A Lactobacillus sp. associated with Korean koji and its application in Ganoderma lucidum spore fermentation

Abstract

The invention discloses a strain of Korean koji-associated lactobacillus and its application in the fermentation of ganoderma lucidum spores. The strain is separated from Guizhou fermented food, is non-hemolytic, is sensitive to multiple antibiotics, has strong gastrointestinal environment adaptability, has strong inhibitory ability against pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella paratyphi B and Shigella dysenteriae, and has strong antioxidant effect. The fermentation of broken wall ganoderma lucidum spore powder with the strain can greatly increase the content of multiple bioactive substances such as amino acids and peptides, eicosanoids, amine compounds, etc. in the fermentation supernatant, and the fermentation supernatant has obvious effects on DSS-induced colitis in mice, and the level of inflammatory factors is significantly reduced. The strain not only has broad application prospects in the field of probiotics for humans and animals, but also opens up new ways for the further development and utilization of ganoderma lucidum spores.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of lactobacillus associated with Korean koji and application thereof in fermentation of wall-broken ganoderma spore powder. Background Art

[0002] Lingzhi, as a traditional Chinese medicinal material, is widely used in the treatment and health care of many diseases. The spores released by Lingzhi are rich in polysaccharides, peptides and other bioactive substances, which can enhance the function of the human immune system and help the body better resist diseases. However, Lingzhi spores have two layers of chitinous outer walls that are extremely difficult to be digested by human gastric acid. Only when the walls are broken can the effective ingredients be absorbed and utilized by the human body.

[0003] Companilactobacillus nuruki is a beneficial lactic acid bacteria that is often found in traditional fermented foods. Public data show that the HHP001 strain of Companilactobacillus nuruki can effectively inhibit the expression of inflammatory factors in human colon cancer cells, thereby inhibiting the development of tumors, and has potential application value. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a strain of lactobacillus associated with Korean koji and application thereof in the fermentation of broken-wall ganoderma spore powder.

[0005] The technical solution of the present invention is: a strain of Korean koji-associated lactobacillus (Companilactobacillus nuruki) GS0904, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 5, 2023, with a deposit number of CGMCC NO: 27806.

[0006] A biological preparation containing the Korean koji-associated lactobacillus GS0904 or its fermentation liquid.

[0007] The Korean koji-associated lactobacillus GS0904 is used for the purpose of inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Shigella dysenteriae for non-disease treatment purposes.

[0008] The application of the Korean koji-associated lactobacillus GS0904 in fermenting broken-wall Ganoderma lucidum spore powder.

[0009] Application of ganoderma lucidum spore powder fermentation liquid in preparing anti-colitis drugs, wherein the ganoderma lucidum spore powder fermentation liquid is obtained by fermenting the wall-broken ganoderma lucidum spore powder with the Korean koji-associated lactobacillus GS0904.

[0010] Furthermore, the method for fermenting and breaking the cell walls of Ganoderma lucidum spores is as follows: The broken Ganoderma lucidum spores are made into a culture medium by adding water, and after sterilization, Lactobacillus nuruki GS0904 is inoculated and cultured at 37°C for 96 hours.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] A strain of Lactobacillus nuruki GS0904 provided by the present invention is isolated from Guizhou fermented food, is non-hemolytic, sensitive to a variety of antibiotics, has strong gastrointestinal environment adaptability, and has strong inhibitory ability against pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Salmonella paratyphi B, and Shigella dysenteriae, and has strong antioxidant effects. Fermenting the broken Ganoderma lucidum spores with this strain can greatly increase the contents of various bioactive substances such as amino acids and peptides, eicosanoids, and amine compounds in the fermentation supernatant. The fermentation supernatant has an obvious alleviating effect on DSS-induced murine colitis, and the levels of inflammatory factors are significantly reduced. This strain not only has broad application prospects in the fields of human and animal probiotics, but also opens up a new way for the further development and utilization of Ganoderma lucidum spores.

[0013] Preservation information:

[0014] Lactobacillus nuruki GS0904 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 5, 2023. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC NO: 27806. Description of the drawings

[0015] Figure 1 It is a phylogenetic relationship diagram of Lactobacillus nuruki GS0904 of the present invention and other strains;

[0016] Figure 2 It is a volcano plot of differential metabolites of fermented broken Ganoderma lucidum spores by Lactobacillus nuruki GS0904 strain of the present invention. Detailed implementation manners

[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0018] The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC 53103.

[0019] Example 1: Isolation, screening and safety of Lactobacillus nuruki GS0904 associated with Korean koji

[0020] 1.1 Isolation and screening

[0021] The strain was isolated and screened from Guizhou fermented food; 2 g of the sample was taken and placed in 20 mL of MRS broth, shaken well, and cultured in a constant temperature shaker at 37 °C for 24 h. Serial 10-fold dilutions were made and spread on MRS agar medium. After culturing at 37 °C for 24 h, single colonies were picked and purified by single colony purification three times. The purified strain was inoculated into 600 μL of MRS broth medium and cultured in a shaker at 37 °C for 18 h. Then 400 μL of sterile glycerol with a concentration of 50% (V / V) was added and stored at -80 °C in an ultra-low temperature freezer for later use. After purification, the strain was amplified in MRS broth, and then the genomic DNA of the strain was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit. PCR amplification was performed using the 16S rRNA universal primers 27F and 1492R. The primer sequences are as follows:

[0022] 27F: 5-AGAGTTTGATCMTGGCTCAG-3,

[0023] 1492R: 5-GGTTACCTTGTTACGACTT-3

[0024] The PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the obtained target gene sequence consisting of 1,176 base pairs (bp) (SEQ ID No. 1) was aligned with BLAST in NCBI. The similarity rate of one strain to the standard strain Companilactobacillus nuruki strain SYF10-1a reached 97.29%. This strain could be preliminarily identified as Lactobacillus nuruki, named GS0904. The phylogenetic relationship between this strain and other strains is shown in the appendix Figure 1 .

[0025] The Gram staining of Lactobacillus nuruki GS0904 was performed using a kit method, and the morphology of the stained bacteria was observed and recorded under a microscope. Lactobacillus nuruki GS0904 grew well on MRS agar medium. The colony morphology was milky white, round and raised, with a flat edge and a smooth surface. Under microscopic examination, the bacteria were rod-shaped and purple, conforming to the morphological characteristics of Lactobacillus nuruki. This strain was deposited in the China General Microbiological Culture Collection Center on July 5, 2023, at No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO. 27806.

[0026] 3.2 Safety

[0027] After reviving and activating the Lactobacillus parafarraginis GS0904 strain from Korean koji for 3 generations, it was streaked on Columbia blood agar plates. After culturing at 37°C for 24 h, whether a hemolytic zone appeared around the colonies to be tested was observed, and Staphylococcus aureus was used as a positive control. The disk agar diffusion method was used to test the drug sensitivity of the strain to common major antibiotics. The cell concentration of the Lactobacillus parafarraginis GS0904 strain from Korean koji was adjusted to 1×10 6 CFU / mL. The cell suspension was evenly spread on the surface of MRS agar plates with a sterile cotton swab. After 10 min at room temperature, drug sensitivity disks were placed, and after culturing at 37°C for 24 h, the diameter of the inhibition zone around each drug sensitivity disk was measured with a vernier caliper. Each antibiotic was repeated 3 times. The test results were judged according to the standards of the National Committee for Clinical Laboratory Standards (NCCLS) of the United States, and the results were expressed as sensitive (S), intermediate (I), and resistant (R).

[0028] The hemolytic test results showed that no hemolytic zone appeared around the colonies; it was sensitive (S) to the 4 tested antibiotics, indicating the safety of the Lactobacillus parafarraginis GS0904 strain (Table 1).

[0029] Table 1 Sensitivity of Lactobacillus parafarraginis GS0904 strain from Korean koji to 4 antibiotics

[0030] Number Tetracycline Ampicillin Gentamicin Chloramphenicol GS0904 S I S S

[0031] Example 2 Probiotic properties of Lactobacillus parafarraginis GS0904 strain from Korean koji

[0032] 2.1 Tolerance to simulated gastric and intestinal fluids

[0033] The simulated gastric and intestinal fluids were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The artificial gastric juice simulation solution consisted of dilute hydrochloric acid, pepsin, and sodium chloride, with a final pH of 2.5; the artificial intestinal juice simulation solution consisted of potassium dihydrogen phosphate and trypsin, with a final pH of 6.8. The Lactobacillus parafarraginis GS0904 strain to be tested was revived and activated for 3 generations on MRS agar plates, and the initial cell concentration was adjusted to 1×10 6 CFU / mL. 1 mL of the cell suspension of the Lactobacillus parafarraginis GS0904 strain to be tested was inoculated into the simulated gastric juice and cultured at 37°C for 3 h. After culturing, 20 μL of the cell suspension was spread on MRS agar plate medium, with 3 replicates set, and cultured at 37°C for 24 h to observe whether colonies grew on the surface of the MRS plates after culturing. Similarly, 1 mL of the cell suspension of the Lactobacillus parafarraginis GS0904 strain to be tested was inoculated into the simulated intestinal juice, cultured at 37°C for 6 h, then spread on plates and cultured at 37°C for 24 h to check the colony growth.

[0034] The results of the simulated gastric fluid tolerance test showed that Korean koji-associated Lactobacillus GS0904 could still grow normal colonies on MRS agar plates after being treated in simulated gastric fluid for 3 hours; the results of the simulated intestinal fluid tolerance test showed that Korean koji-associated Lactobacillus GS0904 could still grow normal colonies on MRS agar plates after being treated in simulated intestinal fluid for 6 hours, indicating that Korean koji-associated Lactobacillus GS0904 has strong acid and bile salt resistance.

[0035] 2.2. Antibacterial activity against common intestinal pathogens

[0036] Escherichia coli (CMCB 44102), Staphylococcus aureus (CMCB 50094), Salmonella typhimurium ATCC 14028), Pseudomonas aeruginosa (CMCB 10104), Salmonella paratyphi B (CMCB 50094) and Shigella dysenteriae (CMCB 51105) were inoculated into nutrient agar medium, revived and activated three times. An appropriate amount of tryptic soy bean liquid medium was drawn into a centrifuge tube, and the activated pathogenic bacteria were inoculated into the broth medium, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL. Take 1mL of the mixture of the above pathogenic bacteria and broth and add it to 500mL of sterilized nutrient agar medium that has not yet solidified (the temperature has been cooled to about 40°C), mix thoroughly and dispense into culture dishes at a volume of 20mL per dish. After the culture medium has cooled and solidified, use a 6mm diameter puncher to punch holes on the plate to make pathogenic bacteria agar plates. Each plate corresponds to one strain of bacteria, and three holes are set as replicates. Resuscitate and activate the GS0904 strain, and adjust the bacterial solution concentration to 1×10 8 CFU / mL, aspirate 50μL of bacterial solution and add it to the agar plate wells of the pathogenic bacteria, culture at 37℃ for 24h, use a vernier caliper to measure the diameter of the inhibition zone around the punch point and record it. The standard strain LGG was used as the control strain, and the above experimental operations were carried out simultaneously with the strain to be tested.

[0037] The experimental results show that the fermentation liquid of the Korean koji-associated Lactobacillus GS0904 strain has a strong inhibitory activity on the growth of pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Shigella dysenteriae (Table 2).

[0038] Table 2 Evaluation of the antibacterial activity of Lactobacillus paracasei GS0904 strain associated with Korean koji (diameter: mm)

[0039]

[0040]

[0041] 2.3 Antioxidant activity of the fermentation broth of Lactobacillus paracasei GS0904 associated with Korean koji

[0042] More and more studies have shown that antioxidant is an important step in preventing aging, because free radicals or oxidants can decompose cells and tissues, affect metabolic functions, and cause different health problems. If excessive oxidative free radicals can be eliminated, it has a preventive effect on many free radical-related diseases, such as common cancers, arteriosclerosis, diabetes, cataracts, cardiovascular diseases, Alzheimer's disease, arthritis, etc.

[0043] Preparation of the fermentation broth: After reviving and culturing the frozen strain of Lactobacillus paracasei GS0904 associated with Korean koji for 24 h, it was inoculated into MRS liquid medium at an inoculation amount of 2%, cultured at 37 °C for 24 h, the bacterial liquid was centrifuged at 4 °C and 4000 r / min for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the fermentation supernatant (CFS) and stored frozen for later use.

[0044] Determination of antioxidant capacity: The determination of the antioxidant capacity of the fermentation broth was completed by Chengdu Lilai Biotechnology Co., Ltd. The determination method used the kit method. The hydroxyl radical determination kit (A018-1-1), DPPH free radical scavenging capacity kit (A153-1-1), and superoxide anion radical inhibition and generation determination kit (colorimetric method A052-1-1) were all produced by Nanjing Jiancheng Bioengineering Institute.

[0045] Ability to inhibit hydroxyl radicals: The determination results are shown in Table 8. The fermentation broth of Lactobacillus paracasei GS0904 associated with Korean koji has a strong ability to inhibit hydroxyl radicals, which is 2682.88 ± 59.34 U / mL. Hydroxyl radicals are a type of reactive oxygen species and have extremely strong electron-gaining ability, that is, very strong oxidation ability. Hydroxyl radicals can kill red blood cells, degrade DNA, cell membranes, and polysaccharide compounds.

[0046] DPPH free radical scavenging capacity: The DPPH free radical scavenging capacity of the fermentation broth of Lactobacillus paracasei GS0904 associated with Korean koji is 167.30 ± 14.72 μg Trolox / mL, and the scavenging rate is 39.66%, indicating that the fermentation broth of Lactobacillus paracasei GS0904 associated with Korean koji has a certain scavenging ability for DPPH free radicals.

[0047] Superoxide anion resistance: The measurement results showed that its superoxide anion resistance was 694.71 ± 7.95 (U / L). As a free radical produced during the metabolic process of organisms, superoxide anion radicals can attack biological macromolecules such as lipids, proteins, nucleic acids, and polyunsaturated fatty acids, causing them to cross-link or break, resulting in the destruction of cell structure and function, and having a very close relationship with the aging and diseases of the body (Table 3).

[0048] Table 3 Antioxidant capacity of the fermentation broth of Lactobacillus parafarraginis GS0904 associated with Korean koji

[0049]

[0050]

[0051] Example 3 Application of Lactobacillus parafarraginis GS0904 strain in fermenting and breaking the wall of Ganoderma lucidum spores

[0052] 3.1 Fermentation and analysis methods

[0053] Fermentation method: 3 g of broken Ganoderma lucidum spores and 20 mL of water were prepared into a medium. After sterilization and cooling, Lactobacillus parafarraginis GS0904 was inoculated and cultured at 37 °C for 96 hours. The fermentation broth was centrifuged at 4000 rpm and 4 °C for 10 min, 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 control group without inoculating Lactobacillus parafarraginis GS0904 was cultured and analyzed in the same way as the treatment group.

[0054] Analysis method: Based on high-resolution mass spectrometry (HRMS) detection technology, combined with the high-quality mzCloud database constructed by standard products and the mzVault and MassList databases, the molecular characteristic peaks were matched and identified. The original data downloaded from the instrument was preprocessed using CD3.3 data processing software. First, simple screening was performed through parameters such as retention time and mass-to-charge ratio, and at the same time, the peak area was quantified. Then, the high-resolution secondary spectrum database mzCloud and mzVault and the MassList primary database were compared and retrieved (searched) for metabolite identification. Using CD3.3 data processing software, the chromatographic peaks detected in the samples were integrated. The peak area of each characteristic peak represented the relative quantitative value of a metabolite, and the total peak area was used to standardize the quantitative results. Finally, the quantitative results of the metabolites were obtained, and the results were expressed as the multiple of the increase in the content of the corresponding components in the sample compared with the control.

[0055] 3.2 Analysis results

[0056] The results of liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis showed that, in the negative ion mode, the contents of 32 compounds increased significantly compared with the control, and the contents of 85 compounds decreased significantly; in the positive ion mode, the contents of 44 compounds increased significantly compared with the control, and the contents of 70 compounds decreased significantly( Figure 2 ).

[0057] (1) Amino acids and peptide compounds:

[0058] Amino acids are the basic building blocks of proteins and play important roles in human health and normal functions, such as protein synthesis, physiological regulation, immune function, energy supply, and maintaining acid-base balance. The determination results showed that in the fermentation supernatant of Ganoderma lucidum spores fermented by Lactobacillus parafarraginis GS0904 strain from Korean koji, the contents of various amino acids and peptide compounds increased significantly. For example, the content of 8-aminooctanoic acid was 18.71 times that of the control. As a ω-amino fatty acid, it participates in the metabolic process in vivo. Some studies have shown that it can also be used for the preparation of oleanolic acid derivatives with anti-HIV activity. The content of 5-hydroxyindole-2-carboxylic acid was 4.14 times that of the control. According to its structure, it may have biological activities similar to those of indole compounds, such as neurotransmitter regulation and signal transduction (Table 4).

[0059] In addition, the contents of D-glutamine and Prolylleucine also increased significantly. D-glutamine shows potential effects in neuroprotection, immunomodulation, anti-tumor, etc. At the same time, it has practical application value as a medicinal amino acid and nutritional supplement. Studies have shown that it can regulate the release of neurotransmitters, reduce neuronal damage, and promote nerve regeneration. It can affect the proliferation and differentiation of immune cells and regulate the intensity and direction of immune responses. It can affect the metabolic pathways of tumor cells, inhibit the proliferation and migration of tumor cells, providing new ideas for tumor treatment. It is a medicinal amino acid that can penetrate into cells and be used to treat digestive tract ulcers. It can increase the absorption of sodium and chloride in the intestine, and then increase the absorption of water in the intestine, which is beneficial to diarrhea patients. Prolylleucine is a dipeptide containing branched-chain amino acids and has the ability to affect the circadian rhythm of animals. The circadian rhythm is an internal rhythm in organisms that controls many physiological and behavioral processes, such as the sleep-wake cycle, body temperature, metabolism, etc. It can also regulate the behavior of animals (Table 4).

[0060] Table 4 Effects of Ganoderma lucidum spores fermented by GS0904 strain on the contents of amino acids and peptide compounds

[0061]

[0062] (2) Eicosanoids:

[0063] Eicosanoid, also known as eicosanoic acid, is a large class of bioactive unsaturated fatty acids produced by the oxidation of eicosapolyunsaturated fatty acids, including prostaglandins, thromboxanes, and leukotrienes. It is an important inflammatory factor that widely exists in body fluids and tissues and regulates numerous physiological and pathological processes in the body.

[0064] Prostaglandin has two main functions. One is to affect the contraction of smooth muscles, strongly acting on the intestines, blood vessels, bronchi, uterus, etc. The other is to change the activity of adenylate cyclase, generally promoting it, but inhibiting it in adipose tissue, so it has antilipolytic effects and participates in the regulation and control of many physiological processes, promotes inflammatory reactions, participates in reproductive processes (such as ovulation, conception, and uterine contractions during childbirth), and digestion. The analysis results show that after fermenting Ganoderma lucidum spore powder with Lactobacillus kefiranofaciens GS0904 strain for 96 hours, various prostaglandin compounds were detected in the supernatant. Compared with the control, the content increased significantly. For example, the content of 15-Deoxy-Δ12,14-prostaglandin D2 (abbreviated as 15d-PGD2) was 9.09 times that of the control (Table 10). It mainly exerts its biological activity by inhibiting inflammation, regulating immune responses, and affecting cell functions. It may play this role by affecting certain key molecules or signaling pathways during the inflammatory process. It may participate in regulating the activity, proliferation, or differentiation of immune cells, thereby affecting the intensity and direction of immune responses. In the fermentation broth, the content of 6-Keto-prostaglandin F1alpha (abbreviated as 6-Keto-PGF1α) was 7.74 times that of the control. It is a metabolite of prostacyclin (PGI2). As a stable metabolite of PGI2, 6-Keto-PGF1α may, to a certain extent, retain the physiological effects of PGI2, such as vasodilation, reducing peripheral vascular resistance, increasing organ blood flow, and promoting sodium excretion and diuresis. Prostaglandin E3 (PGE3) has significant anti-inflammatory, anti-tumor, and intraocular pressure-lowering biological activities, which provide broad prospects for its application in the medical and scientific research fields (Table 5).

[0065] (+ / -)11(12)-DiHET is a member of the Epoxyeicosatrienoic acids (EETs) family. EETs have various physiological and pathological effects, including vasodilation, anti-inflammation, anti-proliferation, and anti-apoptosis, etc. They play important roles in multiple organs and tissues such as the cardiovascular system, nervous system, and kidneys (Table 5).

[0066] Table 5 Effect of GS0904 fermentation of Ganoderma lucidum spore powder on the content of eicosanoid compounds

[0067]

[0068] (3) Amine compounds:

[0069] Amines are molecules of ammonia (NH 3 ) is a product in which one or more hydrogen atoms in the amine are replaced by hydrocarbon groups. Amine compounds are widely present in the biological world and have extremely important physiological and biological activities. For example, proteins, nucleic acids, many hormones, antibiotics and alkaloids are all derivatives of amines. Many drugs used clinically are also amines or amine derivatives. The analysis results showed that after the broken wall spore powder of Ganoderma lucidum was fermented with Korean koji-associated Lactobacillus GS0904 strain for 96 hours, three amine compounds were detected in the supernatant, and the content was greatly increased compared with the control. For example, the content of Amino-1,3-octadecanediol (also known as DL-erythro-dihydroceramide or D-threo-dihydrosphingosine) is 22.15 times that of the control, and its biological activities include antioxidant, moisturizing and potential anti-cancer effects (Table 6).

[0070] The content of spermidine in the fermentation broth was 9.18 times that of the control. It is a polyamine that can delay aging, maintain cell membrane stability, and increase antioxidant enzyme activity by promoting the synthesis of certain large molecular weight proteins or preventing their degradation. In addition, the content of phytosphingosine also increased significantly (2.91 times). It is a phospholipid that has anti-cancer effects and can inhibit a variety of inflammatory factors. It is a natural inflammatory regulator (Table 6).

[0071] Table 6 Effect of GS0904 strain fermentation of Ganoderma lucidum spore powder on the content of amine compounds

[0072]

[0073]

[0074] Example 4 Effect of Ganoderma lucidum spore fermentation supernatant on mouse colitis model

[0075] 4.1 Preparation of fermentation supernatant: see Example 3. The fermentation broth was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was filtered through a bacterial filter to obtain a sterile supernatant. At the same time, a 4% (w / v) aqueous solution of dextran sulfate sodium salt (DSS) was prepared for use.

[0076] 4.2 Animal experiments: The experimental mice were provided by Chengdu Dashuo Experimental Animal Co., Ltd. Twenty-seven 4-week-old SPF-grade male Kunming mice were randomly divided into 9 cages, with 3 mice in each cage. After adaptive feeding, they were randomly divided into 3 groups, with 3 cages in each group, including a blank control group (CK), a DSS model group, and a probiotic (Lactobacillus parafarraginis GS0904 strain) treatment group. The experimental treatments are shown in Table 7.

[0077] Table 7 DSS-induced murine colitis experiment

[0078]

[0079] 4.3 Detection of serum inflammatory factors: At the end of the experiment, 3 mice were randomly sacrificed from each group, and blood was collected from the orbital cavity. The blood was centrifuged at 3000 r / min for 15 min to obtain serum. An ELISA kit was used to measure the inflammatory factors (IL-1β, IL-6, IL-8, and TNF-α) in the serum, and the steps were carried out according to the kit instructions.

[0080] 4.4 Result analysis

[0081] Animal experiment observations showed that before the end of the experiment, the symptoms of colitis in the DSS model group mice were the most obvious, and symptoms such as loss of appetite, listlessness, diarrhea with blood in the stool were generally present. The symptoms in the GS0904 treatment group were significantly milder, with only individual cases of diarrhea, and the control group had no symptoms. At the end of the experiment, the four inflammatory factor indexes of TNF-α, IL-1β, IL-6, and IL-8 were measured. The results showed that the levels of inflammatory factors in the serum of the DSS model group mice were significantly increased, while the levels of the four inflammatory factors in the serum of the Lactobacillus parafarraginis GS0904 strain treatment group mice were significantly lower than those in the DSS model group, indicating that the Ganoderma lucidum spore fermentation supernatant of Lactobacillus parafarraginis GS0904 strain has an obvious alleviating effect on DSS-induced murine colon inflammation (Table 8).

[0082] Table 8 Determination of inflammatory factor concentrations in the serum of mice in each group (Unit: pg / mL)

[0083]

[0084] Note: Compared with the CK group, *p < 0.05, **p < 0.01; compared with the DSS group, #p < 0.05, ##p < 0.01

Claims

1. A strain of Lactobacillus associated with Korean koji ( Companilactobacillus nuruki ) GS0904, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 5, 2023, with the deposit number CGMCC NO:27806.

2. A biological preparation containing the Korean koji-associated Lactobacillus GS0904 or its fermentation liquid as described in claim 1.

3. The application of the Korean koji-associated lactobacillus GS0904 according to claim 1 in inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Shigella dysenteriae for non-disease treatment purposes.

4. Application of the Korean koji-associated lactobacillus GS0904 according to claim 1 in fermentation of broken-wall Ganoderma lucidum spore powder.

5. Application of Ganoderma lucidum spore powder fermentation liquid in the preparation of anti-colitis drugs, wherein the Ganoderma lucidum spore powder fermentation liquid is obtained by fermenting the broken Ganoderma lucidum spore powder with the Korean koji-associated Lactobacillus GS0904 as claimed in claim 1.

6. The use according to claim 5, characterized in that: The method for fermenting the broken wall ganoderma spore powder is as follows: adding water to the broken wall ganoderma spore powder to prepare a culture medium, inoculating Korean koji-associated lactobacillus GS0904 after sterilization, and culturing at 37°C for 96 hours.

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