A strain of Weissella confusa and its application in producing neuromodulators

Abstract

The present invention discloses a strain of Weissella confusa and its application in producing neuromodulators. The strain is called Weissella confusa W1h, and the deposit number is CGMCC NO.25725. The strain was deposited at the General Microbiology Center of China National Committee for the Preservation and Administration of Microorganisms at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on September 15, 2022. Weissella confusa W1h in the present invention can secrete neuromodulators such as tryptophan, dopamine, short-chain fatty acids, etc., which can be used for the development and utilization of antidepressant probiotic products, and the strain has good tolerance to the gastrointestinal environment, has high intestinal adhesion and antibacterial activity, and can be used to improve the structure of intestinal microbial flora and inhibit the growth and reproduction of pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the field of microbial engineering, and particularly relates to a strain of Weissella confussifolia and application thereof in producing a neuromodulator. Background Art

[0002] Depression is a mental disorder characterized by low mood, loss of pleasure and interest, fatigue and lack of energy, often accompanied by physical and cognitive changes. In 2008, the World Health Organization listed major depressive disorder (MDD) as the third leading cause of global disease burden, and it is expected that MDD will rank first in the global disease burden by 2030.

[0003] At present, there are many studies on the pathological mechanisms of depression, mainly including the monoamine hypothesis, changes in the hypothalamic-pituitary-adrenal (HPA) axis, inflammatory factors, neuroplasticity and neurogenesis, changes in brain structure and function, genes, environmental factors, epigenetics, etc., but there is currently no single model or mechanism that can explain all aspects of the onset of depression.

[0004] Methods for treating depression include Western medicine, psychological therapy, sleep therapy, light therapy, Chinese herbal medicine, and electroconvulsive therapy. In recent years, drug-based treatment has gradually become clear. At present, clinical antidepressant drugs work by increasing the level of neurotransmitters in the central nervous system. Commonly used drugs for treating depression include monoamine oxidase inhibitors (MAOIs), tricyclics (TCAs), selective 5-hydroxytryptamine (5-HT) reuptake inhibitors (SSRIs), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) reuptake inhibitors (SNRIs), etc. However, these drugs have the disadvantages of slow onset, long medication time, incomplete symptom relief, obvious side effects, long treatment process, narrow scope of application, and high recurrence rate. Therefore, it is imperative to seek a non-drug, safe, healthy, and effective antidepressant method.

[0005] In recent years, studies at home and abroad have shown that intestinal flora plays an important role in the occurrence and development of depression. Dysfunction of gut-brain communication may be an important pathological mechanism of depression. The "microbiome-gut-brain" (MGB) axis is a pathway for two-way information exchange between the intestine and the brain involving intestinal flora.

[0006] The lack or reduced function of monoamine neurotransmitters is considered to be the biological basis of depression. The regulation of brain function by intestinal flora can be achieved through intestinal flora metabolites. There have been a large number of studies on antidepressant psychobiotics at home and abroad, showing that probiotics and their functional metabolites can improve depressive behavior, such as dopamine, tryptophan and short-chain fatty acids. However, so far, there are no reports on Weissella confusa having the function of relieving depression. Summary of the invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Weissella fusca.

[0008] Another object of the present invention is to provide the application of the confused Weissella.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A strain of Weissella confusa, named Weissella confusa W1h, with the accession number CGMCC NO.25725. The strain was deposited at the China General Microbiological Culture Collection Center (CMGCC) at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on September 15, 2022.

[0011] A method for culturing the confused Weissella comprises the following specific steps: inoculating the confused Weissella into a culture medium and culturing the culture medium at 28°C to 37°C.

[0012] The culture medium is MRS culture medium, including MRS liquid culture medium and MRS solid culture medium.

[0013] The pH value of the culture medium is 3-6.2, preferably 6.2.

[0014] The culture time is 24 to 48 hours, preferably 48 hours.

[0015] The application of the confused Weissella in the preparation of antidepressant products.

[0016] The products include microbial preparations, health foods or medicines.

[0017] The Weissella confusa can secrete neuromodulators to achieve the purpose of treating or relieving depression.

[0018] The neuromodulator includes at least one of tryptophan, dopamine and short-chain fatty acids.

[0019] The short-chain fatty acid includes at least one of acetic acid, propionic acid and butyric acid.

[0020] The application of the confounding Weissella in the production of neuromodulators.

[0021] The neuromodulator includes at least one of dopamine, tryptophan and short-chain fatty acids.

[0022] The short-chain fatty acid includes at least one of acetic acid, propionic acid and butyric acid.

[0023] The application of the confused Weissella in preparing products for improving the structure of intestinal microbial flora.

[0024] The Weissella confusa has good tolerance, high intestinal adhesion and antibacterial activity, can inhibit the growth and reproduction of pathogenic bacteria, and is beneficial to maintaining the balance of normal intestinal microbial flora.

[0025] The pathogenic bacteria include at least one of Escherichia coli, Staphylococcus aureus, Listeria and Salmonella.

[0026] The biological characteristics of the Weissella confusa are as follows: when Weissella confusa is cultured in an MRS liquid medium for 72 hours, the growth of the colony conforms to an S-shaped curve, the strain begins to enter a logarithmic growth phase after 3 hours and produces a large amount of acid, then rapidly grows to about 12 to 15 hours to enter a stable phase, the pH value also tends to be flat, and finally stabilizes at about 3.5. After being cultured on an MRS agar medium at 37°C for 48 hours, the strain grows well, the colony is milky white and round, the colony diameter is 0.5 to 1.0 mm, the colony has protrusions, and the edges are neat; the strain is Gram-positive, the cell morphology under a microscope is mostly short rod-shaped, and tends to form chains, has no flagella, and does not move.

[0027] Compared with the prior art, the present invention has the following advantages and effects:

[0028] (1) The Weissella confusa in the present invention is 195 probiotic strains screened out from fermented kimchi and adult feces, and 4 probiotic strains producing neuromodulators are preliminarily screened out by high performance liquid chromatography, and then further screened out by tests such as acid resistance, bile salt resistance, gastrointestinal environment resistance, and intestinal adhesion ability. The strain has probiotic properties such as producing neuromodulators, gastrointestinal environment resistance, and high adhesion, and has broad application prospects in the development and utilization of anti-depressant probiotic products. The screening method in the present invention can provide a new theoretical basis and method for the screening of probiotics in probiotic products.

[0029] (2) The Weissella confusa in the present invention has advantages over other strains in intestinal adhesion and antibacterial activity, and still has the ability to proliferate in an acidic environment, providing a solid foundation for the development of probiotic preparations, and can be used to develop anti-depressant functional products, such as microbial preparations, health foods or medicines, etc. Long-term use can improve the structure of intestinal microbial flora, increase the levels of neurotransmitters and short-chain fatty acids, and prevent and relieve depressive symptoms.

[0030] (3) The Weissella confusa in the present invention can secrete neuromodulators such as tryptophan, dopamine, and short-chain fatty acids, and promote the ability of other probiotics that produce neuromodulators to secrete neuromodulators. It has the effect of relieving depression, is non-pathogenic and hemolytic, can be used safely, and has good tolerance to the gastrointestinal environment. It has high intestinal adhesion and antibacterial activity, which is beneficial to maintaining the balance of normal intestinal microbial flora. Therefore, it can be used to improve the structure of intestinal microbial flora and inhibit the growth and reproduction of pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a comparison chart of the neuromodulator production of 195 bacterial strains.

[0032] Figure 2 This is a 16srDNA electrophoresis identification diagram of the virulence factors of 9 probiotic strains (in the figure: 0 is the positive control, 1 is N1g, and 2 is W1-5).

[0033] Figure 3 This is the growth curve of 4 strains of probiotics.

[0034] Figure 4 This is a comparison chart of the acid production capacity of 4 strains of probiotics.

[0035] Figure 5 This is a comparison chart of the acid resistance of 4 strains of probiotics.

[0036] Figure 6 This is a comparison chart of the bile salt tolerance of 4 probiotic strains.

[0037] Figure 7 This is a graph showing the surface hydrophobicity of four probiotic strains.

[0038] Figure 8 This is a graph showing the self-aggregation properties of four probiotic strains.

[0039] Fig. 9 This is a graph showing the cross-agglutination of four probiotic strains.

[0040] Fig.10 These are the cell adhesion morphologies of four probiotic strains.

[0041] Fig.11This is a comparison chart of the cell adhesion of four probiotic strains.

[0042] Fig.12 This is a comparison chart of the neuromodulator production of compound probiotics.

[0043] Fig.13 It is a colony morphology diagram of the probiotic bacteria (Weissella confusa) W1h of the present invention.

[0044] Fig.14 It is the 16s rDNA electrophoresis identification diagram of the probiotic bacteria (Weissella confusa) W1h of the present invention. DETAILED DESCRIPTION

[0045] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0046] Example 1: Preliminary screening of probiotics producing neuromodulators

[0047] 1.1 Required Materials

[0048] ① Probiotic solid culture medium (MRS): peptone 10g, beef extract 5g, yeast powder 4g, glucose 20g, Tween 801mL, anhydrous sodium acetate 5g, ammonium citrate 2g, K2HPO4 2g, MgSO47H2O 0.2g, MnSO4H2O 0.05g, calcium carbonate 20g, agar 20g, distilled water 1000mL, pH 6.2, 121℃, sterilize for 20min.

[0049] ② Probiotic liquid culture medium (MRS): peptone 10g, beef extract 5g, yeast powder 4g, glucose 20g, Tween 801mL, anhydrous sodium acetate 5g, ammonium citrate 2g, K2HPO4 2g, MgSO47H2O 0.2g, MnSO4H2O 0.05g, calcium carbonate 20g, distilled water 1000mL, pH 6.2, 121℃, sterilize for 20min.

[0050] ③PBS solution: 0.8% (w / v) NaCl, 0.02% (w / v) KH2PO4, 0.115% (w / v) Na2HPO4, adjusted to pH 7.2.

[0051] ④Kimchi juice: purchased from a market in Guangzhou City, Guangdong Province.

[0052] ⑤Feces: feces of healthy adults.

[0053] 1.2 Isolation and purification of strains

[0054] 1.2.1 Isolation of strains

[0055] ① Pipette 1mL of kimchi juice into a 1.5mL centrifuge tube, then pipette 100μL from the centrifuge tube and inject it into another 900μL PBS solution, and shake to mix. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Select appropriate gradients, pipette 100 μL of dilutions on MRS solid medium, repeat three times, and culture at 37°C for 24-48 hours.

[0056] ② Take 0.3g of fresh feces into a 1.5mL centrifuge tube, then take 100μL from the centrifuge tube and inject it into another 900μL PBS solution, shake and mix. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Select appropriate gradients, pipette 100 μL of dilutions on MRS solid medium, repeat three times, and culture at 37°C for 24-48 hours.

[0057] 1.2.2 Purification of strains

[0058] 10 to 15 single colonies of different sizes, shapes and colors were selected from each plate, and after two streaking purifications, the obtained single strain was transferred to MRS liquid culture medium and cultured at 37°C for 24 to 48 hours. A total of 195 strains were obtained and numbered according to their sources. The bacterial liquid was mixed with 50% (v / v) glycerol in a volume ratio of 1:1 and stored at -80°C.

[0059] 1.3 Preliminary screening of probiotics producing neuromodulators

[0060] 1.3.1 Preparation of standard curve

[0061] (1) 1 mM dopamine standard solution: Accurately weigh 0.5 mmol dopamine hydrochloride (DA) and dissolve it in MRS liquid culture medium and dilute to 50 mL. Use a pipette to transfer 0.1 mL of the solution and dilute to 10 mL to obtain DA standard solution. Then, place the solution in a sterile vial and store in a refrigerator at 4°C.

[0062] (2) 1 mM tryptophan standard solution: Accurately weigh 0.5 mmol L-tryptophan (L-Trp) and dissolve it in MRS liquid culture medium and dilute to 50 mL. Use a pipette to transfer 0.1 mL of the solution and dilute to 10 mL to obtain the Trp standard solution. Then, place the solution in a sterile vial and store in a refrigerator at 4°C.

[0063] (3) Take 0, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL of 1 mM DA standard solution and add them into 10 mL clean test tubes respectively. Then add 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, and 0 mL of MRS liquid culture medium in sequence. Select water (containing 0.1% (v / v) formic acid): methanol = 93:7 (volume ratio) as the mobile phase. The chromatographic column is Agilent ZORBAX SB-C 18 The chromatographic column (4.6×250mm, 5μm), column temperature 30℃, flow rate 1.0ml / min, injection volume 20μL, HPLC was used to determine the sample absorption peak under UV at 280nm, and a standard curve was drawn with dopamine concentration as the horizontal axis and peak area as the vertical axis.

[0064] (4) Take 0, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL of Trp standard solution with a concentration of 1 mM and add them to a 10 mL clean test tube, respectively. Then add 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, and 0 mL of MRS liquid culture medium in sequence. Select water (containing 0.1% (v / v) formic acid): methanol = 93:7 (volume ratio) as the mobile phase. The chromatographic column is Agilent ZORBAX SB-C 18 The chromatographic column (4.6×250mm, 5μm), column temperature 30℃, flow rate 1.0ml / min, injection volume 20μL, HPLC was used to determine the sample absorption peak under UV at 280nm, and a standard curve was drawn with dopamine concentration as the horizontal axis and peak area as the vertical axis.

[0065] 1.3.2 Preliminary screening of probiotics producing neuromodulators

[0066] (1) Inoculate the glycerol-preserved strains in step 1.2.2 into fresh MRS liquid culture medium at a 2% (v / v) inoculation rate, and culture at 37°C and 180 r / min. Inoculate 200 μL of the overnight cultured seed solution into 800 μL of MRS medium and culture at 37°C for 24 h.

[0067] (2) Take 0.2 mL of the bacterial solution cultured for 24 h, centrifuge at 8000 r / min for 1 min, take the supernatant and filter it. Select water (containing 0.1% (v / v) formic acid): methanol = 93:7 (volume ratio) as the mobile phase. Use high performance liquid chromatography (the determination method is the same as 1.3.1) to determine the sample absorption peak under UV at 280 nm. Calculate the ability of the sample to secrete neuromodulators (dopamine, tryptophan) according to the standard curve, and screen out the 9 probiotic strains with the best ability (A6, C6, H9, W1h, W2a, N3b, N2-9, W1-5, N1g). The results are shown in Figure 1 .

[0068] Example 2: Further screening of probiotics producing neuromodulators

[0069] 2.1 Strain safety

[0070] 2.1.1 Virulence factors

[0071] After the 9 preferred strains screened in Example 1 were activated and expanded, 11 common virulence factors (ace, cylA, cylB, cylM, cylL1, efaA, esp, gelE, AS, asal, hyl) were selected and PCR amplified using the genomic DNA of the test strains as templates. The primers were designed as shown in Table 1. 5 μL of PCR product was spotted on agarose gel (1.0%) spotting wells, the voltage was 160V, and the electrophoresis was performed for 20 minutes. After the electrophoresis was completed, it was observed under ultraviolet light and photographed. The results are shown in Table 1. Figure 2 Among them, strains N1g and W1-5 both contain virulence factors efaA and Gel-E, and are conditionally pathogenic bacteria, so the remaining strains without virulence factors were selected for the next test.

[0072] Table 1 Primer information used for virulence gene detection

[0073]

[0074] 2.1.2 Hemolytic

[0075] 2% rat erythrocytes (Nanjing Senbeijia Biotechnology Co., Ltd.) were added to an equal volume of bacterial solution, and distilled water was used as a positive control and physiological saline as a negative control. After incubation in a 37°C incubator for 2 hours, the cells were centrifuged at 10,000 rpm for 5 minutes, the supernatant was taken, the absorbance was measured at 545 nm, and the hemolysis rate was calculated. Where, hemolysis rate = (At -A nc ) / (A pc -A nc )*100%; Where: A t is the test absorbance, A nc is the absorbance of negative control, A pc is the absorbance of the positive control; judgment method: if the hemolysis rate is >5%, the hemolysis test is positive.

[0076] The results are shown in Table 2: The four strains with the lowest hemolytic activity (A6, C6, H9, and W1h) were selected for the next test.

[0077] Table 2 Hemolytic activity of probiotics

[0078] strains Hemolytic % N2-9 4.96±0.9 A6 0.42±0.34 C6 0.24±0.12 H9 0.74±0.42 Wl 1.05±1.82 N3B 4.51±0.45 Wlq 4.51±1.56

[0079] 2.2 Growth curve and acid production curve

[0080] (1) After activating and expanding the culture of the four preferred strains, they were inoculated into MRS liquid culture medium at an inoculum size of 2% (v / v), cultured statically at 37°C, and samples were taken every 2 hours to measure the absorbance at 600 nm and pH value of the culture medium. This was repeated three times, and then growth curves and acid production curves were drawn.

[0081] (2) The growth curve of probiotics is as follows Figure 3 As shown: The growth of the four probiotics all conformed to the S-shaped curve. Because the OD value of the strain growth was measured, the decay period was not observed. Each strain began to enter the logarithmic growth phase after 3 hours, and then grew rapidly to about 12 to 15 hours to enter the stable phase. Different strains have different biomass.

[0082] (3) The acid-producing ability of probiotics Figure 4 As shown: All strains produced a large amount of acid after 5 hours, and the acid value dropped rapidly to about 4.0, then the acid production curve flattened and finally stabilized at about 3.7.

[0083] 2.3 Acid resistance test

[0084] (1) Adjust the pH of MRS liquid culture medium to analyze the acid resistance of probiotics. The pH of MRS liquid culture medium was adjusted to 3.0, 4.0, and 5.0 with 0.1 M HCl solution, and the natural pH of MRS liquid culture medium (pH 6.2) was used as the control group. Each test was repeated 3 times.

[0085] (2) The bacterial suspensions of the four preferred strains were inoculated at 2% (v / v) in MRS culture medium with different pH values, and cultured at 37°C. Samples were taken after 0, 2, 4, 6, and 8 hours, and the OD values ​​in different culture media were measured using the MRS liquid culture medium without the inoculation of the strains as a reference. 600 The acid resistance of the strain was calculated by taking the average value of 3 parallel samples. 600 nm / OD of natural pH control group 600 nm value)×100%.

[0086] (3) The survival rate of probiotics under different pH conditions Figure 5 As shown in Table 3, Table 3 shows the survival rate of probiotics after 2 hours of treatment under different pH conditions. The acid-resistant survival rate of the four probiotic strains continued to decrease with the decrease of pH value. There were certain differences in the acid resistance of each strain. When the pH value was 3.0, all four strains could grow.

[0087] Table 3 Acid resistance of probiotics

[0088]

[0089]

[0090] 2.4 Bile salt tolerance test

[0091] (1) The tolerance of probiotics to bile salts was determined by analyzing the growth of probiotics in a medium containing bile salts. 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (w / v) of porcine bile salts were added to MRS-THIO liquid medium (0.2% (w / v) sodium thioglycolate was added to MRS liquid medium), and the pH was adjusted to 7.2. MRS liquid medium without bile salts was used as a control.

[0092] (2) The probiotic suspension was added to MRS liquid culture medium containing different bile salt concentrations at an inoculum volume of 2% (v / v). Each experiment was repeated 3 times. After culturing at 37°C for 24 h, the culture medium was shaken and the turbidity (absorbance OD) of different strains grown in different bile salt concentrations was measured. 600 ) to analyze its tolerance level. Wherein: Bile salt tolerance (%) = (OD value of medium containing bile salts / OD value of medium without bile salts) × 100%.

[0093] (3) The survival rate of probiotics under different bile salt concentrations Figure 6As shown in the figure, the bile salt tolerance of the four probiotic strains is not much different. When the bile salt concentration is in the range of 0-0.1%, the bile salt tolerance of the probiotics decreases rapidly with the increase of bile salt concentration. When the bile salt concentration rises to 0.1-0.2%, the bile salt tolerance of the probiotics is almost 0. Among them, when the bile salt concentration is in the range of 0-0.1%, the bile salt tolerance of C6 is relatively good.

[0094] 2.5 Intestinal Adhesion

[0095] The glycerol-preserved probiotics were inoculated in an MRS liquid medium at an inoculum size of 2% (v / v) and cultured at 37°C, 180r / min for 24h, the bacterial culture was centrifuged at 8000r / min, 4°C for 10min, the bacteria were collected, washed twice with PBS solution, centrifuged again at 8000r / min for 10min, the supernatant was discarded, and the bacterial precipitate was resuspended with PBS solution to obtain a bacterial suspension. The bacteria were used as a blank control with PBS solution, and the concentration of the bacterial solution was adjusted with PBS solution so that the OD value was 0.5±0.02 at a wavelength of 600nm on a spectrophotometer, which was used for the subsequent determination of the cell surface hydrophobicity, self-aggregation and cross-aggregation of the probiotics.

[0096] 2.5.1 Surface hydrophobicity

[0097] The affinity of the test strain for hydrocarbons reflects the surface hydrophobicity of the strain, that is, the microbial adhesion to hydrocarbons method (Bacteria Adhesion To Hydrocarbons, BATH) is used to determine the surface hydrophobicity of the probiotic cells. The specific steps are as follows: 2mL of the adjusted bacterial solution is added with 2mL of xylene and 2mL of chloroform, and no solvent is added to the control group. The two-phase system is thoroughly mixed by vortexing for 2min and incubated at room temperature for 1h to separate the water and organic phases. Carefully aspirate the water phase, use PBS solution as a blank control, and measure the OD 600 Each group has 3 parallel tubes and records. Among them, the surface hydrophobicity rate of the strain (%) = (OD 600 - Experimental group OD 600 ) / control group OD 600 ×100%.

[0098] The results are as follows Figure 7 As shown: the surface hydrophobicity of the four probiotic strains was different, and the adsorption capacity of chloroform was significantly higher than that of xylene. Among them, C6 had the best surface hydrophobicity for chloroform, which was 25.85±0.64%; A6 was second, which was 18.2±0.6%; H9 and W1h had the lowest.

[0099] 2.5.2 Self-agglutination

[0100] Take 2 mL of the adjusted bacterial solution and vortex for 10 seconds, then let it stand at 37°C for 1, 2, and 3 hours. Carefully aspirate 1 mL of the supernatant after standing, and use PBS solution as a blank control to measure the OD 600 , 3 tubes in each group, record. Among them, the self-agglutination rate (%) = 1-(ODt / OD0) × 100%; in the formula, ODt represents the absorbance at t = xh; OD0 represents the absorbance at t = 0h.

[0101] The results are as follows Figure 8 As shown: The self-aggregation of the four probiotic strains is quite different. 600 The values ​​showed a downward trend with the increase of static time, while the self-aggregation rate of the bacterial solution showed an upward trend, reaching the highest at 3h. C6 had the best self-aggregation property, which was 25.78±0.04%; A6 and W1h were followed by 20.44±0.17% and 23.75±0.2%, and H9 was the worst, which was 10.24±0.18%.

[0102] 2.5.3 Interaction Agglutination

[0103] 2 mL of the adjusted concentration of probiotics and 2 mL of pathogenic bacteria suspension (OD 600 =0.5) and placed at 37°C (do not shake) for 4 h. Take the supernatant and use PBS solution as blank control to measure OD 600 Each group has 3 parallel tubes and records are kept. Among them, the pathogens are Escherichia coli ATCC 25922, referred to as E.coli, Staphylococcus aureus ATCC 51650, referred to as SA, Listeria monocytogenes CMCC 54002, referred to as LM, and Salmonella enterica GIM 1.237, referred to as SE. All of the above pathogens were purchased from Guangdong Microbiological Culture Center (GDMCC).

[0104] Cross-agglutination rate (%) = [(OD pat +OD lab )-2×OD mix ]×100 / (OD pat +OD lab );

[0105] Where, OD pat OD value of pathogen at 0h; OD lab : OD value of probiotics at 0h; OD mix : OD value of the mixed solution at 4h.

[0106] The coagulation ability of 4 probiotic strains with four pathogenic bacteria, Escherichia coli, Staphylococcus aureus, Listeria and Salmonella, was tested. Fig. 9 As shown: the four probiotics have the strongest coaggregation ability with Escherichia coli, among which C6 has the strongest ability, which is 22.53±0.39%, followed by Listeria and Staphylococcus aureus. The coaggregation rates of the four probiotics with the four pathogens are all higher than the self-aggregation rates of the pathogens, which shows that the four probiotics have the ability to promote the aggregation of the four pathogens.

[0107] 2.6 Adhesion effect of probiotics on Caco-2 cells

[0108] 2.6.1 Caco-2 cell culture

[0109] Transfer 2 mL of Caco-2 cell suspension (cell bank of Type Culture Collection Committee of Chinese Academy of Sciences) to a 25T cell culture flask, add 2-4 mL of 20% FBS-DMEM medium (DMEM medium with 20% (v / v) fetal bovine serum), shake the cell culture flask using the cross method to evenly distribute the cells, place at 37°C, 5% (v / v) CO2 for adherent culture, and change the medium every other day.

[0110] 2.6.2 Gram staining to observe the adhesion effect of probiotics

[0111] When the cell density in the culture flask reaches 80% to 90%, the cells are digested and collected, and a 12-well cell culture plate is placed on the cell slide, at a rate of approximately 1×10 5 Add cells / well to the cell culture plate and place in a 37°C, 5% (v / v) CO2 incubator for overnight culture. After the cells adhere, discard the original culture medium, wash 2 to 3 times with DPBS buffer, add 1 mL of the adjusted concentration of bacterial suspension to each culture well, place in a 37°C, 5% (v / v) CO2 incubator for 2 hours, aspirate and discard the bacterial suspension, wash 2 to 3 times with DPBS buffer to remove non-adherent probiotics. After carefully removing the cell slide with tweezers, fix it with 0.4% paraformaldehyde solution for 30 minutes, place it at room temperature to dry naturally, then perform Gram staining, and observe the adhesion of probiotics to Caco-2 cells under a microscope, take pictures and record. The results are as follows Fig.10 shown.

[0112] 2.6.3 Plate count method to test the adhesion effect of probiotics

[0113] When the cell density in the culture flask reaches 80% to 90%, the cells are digested and collected, and a 12-well cell culture plate is placed on the cell slide, at a rate of approximately 1×10 5Cells / well were added to the cell culture plate and cultured overnight in a 37°C, 5% (v / v) CO2 incubator. 1 mL of the bacterial suspension after adjusting the concentration was added to the culture wells where Caco-2 cells were cultured, and DPBS buffer was used as the blank control. After incubation for 2 hours, the bacterial suspension was discarded and gently washed 2 to 3 times with DPBS buffer to remove non-adherent probiotics. The adherent cells were gently scraped with the tip of the pipette to make a cell suspension. The number of adherent colonies was calculated by the spread plate method, and the number of Caco-2 cells in the blank group was calculated by the hemocytometer counting method. The adhesion effect of probiotics on Caco-2 cells was calculated according to the formula; wherein, cell adhesion rate (CFU / 100 cells) = N2×100 / N0; wherein, N2 represents the number of adherent probiotics; N0 represents the number of Caco-2 cells in the control group. The results are as follows Fig.11 shown.

[0114] 2.7 Ability of probiotics to secrete neuromodulators

[0115] 2.7.1 Preparation of standard curve

[0116] ①1mM acetic acid standard solution: Accurately pipette 0.5mmol acetic acid into MRS liquid culture medium and dilute to 50mL. Use a pipette to transfer 0.1mL of the solution and dilute to 10mL. Then put it into a sterile vial and store it in a refrigerator at 4℃.

[0117] ②1mM propionic acid standard solution: Accurately pipette 0.5mmol propionic acid into MRS liquid culture medium and dilute to 50mL. Use a pipette to transfer 0.1mL of the solution and dilute to 10mL. Then put it into a sterile vial and store it in a 4℃ refrigerator.

[0118] ③1mM butyric acid standard solution: Accurately pipette 0.5mmol butyric acid into MRS liquid culture medium and dilute to 50mL. Use a pipette to transfer 0.1mL of the solution and dilute to 10mL. Then put it into a sterile vial and store it in a 4℃ refrigerator.

[0119] ④ Take 0, 20μL, 40μL, 60μL, 80μL, 100μL of 1mM short-chain fatty acid (SCFA) (acetic acid, propionic acid, butyric acid) standard solution and add them to 10mL clean test tubes respectively, and add 1mL, 0.9mL, 0.8mL, 0.7mL, 0.6mL, 0.5mL, 0.4mL, 0.3mL, 0.2mL, 0.1mL, 0mL of MRS liquid culture medium in sequence, and use 0.1% (v / v) phosphoric acid solution (A) and methanol (B) as mobile phases. The gradient elution program is as follows: 0-9min, 5% B; 9-25min, 50%-80% B; 25-30min, 5% B, and the chromatographic column is Agilent ZORBAX SB-C18 The chromatographic column was (4.6×250 mm, 5 μm), the column temperature was 40°C, the flow rate was 0.6 ml / min, the injection volume was 20 μL, and the chromatographic data were recorded at 210 nm by HPLC. The standard curve was drawn with SCFA concentration as the horizontal axis and peak area as the vertical axis.

[0120] 2.7.2 Ability of probiotics to secrete neuromodulators

[0121] The glycerol-preserved strains were inoculated in fresh MRS liquid medium at 2% (v / v) inoculation volume, and cultured at 37°C, 180r / min. 200μL of the overnight cultured seed solution was inoculated into 800μL of MRS liquid medium, and cultured at 37°C for 24h. 0.2mL of the cultured 24h solution was taken, centrifuged at 8000r / min for 1min, and the supernatant was passed through a membrane. The sample absorption peak was determined by high performance liquid chromatography (the determination method is the same as 2.7.1), and the ability of the sample to secrete neuromodulators was calculated according to the standard curve.

[0122] Results Fig.12 :Probiotics can secrete SCFA.

[0123] Example 3: Identification of probiotics with the best ability to secrete neuromodulators

[0124] 3.1 Colony characteristics

[0125] According to the results of Example 2, the probiotic W1h was further identified: after the W1h strain was inoculated on MRS solid medium and cultured at 37°C for 48 hours, the strain grew well, the colonies were milky white and circular, the colony diameter was 0.5-1.0 mm, the colonies had protrusions, and the edges were neat.

[0126] 3.2 Morphology under microscope

[0127] The W1h strain was Gram-positive, and under the microscope, the cells were mostly rod-shaped, did not produce spores, and tended to form chains, had no flagella, and did not move ( Fig.13 ).

[0128] 3.3 16S rDNA electrophoresis identification

[0129] The 16s rDNA of the W1h strain was sequenced and BLAST was performed in NBCI. The electrophoresis of the 16s rDNA of the W1h strain is shown in Fig.14 As shown, the gene sequence is shown in SEQ ID NO.1.

[0130] Combining the sequence comparison results and morphological observation results of the W1h strain, it was determined that the strain belongs to Weissella confusa. The strain was named Weissella confusa W1h and was deposited on September 15, 2022 at the General Microbiology Center of the Chinese Academy of Sciences, Institute of Microbiology, No. 3, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.25725.

[0131] The Weissella confusa W1h provided by the present invention has been proven to have the ability to produce neuromodulators through experiments. The strain meets the basic conditions of being a probiotic, has good tolerance to the gastrointestinal environment, can maintain a certain survival rate, and has strong intestinal adhesion, has a certain effect on maintaining the balance of normal intestinal microbial flora, and can better adhere to intestinal epithelial cells and play a role, can be used to develop microbial preparations, health foods or medicines, and long-term use can improve the intestinal microbial flora structure and prevent and relieve depression symptoms.

[0132] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Weissella fusella, characterized in that: Weissella confuses Weissella confusa )W1h, the deposit number is CGMCC NO. 25725. The strain was deposited at the General Microbiology Center of China Culture Collection Administration at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on September 15, 2022.

2. A method for culturing the Weissella fusella according to claim 1, characterized in that: The specific steps are as follows: inoculate the confused Weissella into the culture medium and culture it at 28°C to 37°C.

3. The method according to claim 2, characterized in that: The culture medium is MRS culture medium; The pH value of the culture medium is 3 to 6.2; The culture time is 24 to 48 hours.

4. The use of the Weissella confusa according to claim 1 in the production of neuromodulators, characterized in that: The neuromodulators are dopamine, tryptophan and short-chain fatty acids; The short-chain fatty acids are acetic acid, propionic acid and butyric acid.

5. The use of the Weissella confusa according to claim 1 in the preparation of a microbial preparation for improving the structure of intestinal microbial flora, characterized in that: The Weissella confusa is used to inhibit the growth and reproduction of pathogenic bacteria and maintain the balance of normal intestinal microbial flora; The pathogenic bacteria is at least one of Escherichia coli, Staphylococcus aureus, Listeria and Salmonella.

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