Application of Streptococcus salivarius subsp. thermophilus DY-001 and its bacteriocins and lyophilized powder in the treatment of upper respiratory tract infections

Abstract

This invention provides the application of *Streptococcus thermophilus* subsp. DY-001 and its bacteriocins and lyophilized powder in the treatment of upper respiratory tract infections, belonging to the field of microbial technology. This invention provides *Streptococcus thermophilus* subsp. DY-001, with accession number CGMCC No. 33409. This invention further provides bacteriocins or lyophilized powders obtained based on *Streptococcus thermophilus* subsp. DY-001, and methods for preparing said bacteriocins or lyophilized powders. The bacteriocins and lyophilized powders of *Streptococcus thermophilus* subsp. DY-001 described in this invention have significant antibacterial effects and can be used to alleviate or treat upper respiratory tract infections, and as an adjunct treatment for adenoid hypertrophy in children.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Streptococcus salivarius subsp. thermophilus DY-001 and its bacteriocins and lyophilized powder in the treatment of upper respiratory tract infections. Background Technology

[0002] Upper respiratory tract infections (such as colds, pharyngitis, and sinusitis) are mainly caused by viruses (such as rhinovirus and influenza virus) or bacteria (such as Streptococcus pneumoniae). Children are more susceptible to these infections and are prone to complications such as otitis media and sinusitis. Traditional treatments include antibiotics (for bacterial infections), antiviral drugs, and symptomatic treatments (such as antipyretics, analgesics, and cough suppressants). However, antibiotic overuse can lead to a surge in drug-resistant bacteria, and long-term use may cause side effects such as gastrointestinal irritation and liver and kidney damage.

[0003] Adenoid hypertrophy is most common in children, mainly caused by repeated nasopharyngeal inflammation (such as rhinitis, sinusitis, upper respiratory tract infection) or allergic reactions, leading to pathological hyperplasia of lymphoid tissue, causing symptoms such as nasal congestion, snoring, and sleep apnea. In severe cases, adenoid facies or cardiopulmonary dysfunction may occur. Current treatments primarily involve medication (such as nasal corticosteroids and antihistamines), traditional Chinese medicine (massage, herbal medicine), and surgery (adenoidectomy). Long-term medication may cause side effects; traditional Chinese medicine treatment is generally ineffective, expensive, and may delay optimal treatment time; surgery carries invasive risks; and the harms of adenoidectomy are currently not fully understood by the medical community.

[0004] In recent years, probiotics, as a novel biological agent, have gradually become a research hotspot in the prevention and treatment of respiratory infections due to their immune-regulating and pathogen-inhibiting properties. Multiple clinical studies have shown that probiotics can competitively inhibit pathogens, regulate mucosal immunity, and enhance epithelial barrier function. For example, *Streptococcus thermophilus* subsp. *salivarius* Biohalo23 has been shown to alleviate upper respiratory tract infection symptoms by inhibiting harmful bacteria (such as *Campylobacter conjunctivae* and *Neisseria oralis*), increasing the abundance of beneficial bacteria, and maintaining gut microbiota balance. Despite the potential of probiotics in the prevention and treatment of respiratory infections, their clinical application still faces the following challenges: the mechanisms of action and effects of different probiotic strains vary significantly; traditional liquid probiotic preparations are easily affected by temperature and humidity, resulting in low viable bacterial survival rates; most probiotic preparations are administered orally, requiring passage through the gastrointestinal environment (gastric acid, bile) before reaching the respiratory tract, leading to a significant reduction in viable bacterial counts and low respiratory tract colonization rates. Therefore, continued screening of probiotics with significant efficacy in alleviating upper respiratory tract infections and the development of related preparations remain crucial research areas in this field. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide a thermophilic subspecies of Streptococcus salivarius (Salivarius). Streptococcus salivarius subsp. thermophilusDY-001, with accession number CGMCCNo.33409.

[0006] The second objective of this invention is to provide a bacteriocin isolated from a fermentation culture of Streptococcus salivarius subsp. DY-001, which has a significant antibacterial effect.

[0007] The third objective of this invention is to provide a freeze-dried powder and its preparation method, which has a good therapeutic effect on relieving upper respiratory tract infections.

[0008] The fourth objective of this invention is to provide the use of the aforementioned Streptococcus salivarius thermophilus subsp. DY-001 or its bacteriocin or its lyophilized powder in the preparation of products that inhibit pathogenic bacteria, drugs that relieve or treat upper respiratory tract infections, and drugs that assist in the treatment of adenoid hypertrophy in children.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a thermophilic subspecies of Streptococcus salivarius ( Streptococcus salivarius subsp. thermophilus The preservation number of the thermophilic subspecies of Streptococcus salivarius DY-001 is CGMCC No. 33409.

[0011] This invention provides a method for preparing bacteriocins, comprising the following steps: fermenting and culturing the above-mentioned Streptococcus salivarius thermophilic subsp. DY-001, and isolating bacteriocins from the fermentation culture product.

[0012] This invention provides bacteriocins prepared by the above-described preparation method.

[0013] This invention provides a method for preparing freeze-dried powder, comprising the following steps: fermenting and culturing the above-mentioned Streptococcus thermophilus subsp. DY-001 to obtain a fermentation culture product, and preparing freeze-dried powder of Streptococcus thermophilus subsp. DY-001 from the fermentation culture product.

[0014] Preferably, the fermentation culture medium comprises 10-15 g / L glucose, 10-15 g / L lactose, 18-22 g / L yeast extract, 10-20 g / L malt extract, 20-25 g / L soybean peptone, 3-7 g / L sodium acetate, 1-3 g / L diammonium hydrogen citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate, and 0.02-0.08 g / L manganese sulfate.

[0015] Preferably, the culture medium for fermentation includes 100-130 g / L glucose, 100-130 g / L lactose, 80-110 g / L honeysuckle powder, 80-110 g / L lily powder, 80-110 g / L loquat powder, and 80-110 g / L jujube powder.

[0016] This invention provides a freeze-dried powder prepared by the above-described preparation method.

[0017] This invention provides the application of the above-mentioned Streptococcus salivarius thermophilus subsp. DY-001 or bacteriocin or lyophilized powder in the preparation of products that inhibit pathogenic bacteria, including Streptococcus pneumoniae, β-hemolytic streptococci, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus.

[0018] This invention provides the use of the above-mentioned Streptococcus salivarius thermophilic subsp. DY-001 or bacteriocins or lyophilized powder in the preparation of drugs for relieving or treating upper respiratory tract infections.

[0019] This invention provides the use of the above-mentioned Streptococcus salivarius thermophilic subsp. DY-001 or bacteriocin or lyophilized powder in the preparation of a drug for the adjunctive treatment of adenoid hypertrophy in children.

[0020] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0021] This invention provides the first known strain of *Streptococcus thermophilus* subsp. DY-001, which can colonize the upper respiratory tract mucosa. Based on *Streptococcus thermophilus* subsp. DY-001, this invention isolates and obtains *Streptococcus thermophilus* subsp. DY-001 bacteriocin, and prepares a lyophilized powder based on *Streptococcus thermophilus* subsp. DY-001. The *Streptococcus thermophilus* subsp. DY-001, its bacteriocin, and the lyophilized powder described in this invention exhibit good antibacterial activity, significantly inhibiting the growth of *Streptococcus pneumoniae*, β-hemolytic streptococci, *Streptococcus pyogenes*, *Escherichia coli*, and *Staphylococcus aureus*, maintaining respiratory flora stability, inhibiting the excessive growth of harmful bacteria, and achieving a significant effect in relieving or treating upper respiratory tract infections. It can also be used as an adjunct treatment for adenoid hypertrophy in children.

[0022] Biological Preservation Instructions

[0023] Streptococcus salivarius subsp. thermophilus DY-001, classified as Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus (), deposited by: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 33409, deposit date: January 14, 2025. Attached Figure Description

[0024] Figure 1 Macroscopic morphology of colonies of Streptococcus salivarius subsp. DY-001 in Petri dishes;

[0025] Figure 2Microscopic morphology of Streptococcus salivarius subsp. thermophilus strain DY-001;

[0026] Figure 3 Adhesion results of different probiotics to oral keratinocytes (HOK);

[0027] Figure 4 The results of an orthogonal experiment on the fermentation of Streptococcus salivarius subsp. thermophilus DY-001 using yeast extract, malt extract, soybean peptone, and a mixture of glucose and lactose. Detailed Implementation

[0028] This invention provides a thermophilic subspecies of Streptococcus salivarius ( Streptococcus salivarius subsp. thermophilus The thermophilic subspecies of Streptococcus salivarius, DY-001, has the accession number CGMCC No. 33409. The thermophilic subspecies of Streptococcus salivarius DY-001 described in this invention was isolated and purified from the oral cavity of a healthy child, and identification confirmed that it belongs to the thermophilic subspecies of Streptococcus salivarius (…). Streptococcus salivarius subsp. thermophilus It was deposited on January 14, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0029] The *Streptococcus thermophilus* subsp. DY-001 described in this invention is sensitive to multiple antibiotics, can colonize the upper respiratory tract mucosa, competes with pathogenic bacteria for nutrients and adhesion sites, reduces pathogen proliferation, activates the mucosal immune system, promotes IgA secretion and immune cell activity, enhances local defense capabilities, thereby maintaining respiratory flora stability, inhibiting the overgrowth of harmful bacteria, and alleviating upper respiratory tract infections. The fermentation medium for *Streptococcus thermophilus* subsp. DY-001 described in this invention can be M17 liquid medium. As an optional implementation, *Streptococcus thermophilus* subsp. DY-001 is activated and cultured in M17 liquid medium. After activation, it is inoculated into M17 liquid medium at an inoculum of 2-4% (v / v) for expansion and fermentation culture, preferably 3%. The fermentation culture temperature is 36-38℃, preferably 37℃; the fermentation culture time is 14-48 hours, preferably 18-24 hours.

[0030] Preferably, the fermentation medium for *Streptococcus thermophilus* subsp. DY-001 of the present invention comprises 10-15 g / L glucose, 10-15 g / L lactose, 18-22 g / L yeast extract, 10-20 g / L malt extract, 20-25 g / L soybean peptone, 3-7 g / L sodium acetate, 1-3 g / L diammonium citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate, and 0.02-0.08 g / L manganese sulfate; more preferably, it comprises 12.5 g / L glucose, 12.5 g / L lactose, 20 g / L yeast extract, 15 g / L malt extract, 23 g / L soybean peptone, 5 g / L sodium acetate, 2 g / L diammonium citrate, 2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, and 0.05 g / L manganese sulfate.

[0031] Preferably, the fermentation medium for *Streptococcus salivarius* subsp. *thermophilus* DY-001 of the present invention comprises 100-130 g / L glucose, 100-130 g / L lactose, 80-110 g / L honeysuckle powder, 80-110 g / L lily powder, 80-110 g / L loquat powder, and 80-110 g / L jujube powder; more preferably, it comprises 110-120 g / L glucose, 110-120 g / L lactose, 90-100 g / L honeysuckle powder, 90-100 g / L lily powder, 90-100 g / L loquat powder, and 90-100 g / L jujube powder.

[0032] The culture medium of this invention is prepared with water, preferably purified water. This invention, by improving the composition of the culture medium, can increase the viable count of Streptococcus salivarius subspecies DY-001.

[0033] The present invention also provides a method for preparing bacteriocins, comprising the following steps: fermenting the above-mentioned Streptococcus salivarius thermophilic subsp. DY-001, and separating bacteriocins from the fermentation culture product.

[0034] The fermentation culture temperature described in this invention is 36-38℃, preferably 37℃; the fermentation culture time is 14-48h, preferably 18-20h. The method for isolating bacteriocins described in this invention is preferably ammonium sulfate precipitation. In this invention, the fermentation culture product (fermentation broth) is centrifuged to obtain the supernatant. The centrifugation conditions are preferably 4℃, 12000r / min for 10-15min. In this invention, ammonium sulfate is added to the centrifuged supernatant to achieve a saturation of 50-65%, preferably 55-60%; the solution after adding ammonium sulfate is magnetically stirred overnight at low temperature, and the precipitate is collected by centrifugation to obtain bacteriocins. The centrifugation conditions are preferably 4℃, 8000r / min for 15min.

[0035] The present invention also provides a bacteriocin prepared by the above preparation method, wherein the bacteriocin can significantly inhibit the growth of Streptococcus pneumoniae, β-hemolytic streptococci, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus.

[0036] The present invention also provides a method for preparing freeze-dried powder, comprising the following steps: fermenting and culturing the above-mentioned Streptococcus salivarius subsp. thermophilicus DY-001 to obtain a fermentation culture product, and preparing freeze-dried powder of Streptococcus salivarius subsp. thermophilicus DY-001 from the fermentation culture product.

[0037] Preferably, the fermentation culture medium comprises 10-15 g / L glucose, 10-15 g / L lactose, 18-22 g / L yeast extract, 10-20 g / L malt extract, 20-25 g / L soybean peptone, 3-7 g / L sodium acetate, 1-3 g / L diammonium hydrogen citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate, and 0.02-0.08 g / L manganese sulfate. When using this culture medium, the fermentation product is centrifuged to obtain bacterial sludge. The bacterial sludge is mixed with a protectant and then freeze-dried to obtain freeze-dried powder of *Streptococcus thermophilus* subsp. DY-001. Alternatively, in this invention, *Streptococcus thermophilus* subsp. DY-001 is activated and cultured in M17 liquid medium. After activation, it is inoculated into the fermentation culture medium at an inoculum rate of 2-4% (v / v) for expansion and fermentation. The fermentation temperature of this invention is 36-38℃, preferably 37℃; the fermentation time is 14-48h, preferably 18-20h. The culture product is centrifuged, preferably at 4℃ and 8000r / min for 20min. After centrifugation, the supernatant is discarded to obtain bacterial sludge. The bacterial sludge is thoroughly mixed with a preservative at a mass ratio of 1:2. The preservative is an aqueous solution containing skim milk powder, trehalose, sucrose, and lactose, which is then sterilized to prepare a freeze-dried preservative. The preservative composition includes 10-15g / L skim milk powder, 10-15g / L trehalose, 2-6g / L sucrose, and 6-10g / L lactose, preferably including 12-13g / L skim milk powder, 12-13g / L trehalose, 3-5g / L sucrose, and 7-8g / L lactose. The sterilization condition is preferably high-temperature sterilization. The preferred method of this invention is to pre-freeze for 3 hours followed by freeze-drying at a cold trap temperature of -50°C for 48 hours. The freeze-dried powder provided by this invention has a viable bacterial count of 2.0 × 10⁻⁶. 11 ~5×10 11 CFU / g, the 1L fermentation culture product of the present invention is preferably obtained to obtain 8~14g of freeze-dried powder.

[0038] Preferably, the fermentation culture medium comprises 100-130 g / L glucose, 100-130 g / L lactose, 80-110 g / L honeysuckle powder, 80-110 g / L lily powder, 80-110 g / L loquat powder, and 80-110 g / L jujube powder. When using this culture medium, the fermentation product is directly freeze-dried to obtain freeze-dried powder of *Streptococcus thermophilus* subsp. *salivarius* DY-001. As an optional embodiment, *Streptococcus thermophilus* subsp. *salivarius* DY-001 is activated and cultured in M17 liquid medium, and then inoculated into the fermentation culture medium at an inoculum rate of 2-4% (v / v) for fermentation. The fermentation temperature of this invention is 36-38℃, preferably 37℃; the fermentation time is 14-48 h, preferably 35-40 h. Preferably, the fermentation culture product is pre-frozen for 3 hours and then freeze-dried at a cold trap temperature of -50°C for 48 hours. The freeze-dried powder provided by this invention has a viable bacterial count of 2.0 × 10⁻⁶. 10 ~4×10 10 CFU / g, the 1L fermentation culture product of the present invention is preferably obtained to obtain 8~14g of freeze-dried powder.

[0039] This invention also provides the application of the above-mentioned *Streptococcus thermophilus* subsp. DY-001, bacteriocins, or lyophilized powder in the preparation of products that inhibit pathogenic bacteria, drugs for relieving or treating upper respiratory tract infections, and drugs for adjuvant treatment of adenoid hypertrophy in children. *Streptococcus thermophilus* subsp. DY-001 can competitively inhibit the colonization of pathogenic bacteria (including *Streptococcus pneumoniae*, β-hemolytic streptococci, *Streptococcus pyogenes*, *Escherichia coli*, and *Staphylococcus aureus*), enhance mucosal barrier function, and regulate the Th1 / Th2 balance. The bacteriocins and lyophilized powders obtained by this invention have significant inhibitory effects on *Streptococcus pneumoniae*, β-hemolytic streptococci, *Streptococcus pyogenes*, *Escherichia coli*, and *Staphylococcus aureus*, and can effectively inhibit upper respiratory tract infections caused by these pathogens. The present invention describes that Streptococcus salivarius thermophilus subsp. DY-001, bacteriocins, or lyophilized powder can reduce recurrent nasopharyngeal inflammation, relieve or treat upper respiratory tract infections, alleviate pathological hyperplasia of adenoids, indirectly help shrink adenoids, and assist in the treatment of adenoid hypertrophy in children.

[0040] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] In this embodiment of the invention, the formula for M17 solid culture medium is as follows: 5.0g soybean peptone, 2.5g bovine bone peptone, 2.5g casein peptone, 2.5g yeast extract, 5.0g beef extract, 5.0g lactose, 0.5g sodium ascorbate, 19.0g sodium β-glycerophosphate, 0.25g magnesium sulfate, 13g agar, and 1000g water; the preparation method is as follows: after mixing all raw materials, adjust the pH to 7.2±0.2 with 20% sodium hydroxide solution; The formula for M17 liquid culture medium is as follows: 5.0g soybean peptone, 2.5g bovine bone peptone, 2.5g casein peptone, 2.5g yeast extract, 5.0g beef extract, 5.0g lactose, 0.5g sodium ascorbate, 19.0g sodium β-glycerophosphate, 0.25g magnesium sulfate, and 1000g water, with a pH of 7.2±0.2 and a temperature of 25℃. The preparation method is as follows: after mixing all the raw materials, adjust the pH to 7.2±0.2 with 20% sodium hydroxide solution.

[0042] In this embodiment of the invention, *Streptococcus salivarius* subsp. *thermophilus* Biohalo23 was purchased from Zhiyu Medical, *Streptococcus salivarius* subsp. *thermophilus* ST-21 was purchased from DuPont-Dannisco, and *Streptococcus salivarius* subsp. *thermophilus* STN26 was purchased from Zhongke Jiayi. *Streptococcus pneumoniae* ATCC49619 was purchased from Shanghai Preservation Biotechnology Center, *Streptococcus β-hemolyticus* CMCC(B)32210 was purchased from Qingdao Haibo Biotechnology, *Streptococcus pyogenes* BNCC337110 was purchased from Beina Chuanglian Biotechnology, *Escherichia coli* BNCC337304 was purchased from Beina Chuanglian Biotechnology, and *Staphylococcus aureus* ATCC29213 was purchased from Shanghai Preservation Biotechnology Center.

[0043] Unless otherwise specified, the following embodiments are all conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] Example 1

[0046] Isolation and identification of Streptococcus salivarius subsp. thermophilus DY-001:

[0047] Samples were collected from the oral cavities of healthy children, rapidly refrigerated, and sent to the laboratory for isolation. The samples were serially diluted with 0.85% physiological saline, and 1 mL was spread onto M17 agar plates and incubated at 37°C for 48 hours. After incubation, single colonies with different characteristics were selected from the plates and streaked onto M17 medium until pure cultures were obtained. A single strain was then selected, which was identified as *Streptococcus salivarius* subsp. *thermophilus* DY-001.

[0048] Streptococcus salivarius subsp. thermophilus DY-001 was inoculated onto M17 solid medium and incubated at 37°C for 72 hours. Colony morphology was observed using petri dishes. The results are as follows: Figure 1As shown in the figure. The results showed that the thermophilic subspecies of Streptococcus salivarius, strain DY-001, was round, milky white, opaque, with a raised, smooth, and moist surface and neat edges.

[0049] Gram staining and microscopic examination, as well as a catalase test, were performed on *Streptococcus salivarius* subsp. *thermophilus* DY-001. After Gram staining and microscopic examination, the strain was Gram-positive (G). + The bacteria exist in a spherical, long chain shape, as shown in the following results. Figure 2 As shown, the peroxidase test result was negative.

[0050] Streptococcus salivarius subsp. thermophilus DY-001 was inoculated into M17 liquid medium and cultured at 37℃ for 18 h. The cultured bacterial suspension was then inoculated into micro-biochemical tubes. After culture, the changes in the biochemical tubes were observed, and physiological and biochemical identification was performed. The results are shown in Table 1.

[0051] Table 1. Physiological and biochemical results of Streptococcus salivarius subsp. DY-001

[0052]

[0053] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0054] The results showed that the isolated single strains showed positive reactions in reaction tubes containing glucose, lactose, galactose, fructose, starch, trehalose, sorbitol, arginine, and sucrose.

[0055] The *Streptococcus salivarius* subsp. *thermophilus* DY-001 strain was sent to Shanjun (Xi'an) Biomedical Technology Co., Ltd. for whole-genome sequencing. The 16S rDNA sequence is shown in SEQ ID No. 1, and the result identified it as *Streptococcus salivarius* subsp. *thermophilus*. Streptococcus salivarius subsp. thermophilus ).

[0056] Streptococcus salivarius thermophilus subsp. Streptococcus salivarius subsp. thermophilus DY-001 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33409 and deposit date of January 14, 2025.

[0057] Example 2

[0058] Resistance of Streptococcus salivarius subsp. DY-001 to 22 antibiotics was determined using the disk agar diffusion method for drug susceptibility testing.

[0059] Streptococcus salivarius subsp. thermophilus DY-001 was inoculated into M17 liquid medium and incubated at 37°C for 16 h. OD 600A culture medium of *Streptococcus salivarius* subsp. *thermophilus* DY-001 was obtained and prepared for use. 100 μL of the culture medium was spread onto an MRS solid culture plate, and antimicrobial susceptibility testing discs were attached to the agar plate. After anaerobic incubation at 37°C for 24 h, the diameter of the inhibition zone was measured and compared with antibiotic resistance standards. The statistical results of the dosage and inhibition zone diameter of the 22 antibiotics are shown in Table 2.

[0060] Table 2. Results of drug resistance tests on Streptococcus salivarius subsp. thermophilus DY-001

[0061]

[0062] Note: S represents sensitive; M represents moderately sensitive; R represents resistant.

[0063] The results showed that *Streptococcus salivarius* subsp. *thermophilus* DY-001 was sensitive to 14 antibiotics, moderately sensitive to 7 antibiotics, and resistant to polymyxins.

[0064] Example 3

[0065] Antibacterial experiment of Streptococcus salivarius subsp. thermophilus DY-001:

[0066] Streptococcus thermophilus subsp. DY-001, Streptococcus thermophilus subsp. Biohalo23, Streptococcus thermophilus subsp. ST-21, and Streptococcus thermophilus subsp. STN26 were activated and cultured in M17 medium, and then inoculated at 3% (v / v) into M17 medium and cultured at 37°C for 18 hours for later use.

[0067] Streptococcus pneumoniae ATCC49619 was inoculated into brain and heart infusion broth and cultured at 37°C for 20 hours for later use.

[0068] β-hemolytic streptococci CMCC(B)32210 were inoculated into brain and heart broth medium (with 5% fetal bovine serum added) and cultured at 37°C for 20 hours for later use.

[0069] Streptococcus pyogenes BNCC337110 was inoculated into Columbia broth medium (with 5% fetal bovine serum added) and cultured at 37°C for 20 hours for later use.

[0070] Escherichia coli BNCC337304 and Staphylococcus aureus ATCC29213 were inoculated into nutrient broth medium and incubated at 37°C for 20 hours for later use.

[0071] Brain heart and brain extract broth solid medium, brain heart and brain extract broth solid medium containing 5% fetal bovine serum, Columbia broth solid medium containing 5% fetal bovine serum, and nutrient broth solid medium were cooled to 55°C respectively, and 10 were taken. 9Add 1 mL of each of the following bacterial suspensions (CFU / mL): Streptococcus pneumoniae ATCC49619, β-hemolytic streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213. Shake well, avoiding air bubbles, to maintain a viable count of 10-1. 7 The concentration was on the order of CFU / mL. The culture was then rapidly poured into plates pre-filled with Oxford cups. After the medium cooled and solidified, the Oxford cups were removed, and 200 μL of the culture medium of four Streptococcus salivarius subsp. thermophilus DY-001, Streptococcus salivarius subsp. thermophilus Biohalo23, Streptococcus salivarius subsp. thermophilus ST-21, and Streptococcus salivarius subsp. thermophilus STN26 was injected into each well. M17 liquid medium was used as a blank control. The plates were gently covered and placed upright in a 37°C tri-gas incubator for 2 days. The diameter of the inhibition zone was measured using calipers. Three replicates were performed for each strain. The results are shown in Table 3.

[0072] Table 3. Results of antibacterial experiments on four strains

[0073]

[0074] The results showed that four strains of Streptococcus salivarius—DY-001, ST-21, Biohalo23, and STN26—were effective against Streptococcus pneumoniae ATCC49619, β-hemolytic streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, and Escherichia coli BNCC337. 304 and Staphylococcus aureus ATCC29213 all showed inhibitory effects. Among them, Streptococcus salivarius subsp. thermophilicus DY-001 had the best inhibitory effect on the five pathogenic bacteria. Streptococcus salivarius subsp. thermophilicus Biohalo23 had an inhibitory effect on Escherichia coli BNCC337304 comparable to that of Streptococcus salivarius subsp. thermophilicus DY-001. The inhibitory effects of the other three Streptococcus salivarius subsp. thermophilicus strains were relatively poor.

[0075] Example 4

[0076] Adhesion test of Streptococcus salivarius subsp. thermophilus DY-001:

[0077] Streptococcus salivarius subsp. thermophilus DY-001, Streptococcus salivarius subsp. thermophilus Biohalo23, Streptococcus salivarius subsp. thermophilus ST-21, and Streptococcus salivarius subsp. thermophilus STN26 were activated and cultured in M17 medium, then inoculated at 3% (v / v) into M17 medium and cultured at 37°C for 18 h for later use. The bacterial cells were collected by low-temperature centrifugation, washed twice with PBS, and resuspended in oral keratinocyte complete culture medium (OKM, containing growth factors and antibiotics, purchased from Sciencell) to adjust the bacterial concentration to 1×10⁻⁶. 6 Prepare a bacterial suspension of CFU / mL for later use.

[0078] Oral keratinocytes (HOK) were removed from the liquid nitrogen container, thawed in a 37°C water bath, and then 5 ml of complete oral keratinocyte culture medium was added. The cells were gently shaken to distribute them evenly in the culture flask, and then incubated for 16 hours. After passage culture, the cell number increased to 102. 5 per mL.

[0079] Suspensions of *Streptococcus thermophilus* subsp. DY-001, *Streptococcus thermophilus* subsp. Biohalo23, *Streptococcus thermophilus* subsp. ST-21, and *Streptococcus thermophilus* subsp. STN26 were mixed with a concentration of 10... 5 0.5 mL of HOK (Hyperbacterium oxychloride) per mL was added to 1.5 mL EP tubes, gently pipetted to disperse evenly, and incubated at 37°C and 100 rpm for 2 h. Epithelial cells were then collected by filtering through a 10 μm pore membrane. The membrane was washed three times with DPBS, and the suspension was collected and centrifuged at low temperature. Epithelial cells were prepared into smears, air-dried, heat-fixed, and Gram-stained. Adhesion morphology changes were observed under an oil immersion microscope. One hundred epithelial cells were selected, and the total number of HOKs with four bacterial strains adhering to the surface and the total number of adhering bacteria were counted to calculate the adhesion index. The adhesion index and adhesion rate were calculated using the following formulas: Adhesion index = Number of adhering bacteria / Total number of counted cells; Adhesion rate (%) = (Number of cells with bacteria adhering to the surface / Total number of counted cells) × 100%. At least three independent replicates were performed. Results are as follows: Figure 3 As shown.

[0080] The results showed that the adhesion amount of Streptococcus salivarius thermophilus subsp. DY-001 to oral keratinocytes (HOK) was 39.72±2.15 CFU / cell, which was significantly different from that of Streptococcus salivarius subsp. ST-21. P <0.01), which is different from that of Streptococcus salivarius subsp. biohalo23. P <0.05), showing a significant difference compared to Streptococcus salivarius subsp. thermophilus STN26 ( P<0.01); there was a significant difference between *Streptococcus thermophilus* subsp. *salivarius* Biohalo23 and *Streptococcus thermophilus* subsp. *salivarius* ST-21 ( ). P <0.05); there was no significant difference between Streptococcus salivarius thermophilus subsp. STN26 and Streptococcus salivarius thermophilus subsp. ST-21.

[0081] Example 5

[0082] Isolation and antibacterial experiment of bacteriocin from Streptococcus salivarius subsp. thermophilus DY-001:

[0083] Streptococcus salivarius subsp. thermophilus DY-001 was activated using M17 liquid medium, inoculated at a rate of 3% (v / v) into M17 liquid medium, and incubated at 37°C for 18 hours. OD 600 The concentration was 5.00, and the culture medium of *Streptococcus salivarius* subsp. *thermophilus* DY-001 was obtained. The culture medium was centrifuged at 12000 rpm at 4°C to obtain the supernatant. The supernatant was divided into 4 portions, and ammonium sulfate was slowly added to each portion to achieve ammonium sulfate saturation of 50%, 60%, 70%, and 80%, respectively. The mixture was magnetically stirred at low temperature overnight, centrifuged at 8000 rpm for 15 min at 4°C, the supernatant was discarded, the precipitate was collected, and dried at 40°C to obtain the corresponding products (bacteriocin), which were designated as DY50, DY60, DY70, and DY80, respectively. Based on the precipitate weight, a bacteriocin solution of 0.1 mg / ml was prepared with sterile water for antibacterial experiments.

[0084] The antibacterial experiment procedure was the same as in Example 3, except that 200 μL of the prepared bacteriocin solutions DY50, DY60, DY70, and DY80 were injected into each well, and the results are shown in Table 4.

[0085] Table 4. Results of antibacterial experiments with different bacteriocin solutions

[0086]

[0087] The results showed that the bacteriocins metabolized by *Streptococcus thermophilus* subsp. DY-001 could be effectively isolated when the ammonium sulfate saturation was 60%, and had a good inhibitory effect on *Streptococcus pneumoniae* ATCC49619, *Streptococcus β-hemolyticus* CMCC(B)32210, *Streptococcus pyogenes* BNCC337110, *Escherichia coli* BNCC337304, and *Staphylococcus aureus* ATCC29213. When the ammonium sulfate saturation reached 80%, the bacteriocins could not be isolated or were destroyed, resulting in serious losses.

[0088] Example 6

[0089] 1. Culture medium screening for Streptococcus salivarius subsp. thermophilus DY-001:

[0090] (1) Carbon source: *Streptococcus thermophilus* subsp. DY-001 was treated with glucose, sucrose, lactose, mannose, arabinose, and trehalose. The carbon source was removed from M17 medium, while other components remained unchanged. The carbon source addition was 2% (g / mL). *Streptococcus thermophilus* subsp. DY-001 was activated using M17 medium. It was inoculated at 3% (v / v) into M17 medium supplemented with different carbon sources and incubated at 37℃ for 18 h. Viable cell counts were then determined. The results are shown in Table 5.

[0091] Table 5. Viable counts of Streptococcus salivarius subsp. thermophilus DY-001 with different carbon sources

[0092]

[0093] The table above shows that *Streptococcus thermophilus* subspecies DY-001 can effectively utilize glucose, sucrose, and lactose, but its utilization of mannose, arabinose, and trehalose is not high. Glucose and lactose provide the best growth for *Streptococcus thermophilus* subspecies DY-001. Therefore, glucose and lactose will be selected as the two carbon sources for the growth of *Streptococcus thermophilus* subspecies DY-001, with a glucose-to-lactose ratio of 1:1.

[0094] (2) Nitrogen source: *Streptococcus thermophilus* subsp. DY-001 was treated with the following nitrogen sources: yeast extract, malt extract, tryptone, bovine bone peptone, soybean peptone, and beef extract. The nitrogen source was removed from M17 medium, while other components remained unchanged. The nitrogen source addition was 2% (g / mL). *Streptococcus thermophilus* subsp. DY-001 was activated using M17 medium. It was inoculated at 3% (v / v) into M17 medium supplemented with different nitrogen sources and incubated statically at 37℃ for 18 h. Viable cell counts were then determined. The results are shown in Table 6.

[0095] Table 6. Viable counts of Streptococcus salivarius subsp. thermophilus DY-001 under different nitrogen sources

[0096]

[0097] As shown in the table above, Streptococcus salivarius subsp. thermophilus DY-001 grows well and reproduces rapidly in yeast extract, malt extract, and soybean peptone. These three nitrogen sources will be selected as the growth nitrogen sources for Streptococcus salivarius subsp. thermophilus DY-001 in the future.

[0098] (3) Orthogonal Experiment: An orthogonal experiment was conducted using the following per liter concentrations of *Streptococcus thermophilus* subsp. *salivarius* DY-001: yeast extract (15 g / L, 20 g / L, 25 g / L), malt extract (10 g / L, 15 g / L, 20 g / L), soybean peptone (17 g / L, 20 g / L, 23 g / L), and glucose-lactose mixture (20 g / L, 25 g / L, 30 g / L). Glucose and inorganic salts remained unchanged. The results are as follows:Figure 4 As shown in the figure, "glucose-lactose mixture" represents a glucose-lactose mixture (glucose to lactose ratio is 1:1).

[0099] The results showed that the optimal addition ratios of glucose and lactose 25 g / L, yeast extract 20 g / L, malt extract 15 g / L, and soybean peptone 23 g / L were used in the fermentation of Streptococcus salivarius subsp. thermophilus DY-001.

[0100] The fermentation medium for Streptococcus salivarius subsp. thermophilus DY-001 was prepared as follows: glucose 12.5 g / L, lactose 12.5 g / L, yeast extract 20 g / L, malt extract 15 g / L, soybean peptone 23 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.1 g / L, and manganese sulfate 0.05 g / L.

[0101] 2. Preparation of freeze-dried powder of Streptococcus salivarius subsp. thermophilus DY-001:

[0102] After activating Streptococcus thermophilus subsp. DY-001 for two generations in M17 liquid medium, it was inoculated into the fermentation medium of Streptococcus thermophilus subsp. DY-001 from step 1 at an inoculum rate of 3% (v / v) and cultured at 37℃ for 18 hours. The activated and expanded seed culture was centrifuged at 8000 rpm for 20 minutes at 4℃, and the supernatant was discarded to collect the bacterial cells. Skim milk powder, trehalose, sucrose, and lactose (12 g / L skim milk powder, 12 g / L trehalose, 5 g / L sucrose, and 8 g / L lactose) were added to the aqueous solution, sterilized (110℃ for 20 minutes), and cooled to 4℃ to prepare a freeze-drying protectant. The bacterial sludge and freeze-drying protectant were thoroughly mixed at a mass ratio of 1:2, pre-frozen for 3 hours, and then freeze-dried at -50℃ for 48 hours. The freeze-dried bacterial powder was collected and named DYGH.

[0103] The viable bacteria count of the lyophilized powder was 2.0 × 10⁻⁶. 11 ~5×10 11 CFU / g.

[0104] 3. Screening of medicinal and edible raw materials from fermentation of Streptococcus salivarius subsp. thermophilus DY-001

[0105] Culture media were prepared using 12.5% ​​glucose + 12.5% ​​lactose, with 10% honeysuckle powder, 10% dandelion powder, 10% lily powder, 10% loquat powder, 10% jujube powder, and 10% ginger powder added respectively. The effects of these six medicinal and edible ingredients on the growth promotion of Streptococcus salivarius subsp. thermophilus DY-001 were then investigated. The preparation methods for the six culture media are as follows: Weigh 12.5g glucose, 12.5g lactose, and 10g honeysuckle powder, add 65g water, mix thoroughly, and sterilize at 121℃ for 15 minutes; Weigh 12.5g glucose, 12.5g lactose, and 10g dandelion powder, add 65g water, mix thoroughly, and sterilize at 121℃ for 15 minutes; Weigh 12.5g glucose, 12.5g lactose, and 10g lily powder, add 65g water, mix thoroughly, and sterilize at 121℃ for 15 minutes. Set aside. Weigh 12.5g glucose, 12.5g lactose, and 10g loquat powder, add 65g water, mix thoroughly, and sterilize at 121℃ for 15 minutes. Set aside. Weigh 12.5g glucose, 12.5g lactose, and 10g jujube powder, add 65g water, mix thoroughly, and sterilize at 121℃ for 15 minutes. Set aside. Weigh 12.5g glucose, 12.5g lactose, and 10g ginger powder, add 65g water, mix thoroughly, and sterilize at 121℃ for 15 minutes. Set aside.

[0106] After activating Streptococcus thermophilus subsp. DY-001 for two generations in M17 liquid medium, it was inoculated into six different culture media at a 3% (v / v) inoculum and cultured at 37°C for 18 hours. The viable count was then measured, and the results are shown in Table 7.

[0107] Table 7. Viable counts of Streptococcus salivarius subsp. thermophilus DY-001 in different fermented medicinal and edible raw materials.

[0108]

[0109] The results showed that the six medicinal and edible ingredients significantly affected the growth of *Streptococcus thermophilus* subsp. DY-001. Honeysuckle powder, lily powder, loquat powder, and jujube powder promoted the growth of the strain, while dandelion powder and ginger powder inhibited its growth, with ginger powder showing the most significant inhibitory effect. Therefore, honeysuckle powder, lily powder, loquat powder, and jujube powder were used to culture *Streptococcus thermophilus* subsp. DY-001.

[0110] The culture medium containing both medicinal and edible ingredients was prepared as follows: glucose 125g / L, lactose 125g / L, honeysuckle powder 100g / L, lily powder 100g / L, loquat powder 100g / L, and jujube powder 100g / L.

[0111] 4. Preparation of freeze-dried powder of Streptococcus salivarius subsp. thermophilus DY-001 (containing medicinal and edible materials)

[0112] After activating Streptococcus salivarius subsp. thermophilicus DY-001 for two generations in M17 liquid medium, it was inoculated into the food-medicine homology medium of step 3 at an inoculation rate of 3% (v / v), cultured at 37℃ for 36h, pre-frozen for 3h and then freeze-dried at -50℃ for 48h. The freeze-dried bacterial powder was collected and named DYGN.

[0113] The viable bacteria count of the lyophilized powder was 2.0 × 10⁻⁶. 10 ~4×10 10 CFU / g.

[0114] 5. Antibacterial experiment of two freeze-dried powders of Streptococcus salivarius subsp. thermophilus DY-001

[0115] A certain sample of freeze-dried Streptococcus salivarius subsp. thermophilus DY-001 (DYGH and DYGN) was added to sterile physiological saline to prepare a final viable count concentration of 10⁻⁶. 9 CFU / mL was used to obtain DYGH solution and DYGN solution for later use.

[0116] The antibacterial experiment procedure was the same as in Example 3, except that 200 μL of the prepared DYGH solution and DYGN solution were injected into each well, and the results are shown in Table 8.

[0117] Table 8. Antibacterial test results of DYGH and DYGN

[0118]

[0119] The results showed that the freeze-dried powders DYGH and DYGN of Streptococcus salivarius subsp. thermophilus DY-001 had inhibitory effects on five strains: Streptococcus pneumoniae ATCC49619, β-hemolytic streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213. The inhibitory effects were not significantly different from those of liquid-cultured Streptococcus salivarius subsp. thermophilus DY-001 and its bacteriocins.

[0120] Example 7

[0121] Animal experiments on respiratory tract infection of mice with Streptococcus salivarius subsp. thermophilus DY-001 used the lyophilized powder prepared in Example 6.

[0122] Ninety healthy 8-week-old SPF-grade BABL / c mice, half male and half female, were purchased from the Animal Experiment Center of Hebei Medical University.

[0123] Mice were randomly divided into a blank control group, a model group, a positive control group, experimental group 1 (high-dose DYGH), experimental group 2 (medium-dose DYGH), experimental group 3 (low-dose DYGH), experimental group 4 (high-dose DYGN), experimental group 5 (medium-dose DYGN), and experimental group 6 (low-dose DYGN), with 10 mice in each group. Following the modeling method of Standiford TJ et al., except for the blank control group, the other eight groups of mice were anesthetized with ether, and 50 μL of a mixture of five strains (Streptococcus pneumoniae ATCC49619, β-hemolytic streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213) was instilled into their nasal cavity (bacterial concentration 1×10⁻⁶). 8 (CFU / mL, mixed in equal proportions of each bacterium) to be automatically inhaled. Administer the drug starting on the day of infection (dissolve the drug in sterile saline and administer by gavage), and continue for 7 days. Dosage is shown in Table 9.

[0124] Table 9 Dosage

[0125]

[0126] Mice were weighed before and after the experiment to compare changes. On day 7, mice were sacrificed, and lung tissue was removed. The wet weight of the lung tissue was measured, and then it was baked in a 60℃ constant-temperature dry oven for 72 hours. The dry weight was measured, and the wet / dry weight ratio was calculated. Lung index = mouse lung weight (g) / mouse body weight (g) × 100%. Results are shown in Table 10.

[0127] Table 10 Changes in body weight and lung tissue of mice before and after the experiment in each group

[0128]

[0129] Note: *: indicates comparison with the control group P <0.05; #: indicates that compared to the model group, P <0.05.

[0130] The results showed that after mice were instilled with a mixture of five bacterial strains through their nasal cavity, the model group exhibited poor mental state, decreased appetite, reduced water intake, decreased activity, increased nasal secretions, rapid breathing, and coughing symptoms. After 7 days, their body weight decreased significantly. P <0.05). The control group, however, was in good spirits, drank and ate normally, was active, had stable breathing, and no cough symptoms. Compared with the model group, all experimental groups showed significant improvement in the above symptoms ( P <0.05), and significantly different from the control group. The wet / dry weight ratio of the model group was significantly higher than that of the normal control group ( P <0.05), the experimental group was significantly lower than the model group (P <0.05), there was no significant difference between the experimental group and the control group, and the trend of lung index change in each group of mice was consistent with the wet / dry weight ratio results.

[0131] On day 7 of the experiment, mice were sacrificed, and lung tissue was removed. The lung tissue was rinsed with pre-cooled PBS to remove blood. Approximately 100 mg of lung tissue was weighed, dried on filter paper, placed in a beaker, and PBS (9 times the weight and volume of the lung tissue block) was added. The tissue was then cut, homogenized, and ground. The mixture was centrifuged at 5000 rpm for 10 minutes in a low-temperature centrifuge, and the supernatant was collected. The IL-6 and TNF-α levels in the lung tissue of each group were detected using an ELISA kit (Bailingke Biotechnology). The results are shown in Table 11.

[0132] Table 11 IL-6 and TNF-α levels in mice of each group

[0133]

[0134] Note: *: indicates comparison with the control group P <0.05; #: indicates that compared to the model group, P <0.05.

[0135] The results showed that 7 days after infection, the levels of IL-6 and TNF-α in the lung tissue homogenate of the model group mice were significantly increased and significantly higher than those in the control group. P <0.05). The levels of IL-6 and TNF-α in the positive control group and each experimental group were significantly different from those in the model group. P <0.05). The results showed that Streptococcus salivarius subsp. thermophilus DY-01 could alleviate upper respiratory tract symptoms in mice with bacterial infection, and both lyophilized bacterial powders showed good antibacterial effects and enhanced immune function.

[0136] Example 8

[0137] The lyophilized powder (DYGH) prepared in Example 6 was used in a trial for children with adenoid hypertrophy.

[0138] 1. Research Subjects

[0139] Inclusion criteria: Age 3-10 years, diagnosed with adenoid hypertrophy (grade II-III), and not having undergone surgical treatment.

[0140] Exclusion criteria: Comorbid severe cardiopulmonary disease or immunodeficiency.

[0141] Grouping: The adenoid hypertrophy control group (conventional treatment) and the adenoid hypertrophy experimental group (conventional treatment + oral preparation of Streptococcus thermophilus subsp. saliva).

[0142] 2. Intervention Plan

[0143] The adenoid hypertrophy experimental group received additional treatment with Streptococcus salivarius subsp. thermophilus DY-001, with a treatment course of 3 months.

[0144] 3. Observation indicators

[0145] Key indicators: changes in adenoid volume (assessed by lateral nasopharyngeal X-ray or endoscopy), and apnea-hypopnea index (AHI).

[0146] Secondary indicators: nasal congestion score, snoring frequency, number of upper respiratory tract infections, and inflammatory factor levels.

[0147] Safety: Record adverse events such as gastrointestinal reactions and allergies.

[0148] The following are children with adenoid hypertrophy who volunteered to try the treatment. They were randomly divided into two groups: one group received conventional treatment, and the other group received conventional treatment plus DY-001 (a probiotic containing Streptococcus thermophilus). The dosage was 1 gram per sachet per dose, containing at least 3 billion active probiotics of DY-001, twice a day, taken orally half an hour after meals without water (do not drink water for 2 hours after taking the sachet). The effects were observed after 90 days of treatment according to different stages of symptoms. The results are shown in Table 12.

[0149] Table 12 Results of the trial of oral medication for adenoid hypertrophy in children

[0150]

[0151] The results showed that Streptococcus thermophilus subsp. DY-001, as an adjunct therapy, can effectively reduce the inflammatory response and clinical symptoms of adenoid hypertrophy in children, with good safety. Compared with drug therapy alone, the combination with probiotics can significantly improve symptoms and reduce the recurrence rate, and avoid irreversible complications caused by surgical trauma and organ removal.

[0152] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thermophilic subspecies of Streptococcus salivarius ( Streptococcus salivarius subsp .thermophilus DY-001, characterized in that, The preservation number of the thermophilic subspecies of Streptococcus salivarius DY-001 is CGMCC No. 33409.

2. A method for preparing bacteriocin, characterized in that, The process includes the following steps: fermenting and culturing the thermophilic subspecies of Streptococcus salivarius DY-001 as described in claim 1, and isolating bacteriocins from the fermentation culture product.

3. A method for preparing a freeze-dried powder, characterized in that, The process includes the following steps: fermenting and culturing Streptococcus thermophilus subsp. DY-001 as described in claim 1 to obtain a fermentation culture product, and preparing freeze-dried powder of Streptococcus thermophilus subsp. DY-001 from the fermentation culture product.

4. The preparation method according to claim 3, characterized in that, The fermentation culture medium comprises 10-15 g / L glucose, 10-15 g / L lactose, 18-22 g / L yeast extract, 10-20 g / L malt extract, 20-25 g / L soybean peptone, 3-7 g / L sodium acetate, 1-3 g / L diammonium hydrogen citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate, and 0.02-0.08 g / L manganese sulfate.

5. The preparation method according to claim 3, characterized in that, The culture medium for fermentation includes 100-130 g / L glucose, 100-130 g / L lactose, 80-110 g / L honeysuckle powder, 80-110 g / L lily powder, 80-110 g / L loquat powder, and 80-110 g / L jujube powder.

6. The freeze-dried powder prepared by the preparation method according to any one of claims 3 to 5.

7. The application of the bacteriocin prepared by the method described in claim 1 (specifically, *Streptococcus salivarius* subsp. *thermophilus* DY-001) or the bacteriocin prepared by the method described in claim 2, or the lyophilized powder described in claim 6, in the preparation of products that inhibit pathogenic bacteria, characterized in that... The pathogens are Streptococcus pneumoniae, β-hemolytic streptococci, Streptococcus pyogenes, Escherichia coli, and Staphylococcus aureus.

8. The use of the bacteriocin prepared by the method described in claim 1 (specifically, *Streptococcus salivarius* subsp. DY-001) or the bacteriocin prepared by the method described in claim 2 or the lyophilized powder described in claim 6 in the preparation of a drug for relieving or treating upper respiratory tract infections caused by pathogens, wherein the pathogens are *Streptococcus pneumoniae*, *β-hemolytic streptococci*, *Streptococcus pyogenes*, *Escherichia coli*, and *Staphylococcus aureus*.

9. The use of the bacteriocin prepared by the method described in claim 1 (Salinomyces thermophilic subsp. DY-001), the bacteriocin prepared by the method described in claim 2, or the lyophilized powder described in claim 6 in the preparation of a drug for the adjunctive treatment of adenoid hypertrophy in children.

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