Lactobacillus delbrueckii subsp. Bulgaricus and application of lactobacillus delbrueckii subsp. Bulgaricus in fermentation of periplaneta americana

Abstract

The invention discloses lactobacillus delbrueckii subsp. Bulgaricus and an application of the lactobacillus delbrueckii subsp. Bulgaricus in fermentation of periplaneta americana. The strain is separated and screened from traditional fermented yak milk, and the preservation number is CGMCC No.31143. The strain does not hemolysis, is sensitive to various antibiotics, has certain tolerance to acid, cholate, artificial gastric juice and artificial intestinal juice, and has a remarkable inhibition effect on various animal enteropathogenic bacteria. When the strain is used for fermenting periplaneta americana, the content of bioactive substances such as amino acids, indoles and steroids can be greatly increased, and fermented supernate has an obvious growth inhibition effect and a cancer cell apoptosis promoting effect on human colon cancer cells and has a good prevention and treatment effect on a DSS-induced mouse colitis model; the inflammation index is obviously reduced. The strain not only has significant application value in functional food and biological agents, but also opens up a new way for further development of the traditional Chinese medicinal material periplaneta americana.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus delbrueckii subsp. bulgaricus and an application thereof. Background Art

[0002] With the continuous deepening of intestinal microbial research, the application of probiotics in promoting animal and human health has received increasing attention. They play an important role in maintaining the health of the host by improving the balance of intestinal microorganisms, enhancing immune function, and inhibiting the growth of harmful bacteria. Lactobacillus delbrueckii subsp.bulgaricus is one of the important probiotics. As a traditional Chinese medicine, American cockroach has multiple effects such as clearing away heat and detoxification, dispersing blood stasis and removing accumulation, and diuresis and swelling. Kangfuxin liquid made from the ethanol extract of the dried insect body of American cockroach has the effects of promoting blood circulation, nourishing yin and promoting muscle growth, promoting granulation tissue growth, and promoting angiogenesis, and is widely used in clinical practice. If Lactobacillus delbrueckii subsp.bulgaricus can be used to ferment American cockroach powder to increase the content of active substances or produce new compounds, it will provide new ideas for the further utilization of traditional Chinese medicine American cockroach. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus delbrueckii subspecies bulgaricus and application thereof in the fermentation of American cockroaches.

[0004] The technical solution of the present invention is: a strain of Lactobacillus delbrueckii subsp. bulgaricus SN22, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 2, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number being CGMCC No. 31143.

[0005] A biological preparation containing the above-mentioned Lactobacillus delbrueckii subsp. bulgaricus SN22 or its fermentation liquid.

[0006] The above-mentioned Lactobacillus delbrueckii subsp. bulgaricus SN22 or biological preparation is used for the purpose of inhibiting Yersinia enterocolitica, Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Salmonella typhimurium or Shigella dysenteriae for non-disease treatment purposes.

[0007] The application of the above-mentioned Lactobacillus delbrueckii subsp. bulgaricus SN22 in fermenting American cockroaches.

[0008] The invention discloses an American cockroach powder fermentation liquid, which is obtained by fermenting the American cockroach powder with the above-mentioned Lactobacillus delbrue ckii subsp. bulgaricus SN22.

[0009] Furthermore, the fermentation method is as follows: American cockroach powder and oligofructose are prepared into a culture medium with water, sterilized and cooled, inoculated with Lactobacillus delbrueckii subsp.bulgaricus SN22, cultured at 37°C for 96 hours, centrifuged the fermentation liquid at 4000rpm and 4°C for 10 minutes, and filtered the supernatant through a bacterial filter membrane to obtain a sterile supernatant.

[0010] The application of the above-mentioned American cockroach powder fermentation liquid in the preparation of medicine for treating or assisting in treating colon cancer.

[0011] An anti-colon cancer drug or an anti-colon cancer auxiliary drug contains the American cockroach powder fermentation liquid mentioned above.

[0012] The application of the American cockroach powder fermentation liquid in the preparation of medicine for preventing or treating colitis.

[0013] A medicine for preventing or treating colitis contains the American cockroach powder fermentation liquid mentioned above.

[0014] The application of the above-mentioned American cockroach powder fermentation liquid in the preparation of functional food.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention discloses a strain of Lactobacillus delbrueckii subspecies bulgaricus and its application in the fermentation of American cockroaches. The strain is separated and screened from traditional fermented yak milk, and the preservation number is CGMCC No.31143. The strain has no hemolysis phenomenon, is sensitive to multiple antibiotics, has a certain tolerance to acid, bile salts, artificial gastric juice and artificial intestinal juice, and has a significant inhibitory effect on multiple animal intestinal pathogens. The fermentation of American cockroaches with the strain can greatly increase the content of bioactive substances such as amino acids, indoles and steroids, and the fermentation supernatant has a significant growth inhibitory effect and a cancer cell apoptosis-promoting effect on human colon cancer cells. The fermentation supernatant has a good preventive and therapeutic effect on a DSS-induced mouse colitis model, and the inflammatory index is significantly reduced. The strain not only has a significant application value in functional foods and biological preparations, but also opens up a new way for the further development of the traditional Chinese medicine American cockroach.

[0017] Collection information:

[0018] Lactobacillus delbrueckii subsp.bulgaricus SN22 was deposited on July 2, 2024 at the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCCNo.31143. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a phylogenetic relationship diagram of the Lactobacillus delbrueckii subspecies bulgaricus SN22 strain in the present invention and other strains in GenBank.

[0020] Figure 2 This is a volcano plot of differential metabolites from the mass spectrometry analysis of the supernatant of American cockroach powder fermented by Lactobacillus delbrueckii subspecies bulgaricus SN22 strain in the present invention.

[0021] Figure 3 It is the number of cloned cells formed by HT-29 cells after being treated with the fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 in the present invention. A is the control group and B is the SN22 treatment group.

[0022] Figure 4 The expression level of ROS in colon cancer cells after being treated with the fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 in the present invention, wherein A is the control group and B is the SN22 treatment group.

[0023] Figure 5 This is the apoptosis-promoting effect of the fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 on HT-29 cells treated with the fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 in the present invention (control group on the left, SN22-treated group on the right). DETAILED DESCRIPTION

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from commercial channels unless otherwise specified. In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the principles of the present invention should be included in the protection scope of the present invention.

[0025] The methods used in the following examples are conventional methods unless otherwise specified, and the percentages involved in the culture medium are all mass-to-volume ratios. The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC 53103.

[0026] Experimental Example 1 Isolation and Identification of Lactobacillus delbrueckii subspecies bulgaricus SN22

[0027] 1.1 Material preparation

[0028] Traditional fermented yak yogurt samples were obtained from Nagqu City, Tibet;

[0029] 16S rRNA universal sequencing primers 27F and 1492R were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the sequences are as follows (5'→3'):

[0030] 27F:AGAGTTTGATCMTGGCTCAG

[0031] 1492R:GGTTACCTTGTTACGACTT

[0032] MRS broth medium formula (per liter): casein digest 10.0g, beef extract powder 10.0g, yeast extract powder 4.0g, triammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate tetrahydrate 0.05g, dipotassium hydrogen phosphate 2.0g, glucose 20.0g, Tween-80 1.08g, final pH is about 5.7. Weigh each component according to the above ratio, add distilled water or deionized water to make up to 1L, dispense into conical flasks, and sterilize under high pressure conditions at 121℃ for 20min.

[0033] MRS agar medium formula (per liter): peptone 10.0g, beef powder 5.0g, yeast powder 4.0g, glucose 20.0g, Tween 80 1.0mL, potassium dihydrogen phosphate heptahydrate 2.0g, sodium acetate trihydrate 5.0g, ammonium citrate trihydrate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate tetrahydrate 0.05g, agar 15.0g, the final pH is about 6.2. Weigh each component according to the above ratio, add distilled water or deionized water to make up to 1L, dispense into conical flasks, and sterilize under high pressure conditions at 121℃ for 20min.

[0034] 1.2 Screening and isolation of strains

[0035] Take a small amount of yogurt sample, inoculate it in 100mL MRS broth, shake it thoroughly and mix it, and place it in a constant temperature shaker at 37℃ for 24h. Take 1mL of the culture solution, dilute it by 10 times, take 20μL respectively, and spread it on MRS agar medium containing calcium carbonate (calcium carbonate 1.5g / 100mL). After culturing at 37℃ for 24h, pick a single colony with a large calcium dissolution circle, and separate and purify it three times in a row using the same method. Inoculate the purified strain into 600μL MRS broth medium, culture it in a shaker at 37℃ for 18h, add 400μL of 50% (V / V) sterile glycerol, and freeze it in a -80℃ ultra-low temperature refrigerator for later use.

[0036] 20 μL of the frozen bacterial strain was inoculated into MRS liquid medium for proliferation culture, and the strain DNA was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit. PCR amplification was performed using the bacterial 16S rRNA universal primer 27F / 1492R with its total genomic DNA as a template to obtain a target gene sequence consisting of 1162 base pairs (bp), and the sequence is shown in SEQ ID No. 1.

[0037] The gene sequence obtained by sequencing was input into the NCBI database for comparison. The similarity between the strain and the strain Lactobacillus delbrueckii subsp.bulgaricus strain DSM 20080 in GenBank: CP019120.1 was 98.35%, and the strain was preliminarily identified as Lactobacillus delbrueckii subsp.bulgaricus. The phylogenetic relationship between this strain and other strains is shown in Figure 2. Figure 1 The strain was inoculated on MRS agar medium and cultured at 37°C for 24 hours. The morphology of the single colonies was observed and recorded. Lactobacillus delbrueckii subspecies bulgaricus SN22 grew well on the MRS agar medium. It was round, smooth and convex, light yellow, opaque, and Gram-positive, which was consistent with the characteristics of Lactobacillus delbrueckii subspecies bulgaricus.

[0038] The strain was named Lactobacillus delbrueckii subsp. bulgaricus SN22, and was deposited in the General Microbiology Center of China Culture Collection Administration on July 22, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 31143.

[0039] Experimental Example 2 Hemolytic and antibiotic sensitivity determination of Lactobacillus delbrueckii subspecies bulgaricus SN22

[0040] Hemolysis is one of the important indicators for evaluating the safety of probiotics. Only when the strain shows non-hemolytic properties can it be considered safe. After the SN22 strain isolated and identified in Experimental Example 1 was revived and activated for 3 generations, it was streaked on a Columbia blood plate and cultured at 37°C for 24 hours to observe whether a hemolytic ring appeared around the test colony. The experiment was repeated 3 times. No hemolysis occurred around the Lactobacillus delbrueckii subspecies bulgaricus SN22 colony.

[0041] As the problem of antibiotic resistance becomes increasingly serious, it is crucial to select probiotics that are sensitive to antibiotics in order to prevent the spread of resistance genes. The sensitivity of the strains to common antibiotics was detected by the disc agar diffusion method (KB method). The Lactobacillus delbrueckii subspecies bulgaricus SN22 strain isolated and identified in Experimental Example 1 was activated and cultured, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL, use a sterile cotton swab to evenly apply the bacterial solution on the surface of the MRS culture medium plate, place it at room temperature for 10 minutes, then put in the drug sensitivity paper, and culture it at 37°C for 18 hours. Use a vernier caliper to measure the diameter of the inhibition zone around each drug sensitivity paper. Repeat each antibiotic 3 times. The test results refer to the standards of the National Committee for Clinical Laboratory Standards (NCCLS) to judge the drug sensitivity of the strain. The results are expressed as sensitive (S), intermediate (I) and resistant (R). As shown in Table 1, Lactobacillus delbrueckii subspecies bulgaricus SN22 is sensitive (S) to the six antibiotics tested, including tetracycline, ampicillin, ceftriaxone, clarithromycin, chloramphenicol, gentamicin, and ciprofloxacin, indicating that Lactobacillus delbrueckii subspecies bulgaricus SN22 has good safety (Table 1).

[0042] Table 1 Sensitivity of Lactobacillus delbrueckii subsp. bulgaricus SN22 to 6 antibiotics

[0043]

[0044] Experimental Example 3 Evaluation of acid and salt resistance of Lactobacillus delbrueckii subspecies bulgaricus SN22

[0045] 3.1 Acid resistance and bile salt resistance evaluation:

[0046] The SN22 strain isolated and identified in Experimental Example 1 was revived and activated, and the initial concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL. MRS broth medium with pH 3.0 and bile salt concentration of 0.3% was prepared with hydrochloric acid and porcine bile salt, respectively. The strain Lactobacillus delbrueckii subsp. bulgaricus SN22 was inoculated into the broth medium with pH 3.0 and bile salt concentration of 0.3%, and the initial concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, after culturing at 37℃ for 18h, 20μL of bacterial solution was spread on MRS agar plate medium, and after culturing at 37℃ for 18h, the surface of the MRS plate was observed to see whether there was colony growth. The experiment was repeated 3 times. According to the results of the acid resistance and bile salt resistance test, Lactobacillus delbrueckii subspecies bulgaricus SN22 can still grow normal colonies on the MRS agar plate after being treated in MRS broth medium with pH 3.0 and bile salt concentration of 0.3% for 18h, indicating that the strain has strong acid resistance and bile salt resistance.

[0047] 3.2 Simulated gastric juice and intestinal juice tolerance test:

[0048] Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the main components of artificial gastric juice simulation fluid were sodium chloride, dilute acid and pepsin, and the final pH was 2.5; the main components of artificial intestinal juice simulation fluid were phosphate and trypsin, and the final pH was 6.8. The Lactobacillus delbrueckii subspecies bulgaricus SN22 strain isolated and identified in Experimental Example 1 was revived and activated, and the bacterial solution was centrifuged at 4°C and 3500r / min for 10min, then washed 2-3 times with artificial gastric juice and resuspended, and the bacterial concentration was adjusted to 1×10 8 CFU / mL, 300μL of bacterial suspension was added to 2.7mL artificial simulated gastric fluid, and 20μL of the mixture was diluted 10 times to count the live bacteria as the initial value of tolerance to artificial gastric fluid; the simulated gastric fluid with live bacteria was cultured at 37℃ for 3h and the live bacteria were counted by the same method as the final value of tolerance to artificial gastric fluid. At the same time, 300μL of the above mixture was added to 2.7mL simulated intestinal fluid, and the above method was used to count the initial live bacteria. After culturing at 37℃ for 3h, the live bacteria were counted again. The experiment was repeated 3 times, with LGG as the control.

[0049] As shown in Table 2, Lactobacillus delbrueckii subspecies bulgaricus SN22 has a certain tolerance to artificial simulated gastric and intestinal fluids, which is conducive to the survival of the strain in the gastrointestinal environment of humans and animals (Table 2).

[0050] Table 2 Tolerance of Lactobacillus delbrueckii subspecies bulgaricus SN22 strain to artificial simulated gastric and intestinal fluids (%)

[0051]

[0052] Experimental Example 4 Inhibitory ability of Lactobacillus delbrueckii subspecies bulgaricus SN22 strain against common intestinal pathogens

[0053] The proliferation of pathogenic bacteria in the intestine will cause the disorder of the intestinal flora system, thus causing a variety of diseases. Escherichia coli (CMCB 44102), Salmonella entericaserovar Paratyphi B (CMCB 50094), Staphylococcus aureus (CMCB26003), Salmonella typhimurium ATCC14028), Shigella dysenteriae (CMCB 51105), and Yersinia enterocolitica subspenterocolitica (CMCB 52204) were revived and purified three times, and the colonies were inoculated into TSB liquid culture medium and cultured at 37°C for 18 hours. The bacterial solution concentration was adjusted to 1×10 8 CFU / mL, then sterilize 400mL of NA solid culture medium using a conical flask, wait until it gradually cools to 40-45°C, pipette 1mL of the cultured indicator bacteria solution into the NA culture medium, mix the culture medium and prepare the pathogen plate. At room temperature, place the plate for 10 minutes and then use a sterile puncher to evenly punch holes (hole diameter ± 6mm), select three holes as replicates for each probiotic to be tested, and pipette 50μL of the prepared CFS into parallel holes. After placing it horizontally for 2 hours, place the plate at 37°C and culture it for 18 hours, measure the diameter of the inhibition zone and count it. The diameter of the inhibition zone is the average of the diameters of the three transparent circles. Use LGG as a standard strain to provide a reference for comparison

[0054] The SN22 strain isolated and identified in Experimental Example 1 was revived and the bacterial solution concentration was adjusted to 1×10 8CFU / mL, 50 μL of the test bacterial solution was added to the above-mentioned pathogenic bacteria agar plate well, and the diameter of the inhibition zone around the punch point was measured with a vernier caliper after culturing for 18 hours at 37°C and recorded. LGG was used as a control. The results of the antibacterial activity evaluation of Lactobacillus delbrueckii subspecies bulgaricus SN22 are shown in Table 3 below. The Lactobacillus delbrueckii subspecies bulgaricus SN22 strain has a strong inhibitory activity on the growth of Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica, and the overall antibacterial effect is comparable to that of the LGG standard strain (Table 3).

[0055] Table 3 Evaluation of antibacterial activity of Lactobacillus delbrueckii subspecies bulgaricus SN22 strain (diameter: mm)

[0056]

[0057]

[0058] Example 5 Application of Lactobacillus delbrueckii subspecies bulgaricus SN22 strain in fermentation of American cockroach powder

[0059] 5.1 Metabolomic analysis of fermentation supernatant

[0060] 5.1.1 Preparation of fermentation supernatant: 10 grams of American cockroach powder, 2 grams of oligofructose, and 100 mL of water were prepared into a culture medium. After sterilization and cooling, Lactobacillus delbrueckii subspecies Bulgaria SN22 was inoculated and cultured at 37 degrees for 96 hours. The fermentation broth was centrifuged at 4000 rpm and 4°C for 10 minutes, and the supernatant was filtered through a bacterial filter membrane and sent to the company for liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis. The experiment was set up with three replicates, and the control group without probiotics was used. The culture and analysis methods were the same as those of the treatment group.

[0061] 5.1.2 Analysis method: Based on high-resolution mass spectrometry (HRMS) detection technology, the high-quality mzCloud database constructed by standards is combined with the mzVault and MassList databases to match and identify molecular characteristic peaks. The raw data is preprocessed using CD3.3 data processing software. First, simple screening is performed by parameters such as retention time and mass-to-charge ratio, and the peak area is quantified at the same time. Then, the high-resolution secondary spectrum databases mzCloud and mzVault and the MassList primary database are compared (searched) to identify metabolites. Using CD3.3 data processing software, the chromatographic peaks detected in the sample are integrated, where the peak area of ​​each characteristic peak represents the relative quantitative value of a metabolite. The total peak area is used to standardize the quantitative results, and finally the quantitative results of the metabolites are obtained. The results are expressed as the multiple increase in the content of the corresponding component in the sample compared with the control.

[0062] 5.1.3 Analysis results

[0063] Metabolomics analysis showed that after 96 hours of fermentation of American cockroach powder with Lactobacillus delbrueckii subspecies bulgaricus SN22, a total of 106 compounds were detected in positive ion mode with significant increases in content, 164 compounds with significant decreases in content, and 659 compounds with no significant differences in content. In negative ion mode, a total of 66 compounds were detected with significant increases in content, 126 compounds with significant decreases in content, and 488 compounds with no significant differences in content. The metabolite difference volcano plot is shown in the figure below. Figure 2 .

[0064] (1) Amino acids and peptide compounds: The analysis results show that after 96 hours of fermentation of American cockroach powder with Lactobacillus delbrueckii subspecies bulgaricus SN22, the contents of various amino acids and their derivatives in the supernatant increased significantly, especially the contents of vitamin U and cysteine, which were 180.89 times and 7.26 times of the control, respectively. Amino acids are the basic building blocks of proteins and play an important role in the health and normal functions of the human body, such as protein synthesis, physiological regulation, immune function, energy supply, and maintaining acid-base balance. The main function of Vitamin U is to help treat gastric ulcers and duodenal ulcers. It can promote the secretion of mucin and accelerate the healing of ulcer wounds, thereby relieving stomach discomfort. In addition, Vitamin U can also participate in the detoxification of the liver, has the potential to reduce blood lipids, and provides a certain degree of radiation protection and anti-inflammatory effects. L-cysteine ​​is a sulfur-containing semi-essential amino acid that plays a key role in human health. It is not only the main component of glutathione, an important antioxidant that helps resist free radicals and promotes cellular antioxidant function, but also has a positive effect on protein synthesis, cell structure stability, immune function regulation, hair strengthening, mucolysis, and bone health. Appropriate intake of cysteine ​​is of great significance for maintaining physiological functions and promoting physical health (Table 4).

[0065] Table 4 Increase in the content of amino acids and peptides after SN22 strain fermented American cockroach powder

[0066]

[0067] (2) Indole compounds: The results showed that after 96 hours of fermentation of American cockroach powder with Lactobacillus delbrueckii subspecies bulgaricus SN22, the content of some indole derivatives in the supernatant increased significantly, especially the content of 5-hydroxytryptamine (5-Hydroxytryptophan, also known as serotonin) was 4.07 times that of the control. Indole and its derivatives are closely related to intestinal microbiota and human health, such as gastrointestinal motility, intestinal barrier, and neural regulation. Serotonin is an important neurotransmitter that has a wide range of effects on physiological functions such as mood regulation, sleep cycle, pain perception, and appetite (Table 5).

[0068] Table 5 Growth of indole content after SN22 strain fermented American cockroach powder

[0069]

[0070]

[0071] (3) Steroid compounds

[0072] Steroid metabolites play a variety of important physiological roles in the human body and are key factors in maintaining normal body functions. These substances, including sex hormones and adrenocortical hormones, are derived from cholesterol, are fat-soluble, and can cross the cell membrane and bind to intracellular receptors, thereby regulating gene expression. The analysis results show that after 96 hours of fermentation of American cockroach powder with Lactobacillus delbrueckii subspecies bulgaricus SN22, the content of various steroid compounds in the supernatant increased significantly, especially the increase in the content of trigoneoside Xb, testosterone sulfate and aldosterone. The biological activity of trigoneoside Xb is not very clear, but testosterone sulfate (or testosterone sulfate) as a metabolite of testosterone may have specific biological activities in the body, such as participating in hormone regulation and affecting growth and development, and has broad application prospects in the fields of biology and medicine. Aldosterone is a steroid hormone secreted by the adrenal cortex and belongs to the mineralocorticoid family. It maintains electrolyte and water balance by regulating the reabsorption of sodium and water by the kidneys, and plays a key role in the stability of blood pressure and normal blood volume. At the same time, the secretion and regulation of aldosterone are also involved in a variety of physiological and pathological processes (Table 6).

[0073] Table 6 Growth of steroid content after SN22 strain fermented American cockroach powder

[0074]

[0075] 5.2 Growth inhibition and apoptosis-promoting effects of fermentation supernatant on human colon cancer cells

[0076] 5.2.1 Growth inhibition ability of fermentation supernatant on HT-29 cells:

[0077] (1) Cell culture: Take out the HT-29 culture dish of human colon cancer cells, remove the culture medium, add 1 mL of PBS for washing, add 1 mL of trypsin after removal, mix gently, digest for 2 min, until the cells are observed to be round under an optical microscope, then remove the trypsin, add 1 mL of culture medium, use a pipette to suspend the cells, and transfer them to a 1.5 mL EP tube. Centrifuge at 1200 rpm / min for 3 min, remove the supernatant, add 300 μL of culture medium and mix thoroughly, pipette 150 μL into the culture dish containing 4 mL of culture medium, mix gently, place in a CO2 incubator for continued culture, and operate according to the cell passaging method until centrifugation after 48 h. Discard the supernatant, mix the cells with 1 mL of culture medium, and then count them with a hemocytometer, and dilute the cell suspension to 3×10 4 CFU / mL. Add 100 μL of cell suspension to each well of the 96-well cell plate and place in a CO2 incubator for culture. After the cells adhere to the wall and grow well, remove the culture medium for later use.

[0078] (2) Cytotoxicity test: The preparation method of the American cockroach fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 strain is as described in 5.1.1. It is added to the serum-free cell culture medium at 4% (v / v), and 100 μL is added to each cell culture well, and cultured for 24 h, 48 h and 72 h respectively. The serum-free cell culture medium without fermentation supernatant is used as the control, and 3 replicates are set.

[0079] (3) CCK-8 detection of cell proliferation: aspirate the culture medium of the cytotoxicity experiment, add 100 μL of CCK-8 solution to each well, continue to culture in the incubator for 1.5 h, and measure the OD with an enzyme marker. 562 The values ​​represent the cell growth activity.

[0080] (4) Results analysis

[0081] The results are shown in Table 4. 4% of the fermentation supernatant in the culture medium has a significant growth inhibitory effect on human colon cancer HT-29 cells. After treatment for 24h, 48h or 72h, the growth inhibition rates were 23.22%, 24.47% and 20.83%, respectively (Table 7).

[0082] Table 7 Inhibitory effect of fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 on the growth of HT-29 cells (%)

[0083]

[0084] 5.2.2 Effect of fermentation supernatant on HT-29 cell colony formation ability:

[0085] Resuscitate and subculture colon cancer cell HT-29 cells, plate at a density of 2000 cells per well in a 6-well plate, and culture for 3 weeks in a serum-free medium containing 1% fermentation supernatant (the preparation method of fermentation supernatant is described in 5.1.1.), and replace fresh medium every two days to ensure good cell growth. After the formation of visible clones in the cell culture plate, discard the medium, wash with PBS, fix with methanol for 30min-2h, add 1% crystal violet staining for 30min, wash off the residual crystal violet staining solution, dry the culture plate, count under a microscope, and set up 3 replicates of the experiment.

[0086] The results showed that 1% of the fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 strain American cockroach had a significant effect on the colony formation ability of HT-29 cells. The number of cell colonies in the treatment group was only 71.93% of that in the control group, indicating that its fermentation broth had a significant inhibitory effect on the colony formation of human colon cancer cells (Table 8, Figure 3 ).

[0087] Table 8 Number of cloned cells after fermentation supernatant of Lactobacillus delbrueckii subspecies bulgaricus SN22 was treated

[0088]

[0089]

[0090] Note: Compared with the control group, **p<0.01

[0091] 5.2.3 Effect of fermentation supernatant on reactive oxygen species (ROS) levels in HT-29 cells:

[0092] Cell collection: After incubating SN22 fermentation supernatant (the preparation method of fermentation supernatant is described in 5.1.1.) with adherent cultured HT-29 cells for 48 hours, the fermentation supernatant was aspirated and repeatedly blown with serum-free culture medium. The bottom of the well plate (bottom of the bottle) changed from translucent (cell monolayer connected into a sheet) to transparent by naked eye observation, and almost all the cell layer was blown into the culture medium. Collect all the cell suspension into a 1.5 mL centrifuge tube. Wash twice with serum-free culture medium, centrifuge at 1000r / min for 5min, aspirate the supernatant, and keep the cell pellet for determination.

[0093] Loading probe: Dilute DCFH-DA with serum-free culture medium at 1:1000 to a final concentration of 10μmol / L. After collecting cells, suspend them in the diluted DCFH-DA at a cell concentration of 1 million to 20 million / mL and incubate in a 37°C cell culture incubator for 20 minutes. Invert and mix every 3-5 minutes to allow the probe and cells to fully contact. Wash the cells 3 times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells.

[0094] Detection: Use an inverted fluorescence microscope to detect the intensity of fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Use the culture medium supernatant without bacteria as the negative control group, and set up 3 replicates of the experiment.

[0095] The level of reactive oxygen species (ROS) reflects the state of cellular metabolites. Under normal circumstances, the generation and removal of ROS are in a dynamic balance. If the ROS level exceeds the normal range, it may cause oxidative damage or cell death. The number of cells expressing reactive oxygen species and the fluorescence intensity of HT-29 cells treated with SN22 fermentation supernatant were significantly higher than those in the control group, indicating that SN22 fermentation supernatant may have a pro-apoptotic effect on colon cancer cells (Table 9, Figure 4 ).

[0096] Table 9 Average number of cells expressing ROS

[0097]

[0098] Note: Compared with the Control group, **p<0.01

[0099] 5.2.3 Pro-apoptotic ability of fermentation supernatant on human colon cancer HT-29 cells

[0100] One-step TUNEL cell apoptosis detection was used. After incubating the SN22 fermentation supernatant (the preparation method of the fermentation supernatant is described in 5.1.1) with the adherent cultured HT-29 cells for 48 hours, the fermentation supernatant was aspirated and washed once with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes and washed again with PBS. Add PBS containing 0.3% Triton X-100 and incubate at room temperature for 5 minutes. After preparing the TUNEL detection solution, mix it thoroughly, add 50μL TUNEL detection solution to the HT-29 cells, and incubate at 37℃ in the dark for 60min. Wash 3 times with PBS, then seal the slide with anti-fluorescence quenching sealing solution and observe under an inverted fluorescence microscope, with an excitation wavelength of 550nm and an emission wavelength of 570nm. The supernatant of the liquid culture medium of American cockroach was used as the negative control group, and the experiment was repeated 3 times.

[0101] The results showed that after treatment with SN22 fermentation supernatant, the number of apoptotic HT-29 cells increased significantly and the fluorescence intensity of the marker increased, further proving that SN22 fermentation supernatant has a significant effect in promoting HT-29 cell apoptosis (Table 10, Figure 5 ).

[0102] Table 10 Pro-apoptotic ability of SN22 fermentation supernatant on human colon cancer cells

[0103]

[0104] Note: Compared with the Control group, **p<0.01

[0105] 5.2.4 Preventive effect of fermentation supernatant on colitis model in mice

[0106] DSS aqueous solution: Dextran sulfate sodium salt (DSS) was used to prepare an aqueous solution with a concentration of 4% (w / v).

[0107] Fermentation supernatant: The preparation method of the American cockroach fermentation supernatant of Lactobacillus delbrueckii subsp. bulgaricus SN22 strain is as described in 5.1.1.

[0108] Experimental animals: 27 4-week-old SPF male Kunming mice were provided by Chengdu Dashuo Experimental Animal Co., Ltd. and randomly divided into 9 cages, 3 mice in each cage. After adaptive feeding, they were randomly divided into 3 groups, 3 cages in each group, including a blank control group (CK), a DSS model group, and a probiotic (Lactobacillus delbrueckii subsp. bulgaricus SN22) treatment group. The experimental treatments are shown in Table 11.

[0109] Table 11DSS-induced colitis experiment in mice

[0110]

[0111] Serum inflammatory factor detection: At the end of the experiment, three mice were randomly killed in each group, and blood was collected from the eye sockets. The blood was centrifuged at 3000 r / min for 15 min to obtain serum. The inflammatory factors (IL-1β, IL-6, IL-8 and TNF-α) in the serum were determined using ELISA kits according to the instructions of the kit.

[0112] Organ index and colorectal length: After the modeling period, three mice in each group were killed and dissected, and their spleens and livers were quickly removed, rinsed with physiological saline, dried with filter paper, and weighed. The colorectal length was measured with a ruler.

[0113] Organ index = spleen or liver mass (g) / mouse body weight (g) × 100%

[0114] 7.2 Results Analysis

[0115] The results of blood inflammatory factor detection showed that the four inflammatory factor indicators of IL-1β, IL-6, IL-8 and TNF-α in the DSS model group were significantly higher than those in the control group. Although the levels of the four inflammatory factors in the serum of mice in the SN22 strain treatment group were higher than those in the control group, they were significantly lower than those in the DSS model group, indicating that the supernatant of Lactobacillus delbrueckii subsp. bulgaricus SN22 strain after fermentation of American cockroaches had a significant alleviating effect on DSS-induced colitis in mice (Table 12).

[0116] Table 12 Changes in the concentration of each inflammatory factor in the serum of each group of mice (x±SD) (unit: pg / mL)

[0117]

[0118] Note: Compared with CK group, *p<0.05, **p<0.01; compared with DSS group, #p<0.05, ##p<0.01

Claims

1. A strain of Lactobacillus delbrueckii subsp.bulgaricus SN22, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31143.

2. A biological preparation containing Lactobacillus delbrueckii subsp. bulgaricus SN22 or its fermentation liquid according to claim 1.

3. Use of Lactobacillus delbrueckii subsp. bulgaricus SN22 according to claim 1 in fermenting American cockroaches.

4. An American cockroach powder fermentation liquid, characterized in that: The method is obtained by fermenting American cockroach powder with Lactobacillus delbrueckii subsp.bulgaricus SN22 as described in claim 1.

5. The American cockroach powder fermentation liquid according to claim 4, characterized in that: The fermentation method comprises the following steps: preparing a culture medium with American cockroach powder and oligofructose with water, sterilizing and cooling the culture medium, inoculating Lactobacillus delbrueckii subsp.bulgaricus SN22, culturing the culture medium at 37°C for 96 hours, centrifuging the fermentation liquid at 4000 rpm and 4°C for 10 minutes, and filtering the supernatant with a bacterial filter membrane to obtain a sterile supernatant.

6. Use of the American cockroach powder fermentation liquid according to claim 4 or 5 in preparing a medicine for treating or assisting in treating colon cancer.

7. An anti-colon cancer drug or an anti-colon cancer auxiliary drug, characterized in that: Contains the American cockroach powder fermentation liquid according to claim 4 or 5.

8. Use of the American cockroach powder fermentation liquid according to claim 4 or 5 in the preparation of a medicament for preventing or treating colitis.

9. A drug for preventing or treating colitis, characterized in that: Contains the American cockroach powder fermentation liquid according to claim 4 or 5.

10. Use of the American cockroach powder fermentation liquid according to claim 4 or 5 in preparing functional foods.

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