Streptococcus salivarius subsp. Thermophilus DY-001 and application of bacteriocin and freeze-dried powder thereof in treatment of upper respiratory infection

Abstract

The invention provides streptococcus salivarius subsp. Thermophilus DY-001 and application of bacteriocin and freeze-dried powder thereof in treatment of upper respiratory tract infection, and belongs to the technical field of microorganisms. The invention provides a streptococcus salivarius subsp. Thermophilus DY-001, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.33409. The invention further provides bacteriocin or freeze-dried powder obtained based on the streptococcus salivarius subsp. Thermophilus DY-001, and a preparation method of the bacteriocin or freeze-dried powder. The bacteriocin of the streptococcus salivarius subsp. Thermophilus DY-001 and the freeze-dried powder of the streptococcus salivarius subsp. Thermophilus DY-001, disclosed by the invention, have a remarkable bacteriostatic effect and can be used for relieving or treating upper respiratory tract infection and assisting in treating adenoidal hypertrophy of children.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of thermophilic Streptococcus salivarius subspecies DY-001 and bacteriocin and lyophilized powder thereof in treating upper respiratory tract infection. Background Art

[0002] Upper respiratory tract infections (such as colds, pharyngitis, sinusitis, etc.) are mainly caused by viruses (such as rhinoviruses, influenza viruses) or bacteria (such as Streptococcus pneumoniae, etc.). Children have a higher incidence rate 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), but the abuse of antibiotics 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 mostly seen in children. It is mainly caused by repeated nasopharyngeal inflammation (such as rhinitis, sinusitis, upper respiratory tract infection) or allergic reaction, which leads to pathological proliferation of lymphoid tissue, causing nasal congestion, snoring, sleep apnea and other symptoms. In severe cases, adenoid facies or abnormal cardiopulmonary function may occur. At present, the treatment methods are mainly drugs (such as nasal spray hormones, antihistamines), traditional Chinese medicine (massage, Chinese medicine) and surgical treatment (adenoidectomy). Long-term medication may have side effects. Traditional Chinese medicine treatment is generally effective. The cycle cost is expensive and delays the best treatment time. There is a risk of trauma in surgery. The harm of adenoidectomy to the human body has not yet been deciphered by medicine.

[0004] In recent years, probiotics, as a new type of biological agent, have gradually become a research hotspot for the prevention and treatment of respiratory tract infections due to their characteristics of regulating immunity and inhibiting pathogens. Many clinical studies have shown that probiotics can competitively inhibit pathogens, regulate mucosal immunity, and enhance epithelial barrier function. For example, the thermophilic subspecies of Streptococcus salivarius Biohalo23 has been shown to relieve the symptoms of upper respiratory tract infections by inhibiting harmful bacteria in the upper respiratory tract (such as Campylobacter conjunctivae and Neisseria oralis), increasing the abundance of beneficial bacteria, and maintaining the balance of the flora. Although probiotics have shown potential in the prevention and treatment of respiratory tract infections, their clinical application still faces the following challenges: the mechanism of action and effect of different probiotic strains vary significantly, traditional liquid probiotic preparations are easily affected by temperature and humidity, and the survival rate of live bacteria is low. Most probiotic preparations are administered orally and need to pass through the gastrointestinal environment (gastric acid, bile) to reach the respiratory tract, resulting in a significant reduction in the number of live bacteria and a low rate of respiratory colonization. Therefore, continuing to screen probiotics with significant efficacy in relieving upper respiratory tract infections and developing related preparations remains the key to research in this field. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the first object of the present invention is to provide a thermophilic Streptococcus salivarius subsp. 嗜热唾液链球菌)DY-001, the deposit number is CGMCCNo.33409.

[0006] The second object of the present invention is to provide a bacteriocin separated from the fermentation culture of Streptococcus salivarius thermophilic subspecies DY-001, which has a significant antibacterial effect.

[0007] The third object of the present invention is to provide a freeze-dried powder and a preparation method thereof, which have a good therapeutic effect on alleviating upper respiratory tract infections.

[0008] The fourth object of the present invention is to provide the use of the thermophilic Streptococcus salivarius subspecies DY-001 or its bacteriocin or its lyophilized powder in the preparation of products for inhibiting pathogens, drugs for relieving or treating upper respiratory tract infections, and drugs for assisting the treatment of adenoid hypertrophy in children.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a Streptococcus salivarius thermophilic subspecies ( 唾液链球菌 嗜热亚种 ) DY-001, the preservation number of the thermophilic Streptococcus salivarius subspecies DY-001 is CGMCC No.33409.

[0010] The present invention provides a method for preparing bacteriocin, comprising the following steps: fermenting and culturing the thermophilic subspecies of Streptococcus salivarius DY-001, and separating and obtaining bacteriocin from the fermentation and culturing product.

[0011] The present invention provides a bacteriocin prepared by the above preparation method.

[0012] The present invention provides a method for preparing a freeze-dried powder, comprising the following steps: fermenting and culturing the thermophilic Streptococcus salivarius subspecies DY-001 to obtain a fermentation culture product, and preparing the thermophilic Streptococcus salivarius subspecies DY-001 freeze-dried powder from the fermentation culture product.

[0013] Preferably, the culture medium for the fermentation culture 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 soy 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.

[0014] Preferably, the culture medium for the fermentation culture comprises 100-130 g / L of glucose, 100-130 g / L of lactose, 80-110 g / L of honeysuckle pollen, 80-110 g / L of lily powder, 80-110 g / L of loquat powder and 80-110 g / L of red date powder.

[0015] The invention provides freeze-dried powder prepared by the preparation method.

[0016] The present invention provides the use of the above-mentioned Streptococcus salivarius thermophilus subspecies DY-001 or bacteriocin or lyophilized powder in preparing products for inhibiting pathogens, wherein the pathogens include Streptococcus pneumoniae, beta-hemolytic Streptococcus, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus.

[0017] The present invention provides the use of the thermophilic Streptococcus salivarius subspecies DY-001 or bacteriocin or lyophilized powder in preparing a medicine for relieving or treating upper respiratory tract infection.

[0018] The present invention provides the use of the thermophilic Streptococcus salivarius subspecies DY-001 or bacteriocin or lyophilized powder in preparing a medicament for auxiliary treatment of children's adenoid hypertrophy.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The present invention obtains a strain of thermophilic Streptococcus salivarius subspecies DY-001 for the first time, which can colonize the upper respiratory tract mucosa. The present invention separates and obtains thermophilic Streptococcus salivarius subspecies DY-001 bacteriocin based on thermophilic Streptococcus salivarius subspecies DY-001, and prepares lyophilized powder based on thermophilic Streptococcus salivarius subspecies DY-001. The thermophilic Streptococcus salivarius subspecies DY-001 and its bacteriocin and lyophilized powder of the present invention have good antibacterial effect, can significantly inhibit the growth of Streptococcus pneumoniae, β-hemolytic Streptococcus, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus, maintain the stability of respiratory flora, inhibit the excessive growth of harmful bacteria, achieve the effect of significantly alleviating or treating upper respiratory tract infection, and can be used for auxiliary treatment of adenoid hypertrophy in children.

[0020] Biological Deposit Description Streptococcus salivarius thermophilus subsp. DY-001, classified as Streptococcus salivarius thermophilus subsp. 链球菌 嗜热唾液链球菌 ), depository: China General Microbiology Center of Microbiological Culture Collection (CGMCC for short), depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No.33409, deposit date January 14, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :The macroscopic morphology of Streptococcus salivarius thermophilus subsp. DY-001 colonies in the plate; Figure 2 :Microscopic morphology of Streptococcus salivarius subsp. thermophilus strain DY-001; Figure 3:Adhesion results of different probiotics to oral epithelial keratinocytes (HOK); Figure 4 : Results of orthogonal test of fermentation of Streptococcus salivarius thermophilus subsp. DY-001 with yeast extract, malt extract, soy peptone and glucose-lactose mixture. DETAILED DESCRIPTION

[0022] The present invention provides a Streptococcus salivarius thermophilic subspecies ( 唾液链球菌 嗜热亚种 ) DY-001, the deposit number of the thermophilic Streptococcus salivarius DY-001 is CGMCC No. 33409. The thermophilic Streptococcus salivarius DY-001 of the present invention is isolated and purified from the oral cavity of a healthy child, and it is proved by identification that it belongs to the thermophilic Streptococcus salivarius ( 嗜热唾液链球菌 ), deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 14, 2025. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] The thermophilic subspecies of Streptococcus salivarius DY-001 of the present invention is sensitive to multiple antibiotics, can colonize the upper respiratory tract mucosa, compete with pathogenic bacteria for nutrition and adhesion sites, reduce pathogen proliferation, activate the mucosal immune system, promote IgA secretion and immune cell activity, enhance local defense capabilities, thereby maintaining the stability of respiratory flora, inhibiting the excessive growth of harmful bacteria, and alleviating upper respiratory tract infections. The fermentation medium of the thermophilic subspecies of Streptococcus salivarius DY-001 of the present invention can be selected as M17 liquid culture medium. As an optional embodiment, the thermophilic subspecies of Streptococcus salivarius DY-001 is activated and cultured in M17 liquid culture medium. After activation, the thermophilic subspecies of Streptococcus salivarius DY-001 is inoculated in M17 liquid culture medium at an inoculum size of 2-4% (v / v) for expansion culture and fermentation culture, and the inoculum size is preferably 3%. The temperature of the fermentation culture of the present invention is 36-38°C, preferably 37°C; the fermentation culture time is 14-48h, preferably 18-24h.

[0024] Preferably, the fermentation medium of the thermophilic Streptococcus salivarius subspecies DY-001 of the present invention comprises 10-15 g / L of glucose, 10-15 g / L of lactose, 18-22 g / L of yeast extract, 10-20 g / L of malt extract, 20-25 g / L of soy peptone, 3-7 g / L of sodium acetate, 1-3 g / L of diammonium hydrogen citrate, 1-3 g / L of dipotassium hydrogen phosphate, 0.05-0.15 g / L of magnesium sulfate and 0.02-0.08 g / L of manganese sulfate; more preferably, it comprises 12.5 g / L of glucose, 12.5 g / L of lactose, 20 g / L of yeast extract, 15 g / L of malt extract, 23 g / L of soy peptone, 5 g / L of sodium acetate, 2 g / L of diammonium hydrogen citrate, 2 g / L of dipotassium hydrogen phosphate, 0.1 g / L of magnesium sulfate and 0.05 g / L of manganese sulfate.

[0025] Preferably, the fermentation medium of the thermophilic Streptococcus salivarius subspecies DY-001 described in the present invention comprises 100-130 g / L of glucose, 100-130 g / L of lactose, 80-110 g / L of honeysuckle pollen, 80-110 g / L of lily powder, 80-110 g / L of loquat powder and 80-110 g / L of red date powder; more preferably comprises 110-120 g / L of glucose, 110-120 g / L of lactose, 90-100 g / L of honeysuckle pollen, 90-100 g / L of lily powder, 90-100 g / L of loquat powder and 90-100 g / L of red date powder.

[0026] The culture medium of the present invention is prepared with water, and the water is preferably purified water. The present invention can increase the number of viable bacteria of Streptococcus salivarius thermophilus subspecies DY-001 by improving the composition of the culture medium.

[0027] The present invention also provides a method for preparing bacteriocin, comprising the following steps: fermenting and culturing the thermophilic Streptococcus salivarius subspecies DY-001, and separating and obtaining bacteriocin from the fermentation and culturing product.

[0028] The temperature of the fermentation culture of the present invention is 36-38°C, preferably 37°C; the fermentation culture time is 14-48h, preferably 18-20h. The method for isolating bacteriocin of the present invention is preferably an ammonium sulfate salting-out method. The present invention centrifuges the fermentation culture product (fermentation broth) to obtain the supernatant, and the centrifugation conditions are preferably centrifuged at 4°C and 12000r / min for 10-15min. In the present invention, ammonium sulfate is added to the centrifuged supernatant to make its saturation reach 50-65%, preferably 55-60%; the solution after adding ammonium sulfate is magnetically stirred at low temperature overnight, and the precipitate is collected by centrifugation to obtain bacteriocin, and the centrifugation conditions are preferably centrifuged at 4°C and 8000r / min for 15min.

[0029] The present invention also provides a bacteriocin prepared by the above preparation method, wherein the bacteriocin can significantly inhibit the growth of Streptococcus pneumoniae, beta-hemolytic Streptococcus, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus.

[0030] The present invention also provides a method for preparing a freeze-dried powder, comprising the following steps: fermenting and culturing the above-mentioned Streptococcus salivarius thermophilus subspecies DY-001 to obtain a fermentation culture product, and preparing the Streptococcus salivarius thermophilus subspecies DY-001 freeze-dried powder from the fermentation culture product.

[0031] Preferably, the fermentation culture medium includes 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 soy 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 this culture medium is used, the fermentation culture product is centrifuged to obtain bacterial mud, which is mixed with a protective agent and then freeze-dried to obtain a lyophilized powder of Streptococcus salivarius thermophilus subspecies DY-001. As an optional embodiment, the present invention activates and cultures Streptococcus salivarius thermophilus subspecies DY-001 in M17 liquid culture medium, and after activation, it is inoculated into the fermentation culture medium at an inoculation rate of 2-4% (v / v) for expansion culture and fermentation culture. The temperature of the fermentation culture of the present invention is 36-38°C, preferably 37°C; the fermentation culture time is 14-48h, preferably 18-20h. The present invention centrifuges the culture product, and the centrifugation conditions are preferably centrifuged at 4°C and 8000r / min for 20min. After centrifugation, the supernatant is discarded to obtain bacterial mud, and the bacterial mud is fully mixed with the protective agent in a mass ratio of 1:2. The protective agent is an aqueous solution in which skim milk powder, trehalose, sucrose, and lactose are added respectively, sterilized, and a freeze-dried protective agent is prepared; the protective agent ingredients include skim milk powder 10-15g / L, trehalose 10-15g / L, sucrose 2-6g / L, and lactose 6-10g / L, preferably skim milk powder 12-13g / L, trehalose 12-13g / L, sucrose 3-5g / L, and lactose 7-8g / L; the sterilization conditions are preferably high temperature sterilization. The present invention preferably freezes and dries the mixture after pre-freezing for 3 hours, with the cold trap temperature at -50°C and freeze drying for 48 hours. The viable bacterial count of the freeze-dried powder provided by the present invention is 2.0×10 11 ~5×10 11 CFU / g, 1L of fermentation culture product of the present invention preferably obtains 8-14g of freeze-dried powder.

[0032] Preferably, the fermentation culture medium includes 100-130 g / L of glucose, 100-130 g / L of lactose, 80-110 g / L of honeysuckle pollen, 80-110 g / L of lily powder, 80-110 g / L of loquat powder and 80-110 g / L of red date powder. When this culture medium is used, the fermentation culture product is directly freeze-dried to obtain the thermophilic subspecies of Streptococcus salivarius DY-001 freeze-dried powder. As an optional embodiment, the present invention activates and cultures the thermophilic subspecies of Streptococcus salivarius DY-001 in M17 liquid culture medium, and after activation, it is inoculated into the fermentation medium at an inoculation rate of 2-4% (v / v) for fermentation culture. The temperature of the fermentation culture of the present invention is 36-38°C, preferably 37°C; the fermentation culture time is 14-48h, preferably 35-40h. The present invention preferably freezes the fermentation culture product for 3 hours before freeze drying, the cold trap temperature is -50°C, and the freeze drying is performed for 48 hours. The viable bacterial count of the freeze-dried powder provided by the present invention is 2.0×10 10 ~4×10 10 CFU / g, 1L of fermentation culture product of the present invention preferably obtains 8-14g of freeze-dried powder.

[0033] The present invention also provides the use of the above-mentioned thermophilic subspecies of Streptococcus salivarius DY-001 or bacteriocin or lyophilized powder in the preparation of products for inhibiting pathogens, drugs for relieving or treating upper respiratory tract infections, and drugs for assisting the treatment of children's adenoid hypertrophy. The thermophilic subspecies of Streptococcus salivarius DY-001 can competitively inhibit the colonization of pathogens (including Streptococcus pneumoniae, β-hemolytic Streptococcus, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus), enhance mucosal barrier function and regulate Th1 / Th2 balance. The bacteriocin and lyophilized powder obtained by the present invention have significant inhibitory effects on Streptococcus pneumoniae, β-hemolytic Streptococcus, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus, and can effectively inhibit upper respiratory tract infections caused by pathogens. The thermophilic Streptococcus salivarius subspecies DY-001 or bacteriocin or lyophilized powder of the present invention can reduce the recurrence of nasopharyngeal inflammation, relieve or treat upper respiratory tract infection, relieve pathological hyperplasia of adenoids, indirectly help adenoids shrink, and assist in the treatment of adenoid hypertrophy in children.

[0034] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] In an embodiment of the present invention, the formula of M17 solid culture medium is: 5.0g soy peptone, 2.5g bovine bone peptone, 2.5g casein peptone, 2.5g yeast extract powder, 5.0g beef extract, 5.0g lactose, 0.5g sodium ascorbate, 19.0g sodium β-glycerophosphate, 0.25g magnesium sulfate, 13g agar, 1000g water; the preparation method is: after mixing all the raw materials, adjust the pH to 7.2±0.2 with 20% sodium hydroxide solution; The formula of M17 liquid culture medium is: 5.0g soy peptone, 2.5g bovine bone peptone, 2.5g casein peptone, 2.5g yeast extract powder, 5.0g beef extract, 5.0g lactose, 0.5g sodium ascorbate, 19.0g sodium β-glycerophosphate, 0.25g magnesium sulfate, 1000g water, pH value 7.2±0.2, 25℃; the preparation method is: after mixing all the raw materials, adjust the pH to 7.2±0.2 with 20% sodium hydroxide solution.

[0036] In the embodiment of the present invention, Streptococcus salivarius thermophilus subspecies Biohalo23 was purchased from Zhiyu Medical, Streptococcus salivarius thermophilus subspecies ST-21 was purchased from DuPont-Danisco, and Streptococcus salivarius thermophilus subspecies STN26 was purchased from Zhongke Jiayi. Streptococcus pneumoniae ATCC49619 was purchased from Shanghai Collection Biotechnology Center, β-hemolytic Streptococcus CMCC (B) 32210 was purchased from Qingdao Haibo Biological, Streptococcus pyogenes BNCC337110 was purchased from Beina Chuanglian Biological, Escherichia coli BNCC337304 was purchased from Beina Chuanglian Biological, and Staphylococcus aureus ATCC29213 was purchased from Shanghai Collection Biotechnology Center.

[0037] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1 Isolation and identification of Streptococcus salivarius thermophilus subspecies DY-001: The samples were collected from the oral cavity of healthy children and quickly refrigerated and sent to the laboratory for separation. 0.85% saline was used for gradient dilution, 1 mL was drawn and spread on the M17 solid plate, and placed in a 37°C incubator for 48 hours. After the culture was completed, single colonies with different characteristics on the plate were selected and streaked on the M17 medium until a pure culture was obtained. A single strain was screened out, which was the thermophilic subspecies of Streptococcus salivarius DY-001.

[0040] Streptococcus salivarius thermophilus subspecies DY-001 was inoculated on M17 solid medium and cultured at 37°C for 72 hours. The colony morphology was observed through the culture dish. The results were as follows: Figure 1The results showed that the thermophilic Streptococcus salivarius subspecies DY-001 strain was round, milky white, opaque, with raised, smooth and moist surfaces and neat edges.

[0041] Gram staining and catalase test were performed on Streptococcus salivarius thermophilus subspecies DY-001. Gram staining and catalase test showed that the strain was Gram-positive (G + ), the bacteria are in the form of spherical long chains, and the results are as follows Figure 2 The peroxidase test result was negative.

[0042] Streptococcus salivarius thermophilus subspecies DY-001 was inoculated into M17 liquid culture medium and cultured at 37°C for 18 hours. The cultured bacterial suspension was inoculated into a microbiochemical tube. After culture, the changes in the biochemical tube were observed and physiological and biochemical identifications were performed. The results are shown in Table 1.

[0043] Table 1 Physiological and biochemical test results of Streptococcus salivarius subsp. thermophilus DY-001

[0044] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0045] The results showed that the isolated single strain showed positive reactions in glucose, lactose, galactose, fructose, starch, trehalose, sorbitol, arginine and sucrose reaction tubes.

[0046] The Streptococcus salivarius thermophilus subsp. DY-001 strain was sent to Shanjun (Xi'an) Biopharmaceutical Technology Co., Ltd. for full genome sequencing. The 16S rDNA sequence is shown in SEQ ID No. 1. The results showed that it was Streptococcus salivarius thermophilus subsp. ( 嗜热唾液链球菌 ).

[0047] Streptococcus salivarius thermophilus subsp. 嗜热唾液链球菌 ) DY-001 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short). The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No.33409 and the deposit date is January 14, 2025.

[0048] Example 2 The drug resistance of Streptococcus salivarius subsp. thermophilus DY-001 to 22 antibiotics was determined using the drug sensitivity paper agar diffusion method: Streptococcus salivarius thermophilus subspecies DY-001 was inoculated into M17 liquid medium and cultured at 37°C for 16 h. 600The concentration of 5% salivarius thermophilic subspecies DY-001 culture fluid was obtained and set aside. 100 μL of the culture fluid was spread on an MRS solid culture plate, and the drug-sensitive paper was pasted on the agar plate. After anaerobic culture at 37°C for 24 hours, the diameter of the inhibition zone was measured and compared with the antibiotic resistance standard. The statistical results of the dosage and inhibition zone diameter of 22 antibiotics are shown in Table 2.

[0049] Table 2 Results of drug resistance experiments of Streptococcus salivarius thermophilus subspecies DY-001

[0050] Note: S stands for sensitive; M stands for moderately sensitive; R stands for resistant.

[0051] The results showed that Streptococcus salivarius thermophilic subspecies DY-001 was sensitive to 14 antibiotics, moderately sensitive to 7 antibiotics, and resistant to polymyxin.

[0052] Example 3 Antibacterial experiment of Streptococcus salivarius thermophilic subspecies DY-001: Streptococcus salivarius thermophilus subsp. DY-001, Streptococcus salivarius thermophilus subsp. Biohalo23, Streptococcus salivarius thermophilus subsp. ST-21, and Streptococcus salivarius thermophilus subsp. STN26 were activated and cultured in M17 medium, and then inoculated into M17 medium at 3% (v / v) and cultured at 37°C for 18 h for use.

[0053] Streptococcus pneumoniae ATCC49619 was inoculated into brain heart infusion broth and cultured at 37°C for 20 h for later use.

[0054] β-hemolytic Streptococcus CMCC(B)32210 was inoculated into brain heart infusion broth (supplemented with 5% fetal bovine serum), cultured at 37°C for 20 h, and set aside.

[0055] Streptococcus pyogenes BNCC337110 was inoculated into Columbia broth (supplemented with 5% fetal bovine serum), cultured at 37°C for 20 h, and set aside.

[0056] Escherichia coli BNCC337304 and Staphylococcus aureus ATCC29213 were inoculated into nutrient broth medium and cultured at 37°C for 20 hours for later use.

[0057] Cool brain heart infusion broth solid medium, brain heart infusion broth solid medium containing 5% fetal bovine serum, Columbia broth solid medium containing 5% fetal bovine serum, and nutrient broth solid medium to 55°C respectively, take 10 91 mL of bacterial suspension of Streptococcus pneumoniae ATCC49619, β-hemolytic Streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213 at CFU / mL was added to the corresponding culture medium and shaken evenly to avoid bubbles. 7 CFU / mL order of magnitude, and then quickly poured into the plate with the Oxford cup placed in advance. After the culture medium is cooled and solidified, take out the Oxford cup, and inject 200μL of culture solution of four strains of Streptococcus salivarius thermophilus subspecies DY-001, Streptococcus salivarius thermophilus subspecies Biohalo23, Streptococcus salivarius thermophilus subspecies ST-21, and Streptococcus salivarius thermophilus subspecies STN26 into each well, respectively. M17 liquid culture medium is used as a blank control. The plate is lightly covered and placed in a 37℃ three-gas incubator for 2 days. The diameter of the inhibition zone is measured with a vernier caliper, and three parallels are made for each bacterium. The results are shown in Table 3.

[0058] Table 3 Antibacterial test results of four strains

[0059] The results showed that the four strains of Streptococcus salivarius thermophilus DY-001, Streptococcus salivarius thermophilus ST-21, Streptococcus salivarius thermophilus Biohalo23, and Streptococcus salivarius thermophilus 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. Among them, Streptococcus salivarius thermophilic subspecies DY-001 had the best inhibitory effect on the five pathogens. The inhibitory effect of Streptococcus salivarius thermophilic subspecies Biohalo23 on Escherichia coli BNCC337304 was comparable to that of Streptococcus salivarius thermophilic subspecies DY-001. The inhibitory effects of the other three Streptococcus salivarius thermophilic subspecies were relatively poor.

[0060] Example 4 Adhesion test of Streptococcus salivarius thermophilus subspecies DY-001: Streptococcus salivarius thermophilus subsp. DY-001, Streptococcus salivarius thermophilus subsp. Biohalo23, Streptococcus salivarius thermophilus subsp. ST-21, and Streptococcus salivarius thermophilus subsp. STN26 were activated and cultured in M17 medium, and then inoculated into M17 medium at 3% (v / v) and cultured at 37°C for 18 h. Bacterial cells were collected by low-temperature centrifuge, washed twice with PBS solution, and resuspended in oral keratinocyte complete medium (OKM, containing growth factors and double antibodies, purchased from Sciencell) to a bacterial concentration of 1×10 6 CFU / mL of bacterial suspension is reserved.

[0061] Oral epithelial keratinocytes (HOK) were taken out of the liquid nitrogen tank and placed in a 37°C constant temperature water bath to melt. 5 ml of oral keratinocyte complete culture medium was added and gently shaken to evenly distribute in the culture bottle. The cells were placed in the incubator for 16 hours and subcultured. The number of cultured cells was expanded to 10 5 Pieces / mL.

[0062] The bacterial suspensions of Streptococcus salivarius thermophilus subsp. DY-001, Streptococcus salivarius thermophilus subsp. Biohalo23, Streptococcus salivarius thermophilus subsp. ST-21, and Streptococcus salivarius thermophilus subsp. STN26 were mixed with a concentration of 10 5 Add 0.5 mL of each HOK of each strain into a 1.5 mL EP tube, blow gently to disperse evenly, incubate in a shaker at 37°C and 100 r / min for 2 h, then filter and collect epithelial cells with a filter membrane with a pore size of 10 μm, wash the filter membrane three times with DPBS to collect the suspension, centrifuge in a low-temperature centrifuge, make a smear of the epithelial cells, air-dry, and heat-fix before Gram staining. Observe the changes in adhesion morphology under an oil immersion lens, select 100 epithelial cells respectively, count the total number of HOK and the total number of bacteria adhered to the surface of the four strains, and calculate the adhesion index. The adhesion index and adhesion rate are calculated using the following formulas: Adhesion index = number of adhered bacteria / total number of counted cells; Adhesion rate (%) = (number of cells with bacteria adhered to the surface / total number of counted cells) × 100%. At least 3 independent repeated experiments were performed. The results are as follows: Figure 3 shown.

[0063] The results showed that the adhesion of Streptococcus salivarius thermophilus subspecies DY-001 to oral epithelial keratinocytes (HOK) was 39.72±2.15 CFU / cell, which was significantly different from that of Streptococcus salivarius thermophilus subspecies ST-21 ( P <0.01), and there was a difference compared with Streptococcus salivarius subspecies Biohalo23 ( P <0.05), and there was a significant difference compared with Streptococcus salivarius thermophilus subspecies STN26 ( P <0.01); the difference between Streptococcus salivarius thermophilus subspecies Biohalo23 and Streptococcus salivarius thermophilus subspecies ST-21 was significant ( P <0.05); there was no significant difference between Streptococcus salivarius thermophilus subspecies STN26 and Streptococcus salivarius thermophilus subspecies ST-21.

[0064] Example 5 Isolation and antibacterial experiment of bacteriocin of Streptococcus salivarius thermophilic subspecies DY-001: Streptococcus salivarius thermophilus subspecies DY-001 was activated with M17 liquid medium, inoculated with 3% (v / v) inoculation volume into M17 liquid medium, and cultured at 37°C for 18 h.600 The concentration of 50% salivarius and 50% salivarius thermophilic subspecies DY-001 culture fluid was obtained. The culture fluid was centrifuged at 12000 rpm at 4°C to obtain a supernatant, which was divided into 4 parts. Ammonium sulfate was slowly added to the supernatant to make the ammonium sulfate saturation in the solution reach 50%, 60%, 70%, and 80%, respectively. The solution was stirred overnight with low temperature magnetic stirring, centrifuged at 4°C and 8000 rpm for 15 minutes, the supernatant was discarded, the precipitate was collected, and the corresponding products (bacteriocin) were obtained after drying at 40°C, which were recorded as DY50, DY60, DY70, and DY80, respectively. According to the weight of the precipitate, a 0.1 mg / ml bacteriocin solution was prepared with sterile water for antibacterial experiments.

[0065] The antibacterial experimental steps were the same as those in Example 3, except that 200 μL of the prepared bacteriocin solutions of DY50, DY60, DY70, and DY80 were injected into each well. The results are shown in Table 4.

[0066] Table 4 Antibacterial test results of different bacteriocin solutions

[0067] The results showed that the bacteriocin metabolized by the thermophilic subspecies of Streptococcus salivarius DY-001 could be effectively separated when the ammonium sulfate saturation was 60%, and had a good inhibitory effect on Streptococcus pneumoniae ATCC49619, β-hemolytic Streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213; when the ammonium sulfate saturation reached 80%, the bacteriocin could not be separated or was destroyed, resulting in serious losses.

[0068] Example 6 1. Culture medium screening of Streptococcus salivarius thermophilic subspecies DY-001: (1) Carbon source: Thermococcus salivarius subsp. DY-001 used the following carbon sources: glucose, sucrose, lactose, mannose, arabinose, and trehalose. The carbon source was removed from the M17 medium, and the other components remained unchanged. The amount of carbon source added was 2% (g / mL). M17 medium was used to activate the thermococcus salivarius subsp. DY-001, and 3% (v / v) was inoculated into the M17 medium with different carbon sources. After static culture at 37°C for 18 hours, the viable bacterial count was detected. The results are shown in Table 5.

[0069] Table 5 The number of viable cells of Streptococcus salivarius subsp. thermophilus DY-001 on different carbon sources

[0070] From the above table, we can see that Streptococcus salivarius thermophilus subsp. DY-001 can make good use of glucose, sucrose and lactose, but has low utilization rates of mannose, arabinose and trehalose. Glucose and lactose are the best for the growth of Streptococcus salivarius thermophilus subsp. DY-001. Subsequently, glucose and lactose were selected as the growth carbon sources for Streptococcus salivarius thermophilus subsp. DY-001, and the ratio of glucose to lactose was set to 1:1.

[0071] (2) Nitrogen source: Thermophyllous Streptococcus salivarius subsp. DY-001 used nitrogen sources: yeast extract, malt extract, tryptone, bovine bone peptone, soy peptone, and beef extract. The nitrogen source was removed from the M17 medium, and the other components remained unchanged. The nitrogen source addition amount was 2% (g / mL). Thermophyllous Streptococcus salivarius subsp. DY-001 was activated using M17 medium and inoculated into M17 medium with different nitrogen sources at 3% (v / v). After static culture at 37°C for 18 hours, the viable bacterial count was detected. The results are shown in Table 6.

[0072] Table 6 The number of viable cells of Streptococcus salivarius subsp. thermophilus DY-001 under different nitrogen sources

[0073] It can be seen from the above table that Streptococcus salivarius thermophilic subspecies DY-001 grows well in yeast extract powder, malt extract, and soy peptone, and has a fast reproduction ability. Subsequently, these three nitrogen sources are selected as the growth nitrogen sources for Streptococcus salivarius thermophilic subspecies DY-001.

[0074] (3) Orthogonal test: Streptococcus salivarius thermophilus subspecies DY-001 was subjected to orthogonal test using yeast extract powder (15g / L, 20g / L, 25g / L), malt extract (10g / L, 15g / L, 20g / L), soy peptone (17g / L, 20g / L, 23g / L), glucose-lactose mixture (20g / L, 25g / L, 30g / L) at each liter. Glucose and inorganic salts were not changed. The results are shown in the figure below. Figure 4 As shown in the figure, lactose represents a mixture of glucose and lactose (the ratio of glucose to lactose is 1:1).

[0075] The results showed that during the fermentation process of Streptococcus salivarius thermophilic subspecies DY-001, the most suitable addition ratios of the culture medium were 25g / L glucose and lactose, 20g / L yeast extract, 15g / L malt extract, and 23g / L soy peptone.

[0076] The fermentation medium of Streptococcus salivarius thermophilic subsp. DY-001 was set as: 12.5 g / L glucose, 12.5 g / L lactose, 20 g / L yeast extract powder, 15 g / L malt extract, 23 g / L soy peptone, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate and 0.05 g / L manganese sulfate.

[0077] 2. Preparation of Lyophilized Powder of Streptococcus salivarius Thermophyllus subsp. DY-001: After the thermophilic subspecies DY-001 of Streptococcus salivarius was activated for two generations in M17 liquid medium, it was inoculated into the fermentation medium of the thermophilic subspecies DY-001 of Streptococcus salivarius in step 1 at a 3% (v / v) inoculation amount and placed at 37°C for constant temperature expansion culture for 18 hours; the seed liquid activated and expanded was centrifuged at 8000rpm for 20 minutes in a 4°C environment, and the supernatant was discarded to collect the bacteria; 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 (sterilized at 110°C for 20 minutes), and cooled to 4°C to prepare a lyophilization protective agent. The bacterial mud and the lyophilization protective agent were fully mixed at a mass ratio of 1:2, pre-frozen for 3 hours, and then freeze-dried. The cold trap temperature was -50°C and freeze-dried for 48 hours. The freeze-dried bacterial powder was collected and named DYGH.

[0078] The viable bacterial count of the freeze-dried powder was 2.0×10 11 ~5×10 11 CFU / g.

[0079] 3. Screening of medicinal and edible raw materials fermented by Streptococcus salivarius thermophilus subspecies DY-001 The culture medium was prepared with 12.5% ​​glucose + 12.5% ​​lactose, and 10% honeysuckle pollen, 10% dandelion powder, 10% lily powder, 10% loquat powder, 10% red date powder and 10% ginger powder were added respectively to detect whether the six medicinal and edible raw materials can promote the growth of thermophilic Streptococcus salivarius subspecies DY-001. The preparation methods of the six culture media were as follows: weigh 12.5 g glucose, 12.5 g lactose, 10 g honeysuckle pollen, add 65 g water, mix thoroughly, sterilize at 121°C for 15 min, and set aside; weigh 12.5 g glucose, 12.5 g lactose, 10 g dandelion powder, add 65 g water, mix thoroughly, sterilize at 121°C for 15 min, and set aside; weigh 12.5 g glucose, 12.5 g lactose, 10 g lily powder, add 65 g water, mix thoroughly, sterilize at 121°C for 15 min, set aside; weigh 12.5g glucose, 12.5g lactose, 10g loquat powder, add 65g water, mix thoroughly, sterilize at 121℃ for 15min, set aside; weigh 12.5g glucose, 12.5g lactose, 10g red date powder, add 65g water, mix thoroughly, sterilize at 121℃ for 15min, set aside; weigh 12.5g glucose, 12.5g lactose, 10g ginger powder, add 65g water, mix thoroughly, sterilize at 121℃ for 15min, set aside.

[0080] After being activated for two generations in M17 liquid culture medium, Streptococcus salivarius thermophilus subspecies DY-001 was inoculated into six culture media at a 3% (v / v) inoculation rate and cultured at 37°C for 18 h. The number of viable bacteria was then detected. The results are shown in Table 7.

[0081] Table 7 The number of viable bacteria of Streptococcus salivarius thermophilus subspecies DY-001 in different fermented medicinal and edible raw materials

[0082] The results showed that the six medicinal and edible raw materials had significant differences in the growth of thermophilic Streptococcus salivarius DY-001. Among them, honeysuckle pollen, lily powder, loquat powder and red date powder promoted the strain, while dandelion powder and ginger powder inhibited the bacteria, among which ginger powder had the most significant inhibitory effect. Therefore, honeysuckle pollen, lily powder, loquat powder and red date powder were used to culture thermophilic Streptococcus salivarius DY-001.

[0083] Set up medicine and food culture medium: 125g / L glucose, 125g / L lactose, 100g / L honeysuckle pollen, 100g / L lily powder, 100g / L loquat powder and 100g / L red date powder.

[0084] 4. Preparation of Lyophilized Powder of Streptococcus salivarius Thermophyllus subsp. DY-001 (Containing Medicinal and Edible Materials) After activating Streptococcus salivarius thermophilic subspecies DY-001 in M17 liquid culture medium for two generations, the culture medium was inoculated at a 3% (v / v) rate into the edible-medicinal medium in step 3, cultured at 37°C for 36 h, pre-frozen for 3 h, and freeze-dried for 48 h at a cold trap temperature of -50°C. The freeze-dried bacterial powder was collected and named DYGN.

[0085] The viable bacterial count of the freeze-dried powder was 2.0×10 10 ~4×10 10 CFU / g.

[0086] 5. Antibacterial test of two freeze-dried powders of Streptococcus salivarius thermophilus subspecies DY-001 Take a certain amount of Streptococcus salivarius thermophilus subspecies DY-001 freeze-dried powder DYGH, DYGN and add it to sterile saline to prepare a final concentration of viable bacteria to 10 9 CFU / mL, and obtain DYGH solution and DYGN solution for use.

[0087] The antibacterial experimental steps were the same as those in Example 3, except that 200 μL of the prepared DYGH solution and DYGN solution were injected into each well, respectively. The results are shown in Table 8.

[0088] Table 8 Antibacterial test results of DYGH and DYGN

[0089] The results showed that the freeze-dried powder of Streptococcus salivarius thermophilic subsp. DY-001 DYGH and DYGN had inhibitory effects on five strains of Streptococcus pneumoniae ATCC49619, β-hemolytic Streptococcus CMCC(B)32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213, and the antibacterial effect was not much different from that of Streptococcus salivarius thermophilic subsp. DY-001 and its bacteriocin in liquid culture.

[0090] Example 7 Animal experiment on respiratory tract infection of Streptococcus salivarius thermophilus subspecies DY-001 in mice, the experiment used the lyophilized powder prepared in Example 6: Ninety 8-week-old SPF-grade BABL / c healthy mice, half male and half female, were purchased from the Animal Experiment Center of Hebei Medical University.

[0091] The mice were randomly divided into blank control group, model group, positive control group, experimental group 1 (DYGH high-dose group), experimental group 2 (DYGH medium-dose group), experimental group 3 (DYGH low-dose group), experimental group 4 (DYGN high-dose group), experimental group 5 (DYGN medium-dose group), and experimental group 6 (DYGN low-dose group), with 10 mice in each group. According to the modeling method of Standiford TJ et al., except for the blank control group, the other 8 groups of mice were anesthetized with ether and 50 μL of a mixture of five strains of Streptococcus pneumoniae ATCC49619, β-hemolytic Streptococcus CMCC (B) 32210, Streptococcus pyogenes BNCC337110, Escherichia coli BNCC337304, and Staphylococcus aureus ATCC29213 were dripped into the nasal cavity (bacterial concentration 1×10 8 CFU / mL, each bacteria is mixed in equal proportions) for automatic inhalation, and medication begins on the day of infection (the drug is dissolved in sterile saline and administered by gavage) and is administered continuously for 7 days. The dosage is shown in Table 9.

[0092] Table 9 Dosage

[0093] The mice were weighed before and after the experiment, and the changes before and after the experiment were compared. On the 7th day of the experiment, the mice were killed and the lung tissue was removed. After the wet weight of the lung tissue was weighed, it was placed in a 60°C electric constant temperature dry oven for 72 hours, the dry weight was weighed, and the wet / dry weight ratio was calculated. Lung index = mouse lung weight (g) / mouse body weight (g) × 100%. The results are shown in Table 10.

[0094] Table 10 Changes in body weight and lung tissue of mice in each group before and after the experiment

[0095] Note: *: Compared with the control group, P <0.05; #: indicates that compared with the model group, P <0.05.

[0096] The results showed that after the mice were dripped with the mixed solution of the five strains into their nasal cavity, they were in poor spirits, had decreased appetite, drank less water, had less activity, had increased nasal secretions, had shortness of breath, and developed coughing symptoms. After 7 days, their body weight decreased significantly ( P <0.05). The control group was in good spirits, drank water and ate normally, was active and lively, had stable breathing, and had no coughing symptoms. Compared with the model group, all experimental groups had significant improvements in the above symptoms ( P <0.05), and there was a significant difference compared with the control group. The wet / dry mass 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 change trend of lung index of mice in each group was consistent with the results of wet / dry mass ratio.

[0097] On the 7th day of the experiment, the mice were killed and the lung tissue was removed. The lung tissue was rinsed with pre-cooled PBS to remove blood. About 100 mg of lung tissue was weighed and wiped dry on filter paper. After being placed in a beaker, PBS with a volume of 9 times the weight of the lung tissue block was added. The tissue was chopped, homogenized, and ground. The supernatant was taken after centrifugation at 5000 r / min in a low-temperature centrifuge for 10 minutes. The ELISA detection kit (Bai Lingke Bio) was used to detect the IL-6 and TNF-α content of the lung tissue of each group. The results are shown in Table 11.

[0098] Table 11 IL-6 and TNF-α levels in each group of mice

[0099] Note: *: Compared with the control group, P <0.05; #: indicates that compared with the model group, P <0.05.

[0100] 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 increased significantly and were 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 thermophilus subspecies DY-01 could alleviate the upper respiratory tract symptoms of mice infected with bacteria, and both freeze-dried bacterial powders showed good antibacterial effects and improved immune function.

[0101] Example 8 The freeze-dried powder (DYGH) prepared in Example 6 was used to conduct a trial test on adenoids hypertrophy in children: 1. Research subjects Inclusion criteria: children aged 3 to 10 years, diagnosed with adenoids hypertrophy (grade II-III), and not receiving surgical treatment.

[0102] Exclusion criteria: patients with severe cardiopulmonary disease or immunodeficiency.

[0103] Grouping: The patients were divided into adenoids hypertrophy control group (conventional treatment) and adenoids hypertrophy experimental group (conventional treatment + oral preparation of Streptococcus salivarius thermophilus subspecies).

[0104] 2. Intervention plan The adenoids hypertrophy experimental group was treated with Streptococcus salivarius thermophilus subspecies DY-001 for 3 months.

[0105] 3. Observation indicators Main Outcome Measures: Changes in adenoid volume (evaluated by lateral nasopharyngeal X-ray or endoscopy), apnea-hypopnea index (AHI).

[0106] Secondary indicators: nasal congestion score, snoring frequency, number of upper respiratory tract infections, and inflammatory factor levels.

[0107] Safety: Adverse events such as gastrointestinal reactions and allergies were recorded.

[0108] The following are children with adenoids hypertrophy who were recruited to take the trial voluntarily. They were randomly divided into 2 groups, and received conventional treatment and conventional treatment plus Streptococcus salivarius thermophilus subsp. DY-001 combined treatment respectively. The dosage was 1 gram / bag / time, containing no less than 3 billion active probiotics of Streptococcus salivarius thermophilus subsp. DY-001, twice a day, half an hour after a meal, orally without water (do not drink water within 2 hours after taking it); the effect was observed for 90 days according to the symptoms of different stages. The results are shown in Table 12.

[0109] Table 12 Results of the trial test on adenoids hypertrophy in children

[0110] The results showed that the thermophilic subspecies of Streptococcus salivarius DY-001 as an adjuvant therapy can effectively reduce the inflammatory response and clinical symptoms of adenoids hypertrophy in children, with good safety. Compared with drug treatment alone, combined with probiotics can more significantly improve symptoms and reduce the recurrence rate, and avoid surgical trauma and irreversible complications after organ resection.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as 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 deposit number of the Streptococcus salivarius thermophilus subspecies DY-001 is CGMCC No.33409.

2. A method for preparing bacteriocin, characterized in that: The method comprises the following steps: fermenting and culturing the thermophilic subspecies of Streptococcus salivarius DY-001 according to claim 1, and separating and obtaining bacteriocin from the fermentation and culturing product.

3. The bacteriocin prepared by the preparation method according to claim 2.

4. A method for preparing a freeze-dried powder, characterized in that: The method comprises the following steps: fermenting and culturing the thermophilic Streptococcus salivarius subspecies DY-001 according to claim 1 to obtain a fermentation culture product, and preparing the thermophilic Streptococcus salivarius subspecies DY-001 freeze-dried powder from the fermentation culture product.

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

6. The preparation method according to claim 4, characterized in that: The culture medium for the fermentation culture comprises 100-130 g / L of glucose, 100-130 g / L of lactose, 80-110 g / L of honeysuckle pollen, 80-110 g / L of lily powder, 80-110 g / L of loquat powder and 80-110 g / L of red date powder.

7. The freeze-dried powder prepared by the preparation method according to any one of claims 4 to 6.

8. Use of the thermophilic Streptococcus salivarius subsp. DY-001 according to claim 1 or the bacteriocin according to claim 3 or the lyophilized powder according to claim 7 in the preparation of a product for inhibiting pathogenic bacteria, characterized in that: The pathogenic bacteria include Streptococcus pneumoniae, beta-hemolytic Streptococcus, Streptococcus pyogenes, Escherichia coli and Staphylococcus aureus.

9. Use of the Streptococcus salivarius thermophilus subsp. DY-001 according to claim 1 or the bacteriocin according to claim 3 or the lyophilized powder according to claim 7 in the preparation of a medicament for relieving or treating upper respiratory tract infection.

10. Use of the thermophilic Streptococcus salivarius subsp. DY-001 according to claim 1 or the bacteriocin according to claim 3 or the lyophilized powder according to claim 7 in the preparation of a medicament for the auxiliary treatment of adenoid hypertrophy in children.

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