Lactococcus lactis subsp. Lactis and application thereof in fermentation of periplaneta americana powder

Abstract

The invention discloses lactococcus lactis subsp. Lactis and application thereof in fermentation of American cockroach powder. The strain is separated and screened from the intestinal contents of the Amolops nobilis, grows well on an MRS agar culture medium, is not hemolyzed, is sensitive to various common antibiotics, has relatively strong adaptive capacity to the intestinal environment of human and animals, has relatively strong lactic acid production capacity, and has strong inhibition capacity to common intestinal pathogens of human and animals. When the strain is used for fermenting American cockroach powder, the content of bioactive substances such as amino acids, peptides, indoles, pyrimidine and purines can be greatly increased, fermented sterile supernate has an obvious effect on mouse colitis induced by DSS, and the inflammatory factor level is remarkably reduced; the strain opens up a new way for further development and utilization 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 Lactococcus lactis subsp. lactis and application thereof in the fermentation of American cockroach powder. Background Art

[0002] As a traditional Chinese medicine, American cockroach has many functions, such as clearing away heat and detoxifying, dispersing blood stasis and removing accumulation, and promoting diuresis and reducing swelling. Among the medicines made from American cockroaches, the most well-known is Kangfuxinye. Kangfuxinye is a pure Chinese medicine preparation, mainly made from ethanol extracts of dried insect bodies of American cockroaches. This medicine has many functions, including promoting blood circulation, nourishing yin and promoting muscle growth, promoting the growth of granulation tissue, and promoting angiogenesis.

[0003] Lactococcus lactis is one of the important intestinal probiotics, which can help the human body digest lactose better, relieve the symptoms of lactose intolerance, and also help the body absorb protein, monosaccharides, calcium, magnesium and other minerals, and produce a large number of beneficial substances such as B vitamins. At the same time, Lactococcus lactis is one of the important strains of dairy fermentation, especially in fermented dairy products such as yogurt, sour cream, soy yogurt, milk drinks, etc.

[0004] If Lactococcus lactis subsp. lactis can be organically combined with American cockroach powder to increase the content of active substances or produce new compounds, it will provide new ideas for the further utilization of the traditional Chinese medicinal material American cockroach. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a strain of Lactococcus lactis subspecies lactis and its application in the fermentation of American cockroach powder.

[0006] The technical solution of the present invention is: a strain of Lactococcus lactis subsp. lactis 12-1, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 5, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 27808.

[0007] A biological preparation containing the above-mentioned Lactococcus lactis subsp. lactis 12-1 or its fermentation liquid.

[0008] The use of the above-mentioned Lactococcus lactis subsp. lactis 12-1 in fermenting American cockroach powder.

[0009] The invention discloses an American cockroach powder fermentation liquid, which is obtained by fermenting the American cockroach powder with the above-mentioned Lactococcus lactis subsp. lactis 12-1.

[0010] Furthermore, the fermentation method comprises: preparing a culture medium with American cockroach powder and oligofructose with water, sterilizing and cooling, inoculating Lactococcus lactis subsp. lactis 12-1, culturing 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.

[0011] Furthermore, the weight ratio of the American cockroach powder to oligofructose is 10:3.

[0012] The use of the American cockroach powder fermentation liquid in preparing a medicine for treating or assisting in treating colitis.

[0013] The use of the American cockroach powder fermentation liquid in preparing functional food.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The invention provides a strain of Lactococcus lactis subsp. lactis 12-1 and its application in the fermentation of American cockroach powder, and the strain deposit number is CGMCC No. 27808. The strain is separated and screened from the intestinal contents of the South China frog, grows well on MRS agar medium, is non-hemolytic, is sensitive to a variety of common antibiotics, has strong adaptability to the intestinal environment of humans and animals, has strong lactic acid production ability, and has strong inhibitory ability to common intestinal pathogens of humans and animals. Fermenting American cockroach powder with the strain can greatly increase the content of bioactive substances such as amino acids, peptides, indoles, pyrimidines and purines, and the fermentation sterile supernatant has a significant effect on DSS-induced colitis in mice, and the level of inflammatory factors is significantly reduced. The strain has opened up a new way for the further development and utilization of the traditional Chinese medicine American cockroach.

[0016] Collection information:

[0017] Lactococcus lactis subsp. lactis 12-1 was deposited at the General Microbiology Center of China Culture Collection Administration on July 5, 2023. 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. 27808. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a phylogenetic relationship diagram of Lactococcus lactis subspecies 12-1 in the present invention.

[0019] Figure 2 This is a volcano plot of differential metabolites of Lactococcus lactis subspecies 12-1 fermenting American cockroach powder in the present invention.

[0020] Figure 3 HE staining (×400) of colorectal tissue sections of normal mice (A), DSS-induced enteritis mice (B) and Lactococcus lactis subsp. lactis 12-1 test group mice (C) in the animal experiment of the present invention. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0022] The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC 53103.

[0023] Experimental Example 1 Isolation and Identification of Lactococcus lactis subspecies 12-1

[0024] 1.1 Material preparation

[0025] Enteric samples of South China turbulent frog (Amolops ricketti) were collected from Xixi Village, Dongcheng Town, Linhai City, Zhejiang Province;

[0026] 16S rRNA universal sequencing primers 27F and 1492R were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the sequences are as follows (5'→3'):

[0027] 27F (SEQ ID No. 1): AGAGTTTGATCMTGGCTCAG

[0028] 1492R(SEQ ID No.2):GGTTACCTTGTTACGACTT

[0029] 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.

[0030] 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.

[0031] 1.2 Screening and isolation of strains

[0032] Take a small amount of enteric sample, inoculate it in 50mL MRS broth, shake it thoroughly and mix it, and place it in a constant temperature shaker at 37℃ for 24h. Take 1mL of culture solution, dilute it by 10 times, take 20μL respectively, spread it on MRS agar medium, and pick a single colony after culturing at 37℃ for 24h. 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.

[0033] 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 1055 base pairs (bp), and the sequence is shown in SEQ ID No. 1.

[0034] The gene sequence obtained by sequencing was input into the NCBI database for comparison. The similarity between the strain and the strain Lactococcus lactis subsp. lactis strain XT7-4 in GenBank: MH891690.1 was 98.19%, and the strain was preliminarily identified as Lactococcus lactis subsp. lactis. The phylogenetic relationship between this strain and other strains is shown in Figure 1. Figure 1 The strain was inoculated on MRS agar medium and cultured at 37°C for 24 hours, and the morphology of the single colonies was observed and recorded. Figure 2 As shown, Lactococcus lactis subspecies 12-1 grows well on MRS agar medium, and is milky white, round, and has smooth convex surfaces. Its Gram staining is positive and spherical, which is consistent with the characteristics of Lactococcus lactis.

[0035] The strain was named Lactococcus lactis subsp. lactis 12-1 and was deposited in the General Microbiology Center of China Culture Collection Administration on July 5, 2023. 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. 27808.

[0036] Experimental Example 2 Hemolytic and Antibiotic Sensitivity Determination of Lactococcus lactis Subspecies 12-1

[0037] The Lactococcus lactis subspecies 12-1 isolated and identified in Experimental Example 1 was activated, streaked on an MRS agar plate and a single colony was selected, streaked on a blood plate and cultured for 24 hours, and then the hemolysis was observed. The experiment was repeated three times. No hemolysis occurred around the Lactococcus lactis subspecies 12-1 colony.

[0038] The sensitivity of the strain to common antibiotics was tested by the paper agar diffusion method. The Lactococcus lactis subsp. lactis 12-1 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 smear the bacterial solution on the surface of the MH medium plate, place it at room temperature for 10 minutes, then put in the drug sensitivity paper, and culture it at 37℃ 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, Lactococcus lactis subspecies 12-1 is sensitive (S) or intermediate (I) to the six antibiotics tested, including tetracycline, ampicillin, ceftriaxone, clarithromycin, chloramphenicol, and ciprofloxacin. Lactococcus lactis subspecies 12-1 has good safety.

[0039] Table 1 Sensitivity of Lactococcus lactis subsp. lactis 12-1 to 6 antibiotics

[0040]

[0041] Experimental Example 3 Evaluation of acid and salt resistance of Lactococcus lactis subspecies 12-1

[0042] 3.1 Acid resistance and bile salt resistance evaluation:

[0043] The Lactococcus lactis subspecies 12-1 isolated and identified in Experimental Example 1 was revived and activated, and the initial concentration of the bacterial solution was adjusted to 1×10 8CFU / 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 Lactococcus lactis subsp. lactis 12-1 was inoculated into the broth medium with pH 3.0 and bile salt concentration of 0.3%, and the initial concentration of the culture medium 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 MRS plate was observed to see if there were colonies growing. The experiment was repeated 3 times.

[0044] According to the results of the acid resistance and bile salt resistance tests, Lactococcus lactis subspecies 12-1 can still grow normal colonies on MRS agar plates after being treated in MRS broth medium with pH 3.0 and a bile salt concentration of 0.3% for 18 hours. This strain has strong acid and bile salt resistance.

[0045] 3.2 Simulated gastric juice and intestinal juice tolerance test:

[0046] Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the main components of gastric juice simulation fluid were sodium chloride, dilute acid and pepsin, and the final pH was 2.5; the main components of intestinal juice simulation fluid were phosphate and trypsin, and the final pH was 6.8. The Lactococcus lactis subsp. lactis 12-1 strain isolated and identified in Experimental Example 1 was revived and activated, and the bacterial solution was centrifuged for 10 minutes and then washed 2-3 times with artificial gastric juice and resuspended. The bacterial concentration was adjusted to 1×10 8 CFU / mL, 300 μL of bacterial suspension was added to 2.7 mL of artificial simulated gastric fluid, and 20 μL of the mixture was diluted 10 times and then counted as the initial value of simulated gastric fluid tolerance. The simulated gastric fluid with live bacteria was cultured at 37°C for 3 hours and then counted using the same method as the final value of tolerance to artificial gastric fluid. At the same time, 300 μL of the above mixture was added to 2.7 mL of simulated intestinal fluid and the above method was used to count live bacteria. The experiment was repeated 3 times, with LGG as the control.

[0047] As shown in Table 2, Lactococcus lactis subspecies 12-1 has good tolerance to artificial simulated gastric and intestinal fluids, especially to simulated gastric fluid, and its comprehensive tolerance is higher than that of LGG strain, which is conducive to the colonization of strains in the gastrointestinal environment of humans and animals (Table 2).

[0048] Table 2 Tolerance of Lactococcus lactis subspecies 12-1 strain to artificial simulated gastric and intestinal fluids (%)

[0049]

[0050] Experimental Example 4 Inhibitory ability of Lactococcus lactis subspecies 12-1 strain against common intestinal pathogens

[0051] 4.1 Lactic acid production capacity of Lactococcus lactis subsp. lactis 12-1

[0052] Lactic acid is an important intermediate product in the metabolism of organisms. It has the effect of reducing the pH value of the intestine and changing the permeability of the cell membrane of Gram-positive bacteria. It also helps inhibit the growth of pathogens and is an important antibacterial compound in the gastrointestinal tract of humans and animals. Lactic acid can generate pyruvate under the action of lactate dehydrogenase and convert NAD + Reduced to NADH and H + , while H + The reduced MTT is transferred to PMSH2 to generate a purple substance with a characteristic absorption peak at 570nm, so that the lactic acid content produced can be measured by instruments such as a spectrophotometer or an enzyme reader.

[0053] After the Lactococcus lactis subspecies 12-1 strain identified and preserved in Experimental Example 1 was activated and cultured, it was inoculated into MRS broth medium, cultured at 37°C for 24h, and centrifuged at 4°C and 12000g for 15min. 100 μL of fermentation supernatant was taken and the lactic acid content was determined according to the instructions of the lactic acid kit. The results showed that the lactic acid content of the Lactococcus lactis subspecies 12-1 fermentation broth was 107.37±0.41 μmol / mL, and its lactic acid production capacity was strong (Table 3).

[0054] Table 3 Lactic acid content of Lactococcus lactis subsp. lactis 12-1 fermentation broth (μmol / mL)

[0055]

[0056] 4.2 Inhibitory effect of 12-1 strain on common intestinal pathogens

[0057] Escherichia coli (CMCB 44102), Salmonella enterica serovar Paratyphi B (CMCB 50094), Pseudomonas aeruginosa (CMCB 10104), Staphylococcus aureus (CMCCB 26003), Salmonella typhimurium ATCC14028), Shigella dysenteriae (CMCB 51105), and Yersinia enterocolitica subsp enterocolitica (CMCB 52204) were revived and purified, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL. Take 10 μL of the above pathogenic bacteria solution and add it to 100 mL of solid agar medium that has been sterilized and cooled to about 40°C. Mix thoroughly and evenly distribute it into culture dishes. After the culture medium cools and solidifies, use a 6 mm diameter puncher to punch holes on the plate. After the Lactococcus lactis subspecies 12-1 isolated and identified in Experimental Example 1 is revived, the bacterial solution concentration is adjusted to 1×10 8 CFU / mL, 50 μL of the fermentation supernatant of the test bacteria was added to the wells of the pathogenic bacteria agar plate, and cultured at 37°C for 18 hours. The diameter of the inhibition zone around the punch point was measured with a vernier caliper (mm) and recorded. The experiment was repeated three times, with LGG as the control.

[0058] The results of the antibacterial activity evaluation of Lactococcus lactis subspecies 12-1 are shown in Table 4 below. The fermentation supernatant of Lactococcus lactis subspecies 12-1 strain has the ability to inhibit the growth of pathogenic bacteria of Salmonella paratyphi B, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica. Among them, the inhibitory ability for Salmonella paratyphi B and Yersinia enterocolitica is relatively strong, and the biological antibacterial agent prepared therefrom can be used to inhibit the growth and reproduction of the above-mentioned pathogenic bacteria (Table 4).

[0059] Table 4 Evaluation of antibacterial activity of Lactococcus lactis subsp. lactis 12-1 strain (diameter: mm)

[0060]

[0061] Example 5 Application of Lactococcus lactis subsp. lactis 12-1 strain in fermentation of American cockroach powder

[0062] 5.1 Fermentation and analytical methods

[0063] Fermentation method: 10 grams of American cockroach powder, 3 grams of oligofructose, and 100 mL of water were prepared into a culture medium. After sterilization and cooling, Lactococcus lactis subspecies 12-1 was inoculated and cultured at 37°C 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 to obtain a sterile supernatant for liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis. The experiment was repeated three times, with the control group not inoculated with Lactococcus lactis subspecies 12-1, and the culture and analysis methods were the same as the treatment group. At the same time, the sterile supernatant was used for animal experiments on mouse colitis models.

[0064] Analytical methods: 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.

[0065] 5.2 Effects of fermentation supernatant on colitis in mice

[0066] DSS aqueous solution: Dextran sulfate sodium salt (DSS) was used to prepare an aqueous solution with a concentration of 4% (w / v).

[0067] 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 Lactococcus lactis subsp. lactis 12-1 treatment group. The experimental treatments are shown in Table 5.

[0068] Table 5 DSS-induced colitis experiment in mice

[0069]

[0070] 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.

[0071] Organ index, colorectal length and tissue sections: 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 and temporarily preserved by immersion in 4% paraformaldehyde fixative. After sectioning, the tissues were stained with HE to observe the tissue condition.

[0072] Organ index = spleen or liver mass (g) / mouse body weight (g) × 100%

[0073] 5.3 Analysis results

[0074] 5.3.1 Metabolomics analysis

[0075] Metabolomics analysis showed that after 96 hours of fermentation of American cockroach powder with Lactococcus lactis subsp. lactis 12-1, a total of 157 compounds were detected in positive ion mode with significant increases in content, 244 compounds were detected with significant decreases in content, and 795 compounds were detected with no significant differences in content. In negative ion mode, a total of 55 compounds were detected with significant increases in content, 217 compounds were detected with significant decreases in content, and 405 compounds were detected with no significant differences in content. The metabolite difference volcano plot is shown in the figure below. Figure 2 .

[0076] (1) Amino acids and peptide compounds:

[0077] Amino acids are the basic building blocks of proteins and play an important role in human health and normal functions, such as protein synthesis, physiological regulation, immune function, energy supply, and maintaining acid-base balance. The analysis results showed that after 96 hours of fermentation of American cockroach powder with Lactococcus lactis subsp. 12-1, the content of various amino acids and their derivatives in the supernatant increased greatly, especially pantetheine, Fmoc-L-isoleucine, and O-Succinyl-L-homoserine, which were 50.97 times, 20.7 times, and 19.14 times that of the control, respectively. Pantetheine is a vitamin B5 derivative with multiple biological activities, mainly used to improve lipid metabolism and anti-platelet aggregation. Clinical studies have shown that within the dosage range of 300 to 600 mg, it has a significant lipid-lowering effect on patients with hyperlipidemia, and can also increase platelet cell membrane fluidity, thereby inhibiting platelet aggregation and helping to prevent thrombosis. As an important biochemical intermediate, Fmoc-L-isoleucine and succinic homoserine play an important role in peptide synthesis and amino acid metabolism (Table 6).

[0078] Table 6 Relative content of amino acids and their derivatives in the supernatant of American cockroach fermentation (fold)

[0079]

[0080] Many active substances exist in the form of peptides. Peptides are involved in the human body's hormones, nerves, cell growth and reproduction. Their importance lies in regulating the physiological functions of various systems and cells in the body, activating relevant enzyme systems in the body, promoting the permeability of intermediate metabolic membranes, or controlling DNA transcription or affecting specific protein synthesis, ultimately producing specific physiological effects. In the fermentation supernatant, the content of several peptide compounds such as S-lactoylglutathione, L-leucine-L-alanine, dipeptide (D-Ala-D-Ala) and reduced glutathione (Reduced glutathione) increased significantly. S-lactoylglutathione can be used as a potential marker for the sensitivity of gastric cancer to neoadjuvant chemotherapy. It is also widely used in biochemical research, drug screening and other fields. Reduced glutathione is a substance with important biological functions, playing an important role in anti-oxidation, detoxification, cell metabolism regulation and skin care (Table 7).

[0081] Table 7 Relative content of peptides in the supernatant of Periplaneta americana fermentation (fold)

[0082]

[0083] (2) Indole compounds:

[0084] Indole and its derivatives are closely related to intestinal microbiota and human health, such as gastrointestinal motility, intestinal barrier, and neural regulation. The analysis results showed that after 96 hours of fermentation of American cockroach powder with Lactococcus lactis subspecies 12-1, the content of various indole derivatives in the supernatant increased significantly, especially 5-hydroxytryptophan (5-Hydroxytryptophan), which was 12.83 times that of the control. Studies have shown that 5-hydroxytryptophan is a precursor of the neurotransmitter 5-hydroxytryptamine (serotonin, 5-HT), which plays an important role in regulating emotions, promoting happiness, affecting appetite, sleep cycles, and pain perception in the human body. In addition, it also has an antidepressant effect, which can increase the concentration of serotonin in the brain, thereby helping to relieve the symptoms of patients with depression (Table 8).

[0085] Table 8 Relative content of indole compounds in the fermentation supernatant of American cockroach (times)

[0086]

[0087] (3) Pyrimidine and purine compounds

[0088] Pyrimidine nucleotides are an important class of compounds in organisms. They play a key role in the composition of nucleic acids (DNA and RNA), energy metabolism, and signal transduction. The analysis results showed that after 96 hours of fermentation of American cockroach powder with Lactococcus lactis subsp. lactis 12-1, the content of various pyrimidine nucleotide substances in the supernatant increased significantly, especially the content of deoxyuridine triphosphate (dUTP) was 48.16 times that of the control. It is a nucleotide that plays an important role in organisms. It participates in biological processes such as DNA repair and nucleotide metabolism, and maintains the stability and accuracy of DNA through interactions with nucleotides such as dTTP. During DNA replication, if dUTP is mistakenly incorporated into the DNA chain, the enzyme system in the cell can recognize and remove these erroneous bases, and then repair them through the DNA repair mechanism. This repair process helps to maintain the stability and accuracy of DNA. It can also be converted into other nucleotides through a series of enzymatic reactions, thereby participating in the energy metabolism and material metabolism of the cell (Table 9).

[0089] Table 9 Relative content of pyrimidine compounds in the supernatant of American cockroach fermentation (times)

[0090]

[0091] Purine and its derivatives play an important role in biochemistry and medicine. They are not only one of the components of DNA and RNA, but also participate in biological processes such as energy metabolism and signal transduction. At the same time, purine derivatives have a wide range of biological activities and pharmacological effects, providing new options for the treatment of diseases. The analysis results showed that after the American cockroach powder was fermented by Lactococcus lactis subsp. lactis 12-1 for 96 hours, the content of various purine and its derivative substances in the supernatant increased significantly, especially the caffeine content was 360.53 times that of the control, which has multiple biological activities and pharmacological effects such as stimulating the central nervous system, improving alertness and reducing fatigue. The second is 1-methylguanine, whose content is 93.24 times that of the control. Methylguanine is an important biomolecular modification, which plays an important role in the structure and function of DNA and RNA and the epigenetic regulation of organisms. The content of xanthine (2,6-Dihydroxypurine) was also 87.87 times that of the control. Xanthine is a product of the purine degradation pathway and is subsequently converted into uric acid by the action of xanthine oxidase. It is often used as a mild stimulant and bronchodilator, especially for the treatment of asthma symptoms (Table 10).

[0092] Table 10 Relative content of purine compounds in the supernatant of American cockroach fermentation (times)

[0093]

[0094] 5.3.2 Animal Experiment Results

[0095] 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 blank control CK group (P<0.01), while the levels of the four inflammatory factors in the serum of mice in the 12-1 strain treatment group were closer to those in the CK group and had no significant difference (P>0.05), but were significantly lower than those in the DSS model group (P<0.01), indicating that the 12-1 strain of Lactococcus lactis subsp. lactis has a significant alleviating effect on DSS-induced colon inflammation in mice (Table 11).

[0096] Table 11 Changes in the concentration of various inflammatory factors in the serum of mice in each group (Unit: pg / mL)

[0097]

[0098] Note: Compared with the CK group, *P<0.05, **P<0.01; compared with the DSS group, #P<0.05, ##P<0.01

[0099] The autopsy revealed that the spleen index of the DSS-treated group increased, the liver index decreased, and the colorectal length was significantly shortened. After intervention with Lactococcus lactis subsp. lactis 12-1, the organ index and colorectal length of the mice were close to those of normal mice (Table 12). The tissue sections showed that colorectal pathological damage could be observed under a microscope in the DSS-treated mice ( Figure 3 ), the colon tissue of mice in the DSS group showed desquamation of mucosal epithelial cells, degeneration and necrosis of the lamina propria, and obvious fibrosis and inflammatory cell infiltration in the necrotic area. Compared with the DSS group, the colon tissue of mice treated with 12-1 showed less necrosis of the lamina propria of the mucosa, lower inflammatory infiltration, and significantly reduced pathological damage.

[0100] Table 12 Changes in organ index and colorectal length in mice

[0101]

Claims

1. A strain of Lactococcus lactis subsp. lactis 12-1, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.27808.

2. A biological preparation comprising the Lactococcus lactis subsp. lactis 12-1 or a fermentation liquid thereof according to claim 1.

3. Use of Lactococcus lactis subsp. lactis 12-1 according to claim 1 in fermenting American cockroach powder.

4. An American cockroach powder fermentation liquid, characterized in that: The method is obtained by fermenting American cockroach powder with Lactococcus lactis subsp. lactis 12-1 as claimed 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 Lactococcus lactis subsp. lactis 12-1, 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. The American cockroach powder fermentation liquid according to claim 4, characterized in that: The weight ratio of the American cockroach powder to oligofructose is 10:

3.

7. Use of the American cockroach powder fermentation liquid according to any one of claims 4 to 6 in the preparation of a medicament for treating or assisting in treating colitis.

8. Use of the American cockroach powder fermentation liquid according to any one of claims 4 to 6 in preparing functional foods.

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