Lactobacillus reuteri hc1602, a bacterial agent and application thereof

Abstract

The present application relates to a lactobacillus reuteri HC1602, a bacterial agent and application thereof, and belongs to the technical field of microorganisms. Limosilactobacillus reuteri The lactobacillus reuteri (HC1602) is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 26890, a preservation date of March 23, 2023, and an address of the preservation agency of No. 3, Beichen West Road, Chaoyang District, Beijing. The present application also provides an application of the lactobacillus reuteri HC1602 in preparing a product for inhibiting and preventing respiratory syncytial virus and an application in preparing a product for reducing cholesterol in blood. The lactobacillus reuteri HC1602 provided by the present application can effectively inhibit the invasion of RSV on A549 lung cells and improve the survival rate of A549 cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a strain of Lactobacillus rhamnosus HC1602, a bacterial agent thereof and application thereof. BACKGROUND

[0002] In recent years, probiotics have been widely studied as a means of preventing and treating respiratory viral infections. The mechanisms of action include stimulating various innate and acquired immune responses in the body, strengthening the barrier function of the respiratory epithelium, producing various anti-pathogen substances, and directly competing with pathogens for ecological niches. For example, deaminated tyrosine (DAT) produced by intestinal flora metabolism can activate the host type I IFN pathway and can resist respiratory infections caused by viruses. Therefore, strains that produce DAT through fermentation metabolism have antiviral potential and can be used to prevent respiratory infections. Research and patent documents have confirmed that some Lactobacillus strains have the effect of inhibiting respiratory syncytial virus. For example, Chinese invention patent CN110721204B discloses a probiotic composition comprising VSL#3, Lactobacillus rhamnosus and Lactobacillus plantarum, which can be effectively used to prevent and treat pneumonia caused by respiratory syncytial virus infection, providing a new approach to the prevention and treatment of respiratory syncytial virus infection. Chinese invention patent CN116829165A discloses a pharmaceutical composition for preventing or treating viral infectious diseases or respiratory diseases, which uses vesicles derived from Lactobacillus paracasei as active ingredients, also showing potential for the prevention and treatment of respiratory-related viral infections.

[0003] As a member of the Lactobacillus genus, Lactobacillus rhamnosus has unique biological properties, especially excellent activity in intestinal flora regulation and mucosal barrier protection. However, in existing published technical documents and patents, there is no report on the application of Lactobacillus rhamnosus as an effective component to inhibit respiratory syncytial virus, and its application value in the field of respiratory syncytial virus infection prevention and treatment needs to be further explored. SUMMARY

[0004] In view of the problem that Lactobacillus rhamnosus is not applied to inhibit or prevent respiratory syncytial virus in the prior art, the present application provides a strain of Lactobacillus rhamnosus HC1602, a bacterial agent thereof and application thereof, to solve the above problems.

[0005] In a first aspect, the present application provides a strain of Lactobacillus rhamnosus HC1602, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) HC1602 is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 26890, the deposit date is March 23, 2023, and the deposit address is No. 3, Beichen West Road, Beijing City, Chaoyang District. Limosilactobacillus reuteri ​

[0006] Further, the 16S rDNA sequence of the Lactobacillus reuteri HC1602 is shown as SEQ ID NO. 3.

[0007] In a second aspect, the present application provides a bacterial agent containing a strain lysate of the Lactobacillus reuteri HC1602 or a fermentation supernatant of the Lactobacillus reuteri HC1602; the strain lysate is obtained by using an ultrasonic disrupter to ultrasonically break the strain fermentation broth; and the fermentation supernatant is obtained by centrifuging the strain fermentation broth.

[0008] Further, the preparation method of the fermentation broth is as follows: the Lactobacillus reuteri HC1602 is cultured in a MRS liquid medium at 37℃ for 24-28h.

[0009] Further, the MRS liquid medium comprises the following components: 10g / L of proteose peptone, 10g / L of beef extract, 5.0g / L of yeast extract, 5g / L of sodium acetate, 5g / L of glucose, 2g / L of KH2PO4, 1.0mL / L of Tween 80, 2.0g / L of citric acid diamine, 20g / L of CaCO3, 0.58g / L of MgSO4·7H2O, 0.25g / L of MnSO4·7H2O, and pure water is added to 1L, and the pH is adjusted to 6.2-6.5.

[0010] In a third aspect, the present application provides an application of the Lactobacillus reuteri HC1602 in preparing a product for reducing blood cholesterol.

[0011] In a fourth aspect, the present application provides an application of the Lactobacillus reuteri HC1602 in preparing a product for inhibiting and preventing respiratory syncytial virus.

[0012] The present application has the following beneficial effects:

[0013] The Lactobacillus reuteri HC1602 provided by the present application can effectively inhibit the invasion of RSV on A549 lung cells and improve the survival rate of A549 cells. The Lactobacillus reuteri HC1602 has a direct inactivation effect on RSV and inhibits the proliferation of RSV in A549 cells. Moreover, the inhibitory effect of the Lactobacillus reuteri HC1602 on RSV is best when used in advance, which is superior to the inactivation effect on RSV during and after the infection. Therefore, the use of the Lactobacillus reuteri HC1602 can produce a direct killing effect on RSV and inhibit the replication and proliferation of RSV in lung cells, which is helpful for preventing or treating respiratory tract infection caused by RSV. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0015] Figure 1 is the colony morphology diagram of Lactobacillus reuteri HC1602 in Embodiment 1 of the present application.

[0016] Figure 2 is the microscope imaging diagram of Lactobacillus reuteri HC1602 in Embodiment 1 of the present application.

[0017] Figure 3 is the RAPD fingerprint map of Lactobacillus reuteri HC1602 in Embodiment 1 of the present application.

[0018] Figure 4 is the rep-PCR fingerprint map of Lactobacillus reuteri HC1602 in Embodiment 1 of the present application.

[0019] Figure 5 is the curve diagram of RSV titer change with time after infection in Embodiment 8 of the present application.

[0020] Figure 6 is the diagram of RSV-F protein mRNA relative expression level change in RSV inactivated group and non-inactivated group in Embodiment 9 of the present application.

[0021] Figure 7 is the diagram of the influence of different treatment methods of supernatant on RSV replication ability in Embodiment 10 of the present application. DETAILED DESCRIPTION

[0022] In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the present application.

[0023] Embodiment 1

[0024] Isolation, screening and identification of Lactobacillus reuteri HC1602

[0025] 1. Sampling: Fermented cheese was collected from a pastoralist family in Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region on September 18, 2022.

[0026] 2. Isolation: MRS (Man Rogosa Sharpe) agar medium was prepared according to the following formula: pure water 1 L, protein peptone 10 g, beef extract 10 g, yeast extract 5.0 g, sodium acetate 5 g, glucose 5 g, KH2PO4 2 g, Tween 80 1.0 mL, citric acid diamine 2.0 g, CaCO320 g, MgSO4·7H2O 0.58 g, MnSO4·7H2O 0.25 g, 15 g agar, pH 6.2-6.5.

[0027] Take 10 g of the fermented cheese collected in step (1) and place it in a sampling bag containing 300 mL of sterile normal saline. Use a patting instrument to repeatedly pat the cheese to fully mix it in the sterile normal saline. Take 100 μL of the mixed liquid and spread it on a MRS agar medium plate. After 10 min, place the plate upside down in an anaerobic bag and place the anaerobic bag in a 37°C constant temperature incubator for anaerobic culture for 48 h. After single colonies grow on the plate, pick off the single clones for further streak culture. After 3 times of purification culture, pick off the single clones for microscopic examination. According to the microscopic examination results, 35 strains of bacilli were screened out. The 35 potential lactic acid bacteria obtained above were inoculated into 10 mL of MRS liquid medium, and after 24 h of static culture at 37°C, they were ready for use.

[0028] 3. Screening

[0029] (1) Medium preparation: M9 medium was prepared, including the following components: pure water 1 L, Na2HPO4 4.78 g, KH2PO4 2.99 g, NaCl 0.5 g, MgSO4 0.24 g, CaCl2 0.01 g, NH4Cl 0.50 mL. High-pressure steam sterilization at 115°C for 20 min. MgSO4 and CaCl2 were sterilized separately. Phloretin was added to the M9 medium at a final concentration of 0.5 mM, and sterilized by filtering with a 0.22 μm organic phase filter.

[0030] (2) Collection and detection of the target product of potential lactic acid bacteria

[0031] The potential lactic acid bacteria culture liquid cultured for 24 h was centrifuged at 4°C and 3400 r / min for 20 min to remove the MRS medium. The bacterial slurry was washed once with sterile PBS buffer, and then centrifuged to obtain the bacterial slurry, which was then added with 10 mL of M9 medium (containing 0.5 mM phloretin) and cultured in a 37°C constant temperature incubator for 24 h.

[0032] After the fermentation culture is finished, centrifuge the bacterial liquid at 7000 r / min for 10 min at 4℃, take the supernatant into a 1.5 mL centrifuge tube, vortex for 10 s, then take 200 μL of the supernatant and add 800 μL of pre-cooled ethanol, mix well, and place on ice for 30 min to precipitate the protein. Centrifuge the mixture at 15000 r / min for 10 min at 4℃, take the supernatant and vacuum concentrate for 2-4 h. After concentration, re-dissolve with 100 μL of methanol / water (4:1), centrifuge at 15000 r / min for 10 min at 4℃, and use ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect the content of desaminotyrosine (DAT) in the supernatant.

[0033] (3) Screening results

[0034] After detection, 12 strains of the 35 potential lactobacillus strains can ferment and metabolize phloretin to produce DAT, with a yield of 12.28-82.43 μg / mL, and the strain numbered HC1602 has the highest yield. Therefore, the strain HC1602 is selected for further study.

[0035] 4. Identification

[0036] (1) Colony morphology identification

[0037] Inoculate the strain HC1602 into MRS agar medium, and after 24 h of culture at 37℃, it can be seen that the single colony of the strain HC1602 is milky white, opaque in texture, smooth and moist on the surface, with a neat edge, and the colony diameter is about 2 mm. The colony morphology of the strain HC1602 is shown in Figure 1 . Under an optical microscope, the strain HC1602 is short rod-shaped, smooth at both ends, arranged in clusters or chains, and the optical microscope image is shown in Figure 2 .

[0038] (2) Physiological and biochemical property identification

[0039] The preparation of the strain HC1602 inoculum is as follows: inoculate the activated strain HC1602 into MRS liquid medium at a 2% inoculation amount, and culture at 37℃ for 24 h until the viable bacterial count reaches 10 9 CFU / mL, to obtain the strain HC1602 inoculum.

[0040] (2.1) Salt tolerance test

[0041] Under sterile conditions, the activated bacterial liquid of strain HC1602 was inoculated into 5 mL MRS liquid medium with NaCl concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% at an inoculation amount of 10%, 5 mL MRS liquid medium without inoculation of the strain was taken as a control, and was placed in a constant temperature shaking incubator at 37°C for 48 h, and whether the medium became turbid was observed.

[0042] The results showed that strain HC1602 grew in the medium with a salt concentration of 1% to 4%, and did not grow in the medium with a salt concentration of 5% or more, and the highest salt tolerance of strain HC1602 was 4%.

[0043] (2.2) Temperature growth range experiment

[0044] The bacterial inoculum of strain HC1602 was inoculated into 10 mL MRS liquid medium at an inoculation amount of 10%, 10 mL MRS liquid medium without inoculation of the strain was taken as a control, and was placed in a constant temperature shaking incubator at 0°C, 15°C, 37°C, 45°C and 60°C for 48 h, and whether the culture liquid became turbid was observed.

[0045] The results showed that after strain HC1602 was cultured in the constant temperature shaking incubator at 0°C, 15°C, 45°C and 60°C for 48 h, the medium remained clear. However, after strain HC1602 was cultured at 37°C for 48 h, the medium became turbid.

[0046] (2.3) Glucose acid and gas production test

[0047] The medium formula used in the glucose acid and gas production test includes the following components: peptone 0.5 g, yeast extract 0.3 g, Tween 80 0.1 mL, salt solution A 0.5 mL, salt solution B 0.5 mL, sodium acetate 0.5 g, glucose 2.5 g, 2% bromocresol green (w / v) 0.05 mL, distilled water 100 mL; pH 6.8-7.0. The prepared medium was divided into large test tubes containing inverted small test tubes, 3 mL / tube, 121°C, high pressure sterilization for 15 min.

[0048] Salt solution A includes the following components: KH2PO4 10 g, K2HPO4 1.0 g, dissolved in distilled water, and made up to 100 mL.

[0049] Salt solution B includes the following components: MgSO4·7H2O 11.5 g, MnSO4·2H2O 2.4 g, FeSO4·7H2O 0.68 g, dissolved in distilled water, and made up to 100 mL.

[0050] In sterile conditions, the strain HC1602 inoculum was inoculated into the above-mentioned medium at an inoculation amount of 2%, and the medium without inoculation of the strain was used as a control, then the top was sealed with 2 mL of sterile liquid paraffin, and the culture was placed at 37°C for 48 h, and the color change of the medium was observed.

[0051] The results showed that the culture medium changed from green to yellow after 48 h of culture, and gas was produced in the small inverted tube, indicating that the strain HC1602 fermented glucose to produce acid and gas.

[0052] (2.4) Antibiotic resistance and hemolytic experiment

[0053] (2.4.1) Antibiotic resistance experiment

[0054] The minimum inhibitory concentration MIC value of antibiotics on the strain HC1602 was determined by micro-broth dilution method. The antibiotics were prepared as follows: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline and vancomycin were prepared into 2048 μg / mL stock solution, and stored at -20°C for standby use. When used, the stock solution was diluted into working solution with MRS liquid medium by 2-fold serial gradient, and the gradient dilution concentration was 1~1024 μg / mL.

[0055] 190 μL of MRS liquid medium without antibiotics was added to 8 empty holes in the first column of the 96-well plate as negative control, 190 μL of MRS liquid medium with gradient dilution concentration of 1~1024 μg / mL of antibiotics was added to 11 holes in the 2~12 columns, and then 10 μL of strain HC1602 inoculum was inoculated. Each group of experiments was repeated 4 times, and the holes without inoculation of the strain HC1602 were used as blank control. 50 μL of sterilized paraffin oil was added to each hole to prevent water evaporation during culture. The 96-well plate was incubated at 37°C, and the OD 600 value was measured every 5 min, and the MIC of different strains was calculated. The specific results are shown in Table 1.

[0056] Table 1 - MIC value of antibiotic resistance experiment of strain HC1602

[0057]

[0058] Note: MIC unit μg / mL; R: resistant; S: sensitive.

[0059] As can be seen from the results in Table 1, the strain HC1602 is sensitive to common antibiotics such as erythromycin, streptomycin, ampicillin, tetracycline and clindamycin, and has good biosafety.

[0060] (2.4.2) Hemolytic experiment

[0061] Prepare TBS basal medium comprising the following components: 1 L distilled water, 17.0 g tryptone, 5.0 g NaCl, 3.0 g soybean peptone, 2.5 g KH₂PO₄, and 2.5 g glucose. After dissolving, autoclave at 121°C for 15 min. Once the TBS medium has cooled to 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour onto plates. Streak strain HC1602 onto the prepared blood cell plates and incubate at 37°C for 24–48 h. Observe whether hemolysis occurs in strain HC1602.

[0062] The results showed that strain HC1602 could not grow and there were no changes on the blood cell plate, indicating that strain HC1602 does not produce hemolysin and cannot lyse blood cells, thus exhibiting good biosafety.

[0063] 3. Carbon source metabolism experiment

[0064] Prepare the phenol red basal medium, comprising the following components: 1.5 g peptone, 0.6 g yeast extract, 0.1 g Tween 80, 0.5 mL salt solution, 18 mg phenol red, 100 mL distilled water, pH = 7.4. The salt solution composition is as follows: 11.5 g MgSO4·7H2O, 8 g MnSO4·4H2O, 100 mL distilled water.

[0065] Prepare solutions of sugars, alcohols, and glycosides at a concentration of 10 g / mL and filter using a 0.22 μm sterile filter. Under aseptic conditions, add 20 μL of the sterilized carbohydrate solution to each well of a 96-well plate. Perform three parallel experiments for each carbohydrate. Then add 170 μL of sterilized basal medium containing phenol red, followed by 10 μL of *Lactobacillus reuteri* HC1602 inoculation solution. Wells without inoculation serve as controls. Add 50 μL of liquid paraffin to each well to prevent water evaporation during culture. Incubate at 37°C and observe the color change of the medium using phenol red as an indicator. The results of the carbon source metabolism experiment are shown in Table 2.

[0066] Table 2 - Results of carbon source metabolism experiments for strain HC1602

[0067]

[0068] 4. MALDI-TOF-MS identification test

[0069] According to the kit instructions, the steps are as follows: pick up the activated strain HC1602 monoclonal to evenly coat the target plate in the form of film, add 1 μL lysis solution to cover the sample, dry, then add 1 μL matrix solution to cover the sample, dry, and then put the sample target into the mass spectrometer for identification. The sample and the matrix formed by the co-crystallization film are irradiated with laser to ionize the protein in the sample. The ions are accelerated to fly through the flight tube under the action of 10-20 KV electric field, and the molecular weight of the protein is detected according to the different flight times to the detector. The protein fingerprint is obtained by using Autofms 1000 analysis software Autof Analyzer v1.0, and the identification result shows that the strain HC1602 is Lactobacillus rhamnosus.

[0070] 5. Molecular biology identification

[0071] The single colony of strain HC1602 on the plate was picked up in MRS liquid medium, and 800 μL of fermentation liquor was taken after 24 hours of culture at 37°C. The genomic DNA of the strain was extracted by using TIANGEN ® The genomic DNA of the strain was extracted by using TIANGEN

[0072] (1) 16s rDNA gene sequence identification

[0073] The 16s rDNA gene of strain HC1602 was amplified by using TIANGEN ® 2×Taq PCR premix kit, and the reaction system and reaction cycle were set according to the kit instructions.

[0074] The upstream primer is 27F: AGAGTTTGATCCTGGCTCA;

[0075] The downstream primer is 1492R: GGTTACCTTGTTACGACTT.

[0076] The size of the PCR amplification product verified by electrophoresis is about 1500 bp, which meets the requirements. The 16s rDNA gene sequencing result shows that the 16s rDNA sequence of strain HC1602 is as shown in SEQ ID NO. 3:

[0077]

[0078] The sequence was compared with BALST on the EzBioCloud website and it matched Lactobacillus reuteri (… Limosilactobacillus reuteri The strain showed the highest similarity to *Lactobacillus reuteri*. Therefore, strain HC1602 was identified as *Lactobacillus reuteri*. Limosilactobacillus reuteri Lactobacillus reuteri HC1602 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26890, deposited on March 23, 2023. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0079] (2) RAPD fingerprint identification

[0080] Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify strain HC1602. The reaction system and reaction cycle settings were performed according to the kit instructions. The M13 primer sequence was: 5'-GAGGGTGGCGGTTCT-3'.

[0081] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a result control. Electrophoresis was performed at a constant voltage of 100V for 80 minutes. Finally, the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of strain HC1602 is shown below. Figure 3 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 3 The matching RAPD fingerprint profiles indicate that *Lactobacillus reuteri* HC1602 is a novel *Lactobacillus reuteri* strain.

[0082] (3) Rep-PCR fingerprint identification

[0083] Using TIANGEN ® The 2×Taq PCR premix kit was used to amplify strain HC1602. The reaction system and reaction cycle settings were performed according to the kit instructions. The rep-PCR primers were: 5'-GTGGTGGTGGTGGTG-3'.

[0084] Prepare 1.5% agarose gel plates, using a DL2000 DNA Marker as a result control. Electrophoresis was performed at 100V for 80 minutes to detect the amplification results. The rep-PCR fingerprint of *Lactobacillus reuteri* HC1602 is shown below. Figure 4 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 4 The matching rep-PCR fingerprint pattern indicates that the selected *Lactobacillus reuteri* HC1602 is a new *Lactobacillus reuteri* strain.

[0085] Comprehensive MALDI-TOF-MS identification test, molecular biology experiment, carbon source metabolism experiment and glucose acid and gas production experiment identification results, it can be known that Lactobacillus mucosus HC1602 is different from the Lactobacillus mucosus currently reported in the public report, and is a new strain of Lactobacillus mucosus.

[0086] Example 2

[0087] Preparation of fermentation supernatant of Lactobacillus mucosus HC1602:

[0088] After taking the activated 3rd generation Lactobacillus mucosus HC1602, it was cultured in MRS liquid medium at 37℃ for 24h, then centrifuged at 6000r / min for 10min, and the supernatant was collected as the fermentation supernatant.

[0089] Preparation of strain lysate containing Lactobacillus mucosus HC1602:

[0090] After taking the activated 3rd generation Lactobacillus mucosus HC1602, it was cultured in MRS liquid medium at 37℃ for 24h, then the fermentation broth was collected, and the fermentation broth was ultrasonically broken for 20min by using an ultrasonic disrupter to obtain the strain lysate containing Lactobacillus mucosus HC1602.

[0091] Example 3

[0092] Antioxidant function determination of Lactobacillus mucosus HC1602

[0093] 1. Determination of the ability of Lactobacillus mucosus HC1602 to scavenge DPPH free radicals and hydroxyl free radicals HRS

[0094] (1) Determination of the ability of Lactobacillus mucosus HC1602 to scavenge DPPH free radicals

[0095] 1mL of fermentation supernatant and lysate of Lactobacillus mucosus HC1602 were taken respectively, 1mL of 0.4mM freshly prepared DPPH free radical solution was added, and then it was mixed uniformly and then placed in a room temperature light-shielded reaction for 30min, then the absorbance A of the fermentation supernatant and lysate sample at a wavelength of 517nm was measured 样本 , and 3 parallel measurements were made. The control sample was mixed with equal volume of PBS solution and DPPH / ethanol mixture, and equal volume of supernatant or lysate and ethanol mixture was mixed to zero. Scavenging rate = [1- (A 样品 -A 空白 ) / A 对照 ] × 100%. Lactobacillus lactis Lr-10 was used as a control strain. The results are shown in Table 3.

[0096] Table 3 - DPPH radical scavenging rate of L. reuteri HC1602

[0097]

[0098] As can be seen from the results of Table 3, the DPPH scavenging rates of the fermentation supernatant and lysate of L. reuteri HC1602 are significantly better than those of the control strain L. reuteri Lr-10.

[0099] (2) Measurement of the ability of L. reuteri HC1602 to scavenge hydroxyl radicals HRS

[0100] After mixing 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water and 200 μL of the fermentation supernatant or lysate of L. reuteri HC1602, 100 μL of hydrogen peroxide solution (3 mM) was added, and the absorbance of the sample was measured at 510 nm after 15 min of 37 °C water bath. The HRS scavenging rate was calculated according to the following formula: Scavenging rate = (A 样品 -A 控制 ) / (A 空白 -A 控制 ) x 100%. Wherein: A 控制 is deionized water instead of the sample, A 空白 is deionized water instead of the sample and H2O2. L. reuteri Lr-10 was used as a control strain. The results are shown in Table 4.

[0101] Table 4 - Hydroxyl radical scavenging rate of L. reuteri HC1602

[0102]

[0103] As can be seen from the results of Table 4, the HRS scavenging rates of the fermentation supernatant and lysate of L. reuteri HC1602 are significantly better than those of the control strain L. reuteri Lr-10.

[0104] 2. Measurement of the ability of L. reuteri HC1602 to resist lipid peroxidation

[0105] Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water. In 0.5 mL of PBS solution, add 1 mL of linoleic acid emulsion, 1 mL of FeSO4(1%), and then add 0.5 mL of fermentation supernatant and lysate of L. reuteri HC1602, respectively, and incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixed solution, and incubate at 100°C for 30 min. Rapidly cool, centrifuge at 4000 r / min for 15 min, collect the supernatant, and measure the OD 532nm The absorbance of the lower layer is A; the control group uses 0.5 mL of distilled water instead of the sample, which is A0. The inhibition rate = (A0-A) / A0x100%, and L. reuteri Lr-10 is used as the control strain. The results are shown in Table 5.

[0106] Table 5 - Determination results of the anti-lipid peroxidation rate of L. reuteri HC1602

[0107]

[0108] As can be seen from the determination results in Table 5, the fermentation supernatant and lysate of L. reuteri HC1602 can both resist lipid peroxidation, and the effect is better than that of the control strain L. reuteri Lr-10.

[0109] In summary, L. reuteri HC1602 has excellent antioxidant levels and helps to improve the body's antioxidant capacity based on its performance in removing DPPH free radicals, removing hydroxyl radicals HRS, and resisting lipid peroxidation.

[0110] Example 4

[0111] Simulation of the intestinal tolerance of L. reuteri HC1602

[0112] Preparation of artificial simulated gastric juice: add 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, and 2 g of NaCl to 1 L of distilled water, mix well, adjust the pH of the system to 3.0, and sterilize at 115°C for 20 min. After sterilization, add 3.2 g of porcine mucosal pepsin powder to the solution when it cools to an appropriate temperature, shake the container to mix the powder thoroughly, and obtain the artificial simulated gastric juice.

[0113] Preparation of artificial simulated intestinal fluid: 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, 6.8 g of potassium dihydrogen phosphate and 3.0 g of choline salt were sequentially added to 1 L of distilled water, and after stirring and dissolving, 77 mL of NaOH solution (0.2 M) was added to adjust the pH of the system to 6.8 ± 0.1, and then sterilized at 115°C for 20 minutes. Before the experiment, 1.0 g of pancreatin powder was added to the cooled and sterilized solution, and the mixture was shaken to obtain the artificial simulated intestinal fluid.

[0114] Experimental process: The absorbance OD 600 nm was adjusted to 1.5. 1 mL of the bacterial solution was centrifuged at 6000 x g at 4°C for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in 1 mL of simulated gastric fluid and cultured in an anaerobic environment at 37°C for 3 h. The sample was plated for colony counting at the beginning (0 h) and end (3 h) of the culture. Then the bacterial solution cultured in the simulated gastric fluid for 3 h was centrifuged again at 6000 x g at 4°C for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in an equal volume of simulated intestinal fluid and cultured at 37°C for 2 h, followed by plate colony counting. The commercially available Lactobacillus reuteri DSM 23878 was used as a control strain, and the survival rate was calculated according to the following formula:

[0115] .

[0116] wherein N0 and N1 are the number of viable bacteria (CFU / mL) before and after the strain is treated with simulated gastrointestinal fluid. The results are shown in Table 6.

[0117] Table 6 - Results of the tolerance of Lactobacillus reuteri HC1602 to simulated artificial gastric fluid and artificial intestinal fluid

[0118]

[0119] As can be seen from the determination results in Table 6, Lactobacillus reuteri HC1602 has high tolerance to simulated artificial gastric fluid and simulated artificial intestinal fluid, with a survival rate of 97.06% after digestion in simulated gastric fluid and a survival rate of 94.56% after digestion in simulated gastric fluid, which is significantly better than the control strain Lactobacillus reuteri DSM 23878. This indicates that Lactobacillus reuteri HC1602 can tolerate the harsh gastric and intestinal acid-base environment in the human body and thus exert its probiotic function.

[0120] Example 5

[0121] Hydrophobic cell surface test of Lactobacillus reuteri HC1602

[0122] Activated Lactobacillus reuteri HC1602 colonies were picked and inoculated into freshly prepared MRS liquid medium. The culture was incubated at 37°C with shaking for 24 h. Then, 1% of the colonies were inoculated into MRS liquid medium and cultured at 37°C with shaking for another 24 h. After centrifugation at 6000×g for 10 min, the bacterial cells were collected and washed twice with sterile physiological saline. The bacterial cells were then resuspended in 1 mL of sterile KNO3 (0.1M) solution as the test solution.

[0123] Add 50 μL of the above bacterial suspension to 2450 μL of KNO3 (0.1 M) and record the OD. 600 For A0, 1.5 mL of bacterial suspension was mixed with 500 μL of xylene and allowed to stand at room temperature for 10 min. The two-phase system was vortexed for 2 min and then allowed to stand for 20 min to reform the aqueous and organic phases. The absorbance A1 of the aqueous phase was measured at 600 nm. Cell hydrophobicity was calculated using the formula: Hydrophobicity = (A0 - A1) / A1 × 100%, and the average value was taken from three measurements. Commercially available Lactobacillus reuteri DSM 23878 was used as a control strain. The results are shown in Table 7.

[0124] Table 7 - Results of hydrophobicity test of cell surface of Lactobacillus reuteri HC1602

[0125]

[0126] As can be seen from the test results in Table 7, the surface hydrophobicity of Lactobacillus reuteri HC1602 is better than that of the control strain Lactobacillus reuteri DSM 23878, indicating that Lactobacillus reuteri HC1602 has greater adhesion potential.

[0127] Example 6

[0128] Adhesion of Lactobacillus reuteri HC1602 to intestinal epithelial cells

[0129] Intestinal epithelial cells (Caco-2) were revived and cultured to the required volume. When the cell density reached approximately 80%, they were digested with trypsin into a single-cell suspension and counted using a hemocytometer to obtain a cell count of 5 × 10⁶ cells. 5 Cells / mL. Then, 500 μL of cell suspension was seeded into 24-well plates with cell spreaders at a seeding density of 2.5 × 10⁻⁶ cells / mL. 5 Cells per well. After overnight culture until fully adhered, discard the culture medium, rinse twice with fresh culture medium, and set aside.

[0130] Fresh Lactobacillus reuteri HC1602 bacterial culture was washed twice with PBS buffer at pH 7.0, then resuspended in an equal volume of RPMI-1640 medium containing 10% fetal bovine serum. The absorbance was adjusted to achieve the desired OD value. 600= 0.8~1.0. The control strain Lactobacillus reuteri DSM 23878 was prepared in the same way to obtain the bacterial suspension.

[0131] The bacterial suspension was added to the prepared 24-well plate containing intestinal epithelial cells Caco-2 at 500 μL, and co-cultured in a CO2 incubator for 2 h. The unadhered bacteria were removed by washing 3 times with PBS buffer at pH = 7.0. The cell sheet was fixed with methanol for 15 min, then stained with Giemsa staining solution for 5 min, washed with PBS buffer at pH = 7.0, and then taken out onto a glass slide and observed under a microscope. The commercially available Lactobacillus reuteri DSM 23878 was used as a control strain. The adhesion ability of the test strain was calculated according to the following formula, and the results are shown in Table 8.

[0132] Adhesion ability (CFU / cell) = total number of adhered bacteria in each culture well / total cells in each culture well.

[0133] Table 8 - Test results of cell adhesion ability of Lactobacillus reuteri mucus HC1602

[0134]

[0135] As can be seen from the test results in Table 8, the adhesion ability of Lactobacillus reuteri mucus HC1602 to intestinal epithelial cells Caco-2 is 25.33±6.96, and the adhesion ability of Lactobacillus reuteri DSM 23878 is 9.78±5.64. The adhesion ability is closely related to the bacterial colonization ability, and strains with good adhesion ability can have an advantage in intestinal colonization. Lactobacillus reuteri mucus HC1602 has excellent adhesion ability to intestinal epithelial cells Caco-2, which is beneficial to its colonization in the intestine to metabolize beneficial substances or inhibit the colonization of pathogenic bacteria.

[0136] Example 7

[0137] In vitro cholesterol degradation test of Lactobacillus reuteri mucus HC1602

[0138] Preparation of cholesterol solution: 1 g of cholesterol was weighed, dissolved in anhydrous ethanol and diluted to 100 mL, sterilized by filtering with a 0.22 μm microporous filter under sterile conditions, and a cholesterol solution was obtained.

[0139] Preparation of liquid medium containing cholesterol: weigh proteose peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, CH3COONa 5.0 g, MgSO4 0.1 g, MnSO4 0.05 g, K2HPO4 2.0 g, distilled water 1000 mL, dissolve and adjust pH to 7.3, sterilize at 115°C for 30 min, then add cholesterol solution to make the final concentration of cholesterol 0.1%, and obtain the liquid medium containing cholesterol.

[0140] Inoculate L. reuteri HC1602 inoculum into the liquid medium containing cholesterol at an inoculation amount of 0.1% (v / v), and incubate at 37°C for 48 h, then take 0.2 mL of bacterial solution and add 1.8 mL of anhydrous ethanol, mix and stand for 10 min, then centrifuge at 3000 rpm for 5 min, take the supernatant and determine the cholesterol content according to the method specified in GB 5009.128-2016 "Determination of cholesterol in food", and calculate the cholesterol degradation rate. Use commercially available L. reuteri DSM 23878 as a control strain. The results are shown in Table 9.

[0141] Table 9 - Cholesterol degradation rate (%) of L. reuteri HC1602

[0142]

[0143] Cholesterol is an essential nutrient for the human body, and long-term high cholesterol levels in the blood can easily induce cardiovascular and cerebrovascular diseases. As can be seen from the test results in Table 9, the cholesterol degradation rate of L. reuteri HC1602 is 65.40%, which is much higher than that of the control strain L. reuteri DSM 23878, indicating that L. reuteri HC1602 helps to reduce the cholesterol level in the blood and maintain cardiovascular and cerebrovascular health.

[0144] Example 8

[0145] Indirect method for detecting the inhibitory effect of L. reuteri HC1602 on RSV

[0146] 1. Preparation of L. reuteri HC1602 fermentation test solution

[0147] According to the preparation method of the fermentation supernatant of L. reuteri HC1602 in Reference Example 2, after fermentation, collect the fermentation supernatant, adjust the pH of the fermentation supernatant to 7.0±0.1 with low-concentration NaOH, then filter with a 0.22 μm filter and store at -20°C for standby use.

[0148] 2. Respiratory syncytial virus (RSV virus) growth curve

[0149] Well-grown human non-small cell lung cancer (A549) cells were digested into 16 mL cell suspensions and evenly seeded into 12-well cell culture plates, 1 mL per well. The cells were incubated overnight (24 h) at 37°C in a CO2 incubator (5% concentration). RSV virus was diluted 10-fold with cell maintenance medium to a final concentration of 10... -1 ~10 -8 Gradient of cells was established. Culture medium was aspirated from each well, and once A549 cells had grown into a dense monolayer, the culture medium was discarded. The cells were washed twice with PBS, and 100 μL of viral dilution (MOI = 0.01) was inoculated into each well. Eight replicates were performed for each dilution, with a negative control included. Cells were incubated in a CO2 incubator for 72 h. The Reed-Muench two-component method was used to calculate the RSV median tissue infection dose (TCID). 50 Plot the number of hours post-RSV infection on the x-axis and the viral titer (TCID) on the y-axis. 50 Plot the RSV titer against the time following infection, using the vertical axis as the ordinate, to determine the time of viral harvesting in the in vitro antiviral test. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, RSV enters the logarithmic growth phase 6 hours after infecting A549 cells, reaches its peak proliferation 24 hours after infection, and gradually begins to inactivate after 24 hours. Therefore, the viral harvesting time for in vitro antiviral assays is 6 to 24 hours after infection.

[0150] 3. CCK8 assay to detect the inhibitory effect of Lactobacillus reuteri HC1602 on respiratory syncytial virus (RSV).

[0151] A549 cells in the logarithmic growth phase were harvested, digested with trypsin, and resuspended into a single-cell suspension. Cell counts were performed using a cell counting chamber, and the cell concentration was adjusted to 1 × 10⁶ cells / cells. 5cells / mL. The cell suspension was inoculated in 96-well plates, wherein the blank group was added with 100 μL of DMEM high glucose medium (complete medium containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin) without cells, the control group was added with 100 μL of cell suspension and 100 μL of basic medium (serum-free cell culture medium). The L. reuteri HC1602 fermentation test solution was diluted by 1:4 with the basic medium, and the test group was added with 100 μL of cell suspension and 100 μL of the above diluted L. reuteri HC1602 fermentation test solution. The 96-well plates were incubated in a 5% CO2 incubator at 37°C for 1.5 h, and then the cells were infected with RSV virus (MOI = 0.01). The cell plates were further incubated in the incubator for 48 h, the supernatant was discarded, and the cells were washed twice with PBS buffer, 10 μL of CCK-8 reagent and 90 μL of basic medium were added to each well, and the 96-well plates were gently shaken for 10 s to mix the reagent and the medium. The 96-well plates were incubated in a 5% CO2 incubator at 37°C for 2 h in the dark. The 96-well plates were taken out and the OD value of each well was detected by a microplate reader at a wavelength of 450 nm. The cell survival rate was used to indirectly replace the inhibition rate of L. reuteri HC1602 on RSV virus, and the inhibition rate (%) = (OD 试验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%. The commercially available L. reuteri DSM 23878 was used as a control strain, and the preparation method of the L. reuteri DSM 23878 fermentation test solution was the same as that of the L. reuteri HC1602 fermentation test solution. The test results are shown in Table 10.

[0152] Table 10 - Results of indirect detection of the inhibition rate of L. reuteri HC1602 on virus

[0153]

[0154] As can be seen from the test results in Table 10, the inhibition rate of L. reuteri HC1602 on RSV virus reached 80.29%, which indicates that it can effectively inhibit the invasion of RSV virus on lung cells, while the inhibition rate of the control strain on RSV virus is only 34.71%, which is weaker than that of the HC1602 strain in resisting the toxicity of RSV virus on lung cells.

[0155] Example 9

[0156] Direct inactivation of L. reuteri HC1602 on RSV virus

[0157] Take the Lactobacillus reuteri HC1602 fermentation test solution prepared in Example 8. The well-grown A549 cells in the cell culture bottle were digested into 16 mL of cell suspension, evenly inoculated into a 24-well cell culture plate, 500 μL per well, and cultured in a 37°C cell incubator with 5% CO2 overnight for 24 h to grow into a dense monolayer of cells. Take 4 1.5 mL EP tubes, respectively numbered A, B, C and D, and the components are as follows:

[0158] Tube A: mixture of 200 μL RSV virus and 200 μL Lactobacillus reuteri HC1602 fermentation test solution (1:4 dilution);

[0159] Tube B: mixture of 200 μL RSV virus and 200 μL DMEM high-sugar medium;

[0160] Tube C: 200 μL DMEM high-sugar medium;

[0161] Tube D: 200 μL Lactobacillus reuteri HC1602 fermentation test solution (1:4 dilution).

[0162] After incubating tubes A-D at 37°C for 1.5 h, mix tubes A and C to obtain the RSV virus inactivation group, and mix tubes B and D to obtain the RSV virus non-inactivation group. Discard the cell culture solution in the 24-well plate and wash it twice with PBS buffer. Infect A549 cells with RSV virus (MOI=0.01) from the RSV virus inactivation group and the RSV virus non-inactivation group, respectively. Place the 24-well plate in a 37°C incubator for 1.5 h, then wash it twice with PBS, and incubate it with fresh maintenance solution for 24 h.

[0163] RT-qPCR was used to detect the expression level of RSV-F protein mRNA in A549 cells to characterize the direct inactivation effect of Lactobacillus reuteri HC1602 on RSV. Total RNA extraction in A549 cells was performed according to the instructions of the Tiangen ® RNA reverse transcription and qPCR were performed using the Tiangen ® FastKing One-step RT-PCR Kit (KR123). The reaction system and reaction program are described in the kit instructions. GAPDH was used as the internal reference gene, and the primer sequences are shown in Table 11.

[0164] Table 11 - Fluorescent quantitative PCR primer sequences

[0165]

[0166] The commercially available Lactobacillus reuteri DSM 23878 was used as a control strain, and the results are shown in Figure 6 Figure 6 ​The results can be seen that, for RSV infected A549 cells treated by L. reuteri HC1602 fermentation test solution, the relative expression level of RSV-F protein mRNA in the inactivated group decreased by 61.33% compared with the non-inactivated group, and the difference was significant (P<0.05). P For RSV virus infected A549 cells treated by L. reuteri DSM 23878 fermentation test solution, the relative expression level of RSV-F protein mRNA in the inactivated group decreased by only 2.67% compared with the non-inactivated group, and the difference was not significant (P>0.05). This shows that L. reuteri HC1602 can directly inactivate RSV virus and inhibit its replication and proliferation in A549 cells. P

[0167] Example 10

[0168] Effect of different treatment methods on RSV virus replication

[0169] Take the L. reuteri HC1602 fermentation test solution prepared in Example 8, and culture A549 cells as in Example 9. After the A549 cells grow into a single layer of dense cells, discard the cell culture solution, wash twice with PBS, and pretreat, co-treat and post-treat A549 cells with L. reuteri HC1602 fermentation test solution, respectively.

[0170] Pretreatment group: add 500 μL of strain fermentation supernatant (dilution ratio of 1:4) to each well, and incubate in a CO2 incubator (5%) at 37°C for 90 min. After incubation, infect the cells with RSV virus (MOI=0.01) and incubate for 90 min, then discard the virus solution and add new maintenance solution, and continue to culture in a CO2 incubator for 24 h;

[0171] Co-treatment group: add L. reuteri HC1602 fermentation test solution (final dilution ratio of 1:4) and RSV virus (final MOI=0.01) to A549 cells at the same time, and incubate in a cell culture incubator for 90 min, then discard the virus solution and add new maintenance solution and continue to culture in a CO2 incubator at 37°C (5%) for 24 h;

[0172] Post-treatment group: first infect A549 cells with RSV virus (MOI=0.01), incubate in a CO2 incubator at 37°C (5%) for 90 min, then discard the virus solution, add 500 μL of L. reuteri HC1602 fermentation test solution (dilution ratio of 1:4) to each well, and incubate in a cell culture incubator for 90 min. After incubation, discard the virus solution, add maintenance solution, and continue to culture in a cell culture incubator for 24 h;

[0173] ​Control group: A549 cells were infected with RSV virus (MOI=0.01), incubated in a 37℃ CO2 incubator (5%) for 90 min, and the virus solution was discarded. Then, 500 μL of serum-free DMEM high glucose medium was added to each well, and the cells were incubated in a cell culture incubator for 90 min. After incubation, the medium was discarded, maintenance medium was added, and the cells were cultured in a cell culture incubator for 24 h.

[0174] Following the testing method in Example 9, the expression of RSV-F protein mRNA in A549 cells was detected using RT-qPCR. The results are as follows: Figure 7 As shown. By Figure 7 The results showed that the relative expression level of RSV-F protein mRNA in the pretreatment group decreased by 67.67% compared with the control group, and the difference was highly significant. P <0.01); In the co-treatment group, the relative expression level of RSV-F protein mRNA decreased by 37.17% compared with the control group, and the difference was highly significant ( P <0.01); In the post-treatment group, the relative expression level of RSV-F protein mRNA decreased by 10.83% compared with the control group, and the difference was statistically significant. P <0.05). This indicates that the fermentation supernatant of *Lactobacillus reuteri* HC1602 not only inhibits RSV replication in A549 cells, but also significantly enhances the antiviral effect of A549 cells after pre-incubation. Furthermore, the *Lactobacillus reuteri* HC1602 fermentation test solution also exhibits a certain antiviral effect after A549 cells are infected with RSV.

[0175] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A Lactobacillus reuteri (L. reuteri) strain HC1602, characterized in that, Limosilactobacillus reuteri ) HC1602, characterized in that, The Lactobacillus reuteri HC1602 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 26890, the preservation date is March 23, 2023, and the address of the preservation agency is No. 3, Beichen West Road, Chaoyang District, Beijing.

2. Lactobacillus muci lagosus HC1602 according to claim 1, characterized in that, The 16S rDNA sequence of the Lactobacillus reuteri HC1602 is shown as SEQ ID NO.

3.

3. An inoculant characterized in that, The bacterial agent contains the strain lysate of the Lactobacillus reuteri HC1602 of claim 1 or the fermentation supernatant of the Lactobacillus reuteri HC1602 of claim 1; the strain lysate is obtained by using an ultrasonic disrupter to ultrasonically break the strain fermentation broth.

4. The bacterial agent of claim 3, wherein The fermentation supernatant is obtained by centrifuging the strain fermentation broth.

5. The bacterial agent of claim 4, wherein The preparation method of the strain fermentation broth is that the Lactobacillus reuteri HC1602 is cultured in MRS liquid medium at 37℃ for 24-28h.

6. The bacterial agent as claimed in claim 5, characterized by The MRS liquid medium comprises the following components: 10g / L of proteose peptone, 10g / L of beef extract, 5.0g / L of yeast extract, 5g / L of sodium acetate, 5g / L of glucose, 2g / L of KH2PO4, 1.0mL / L of Tween 80, 2.0g / L of citric acid diamine, 20g / L of CaCO3, 0.58g / L of MgSO4·7H2O, 0.25g / L of MnSO4·7H2O, and pure water to 1L, and the pH is adjusted to 6.2-6.

5.

7. Use of the Lactobacillus reuteri HC1602 of claim 1 in the preparation of a product for reducing cholesterol in blood.

8. Use of the Lactobacillus reuteri HC1602 of claim 1 in the preparation of a product for inhibiting and preventing respiratory syncytial virus.

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