is observed and recorded under a microscope. Lactobacillus plantarum GZ1806 grows well on MRS agar medium, and the colony form is milky white, round convex, flat in edge and smooth in surface, as shown in FIG. 2. The microscopic examination shows that the thallus is rod-shaped and purple, and accords with the morphological characteristics of lactobacillus plantarum. The strain is preserved in China general microbiological culture Collection center, address: the collection number of the microbiological institute of China academy of sciences is CGMCC 26590, and the collection number of the microbiological institute of China is No. 3 in North Chen West Lu of the Chaoyang area of Beijing city.
EXAMPLE 2 growth inhibition of colon cancer cells by Lactobacillus plantarum GZ1806 fermentation broth
2.1 adhesion of Lactobacillus plantarum GZ1806 to human colon cancer cells
Lactobacillus plantarum GZ1806 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 at 37℃with 5% CO 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 addedInto the cell well, the cell culture plate was slightly shaken, and a small amount of bacterial liquid in the well was aspirated for plate counting, and the result was taken as the initial viable 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. And the standard strain LGG was used as a control.
Adhesion (%) = number of lactic acid bacteria at end period/number of initial lactic acid bacteria inoculation x 100%
The results are shown in Table 1. As a result, it was found that the adhesion rate of Lactobacillus plantarum GZ1806 to human colon cancer cell HT-29 was 18.83%.
TABLE 1 adhesion Rate of Lactobacillus plantarum GZ1806 to human colon cancer cells HT-29 (%)
| Strain | Repeat 1 | Repeat 2 | Repeat 3 | Mean value of |
| GZ1806 | 18.06 | 19.63 | 18.79 | 18.83±0.68 |
| Standard Strain LGG | 15.13 | 16.42 | 15.14 | 15.56±0.94 |
2.2 growth inhibition of colon cancer cells by Lactobacillus plantarum GZ1806 fermentation supernatant
Lactobacillus plantarum GZ1806 fermentation supernatant preparation: resuscitating Lactobacillus plantarum GZ1806, 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, transfer them on a sterile operating table into a prepared 15mL centrifuge tube containing culture medium, centrifuge at 1000r/min for 3min to resuspend the cells, aspirate into a 6cm cell culture dish, and mix with 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.
Cell activity was measured by MTT assay: the influence of lactobacillus plantarum GZ1806 fermentation supernatants with different concentrations on the growth of human colon cancer cells is detected by adopting a Biyundian MTT cell proliferation and cytotoxicity detection kit. The MTT cell proliferation and cytotoxicity detection kit (MTT Cell Proliferation and Cytotoxicity Assay Kit) is a very classical cell proliferation and cytotoxicity detection kit and is widely used for detecting cell proliferation and cytotoxicity. MTT can be reduced by some dehydrogenases in the mitochondria of living cells to form crystalline dark purple product formazan (formazan) which is deposited in cells, but dead cells do not. Dimethyl sulfoxide (DMSO) can solubilize formazan in cells and absorbance is measured at 570nm using a microplate reader. The more and faster the cell proliferation, the higher the absorbance, the greater the cytotoxicity, the lower the absorbance, and the standard curve is shown in fig. 3.
25mg of MTT was dissolved in 5mL of MTT solvent to prepare a 5mg/mL MTT solution. About 2000 resuscitated human colon cancer cells were added per well, and 1. Mu.L, 0.75. Mu.L, 0.5. Mu.L, and 0.25. Mu.L Lactobacillus plantarum GZ1806 fermentation supernatants were added, three replicates per concentration, and a blank control was set at 5% CO 2 Culturing in an incubator at 37 ℃ for 120 hours. Then adding 10 mu LMTT solution into each culture hole, continuously incubating for 4 hours in a cell culture box, adding 100 mu L Formazan solution into each hole, properly mixing, continuously incubating for 4 hours in the cell culture box, observing that Formazan is completely dissolved under a common optical microscope, and measuring absorbance by using 570nm wavelength of an enzyme-labeled instrument.
The measurement results are shown in Table 2. The lactobacillus plantarum GZ1806 fermentation supernatants with different concentrations show obvious inhibition effect on the growth of human colon cancer cells, the effect is most obvious when 1% of fermentation supernatant is added, the inhibition effect on the cell growth is weakened along with the reduction of the concentration of the supernatant, and when the concentration is reduced to 0.25%, the inhibition effect is greatly weakened, as shown in figure 4.
TABLE 2 cell Activity (absorbance) of different concentrations of fermentation supernatants co-cultured with human colon cancer cells HCT116 for 5 days
Example 3 adaptation and safety of Lactobacillus plantarum GZ1806 to human intestinal tract
3.1, evaluation of acid resistance and bile salt resistance: resuscitating Lactobacillus plantarum GZ1806 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. MRS broth medium of acidity (ph=3.0) and bile salt concentration (0.3%) was prepared using hydrochloric acid and porcine bile salt, respectively. 1mL of Lactobacillus plantarum GZ1806 strain solution of the strain to be tested is inoculated into a broth culture medium with pH=3.0, and is cultured for 18h at 37 ℃. After the completion of the culture, 20. Mu.L of the bacterial liquid was spread on MRS agar plate medium, and 3 replicates were set, and the culture was performed at 37℃for 18 hours. After incubation, the MRS plate surface was observed for colony growth. Similarly, 1mL of Lactobacillus plantarum GZ1806 strain to be tested was inoculated into MRS broth with a bile salt concentration of 0.3%, cultured at 37℃for 18 hours, plated, and cultured at 37℃for 24 hours, and the colony growth was observed.
Acid resistance test results show that normal colonies can still grow on an MRS agar plate after lactobacillus plantarum GZ1806 is treated in an MRS broth culture medium with the pH value of=3.0 for 18 hours; salt tolerance test results show that the lactobacillus plantarum GZ1806 can still grow normal colonies on an MRS agar plate after being tolerant for 18 hours in an MRS broth culture medium with a bile salt concentration of 0.3%; the lactobacillus plantarum GZ1806 has stronger acid resistance and cholate resistance.
3.2, simulating gastric juice and intestinal juice 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 and activating Lactobacillus plantarum GZ1806 strain, and regulating bacterial liquid concentration to 1×10 8 CFU/mL, 1mL of bacterial liquid is added into 9mL of simulated artificial gastric fluid, 10 times gradient dilution is carried out, 20 mu L of viable bacteria count of a coated plate is absorbed, and the viable bacteria count is used as an initial viable bacteria value of the tolerance artificial gastric fluid; the simulated gastric juice after inoculation is cultured for 3 hours at 37 ℃, and then the plate is coated again to count the number of viable bacteria, and the number of viable bacteria is taken as the final viable bacteria value of the artificial gastric juice tolerance. Similarly, 1mL was concentrated to 1X 10 8 CFU/mL lactobacillus plantarum GZ1806 fermentation liquor is added into 9mL simulated intestinal fluid, viable bacteria are counted, and viable bacteria are counted again after culture for 6 hours at 37 DEG CThe number of counts and the survival rate calculated.
Survival = number of viable bacteria at end/number of initial viable bacteria 100%.
The results show that the lactobacillus plantarum GZ1806 strain has better tolerance to artificial simulated gastric and intestinal fluids, the survival rate of the lactobacillus plantarum GZ1806 strain in the artificial gastric fluid after 3 hours is 74.40%, and the survival rate of the lactobacillus plantarum strain after 6 hours is 62.64% (Table 3).
TABLE 3 tolerance of Lactobacillus plantarum GZ1806 Strain to artificial simulated gastric and intestinal fluids (%)
3.3 Lactobacillus plantarum GZ1806 Strain hemolytic and antibiotic resistance
After the lactobacillus plantarum GZ1806 strain is revived and activated for 3 generations, streaking is inoculated on a Columbia blood plate, after the culture is carried out for 24 hours at 37 ℃, whether hemolysis rings appear around the colony to be detected is observed, and the hemolytic staphylococcus is used as a positive control. The susceptibility of the strain to common large antibiotics was tested by a paper sheet agar diffusion method. Resuscitating and activating Lactobacillus plantarum GZ1806 strain for 3 generations, and regulating bacterial liquid concentration to 1×10 6 CFU/mL, evenly smearing bacterial liquid on the surface of an MRS culture medium flat plate by using a sterile cotton swab, placing drug sensitive paper sheets after 10min at room temperature, culturing for 24h at 37 ℃, measuring the diameter of a bacteriostasis ring around each drug sensitive paper sheet by using a vernier caliper, repeating each antibiotic 3 times, and judging the drug sensitivity of the strain by referring to the American clinical laboratory standards Committee (NCCLS) standard according to test results, wherein the results are expressed as sensitivity (S), intermediation (I) and drug resistance (R).
The hemolytic experiment result shows that the hemolysis ring phenomenon does not appear around the colony (figure 5); the 6 antibiotics tested showed sensitivity (S), demonstrating the safety of the Lactobacillus plantarum GZ1806 strain (Table 4).
TABLE 4 sensitivity of Lactobacillus plantarum GZ1806 Strain to 6 antibiotics
| Numbering device | Tetracycline | Ampicillin (Amoxicillin) | Ceftriaxone | Clindamycin | Clarithromycin | Chloramphenicol |
| GZ1806 | S | S | S | S | S | S |
Example 4 probiotic Properties of Lactobacillus plantarum GZ1806 Strain
4.1 short chain fatty acid production Capacity
Preparation of fermentation liquor: after the lactobacillus plantarum GZ1806 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 culture is carried out at 37 ℃ for 24 hours 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 5.
TABLE 5 Lactobacillus plantarum GZ1806 fermentation broth short chain fatty acid content (ug/mL)
| Strain number | Strain name | Acetic acid | N-butyric acid | Isobutyric acid | Isopentanoic acid | 2-methylbutyric acid |
| GZ1806 | Lactobacillus plantarum | 8.801 | 0.094 | 0.128 | 0.332 | 0.1 |
The GC-MS detection 5 short chain fatty acids are acetic acid, n-butyric acid, isobutyric acid, isovaleric acid and isocaproic acid respectively, the total ion flow diagram is shown in figure 6, the base line is stable, the peak positions of the internal standard substance and other targets are similar, the separation degree is good, no interference exists between the internal standard substance and other targets, and the result is normal. The calculation result shows that the acetic acid content in lactobacillus plantarum GZ1806 fermentation liquor is the highest and is 8.801 mug/mL, and the lactobacillus plantarum fermentation liquor further contains n-butyric acid, isobutyric acid, isovaleric acid and 2-methyl butyric acid. 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), staphylococcus aureus (Staphylococcus aureus CMCCB 50094), salmonella typhimurium (Salmonella typhimurium ATCC 14028), pseudomonas aeruginosa (Pseudomonas aeruginosa CMCCB 10104), campylobacter jejuni (Campylobacter jejuni ATCC 33291), campylobacter coli (Campylobacter coli ATCC 43478), listeria monocytogenes (Listeria monocytogenes ATCC 19115), staphylococcus hemolyticus (Staphylococcus haemolyticus ATCC 29970), pasteurella multocida (Pasteurella multocida ATCC 51689) and yersinia enterocolitica (Yersinia enterocolitica ATCC 23715) were inoculated to nutrient agar medium, respectively, and resuscitated 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 to prepare pathogenic bacteria agar plates, and each plate corresponds to oneStrain, and three wells were set as replicates. 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, staphylococcus aureus, salmonella typhi B, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli and the like, and has basically equivalent effect to LGG standard strains (Table 6).
The antibacterial activity evaluation and measurement results of the lactobacillus plantarum GZ1806 strain are shown in the following table 6, the antibacterial effect of the strain on 10 common enteropathogenic bacteria is generally better than that of a standard strain LGG, and particularly the antibacterial advantages of the strain on staphylococcus aureus, salmonella typhimurium, listeria monocytogenes and pasteurella multocida are more obvious, so that the microbial inoculum prepared from the lactobacillus plantarum GZ1806 strain can be used for preventing and controlling the pathogenic bacteria.
TABLE 6 evaluation of antibacterial Activity of Lactobacillus plantarum GZ1806 Strain (diameter: mm)
4.3 inhibition of alpha-glucosidase by Lactobacillus plantarum GZ1806
Alpha-glucosidase participates in the decomposition and utilization of carbohydrate, and research shows that inhibiting the activity of alpha-glucosidase can reduce the blood sugar level in human blood. Resuscitating and culturing Lactobacillus plantarum GZ1806 preserved strain for 24h, inoculating 2% of the strain into MRS liquid culture medium, culturing at 37deg.C for 24h, centrifuging at 4deg.C 4000r/min for 15min, and filtering the supernatant with 0.22 μm filter membrane to obtain fermentation supernatant (CFS); 25 μl of the supernatant was taken, 50 μl of PNPG solution of 50 μl and 20mmol/L in PBS buffer (pH=6.8) was added, and the mixture was subjected to water bath at 37deg.C for 10min; adding 20U/ml alpha-glucosidase solution 30 muL, continuing to react for 10min at 37 ℃; 50 mu L of 1mol/L Na was added 2 CO 3 Stopping the reaction by the solution; in 96-well plates (365 μl), 3 replicates were set for each group; and measuring the absorbance at 405nm of the enzyme label instrument, and calculating to obtain the alpha-glucosidase inhibition rate (%). The calculation formula is as follows:
wherein group A is alpha-glucosidase; group B is PBS buffer solution; c is a sample group to be detected and contains alpha-glucosidase and a sample to be detected; group D contained only the samples to be tested.
The detection result shows that lactobacillus plantarum GZ1806 fermentation supernatant has the activity of inhibiting alpha-glucosidase, the inhibition rate is 66.46 percent, and the inhibition activity is obviously higher than that of lactobacillus rhamnosus LGG and other related strains of a standard strain on the alpha-glucosidase (table 7), and the lactobacillus plantarum GZ1806 preparation possibly has the potential of reducing the blood glucose level of animal and human blood.
TABLE 7 inhibition of alpha-glucosidase by Lactobacillus plantarum GZ1806 fermentation broth (%)
4.4 antioxidant Activity of Lactobacillus plantarum GZ1806 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: lactobacillus plantarum GZ1806 frozen strains are resuscitated and cultured for 24 hours, inoculated into MRS liquid culture medium according to 2 percent of inoculum size, cultured for 24 hours at 37 ℃, and the bacterial liquid is centrifuged for 15 minutes at 4 ℃ and 4000r/min, and the supernatant is filtered by a 0.22 mu m filter membrane to obtain fermentation supernatant (CFS) for freezing and storing.
Reagents and instrumentation: 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 built 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.
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 lactobacillus plantarum GZ1806 fermentation liquor has stronger capability of inhibiting hydroxyl radicals, namely 3590.76 +/-50.85U/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 lactobacillus plantarum GZ1806 fermentation liquid are shown in Table 8, and the DPPH radical scavenging ability 192.69 +/-4.29 mug Trolox/mL of the lactobacillus plantarum GZ1806 fermentation liquid is known, so that the lactobacillus plantarum GZ1806 fermentation liquid has certain scavenging ability on DPPH radicals.
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.
Anti-superoxide anion Capacity (U/L) = (control-A assay)/(control-A Standard) ×C Standard×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. The measurement results of the anti-superoxide anion capacity of the lactobacillus plantarum GZ1806 fermentation liquid are shown in the following table 8, and the inhibition capacity of the lactobacillus plantarum GZ1806 fermentation liquid on superoxide anion free radicals is 672.36 +/-8.81 (U/L), which indicates that the inhibition capacity of the lactobacillus plantarum GZ1806 fermentation liquid on superoxide anion free radicals is stronger.
TABLE 8 antioxidant capacity of Lactobacillus plantarum GZ1806 fermentation broth
In conclusion, the GZ1806 fermentation liquor has strong oxidation resistance 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 (8)
1. Lactobacillus plantarumLactobacillus plantarum) GZ1806 is preserved in China general microbiological culture Collection center (CGMCC) in 2023, 02 and 20, and the preservation number is CGMCC NO:26590.
2. Use of lactobacillus plantarum GZ1806 according to claim 1 for the preparation of an anti-colon cancer medicament or an anti-colon cancer adjuvant.
3. Use of lactobacillus plantarum GZ1806 as claimed in claim 1 for inhibiting escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli, listeria monocytogenes, staphylococcus haemolyticus, pasteurella multocida or yersinia enterocolitica for non-disease therapeutic purposes.
4. Use of lactobacillus plantarum GZ1806 according to claim 1 for the manufacture of a medicament for the treatment of diseases caused by escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli, listeria monocytogenes, staphylococcus haemolyticus, pasteurella multocida or yersinia enterocolitica.
5. Use of lactobacillus plantarum GZ1806 according to claim 1 for the preparation of a medicament with hypoglycemic or antioxidant function.
6. An anti-colon cancer drug or an anti-colon cancer adjuvant comprising lactobacillus plantarum GZ1806 as described in claim 1.
7. A hypoglycemic agent comprising lactobacillus plantarum GZ1806 as claimed in claim 1.
8. An antioxidant drug comprising lactobacillus plantarum GZ1806 as claimed in claim 1.
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