Neisseria subflava with high yield and anti-inflammatory and anti-asthmatic effects and application thereof

Abstract

The application discloses a high-yield Neisseria subflava NICE2022-623 with anti-inflammatory and asthma-relieving effects, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 2023605 and belongs to the field of microorganisms. The strain has strong fermentation capacity and large biological yield, and inactivated bacteria powder prepared from the strain has good anti-inflammatory and asthma-relieving effects. The application can significantly reduce the cost of anti-inflammatory and asthma-relieving drugs prepared from the bacteria powder and has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a high-yield strain of Neisseria subflava with anti-inflammatory and bronchodilatory effects and its applications. Background Technology

[0002] *Neisseria subflava*: Belongs to the phylum Proteobacteria, family Neisseriaceae, and genus *Neisseria*. Neisseria is a Gram-negative, spherical bacterium. Common members of the Neisseria family include *Neisseria sicca*, *Neisseria subflava*, *Neisseria mucosa*, *Neisseria meningitidis*, and *Neisseria gonorrhoeae*. Neisseria bacteria are relatively small, kidney-shaped cocci with a diameter of 0.6–1.0 micrometers. They are non-spore-forming, non-flagellated, and a few have capsules. They are aerobic or facultative anaerobic, have weak acidification ability, possess oxidase, do not form indole, do not reduce nitrates, parasitize the mucous membranes of mammals, and can oxidize various sugars, producing acid but not gas. Among them, Neisseria meningitidis and Neisseria gonorrhoeae are pathogens that cause human diseases, while other Neisseria species are mostly normal flora that parasitize the human respiratory tract.

[0003] In the 1970s, Chinese scientists isolated Staphylococcus albus, Neisseria gonorrhoeae, and Bacillus subtilis from the sputum of patients with chronic bronchitis. Combining these three bacteria for the treatment of bronchitis and asthma yielded excellent results. This bacterial agent is now produced and marketed by Shandong Qilu Pharmaceutical Co., Ltd. under the brand name "Staphylococcus albus and Neisseria gonorrhoeae Tablets." The lipopolysaccharide component in the cell wall of Neisseria gonorrhoeae can induce interferon production, directly activating the complement C3 pathway, mobilizing the body's reticuloendothelial system, and enhancing the phagocytic capacity of leukocytes. This improves the body's non-specific immunity and specific immunity of the respiratory mucosa, thus enhancing the body's resistance. Therefore, it can be used to treat diseases such as bronchitis and asthma. This drug is beneficial in reducing antibiotic resistance caused by overuse and has great market potential. However, the fermentation and cultivation of Neisseria gonorrhoeae requires high standards and results in low yields, significantly increasing production costs and limiting its industrial development and further clinical application.

[0004] Therefore, it is particularly important to screen Neisseria species that have good therapeutic effects and high production capacity. This is not only conducive to solving the bottleneck problems encountered in the industrialization of Neisseria species, but also helps to realize the transformation and upgrading of Neisseria species production enterprises, which has great social and economic benefits. Summary of the Invention

[0005] The purpose of this invention is to provide a high-yield strain of Neisseria flavescens with anti-inflammatory and bronchodilatory effects and its applications.

[0006] The applicant screened respiratory swabs from healthy individuals and collected a strain of Gram-negative cocci with strong growth ability. Under the same fermentation conditions, this strain had a stronger fermentation ability than other screened Gram-negative cocci and commercially available Neisseria. The strain was identified as *Neisseria subflava*, which the applicant named *Neisseria subflava* NICE2022-623. On April 24, 2023, it was deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO: M 2023605.

[0007] Then, the applicant expanded the culture and inactivated the NICE2022-623 strain, and finally obtained an inactivated bacterial powder with a yield of up to 24.6 g / L, which greatly reduced the company's production costs and solved the bottleneck problem that restricted the industrialization of the strain.

[0008] Finally, the applicant constructed an animal model of bronchitis using a fumigation method and investigated the efficacy of NICE2022-623 inactivated bacterial powder on bronchitis. The results showed that the bacteria had a good anti-asthmatic effect, prolonging the latency period of asthma in the model animals. Further research revealed that the bacteria possesses strong anti-inflammatory activity, significantly reducing the levels of inflammatory factors in the alveoli and inflammatory infiltration in the lung tissue of the model animals. Therefore, it can be used to prepare drugs and treat bronchitis by exerting anti-inflammatory and anti-asthmatic effects.

[0009] The present invention further provides a method for preparing the bronchitis treatment drug, comprising the following steps:

[0010] 1) The seed culture of Neisseria microflagellate strain NICE2022-623 was fermented to obtain the fermentation broth;

[0011] 2) Formaldehyde was added to the fermentation broth to inactivate the bacteria. After centrifugation, washing, and vacuum freeze-drying, the bacterial powder was obtained.

[0012] The fermentation culture medium consists of: 6 g / L glucose, 25 g / L yeast extract, 15 g / L peptone, 5 g / L sodium chloride, 3 g / L disodium hydrogen phosphate, 0.3 g / L magnesium sulfate, 5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, 2 g / L citric acid, 0.1% trace elements, and water as the balance, with a pH of 7.0–7.6.

[0013] The fermentation conditions are as follows: the culture temperature is 37±1℃, the sugar content is not less than 0.3wt%, and the fermentation time is 18 to 24 hours.

[0014] The final concentration of formaldehyde is 0.6 wt%.

[0015] The inactivated bacterial powder of NICE2022-623 can be used to prepare various preparations suitable for clinical use, including but not limited to oral preparations, injections, and topical preparations. Animal experiments can be used to guide clinical drug use. According to a specific embodiment of the present invention, the preferred drug is an oral preparation.

[0016] The beneficial effects of this invention are:

[0017] (1) Strong fermentation ability and large yield of mycelium powder. The strain of the present invention was obtained by screening the fermentation ability of strains. The yield of mycelium powder is not only higher than that of other strains screened, but also higher than that of commercial strains of the same genus. The present invention is conducive to increasing the yield of Neisseria microflagellate, reducing production costs, and realizing industrialization.

[0018] (2) The drug has good efficacy and high safety. The bacterial strains screened in this invention have strong anti-inflammatory and bronchodilator effects and can be used for the clinical treatment of bronchitis with significant efficacy. *Neisseria flavescens* is a human parasite and is harmless itself; furthermore, this invention inactivates it, thus ensuring high safety. This invention provides a new drug option for the clinical treatment of bronchitis and is of great significance in meeting clinical medication needs. Attached Figure Description

[0019] Figure 1 This is the cell morphology of strain NICE2022-623.

[0020] Figure 2 This is the growth curve of strain NICE2022-623.

[0021] Figure 3 The effect of strain NICE2022-623 on inflammatory factors IL-6 and IL-8 in bronchoalveolar lavage fluid of guinea pigs.

[0022] Figure 4 The effect of strain NICE2022-623 on pathological sections of guinea pig lung tissue. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Strain Screening

[0025] (1) Sample collection: Pharyngeal swabs were aseptically collected from healthy individuals. Twenty students from Huazhong University of Science and Technology (10 males and 10 females) were selected. The subjects opened their mouths and tilted their heads back. The sampling personnel used a tongue depressor to fix the tongue, and the sterile flocked swabs were repeatedly swabbed three times on the posterior and lateral walls of the pharynx to collect pharyngeal secretions.

[0026] (2) Purification: Each sample was streaked onto a Columbia plate and incubated at 37°C for 24 hours. Medium-sized, smooth, moist, and round colonies were picked and purified three times on Columbia plates to obtain purified culture.

[0027] (3) Isolation: The purified culture from step (2) was evenly spread onto a glass slide to prepare a smear. After Gram staining, the bacterial morphology was observed under a microscope. Single Gram-negative bacteria were selected, inoculated into LB medium, and cultured for 12 hours. Then, Gram staining was used to observe the morphology, and Gram-negative cocci with diplococcal morphology were selected. A total of three Gram-negative cocci were obtained in this experiment, and their bacterial morphology is as follows: Figure 1 As shown.

[0028] (4) Screening: The Gram-negative cocci obtained in step (3) were aseptically inoculated into seed culture medium from the cryopreservation tube and cultured at 37±1℃ and 150rpm for 24h to obtain seed liquid. The seed liquid was then inoculated into a fermenter containing fermentation culture medium at an inoculation rate of 5% for fermentation culture. The culture temperature was 37±1℃, the aeration rate was controlled to maintain the dissolved oxygen level at 20%, and the sugar content was controlled to be no less than 0.3% by adding glucose. After fermentation for 24h, the fermentation broth of Neisseria flavipes was obtained.

[0029] Seed culture medium (LB medium): tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0030] Fermentation medium: glucose 6g / L, yeast powder 25g / L, peptone 15g / L, sodium chloride 5g / L, disodium hydrogen phosphate 3g / L, magnesium sulfate 0.3g / L, potassium dihydrogen phosphate 5g / L, ammonium sulfate 2g / L, citric acid 2g / L, trace elements 0.1%, add water to make up to the total volume, and adjust the pH to 7.0-7.6.

[0031] After cultivation, the yield of mycelium powder was measured. The experiment was repeated three times, and the average value was calculated. The results are shown in Table 1. Under the same fermentation conditions, strain 3 had a stronger fermentation capacity, and its mycelium powder yield was increased by 300%, 160%, and 63% compared with the other two strains and the commercial strain.

[0032] Commercial strain: Neisseria thunbergii standard strain ATCC14799.

[0033] Method for determining the yield of bacterial powder: After inactivation, the bacterial solution is centrifuged and washed to obtain bacterial cells. The bacterial cells are then freeze-dried under vacuum to obtain dry bacterial powder. The yield of bacterial powder per liter of culture medium is calculated after weighing.

[0034] Table 1 Comparison of mycelial powder yield of three Gram-negative cocci

[0035] strain 1 6.2g / L 0.323 strain 2 9.3g / L 3.180 strain 3 24.6g / L 2.925 Commercial strains 15.1g / L 1.672

[0036] Example 2: Strain Identification

[0037] Genomic DNA was extracted from the screened strains, and the 16S rDNA gene was amplified using 16S rDNA primers. The universal primers for PCR amplification of 16S rDNA were: F27: 5'-AGAGTTTGATCCTGGCTCAG-3', R1492: 5'-TACGGCTACCTTTGTTACGACTT-3'. The PCR reaction system consisted of 25 μL: 12.5 μL of 2x Phanta Master Buffer, 7.5 μL of ddH2O, 1 μL each of forward and reverse primers (10 mmol / L), and 2 μL of template. The reaction program was 95℃ for 10 min, followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 90 s, and a final extension at 72℃ for 10 min. The PCR amplification product was recovered by gel extraction after 1% agarose gel electrophoresis. The recovered target fragment was sequenced, and the obtained sequence was compared with the corresponding sequence of Neisseria subflava in GenBank. The homology reached 99.8%, so it was identified as Neisseria subflava.

[0038] The sequencing results are as follows:

[0039]

[0040] Example 3: Production of inactivated sterilization powder

[0041] First, the growth curve of the obtained *Neisseria flavescens* was determined. Freshly cultured colonies from Columbia plates were picked and inoculated onto LB medium. Samples were taken every 2 hours from 0 to 22 hours. After sampling, the bacterial suspension was serially diluted, and the optical density of the suspension was measured. A growth curve of *Neisseria flavescens* was plotted based on the OD600nm data. The curve showed that the culture period was a stationary phase from 16 to 22 hours.

[0042] Next, the selected bacterial strains were cultured on a larger scale. The specific steps are as follows:

[0043] 1) Preparation of seed solution:

[0044] Following aseptic requirements, the bacterial culture from the cryopreservation tube was inoculated into the seed culture medium and cultured at 37±1℃ and 150rpm for 18–24 hours to obtain the seed liquid.

[0045] Seed culture medium (LB medium): tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0046] 2) Preparation of fermentation broth:

[0047] A fermentation medium was prepared by adding water to the following ingredients: 6 g / L glucose, 25 g / L yeast powder, 15 g / L peptone, 5 g / L sodium chloride, 3 g / L disodium hydrogen phosphate, 0.3 g / L magnesium sulfate, 5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, 2 g / L citric acid, and 0.1% trace elements. After thorough dissolution, the pH was adjusted to 7.0–7.6 using sodium hydroxide and hydrochloric acid solutions, and the medium was sterilized. The seed culture was inoculated into a fermenter containing the fermentation medium at an inoculation rate of 3–10% (by volume) and fermented at 37 ± 1 °C. The dissolved oxygen level was maintained at 20% by controlling the aeration rate, and the sugar content was controlled to be no less than 0.3 wt% by adding glucose. Fermentation was carried out for 18–24 hours to obtain the *Neisseria flavipes* fermentation broth.

[0048] 3) Preparation of inactivated sterilization powder

[0049] Formaldehyde was added to the obtained *Neisseria flavescens* fermentation broth to achieve a final formaldehyde concentration of 0.6 wt%. The mixture was stirred at 180 rpm for 2 hours at 37°C. After stirring, the bacterial solution was allowed to stand for 2 hours to inactivate the bacteria. After inactivation, samples were taken and plated to test the inactivation effect. Once inactivation was confirmed, the inactivated bacterial solution was centrifuged, washed, and the bacterial cells were obtained. The bacterial cells were then freeze-dried under vacuum to obtain bacterial powder.

[0050] Example 4: Evaluation of therapeutic effect

[0051] 1. Test Methods

[0052] Guinea pigs, half male and half female, weighing 250–500g, were randomly divided into a blank control group, a model group, and a drug treatment group, with 6 guinea pigs in each group. The blank control group received no treatment. The model group and the drug treatment group were first subjected to smoke fumigation for one week to establish a tracheitis model. The specific modeling method was as follows: the guinea pigs were placed in a smoke chamber, and the tobacco leaves in the smoke chamber were lit for fumigation. The fumigation was carried out four times a day from 8:00 to 20:00, with an interval of 3 hours between each fumigation, and each fumigation lasted for 30 minutes. To prevent burns to the guinea pigs, the guinea pigs and the tobacco leaves were separated by a partition with smoke holes.

[0053] One week after modeling, guinea pigs were transferred to an experimental chamber, and a mixture of equal volume of 2% acetylcholine chloride and 0.1% histamine phosphate was sprayed into the air for 15 seconds to induce asthma. The latency period of asthma induction before administration was recorded. After successful modeling, the treatment group was administered Neisseria gonorrhoeae powder (100 mg / kg) by gavage once daily for 5 consecutive days, while the model group and the blank control group were administered the same volume of physiological saline by gavage daily. On the 5th day, 1 hour after administration, a mixture of equal volume of 2% acetylcholine chloride and 0.1% histamine phosphate was sprayed into the air again for 15 seconds to induce asthma. The latency period of asthma induction after administration was recorded.

[0054] After recording the latency period of induced asthma, guinea pigs were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (1 mL / kg), euthanized by cervical dislocation, and the thoracic cavity and trachea were fully exposed. The right lung was ligated, intubated, and lavaged with 6 mL of pre-cooled PBS in three divided doses, with the bronchoalveolar lavage fluid combined. Following the kit instructions, the levels of inflammatory factors IL-6 and IL-8 in the bronchoalveolar lavage fluid were detected by ELISA. Simultaneously, guinea pig lung tissue was isolated, infiltrated and fixed in 4% paraformaldehyde solution, dehydrated, embedded in paraffin, and then sectioned for pathological observation.

[0055] 2. Test Results

[0056] (1) Evaluation of the antiasthmatic effect of inactivated bacterial powder

[0057] The results of the pre- and post-administration asthma latency periods in the three groups of guinea pigs are shown in Table 2 below. The results showed that the pre-administration asthma latency period in the model group and the drug-treated group was significantly shorter than that in the control group (P<0.01), indicating that the tracheitis model was successfully established. The post-administration asthma latency period in the model group was slightly longer than that before administration, but there was no significant difference between the two (P>0.05), which may be related to the self-healing ability of guinea pigs. The post-administration latency period in the drug-treated group was significantly longer than that before administration (P<0.01), and the post-administration latency period in the drug-treated group was significantly different from that in the model group (P<0.05 or P<0.01), indicating that the inactivated Neisseria flavescens powder has a relieving and therapeutic effect on tracheitis.

[0058] Table 2 Evaluation of the antiasthmatic effect of inactivated bacterial powder (n=6)

[0059]

[0060] Note: Compared with the control group, *P<0.05, **P<0.01; compared with before drug administration, #P<0.05, ##P<0.01; compared with the model group, △ P<0.05, △△ P<0.01.

[0061] (2) Evaluation of the anti-inflammatory efficacy of inactivated bacterial powder

[0062] The results of inflammatory factor detection showed that ( Figure 3 The levels of inflammatory factors IL-6 and IL-8 in the alveoli of the model group were significantly higher than those in the blank group (P<0.001), while the NICE2022-623 group reduced the levels of inflammatory factors IL-6 and IL-8 in the bronchoalveolar lavage fluid, showing a significant difference compared with the model group (P<0.001 or P<0.005), and was more effective than the commercial strain.

[0063] Histopathological observation results showed ( Figure 4 In the control group, alveolar inflammatory infiltration was minimal, bronchial structure was normal, and there was no exudate within the lumen. In the model group, guinea pigs showed significant inflammatory infiltration of the tracheal and bronchiolar mucosa, with a large amount of mucus exudate visible in the lumen. In the drug-treated group, guinea pigs showed reduced inflammatory infiltration of the airway and lung tissue compared to the model group, and significantly less exudate in the lumen. Compared to the model group, the drug-treated group showed significantly reduced airway smooth muscle thickening and less inflammatory cell infiltration, and the effect was better than that of the commercially available strain.

[0064] The above results indicate that the inactivated bacterial powder screened and prepared in this invention has anti-inflammatory effects and can be used to treat bronchitis.

Claims

1. A high-yield strain of *Neisseria flavescens* with anti-inflammatory and bronchodilatory effects (… Neisseria subflava The strain NICE2022-623 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2023605.

2. The use of the inactivated Neisseria flavescens powder NICE2022-623 as described in claim 1 in the preparation of a bronchitis treatment drug.

3. A drug for treating bronchitis, wherein the active ingredient in the drug is the inactivated powder of Neisseria flavescens NICE2022-623 as described in claim 1.

4. The bronchitis treatment drug as described in claim 3, wherein the drug is an oral medication.

5. A method for preparing the bronchitis treatment drug according to claim 3 or 4, characterized in that... Includes the following steps: 1) The seed culture of Neisseria microflagellate strain NICE2022-623 was fermented to obtain the fermentation broth; 2) Formaldehyde was added to the fermentation broth to inactivate the bacteria. After centrifugation, washing, and vacuum freeze-drying, the bacterial powder was obtained.

6. The method for preparing the bronchitis treatment drug as described in claim 5, characterized in that, The culture medium for fermentation is as follows: glucose 6 g / L, yeast extract 25 g / L, peptone 15 g / L, sodium chloride 5 g / L, disodium hydrogen phosphate 3 g / L, magnesium sulfate 0.3 g / L, potassium dihydrogen phosphate 5 g / L, ammonium sulfate 2 g / L, citric acid 2 g / L, trace elements 0.1%, water as the balance, pH 7.0–7.

6.

7. The method for preparing the bronchitis treatment drug as described in claim 5, characterized in that, The fermentation conditions are: a culture temperature of 37±1℃, a sugar content of not less than 0.3%, and a fermentation time of 18 to 24 hours.

8. The method for preparing the medicament for treating bronchitis as described in claim 5, characterized in that: The final concentration of formaldehyde is 0.6%.

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