Probiotic agent capable of inhibiting cryptococcus and Kodaea yeast and application of lactobacillus reuteri LR-03

Abstract

The invention belongs to the technical field of biology, and particularly relates to a probiotic agent capable of inhibiting cryptococcus and Kodaea yeast and application of lactobacillus reuteri LR-03. The probiotic agent at least comprises any one of the following strains: (1) lactobacillus rhamnosus GYL-718, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.31642, and (2) lactobacillus rhamnosus GYL-718 is preserved in the China General Microbiological Culture Collection Center (CGMCC); and (2) the lactobacillus helveticus ZY-01 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31641. The probiotic agent further comprises lactobacillus reuteri LR-03 which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the lactobacillus reuteri LR-03 is CGMCC No.31643. The lactobacillus rhamnosus GYL-718 and the lactobacillus helveticus ZY-01 are superior to the known lactobacillus rhamnosus and the known lactobacillus helveticus in the capability of inhibiting candida albicans, cryptococcus and Kodara yeast, and the lactobacillus reuteri LR-03 has the capability of enhancing the capability of inhibiting the cryptococcus and the Kodara yeast of the lactobacillus rhamnosus GYL-718 and the lactobacillus helveticus ZY-01.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a probiotic agent capable of inhibiting cryptococcus and kodak yeast, and application of Lactobacillus reuteri LR-03. Background Art

[0002] In many developed countries, invasive fungal infections are associated with advances in medicine. In addition to the millions of people with human immunodeficiency virus (HIV) worldwide, which constitute a significant host population, infections are also common in individuals with other immunodeficiencies, reflecting the expansion of susceptible host populations due to technological and therapeutic advances. Examples include novel chemotherapies and other antineoplastic therapies, the frequent use of glucocorticoids for a variety of underlying conditions, the expanded use of monoclonal antibodies and other biologics for the treatment of autoimmune diseases, and the widespread use of intravascular catheters and other medical devices that contribute to disease in immunocompetent individuals.

[0003] According to estimates of global fungal infections, the number of patients with severe fungal diseases worldwide exceeds 150 million, and the number of deaths caused by fungal diseases is about 1.6 million per year. The main reasons for the increase in fungal infections worldwide include the increase in high-risk groups such as tuberculosis, chronic obstructive pulmonary disease, tumors and organ transplantation, and glucocorticoid treatment. The global situation of new and emerging pathogenic fungal infections and antifungal resistance is worrying. Invasive fungal diseases are highly harmful and have a high mortality rate, and deserve attention. At the same time, the epidemiological characteristics of invasive fungal diseases have changed in recent years, risk factors have continued to change, and pathogenic fungi have increased their natural insensitivity to antifungal drugs or acquired resistance, posing clinical challenges.

[0004] Chinese patent document CN114585258A discloses a bacterial composition for controlling fungal corruption, which is used to inhibit or delay the growth of fungi in food products. The lactic acid bacteria may be Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus kefiri, etc. The fungi are yeast and / or mold, and the yeast includes Cryptococcus spp. That is, the prior art discloses that Lactobacillus reuteri and Lactobacillus rhamnosus have a certain inhibitory effect on fungi.

[0005] Chinese patent document CN109820044A also discloses a probiotic solid beverage formula for enhancing immunity and assisting in anti-allergy, wherein the probiotic formula includes Lactobacillus rhamnosus R-11, Lactobacillus reuteri HA-188, and Lactobacillus helveticus R-52. The document discloses that the main functions of the formula are to regulate microecological balance, enhance host intestinal resistance, prevent and treat diarrhea, eliminate allergies, etc. Summary of the invention

[0006] The first object of the present invention is to provide a probiotic agent having the ability to inhibit Cryptococcus and Kodak yeast, which has a high activity of inhibiting Cryptococcus and Kodak yeast.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A probiotic agent having the function of inhibiting Cryptococcus and Kodacryocystis, comprising at least any one of the following strains:

[0009] (1) Lactobacillus rhamnosus GYL-718, deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No. 31642;

[0010] (2) Lactobacillus helveticus ZY-01, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31641.

[0011] As a preferred solution, the probiotics also include

[0012] Lactobacillus reuteri LR-03 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC No.31643.

[0013] As a preferred solution, the number of viable bacteria of a single species in the probiotic agent is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0014] As a preferred solution, the probiotics include freeze-dried powder, capsules, tablets or granules.

[0015] As a preferred solution, the probiotic agent is in the form of a freeze-dried powder, and the probiotic agent also includes a freeze-drying protectant and / or auxiliary additives.

[0016] As a preferred embodiment, the lyoprotectant includes any one or more of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0017] As a preferred embodiment, the auxiliary additives include any one or more of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactoprotein or lactoferrin.

[0018] The second object of the present invention is to provide an application of Lactobacillus reuteri LR-03 in promoting the activity of Lactobacillus rhamnosus GYL-718 and / or Lactobacillus helveticus ZY-01 in inhibiting Cryptococcus and Kodacryocystis.

[0019] The Lactobacillus reuteri LR-03 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.31643;

[0020] The Lactobacillus rhamnosus strain GYL-718 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31642;

[0021] The Lactobacillus helveticus ZY-01 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31641.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present application found that Lactobacillus rhamnosus GYL-718 with a preservation number of CGMCC No. 31642 and Lactobacillus helveticus ZY-01 with a preservation number of CGMCC No. 31641 are superior to the known Lactobacillus rhamnosus and Lactobacillus helveticus in the ability to inhibit Candida albicans (Candida genus), Cryptococcus (Cryptococcus genus) and Kodamaea genus, and can be used to prepare foods, health products or medicines with relevant efficacy.

[0024] At the same time, the present application also found that Lactobacillus reuteri LR-03 with a preservation number of CGMCC No.31643 has an enhanced ability to inhibit Cryptococcus (Cryptococcus genus) and Kodamaea (Kodamaea genus) against Lactobacillus rhamnosus GYL-718 with a preservation number of CGMCC No.31642 and Lactobacillus helveticus ZY-01 with a preservation number of CGMCC No.31641, and can be used to prepare foods or health products with relevant functions.

[0025] Biological Deposit Description

[0026] Lactobacillus rhamnosus GYL-718 (Lacticaseibacillus rhamnosus GYL-718), deposited at: General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: August 8, 2024, deposit number: CGMCC No.31642.

[0027] Lactobacillus helveticus ZY-01, deposited at: General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: August 8, 2024, deposit number: CGMCC No.31641.

[0028] Lactobacillus reuteri LR-03 (Limosilactibacilus reuteri LR-03), deposited at: General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: August 8, 2024, deposit number: CGMCC No.31643. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The colony morphology of Lacticaseibacillus rhamnosus GYL-718 on MRS solid culture plates is shown.

[0030] Figure 2 The colony morphology of Limosilactibacilus reuteri LR-03 on MRS solid culture plates is shown.

[0031] Figure 3Shows the colony morphology of Lactobacillus helveticus ZY-01 on MRS solid culture plates.

[0032] Figure 4 Shows the cell morphology of Cryptococcus neoformans ATCC 32045 grown on YPD solid medium.

[0033] Figure 5 Shows the cell morphology of Kodamaea ohmeri ATCC 20282 grown on YPD solid medium.

[0034] Figure 6 The antibacterial effects of the three strains of the patent and the commercial control strain on Cryptococcus are shown.

[0035] Figure 7 Shows the antibacterial effect of the patented three-strain mixed bacterial agent on Omokoda yeast.

[0036] Figure 8 Shows the feeding status of mice. DETAILED DESCRIPTION

[0037] In order to further explain the technical means and effects of the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the embodiments. The raw materials, equipment, etc. used in the following embodiments can be purchased through commercial channels unless otherwise specified.

[0038] Example 1: Isolation and identification of bacterial species

[0039] 1. Isolation of new strains

[0040] (1) Isolation of microorganisms from the human intestine

[0041] Select healthy adults aged 20-30 with no reported diseases, and excrete feces into a clean container. Collect fresh feces from the middle and rear of the excretion, and dig out the feces from the middle and inner part as samples. Use a sterile spoon to dig out the fresh feces and put the feces sample into a sterile storage tube. The sample needs to be about 1g. The collected sample is immediately placed in a -80℃ refrigerator for low-temperature storage.

[0042] When isolating intestinal microorganisms, take 1g of feces, add it to 99g of PBS buffer, and dilute it in a 10-fold gradient. Take 1ml of each dilution gradient and spread it on the surface of MRS solid culture medium (prepared according to the instructions using Oxoid MRS broth dry powder), and culture it anaerobically at 37℃ for 48h until there are 1-2mm colonies.

[0043] (2) Isolation of Microorganisms from Fermented Milk

[0044] 3g of kefir grains (stored in Huzhou Normal University) were inoculated into 97g of fresh pasteurized milk, fermented at 30℃ for 24-36h until pH 4.0, cooled at 4℃, 1g of fermented milk was added to 99g of PBS buffer, and diluted in 10-fold gradients. 1ml of each dilution gradient was spread on the surface of MRS solid culture medium (prepared with Oxoid MRS broth dry powder according to the instructions), and anaerobically cultured at 37℃ for 48h until 1-2mm colonies were formed.

[0045] (3) Isolation of acid-resistant and bile-resistant microorganisms

[0046] For screening of acid-resistant strains, colonies were picked with a sterilized inoculation loop and placed in 96-well plates containing 200 μl of MRS liquid medium. After anaerobic culture at 37°C for 48 hours, the colonies were inoculated with 2% inoculum in 96-well plates containing 200 μl of MRS pH 2.5 liquid medium. The cells were anaerobically fermented at 37°C for 24 hours, and the optimal density (OD600) was measured every hour. As a negative control, the strains were cultured in MRS medium without normal pH. The detection was performed by the triple method. The screening criteria were: strains with an OD600 increase of greater than 0.2 after 24 hours of anaerobic culture.

[0047] Screening of bile salt-tolerant strains: Use a sterilized inoculation loop to pick colonies in 96-well plates containing 200 μl of MRS liquid medium. After anaerobic culture at 37°C for 48 hours, inoculate with a 2% inoculum into 200 μl of a mixture of 0.05% (w / v) glycocholic acid, glycodeoxycholic acid, taurocholic acid and taurochenodeoxycholic acid. The strains were anaerobically fermented at 37°C for 24 hours, and the optimal density (OD600) was measured every hour. As a negative control, the strains were cultured in MRS medium without bile salts, and the detection was performed by the triple method. The screening criteria are: strains with an OD600 increase of greater than 0.2 after 24 hours of anaerobic culture.

[0048] (4) Strain isolation results

[0049] Through acid and bile resistance tests, we isolated two strains of interest from stool samples, numbered GYL-718 and LR-03; and one strain of interest was isolated from kefir fermented milk, numbered ZY-01.

[0050] (5) Molecular identification of strains

[0051] The three strains that are resistant to acid and bile salts were inoculated at 2% in a 96-well plate containing 200 μl of MRS liquid medium. After anaerobic culture at 37°C for 48 hours, 1 μl of the bacterial solution was taken as a PCR template and PCR was performed using universal primers 27F and 1492R to amplify the 16S rDNA sequence. The primer sequences are as follows:

[0052] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0053] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0054] The PCR products were separated by agarose gel electrophoresis, excised and recovered, and subjected to Sanger sequencing. The sequences were compared by Blastn to determine the species of the strain.

[0055] 2. Colony morphology

[0056] MRS medium: Prepare Oxoid MRS broth powder according to the instructions. For solid plates, add 15g / L agar to the liquid medium. Streak GYL-718 and LR-03 bacteria on MRS solid plates, culture anaerobically at 37℃ for 48h, and observe the colony morphology.

[0057] The colony morphology of GYL-718 bacteria is as follows Figure 1 The main morphological characteristics of GYL-718 on MRS solid medium are: milky white, round colonies, smooth, opaque with raised center, and complete colony edges. The morphological characteristics of LR-03 on MRS solid medium are similar to those of GYL-718, as shown in Figure 1. Figure 2 As shown in the figure, the colony is milky white, round, smooth, with an opaque raised center and complete colony edges. Figure 3 As shown, the main morphological characteristics of ZY-01 bacteria on MRS solid culture medium are: white, round, smooth, moist colonies, and complete colony edges.

[0058] 3. Molecular biological verification of strains

[0059] (1) DNA extraction

[0060] Bacterial DNA was extracted using Femto TM Bacterial DNA Quantification Kit (ZymoResearch) was used according to the instruction manual.

[0061] (2) 16S rRNA identification

[0062] The primers used to amplify the 16S rDNA sequences of GYL-718, LR-03 and ZY-01 were 27F and 1492R, and the sequences were as described above.

[0063] The rDNA sequencing result of GYL-718 is shown in SEQ ID NO.1. After Blast comparison, GYL-718 was identified as Lactobacillus rhamnosus.

[0064] The rDNA sequence sequencing result of ZY-01 bacteria is shown in SEQ ID No.2. After Blast comparison, ZY-01 bacteria is Lactobacillus helveticus.

[0065] The rDNA sequence sequencing result of LR-03 bacteria is shown in SEQ ID NO.3. After Blast comparison, LR-03 bacteria is Lactobacillus reuteri.

[0066] Example 2, acid and bile salt resistance test

[0067] 1. Bacterial acid and bile resistance test

[0068] The acid and bile salt resistance in Example 1 is a screening experiment. The acid and bile salt resistance characteristics of the three strains screened are demonstrated below by using different pH environments and different bile salt concentrations.

[0069] The pH value of MRS liquid culture medium was adjusted to 2.0, 3.0 and 4.0 respectively with 0.1 mol / L hydrochloric acid to prepare an acidic culture environment; using MRS liquid culture medium without adding bile salt as a control, different concentrations of ox bile salt were added to the culture medium, and the bile salt concentrations were 0.1%, 0.2% and 0.3%, respectively, to prepare a bile salt environment culture medium; the two culture bases prepared above were sterilized at 121° C. for 20 minutes and cooled for use.

[0070] The activated Lactobacillus rhamnosus GYL-718, Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 were inoculated at a 1% inoculation rate into MRS liquid culture media with pH values ​​of 2.0, 3.0 and 4.0, and culture media with bile salt concentrations of 0.1%, 0.2% and 0.3%, respectively, and cultured at 37°C. Samples were taken at 0, 2, 3 and 4 h, and the number of viable cells at different time periods was determined by the plate count method to calculate the survival rate.

[0071] Survival rate (%) = number of viable bacteria cultured at 2, 3, and 4 hours respectively / initial number of viable bacteria at 0 hours × 100%

[0072] Among them, Table 1 is the survival rate of Lactobacillus rhamnosus GYL-718 in simulated gastric acid, Table 2 is the survival rate of Lactobacillus rhamnosus GYL-718 in bile salts, Table 3 is the survival rate of Lactobacillus reuteri LR-03 in simulated gastric acid, Table 4 is the survival rate of Lactobacillus reuteri LR-03 in bile salts, Table 5 is the survival rate of Lactobacillus helveticus ZY-01 in simulated gastric acid, and Table 6 is the survival rate of Lactobacillus helveticus ZY-01 in bile salts.

[0073] Table 1

[0074] Survival rate 0h 2h 3h 4h pH=2.0 100 93.2 92.6 88.8 pH=3.0 100 97.3 95.2 94.2 pH = 4.0 100 99.1 99.2 98.3

[0075] Table 2

[0076] Survival rate 0h 2h 3h 4h 0.1% Bile Salts 100 99.8 98.9 99.1 0.2% Bile Salts 100 98.7 97.3 96.4 0.3% Bile Salts 100 98.7 98.5 97.6

[0077] Table 3

[0078]

[0079]

[0080] Table 4

[0081] Survival rate 0h 2h 3h 4h 0.1% Bile Salts 100 97.2 96.9 96.1 0.2% Bile Salts 100 96.4 95.3 95.4 0.3% Bile Salts 100 96.1 94.5 91.6

[0082] Table 5

[0083] Survival rate 0h 2h 3h 4h pH=2.0 100 90.2 87.6 85.8 pH=3.0 100 93.8 93.3 91.1 pH = 4.0 100 95.6 95.7 92.9

[0084] Table 6

[0085] Survival rate 0h 2h 3h 4h 0.1% Bile Salts 100 96.8 95.9 95.1 0.2% Bile Salts 100 93.7 92.3 90.4 0.3% Bile Salts 100 91.7 90.5 87.6

[0086] It can be seen from the data in Tables 1 to 6 that the strains were cultured at 37°C in MRS liquid culture media with pH values ​​of 2.0, 3.0 and 4.0, and in culture media with bile salt concentrations of 0.1%, 0.2% and 0.3%, respectively, to simulate the situation in which the strains enter the human intestine. After 4 hours of culture, the survival rates of the three strains were all above 80%, indicating that Lactobacillus rhamnosus GYL-718, Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 all have good acid and bile salt resistance, which enables them to adhere to the human intestine very well in the body and produce a probiotic effect on the body.

[0087] Example 3. Antibiotic resistance

[0088] The antibiotic resistance potential of the strains was determined using the ISO 10932IDF223 method and VetMIC Lact-1 and 2 microdilution plates, which included all antibiotics recommended by FEEDAP updated in 2018. All MIC values ​​were lower than or equal to the European Food Safety Authority Microbial Break Points (MBPs) for Lactobacillus rhamnosus and Lactobacillus reuteri. For the specific process, please refer to Guidance on microorganisms used as feed additives or as productionorganism, EFSA Journal 2018, 16(3): 5206.

[0089] Table 7 Antibiotic resistance

[0090]

[0091] () The brackets are the cut-off values ​​of EFSA for antibiotic resistance of strains of various genera

[0092] As shown in Table 7, the concentrations of Lactobacillus rhamnosus GYL-718, Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 were all lower than the maximum allowable concentration of antibiotic resistance of strains of various genera set by EFSA, proving that these three strains were safe and reliable in terms of antibiotic resistance.

[0093] Example 4: Effect of inhibiting yeast

[0094] Many lactic acid bacteria are reported to be able to inhibit the pathogenic fungus Candida albicans, but there are fewer reports on inhibiting Cryptococcus and Kodamaea ohmeri ATCC 20282. Therefore, we selected Cryptococcus neoformans ATCC 32045 or Kodamaea ohmeri ATCC 20282 as indicator bacteria. It should be understood that these two indicator bacteria are only used as demonstrations, and do not limit the types of pathogenic fungi that the probiotics involved in the present invention can inhibit.

[0095] The antibacterial activity was tested by antibacterial experiment, the steps are as follows:

[0096] (1) Inoculate 5 mL of YPD liquid medium with 1% inoculum of each of the two indicator bacteria;

[0097] (2) Cultivate at 30°C for 18-24 hours until the final viable count reaches 10 7 CFU / mL. The specific inoculation amount of each bacterial agent is as follows: (a) 10 7 CFU / ml of Lactobacillus rhamnosus GYL-718, (b) 10 7CFU / ml of Lactobacillus reuteri LR-03, (c) 10 7 CFU / ml of Lactobacillus helveticus ZY-01, (d) 10 7 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863, (e) 10 7 CFU / ml of commercial Lactobacillus reuteri ATCC 23272, (f) 10 7 CFU / ml of commercial Lactobacillus helveticus ATCC15009, (g) 5×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718 and 5×10 6 CFU / ml of Lactobacillus reuteri LR-03 mixed culture, (h) 5×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718 and 5×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed culture, (i) 5×10 6 CFU / ml of Lactobacillus reuteri LR-03 and 5×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution, (j) 5×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863 and 5×10 6 CFU / ml of commercial Lactobacillus reuteri mixed culture ATCC 23272, (k) 5×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863 and 5×10 6 CFU / ml of commercial Lactobacillus helveticus mixed culture ATCC 15009, (l) 5×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC 23272 and 5×10 6 CFU / ml of commercial Lactobacillus helveticus ATCC15009 mixed culture, (m) 5×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718 and 5×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC 23272 mixed culture, (n) 5×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718 and 5×10 6 CFU / ml of commercial Lactobacillus helveticus ATCC 15009 mixed culture, (o) 5×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863 and 5×10 6 CFU / ml of Lactobacillus reuteri LR-03 mixed culture, (p) 5×10 6CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863 and 5×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution, (q) 5×10 6 CFU / ml of Lactobacillus reuteri LR-03 and 5×10 6 CFU / ml of commercial Lactobacillus helveticus ATCC 15009 mixed culture, (r) 5×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC23272 and 5×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution, (s) 4×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718, 3×10 6 CFU / ml of Lactobacillus reuteri LR-03 and 3×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution, (t) 4×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718, 3×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC 23272 and 3×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution, (u) 4×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718, 3×10 6 CFU / ml of Lactobacillus reuteri LR-03 and 3×10 6 CFU / ml of commercial Lactobacillus helveticus ATCC 15009 mixed culture, (v) 4×10 6 CFU / ml of Lactobacillus rhamnosus GYL-718, 3×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC23272 and 3×10 6 CFU / ml of commercial Lactobacillus helveticus ATCC 15009 mixed culture, (w) 4×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863, 3×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC 23272 and 3×10 6 CFU / ml of commercial Lactobacillus helveticus ATCC 15009 mixed culture, (x) 4×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863, 3×10 6 CFU / ml of Lactobacillus reuteri LR-03 and 3×10 6CFU / ml of commercial Lactobacillus helveticus ATCC 15009 mixed culture, (y) 4×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863, 3×10 6 CFU / ml of commercial Lactobacillus reuteri ATCC 23272 and 3×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution, (z) 4×10 6 CFU / ml of commercial Lactobacillus rhamnosus ATCC 10863, 3×10 6 CFU / ml of Lactobacillus reuteri LR-03 and 3×10 6 CFU / ml of Lactobacillus helveticus ZY-01 mixed bacterial solution.

[0098] (3) Pour a YPD culture plate (dish diameter 90 mm) containing 2% agar;

[0099] (4) Evenly spread 0.2 mL of each indicator bacteria on the plate culture medium;

[0100] (5) A 6 mm diameter circular hole is cut in the center of each plate;

[0101] (6) 60 μL of probiotic or composite probiotic suspension was dripped into each well;

[0102] (7) In the same manner as step (2), the plates were cultured at 30°C for 24 h corresponding to different bacteria;

[0103] (8) The antibacterial activity was evaluated by measuring the radius of the inhibition zone.

[0104] Figure 4 and Figure 5 Shown is the microscopic morphology of two indicator bacteria on YPD plates.

[0105] Figure 6-Figure 7 The following table shows the antibacterial effects of the three strains and the composite probiotics on Cryptococcus ATCC 32045 and Omokoda yeast ATCC 20282 (diameter of the inhibition zone, in mm, with a punch diameter of 6 mm). Table 8 summarizes the antibacterial effects of the three strains and the composite probiotics on Cryptococcus ATCC 32045 and Omokoda yeast ATCC 20282.

[0106] Table 8 Inhibition zone diameter (mm) 1

[0107]

[0108]

[0109] Note: 1 Plate diameter 90mm;2 The numbers in brackets correspond to Figure 6 and Figure 7 The serial number in 3 The total bacterial count of each bacterial agent was controlled to be 10 7 CFU / ml, the ratios of each strain are shown in the table.

[0110] From Table 8 and Figure 6 to Figure 7 It can be seen that the three commercial lactic acid bacteria have no significant inhibitory activity against Cryptococcus and Omerkoda yeast. The Lactobacillus rhamnosus GYL-718 in the present invention has good inhibitory activity against Cryptococcus and Omerkoda yeast, and Lactobacillus helveticus ZY-01 has slightly weaker inhibitory activity against Cryptococcus and Omerkoda yeast, which is unique to these two strains.

[0111] Lactobacillus rhamnosus GYL-718 and Lactobacillus reuteri LR-03 have a positive synergistic effect, and the mixed bacterial suspension of the two has better antibacterial activity than Lactobacillus rhamnosus GYL-718. However, the commercialized Lactobacillus reuteri ATCC 23272 has no promoting effect on GYL-718 and ATCC 10863. The positive synergistic effect of Lactobacillus rhamnosus GYL-718 and Lactobacillus reuteri LR-03 is unique to these two strains.

[0112] Lactobacillus rhamnosus GYL-718 and Lactobacillus helveticus ZY-01 have a positive synergistic effect, and the mixed bacterial suspension of the two has better antibacterial activity than Lactobacillus rhamnosus GYL-718 or Lactobacillus helveticus ZY-01 used alone. However, the commercialized Lactobacillus helveticus ATCC 15009 has no promoting effect on GYL-718 and ATCC 10863. The positive synergistic effect of Lactobacillus rhamnosus GYL-718 and Lactobacillus helveticus ZY-01 is unique to these two strains.

[0113] Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 have a positive synergistic effect, and the mixed bacterial suspension of the two has better antibacterial activity than Lactobacillus reuteri LR-03 or Lactobacillus helveticus ZY-01 used alone, while the commercial Lactobacillus reuteri ATCC 23272 has no promoting effect on Lactobacillus helveticus ZY-01 and Lactobacillus helveticus ATCC 15009. The positive synergistic effect of Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 is unique to these two strains.

[0114] It can be seen that Lactobacillus reuteri LR-03 only has the ability to promote Lactobacillus rhamnosus GYL-718 and Lactobacillus helveticus ZY-01 in the present invention, which is unique to the present invention.

[0115] The composite probiotics composed of three strains of bacteria, GYL-718, LR-03 and ZY-01, have the highest inhibitory activity against Cryptococcus and Omokoda yeast under the same number of viable bacteria. However, replacing one, two or all three of GYL-718, LR-03 and ZY-01 with commercial ATCC 10863, ATCC 23272 and ATCC 15009 cannot achieve the antibacterial effect of the composite probiotics composed of three strains of bacteria, GYL-718, LR-03 and ZY-01, proving that the positive synergistic effect of the three strains is unique to these three strains.

[0116] Example 5: Probiotics improve and alleviate invasive fungal infections caused by Cryptococcus or Kodacryocystis

[0117] The ITS1-based amplicon sequencing technology was used to analyze the changes in Cryptococcus or Omerkoda yeast in the feces of each group of experimental mice, and the abundance of pathogenic yeast was calculated according to the FKPM value, reflecting the improvement of Cryptococcus or Omerkoda yeast infection in mice.

[0118] (1) Animal husbandry and selection

[0119] 35 healthy SPF-grade BALB / c male mice aged 6 weeks were selected, weighing 18-20 g. After 2 weeks of adaptive feeding, the ambient temperature (22±2°C) and humidity (55%±10%) were controlled, the light-dark cycle was 12 hours, and the mice were free to eat and drink. The feeding conditions of mice are shown in Figure 8 .

[0120] (2) Mouse modeling

[0121] After 2 weeks of adaptive feeding, the mice were divided into a blank group (n=7), a Cryptococcus group (n=14), and an Omerkoda yeast group (n=14). The initial weight of the mice in each group was weighed and recorded. The mice in the blank group were fed with ordinary feed and pure water, while the Cryptococcus group or Omerkoda yeast group were fed with 10 mg / kg water every day. 7 CFU / ml of Cryptococcus neoformans ATCC 32045 or Kodamaea ohmeri ATCC 20282, and the corresponding yeast suspension was gavaged every 3 days (10 9 CFU / ml). After two months of continuous feeding, the feces of mice were collected every other day, and the corresponding yeast count in the feces reached 10 7 CFU / g feces, and the number of pathogenic yeasts remained for one week, the model was considered successful. The number of Cryptococcus neoformans and Omerkoda yeast was quantified by real-time quantitative PCR, and the method was as follows:

[0122] The number of target yeasts was quantified by real-time quantitative PCR. The primers for amplification of target Cryptococcus neoformans and Omerkoda yeast were referenced in relevant literature. Fluorescence quantitative PCR was used to amplify the ITS1 fragments of Cryptococcus neoformans and Omerkoda yeast, and the DNA standard samples of target bacteria were used after gel excision and recovery. The operation process was strictly carried out according to the instructions of the agarose gel DNA recovery kit. Sample detection: Dilute the Cryptococcus neoformans and Omerkoda yeast DNA standards to 10 -1 ~10 - 9 10 copies / uL, and denuclease-free water was used as a control. The amplification system was 10uL of fluorescent dye 2×SYBR Green Reaction Mix, 2uL of upstream and downstream primers of target bacteria standard, 2uL of DNA sample extracted from feces, and 8uL of deionized water. Fluorescence quantitative PCR was performed under the reaction conditions of 95℃ pre-denaturation for 5min, 95℃ denaturation for 15s, 60℃ annealing for 1min, and 72℃ extension for 1min, after 40 cycles. The fluorescence signal was collected during the annealing stage, and the result was expressed as the CT value. The CT value refers to the number of amplification cycles corresponding to when the fluorescence signal of the amplified product reaches the set fluorescence threshold during real-time PCR amplification. Therefore, the smaller the CT value, the higher the template DNA content, that is, the higher the content of Cryptococcus neoformans or Omerkoda yeast. If the CT value is ≥40, it means that the template DNA content in the sample to be tested is very small and can be ignored.

[0123] (3) Experimental mouse grouping

[0124] After the mice were modeled, the 14 cryptococcal mice were randomly divided into a control group and a treatment group, with 7 mice in each group. The cryptococcal treatment group added probiotic freeze-dried powder to the drinking water every day, so that the number of live probiotics in the water reached 5×10 7 CFU / ml, the ratio of the number of live bacteria of Lactobacillus rhamnosus GYL-718, Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 was 4:3:3; the Cryptococcus control group was supplemented with an equal weight of auxiliary material maltodextrin in the drinking water every day. The experimental period was 1 month.

[0125] For the Omokoda yeast experimental group, 14 Omokoda yeast group mice were randomly divided into a control group and a treatment group, with 7 mice in each group. The Omokoda yeast treatment group added probiotic freeze-dried powder to the drinking water every day, so that the number of live probiotics in the water reached 5×10 7 CFU / ml, the ratio of the number of live bacteria of Lactobacillus rhamnosus GYL-718, Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 was 4:3:3; the control group of Omokoda yeast was supplemented with an equal weight of auxiliary material maltodextrin in the drinking water every day. The experimental period was 1 month.

[0126] In fact, the ratio of the number of live bacteria of Lactobacillus rhamnosus GYL-718, Lactobacillus reuteri LR-03 and Lactobacillus helveticus ZY-01 is 1:(0.5-10):(0.5-10), and the total number of live bacteria in the composite probiotic is not less than 3×10 6 CFU / mL or 3×10 6 CFU / g, the probiotics have very obvious technical effects. Preferably, in the probiotics, the number of viable bacteria of a single strain is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0127] (4) Collection of mouse fecal samples

[0128] At the beginning of the experiment, 4 g of fresh, naturally excreted feces samples were collected from each group of mice at 0, 2, and 4 weeks, and stored at -80°C. After the fecal samples were collected, the fecal fungal genomic DNA extraction kit was used to extract the total microbial DNA in the mouse intestine. The operation method was strictly carried out in accordance with the instructions of the kit. In addition, the NanoDrop spectrophotometer was used to measure the DNA concentration and purity, and the extracted DNA samples were stored at -20°C for future use.

[0129] (5) Target yeast real-time quantitative PCR detection

[0130] The bacterial counts of Cryptococcus neoformans and Omerkoda pyogenes were quantified by real-time quantitative PCR, using the same method as in (2).

[0131] (6) Results and analysis

[0132] Table 9 Quantitative analysis of Cryptococcus and Omokoda yeast in feces

[0133]

[0134] The residues of Cryptococcus neoformans and Omerkoda yeast in mice are shown in Table 9. After one month of probiotic feeding, the abundance of Cryptococcus neoformans in the feces of mice in the treatment group was significantly lower than that in the control group, as shown by a higher CT value. The data of the Cryptococcus control group and the Cryptococcus treatment group were tested using T-test, and the results were significant, indicating that the composite probiotic can significantly reduce the abundance of Cryptococcus neoformans in the intestines of mice.

[0135] After one month of feeding water with probiotics, the abundance of Omokoda yeast in the feces of the treated group of mice was significantly lower than that of the control group, as shown by a higher CT value. The data of the Omokoda yeast control group and the Cryptococcus treatment group were tested using T-test, and the results were significant, indicating that the composite probiotics can significantly reduce the abundance of Omokoda yeast in the intestines of mice.

[0136] Obviously, the probiotic can be used to prepare food, health products or medicines for preventing and alleviating invasive fungal infections caused by Cryptococcus or Koda yeast. In addition to the freeze-dried powder dosage form used in the above experiment, capsules, tablets or granules can also be used. The probiotic also includes a freeze-dried protective agent and / or auxiliary additives. Among them, the freeze-dried protective agent includes any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol; the auxiliary additive includes any one or a combination of at least two of oligofructose, oligogalactose, oligoxylose, oligomalto-oligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, Yunzhi polysaccharide, stachyose, polydextrose, α-lactoprotein or lactoferrin.

[0137] The above embodiments are only preferred embodiments of the present invention, and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A probiotic agent having the ability to inhibit Cryptococcus and Kodacryocystis, characterized in that: It includes at least one of the following strains: (1) Lactobacillus rhamnosus GYL-718, deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No. 31642; (2) Lactobacillus helveticus ZY-01, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31641.

2. The probiotic agent having the function of inhibiting Cryptococcus and Kodacryocystis as claimed in claim 1, characterized in that: The probiotics also include Lactobacillus reuteri LR-03 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC No.31643.

3. The probiotic agent having the function of inhibiting Cryptococcus and Kodacryocystis according to claim 1 or 2, characterized in that: The number of viable bacteria of a single species in the probiotic is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The probiotic agent having the function of inhibiting Cryptococcus and Kodacryosaccharomyces according to claim 1 or 2, characterized in that: The probiotics include freeze-dried powder, capsules, tablets or granules.

5. The probiotic agent having the function of inhibiting Cryptococcus and Kodacryosaccharomyces according to claim 4, characterized in that: The probiotic agent is in the form of a freeze-dried powder, and the probiotic agent also includes a freeze-drying protective agent and / or auxiliary additives.

6. The probiotic agent having the function of inhibiting Cryptococcus and Kodacryocystis according to claim 5, characterized in that: The lyophilization protective agent includes any one or more of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

7. The probiotic agent having the function of inhibiting Cryptococcus and Kodacryocystis according to claim 5, characterized in that: The auxiliary additives include any one or more of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactoprotein or lactoferrin.

8. Use of Lactobacillus reuteri LR-03 in promoting the activity of Lactobacillus rhamnosus GYL-718 and / or Lactobacillus helveticus ZY-01 in inhibiting Cryptococcus and Kodacryosaccharomyces cerevisiae, characterized in that: The Lactobacillus reuteri LR-03 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.31643; The Lactobacillus rhamnosus strain GYL-718 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31642; The Lactobacillus helveticus ZY-01 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31641.

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