Enterococcus hirae, composition containing same and application

Abstract

The present application relates to the technical field of microorganism, in particular to a strain of Enterococcus hirae QHBMA068, a composition containing the same and use thereof.The Enterococcus hirae is Enterococcus hirae QHBMA068, the preservation number of which is CCTCC NO: M 20252987, and it is preserved in China Center for Type Culture Collection.The composition contains at least one of Pediococcus acidilactici and a culture of the strain.The composition is used for preparing a composition for feed fermentation.The present application has the following advantages: (1) high efficiency in degrading mycotoxins; (2) good probiotic properties, strong growth performance, strong acid tolerance, bile salt tolerance and osmotic pressure tolerance; (3) good hydrophobicity and self-aggregation performance; and (4) high safety.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Enterococcus hirae QHBMA068, compositions containing it, and their uses. Background Technology

[0002] Mycotoxins are secondary metabolites of fungi and are natural contaminants that seriously endanger feed and agricultural products, posing a serious threat to feed safety, food preservation, and human health. Globally, approximately 25% of crops are contaminated with mycotoxins annually. Among them, aflatoxin B1 (AFB1), deoxynivalenol (DON), and zearalenone (ZEA) are widely found in grains such as corn and wheat. Under high temperature and humidity conditions, whether during the field growth period of grains or during feed production and storage, mold growth easily occurs, leading to excessive mycotoxin levels. Furthermore, feed is susceptible to co-contamination by multiple mycotoxins.

[0003] Currently, diseases caused by bacteria and fungi are generally treated with antibiotics. However, the overuse of antibiotics has led to varying degrees of resistance in most bacteria and fungi, resulting in drug residues in food, which has become a serious public health and food safety issue. Since July 1, 2020, my country has implemented a complete ban on antibiotics in livestock and poultry feed. This ban presents a new round of challenges for the livestock and poultry farming industry.

[0004] Fermenting feed with probiotics can not only reduce the content of mycotoxins in feed, but also improve the nutritional value and digestibility of feed, regulate the intestinal microecology of livestock and poultry, enhance the immunity of animals, and have no side effects on feed quality, livestock and poultry growth and livestock product safety.

[0005] Animal gastrointestinal microorganisms play an extremely important role in animal nutrition and health. Functional research on the exploration of probiotic resources in the intestines of animals on the Qinghai-Tibet Plateau will not only help to understand the ecological adaptation mechanism of plateau wild animals, but also bring new opportunities to human health and biotechnology. It is also the key to developing new green, pollution-free, and low-residue feed additives to replace the use of antibiotics, and a guarantee for the healthy development of intensive and high-yield livestock and poultry production models. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient mycotoxin degrading Enterococcus haematobacterium, a probiotic, with strong growth performance, strong acid, bile salt and osmotic pressure resistance, good hydrophobicity and self-aggregation properties, and high safety, as well as a composition containing the same and its applications.

[0007] Preservation instructions:

[0008] Classification and naming: Enterococcus hirae QHBMA068;

[0009] Accession number: CCTCC NO: M 20252987.

[0010] Preservation period: December 22, 2025;

[0011] Depository: China Center for Type Culture Collection;

[0012] Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0013] Strain source: Enterococcus hirae QHBMA068 was isolated from the feces of wild yak and Tibetan wild ass in Qinghai Province, and was previously isolated and purified by the Key Laboratory of Conservation and Innovative Utilization of Genetic Resources of Plateau Livestock in Qinghai Province.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] In a first aspect, the present invention provides a *Enterococcus haematobacterium*.

[0016] A type of Enterococcus hirae, namely Enterococcus hirae QHBMA068, with accession number CCTCC NO: M 20252987, is deposited at the China Center for Type Culture Collection.

[0017] Secondly, the present invention provides a microbial preparation.

[0018] A microbial preparation comprising Enterococcus haematobacterium or a culture of said strain.

[0019] The Enterococcus hirae mentioned is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M20252987, deposited at the China Center for Type Culture Collection.

[0020] Thirdly, the present invention provides a composition.

[0021] A composition comprising at least one of Enterococcus haematobacterium and a culture of said strain;

[0022] The cultures of the strains include, but are not limited to, fermentation broth, fermentation precipitate, or lyophilized powder of Enterococcus hesperidin.

[0023] The Enterococcus hirae mentioned is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M20252987, deposited at the China Center for Type Culture Collection.

[0024] Furthermore, the composition is a microecological preparation or a pharmaceutical preparation.

[0025] Furthermore, the composition is a probiotic agent.

[0026] Furthermore, the composition also contains substances that help maintain the viability of Enterococcus hesperidin; the composition also contains other probiotics and / or prebiotics.

[0027] Fourthly, the present invention provides the use of Enterococcus helicobacter of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect.

[0028] The use of Enterococcus haematobacterium of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect, in the preparation of a composition for degrading mycotoxins.

[0029] Fifthly, the present invention provides another use of the Enterococcus helicobacter of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect.

[0030] The use of Enterococcus helixeri of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect, in the preparation of probiotic preparations and / or probiotic preparation compositions.

[0031] In a sixth aspect, the present invention provides a third use of the Enterococcus helicobacter of the first aspect, the microbial preparation of the second aspect, or the composition of the third aspect.

[0032] The use of Enterococcus haematobacterium of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect, in the preparation of a composition for feed fermentation.

[0033] In the fourth, fifth, and sixth uses, the Enterococcus hirae is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M 20252987, deposited at the China Center for Type Culture Collection.

[0034] In a seventh aspect, the present invention provides an animal feed.

[0035] An animal feed comprising Enterococcus heisenbergii as described in the first aspect;

[0036] And / or the fermentation product of Enterococcus heisenbergii from the first aspect;

[0037] and / or the second aspect of microbial preparations;

[0038] Combinations with and / or third parties.

[0039] The composition of Enterococcus hydatids provided by this invention may contain substances that help maintain the viability of Enterococcus hydatids. Substances that help maintain the viability of Enterococcus hydatids (such as protectants) may be any one or a combination of cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), and vitamin E.

[0040] The composition of Enterococcus haematobacterium provided by the present invention may contain other probiotics, which may be selected from any one or a combination of lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, Enterococcus haematobacterium, Enterococcus haematobacterium, and Enterococcus haematobacterium.

[0041] The Enterococcus helicobacter provided by the present invention may contain prebiotics, which may be selected from any one or a combination thereof of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, and oligosaccharides.

[0042] The *Enterococcus hirae* strain provided in this invention is *Enterococcus hirae* QHBMA068, with accession number CCTCCNO: M 20252987, deposited at the China Center for Type Culture Collection on December 22, 2025. This strain was isolated from the feces of wild yak and Tibetan wild ass in Qinghai. *Enterococcus hirae* (QHBMA068) can efficiently degrade mycotoxins and has probiotic properties; it can be used as a feed additive or as a microbial inoculum for feed fermentation, while also improving feed utilization.

[0043] Compared with the prior art, the advantages of the Enterococcus haematobacterium strain, the composition containing it, and its application provided by the present invention are as follows:

[0044] (1) It can efficiently degrade mycotoxins.

[0045] (2) It has beneficial properties, strong growth performance, and strong resistance to acid, bile salts and osmotic pressure.

[0046] (3) It has good hydrophobicity and self-aggregating properties.

[0047] (4) High security. Attached Figure Description

[0048] Figure 1The degradation rate of AFB1, DON, and ZEA by Enterococcus hirae (QHBMA068) provided by this invention.

[0049] Figure 2 The growth curve and acid production curve of the Enterococcus hirae QHBMA068 strain provided by this invention are shown.

[0050] Figure 3 This invention describes the growth of the Enterococcus hirae (QHBMA068) strain under different pH values, bile salt concentrations, and NaCl concentrations.

[0051] Figure 4 The results of the drug susceptibility test of the Enterococcus hirae QHBMA068 strain provided by this invention are as follows.

[0052] Figure 5 The results of the Columbia blood agar plate test for the Enterococcus hirae QHBMA068 strain provided by this invention are shown.

[0053] Figure 3 In the figure, A is the bile salt tolerance curve; B is the acid tolerance curve; and C is the osmotic pressure tolerance curve. Detailed Implementation

[0054] To enable those skilled in the art to better understand the technical solutions of the present invention, the following embodiments provide a more detailed description of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0055] In a first aspect, the present invention provides a *Enterococcus haematobacterium*.

[0056] A type of Enterococcus hirae, namely Enterococcus hirae QHBMA068, with accession number CCTCC NO: M 20252987, is deposited at the China Center for Type Culture Collection.

[0057] Secondly, the present invention provides a microbial preparation.

[0058] A microbial preparation comprising Enterococcus haematobacterium or a culture of said strain.

[0059] The Enterococcus hirae mentioned is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M20252987, deposited at the China Center for Type Culture Collection.

[0060] Thirdly, the present invention provides a composition.

[0061] A composition comprising at least one of Enterococcus haematobacterium and a culture of said strain;

[0062] The cultures of the strains include, but are not limited to, fermentation broth, fermentation precipitate, or lyophilized powder of Enterococcus hesperidin.

[0063] The Enterococcus hirae mentioned is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M20252987, deposited at the China Center for Type Culture Collection.

[0064] Furthermore, the composition is a microecological preparation or a pharmaceutical preparation.

[0065] Furthermore, the composition is a probiotic agent.

[0066] Furthermore, the composition also contains substances that help maintain the viability of Enterococcus hesperidin; the composition also contains other probiotics and / or prebiotics.

[0067] Fourthly, the present invention provides the use of Enterococcus helicobacter of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect.

[0068] The use of Enterococcus haematobacterium of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect, in the preparation of a composition for degrading mycotoxins.

[0069] Fifthly, the present invention provides another use of the Enterococcus helicobacter of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect.

[0070] The use of Enterococcus helixeri of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect, in the preparation of probiotic preparations and / or probiotic preparation compositions.

[0071] In a sixth aspect, the present invention provides a third use of the Enterococcus helicobacter of the first aspect, the microbial preparation of the second aspect, or the composition of the third aspect.

[0072] The use of Enterococcus haematobacterium of the first aspect, or the microbial preparation of the second aspect, or the composition of the third aspect, in the preparation of a composition for feed fermentation.

[0073] In the fourth, fifth, and sixth uses, the Enterococcus hirae is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M 20252987, deposited at the China Center for Type Culture Collection.

[0074] In a seventh aspect, the present invention provides an animal feed.

[0075] An animal feed comprising Enterococcus heisenbergii as described in the first aspect;

[0076] And / or the fermentation product of Enterococcus heisenbergii from the first aspect;

[0077] and / or the second aspect of microbial preparations;

[0078] Combinations with and / or third parties.

[0079] The composition of Enterococcus hydatids provided by this invention may contain substances that help maintain the viability of Enterococcus hydatids. Substances that help maintain the viability of Enterococcus hydatids (such as protectants) may be any one or a combination of cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), and vitamin E.

[0080] The composition of Enterococcus haematobacterium provided by the present invention may contain other probiotics, which may be selected from any one or a combination of lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, Enterococcus haematobacterium, Enterococcus haematobacterium, and Enterococcus haematobacterium.

[0081] The Enterococcus helicobacter provided by the present invention may contain prebiotics, which may be selected from any one or a combination thereof of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, and oligosaccharides.

[0082] Implementation Examples Basic Methods

[0083] 1. Screening of mycotoxin-degrading strains

[0084] The strain was cultured to the plateau phase. 5 mL of bacterial culture was taken and 1 mg / L AFB1, DON, and ZEA were added simultaneously. The culture was incubated at 210 r / min for 72 h. After centrifugation at 4000 × g for 15 min, the supernatant was collected. The mycotoxin content was detected using an ELISA kit to screen strains with high degradation rates of mixed toxins.

[0085] 2. Study on the beneficial properties of the strain

[0086] 2.1 Growth performance and acid production performance

[0087] The bacterial strain was inoculated into liquid culture medium at a 1% inoculum and incubated at 37°C for 0h, 2h, 4h, 6h, 8h, 12h, 16h, 20h, 24h, 28h, 36h, and 48h, with a blank culture medium as a control. The OD of the bacterial culture was measured. 600 Based on nm and pH values, with 3 replicates per group, growth curves and acid production curves were plotted.

[0088] 2.2 Resistance to acids, bile salts, and osmotic pressure

[0089] The bacterial strains were inoculated into liquid culture media with pH adjusted to 4.0, 3.0, and 2.5; liquid culture media with ox bile salt concentrations of 0, 0.1%, 0.2%, and 0.3%; and liquid culture media with NaCl concentrations of 2%, 4%, 6%, and 8%, respectively. The cultures were incubated at 37°C for 24 h, with the corresponding uninoculated media serving as blank controls. The OD values ​​of the bacterial cultures were measured. 600 nm value.

[0090] 2.3 Hydrophobicity and self-aggregation

[0091] Hydrophobicity and autoagglutination rate were determined by culturing the bacterial suspension at 37°C for 24 h, followed by centrifugation (8000×g, 5 min), washing twice with PBS, and resuspending. The OD value (A0) of the bacterial suspension was measured. 3 ml of the cell suspension was placed in a centrifuge tube, and 1 ml of xylene was added. The mixture was pre-cultured at room temperature for 10 min. The mixture was then rapidly vortexed for 2 min, allowed to stand at room temperature for 15 min to allow for layering, and the lower aqueous phase was collected for OD measurement. 600 Value (A) t Hydrophobicity calculation: A% = (A0 - A) t ) / A0×100%. Add 5 mL of bacterial suspension to a test tube, let it stand at room temperature, and measure the OD value (At) of the supernatant after 20 h. Calculate the autoagglutination rate: A%=[(A0-A0) / A0×100%. t ) / A0]×100%.

[0092] 2.4 Antibacterial properties

[0093] Following GB / T 38483-2020, Salmonella, Staphylococcus aureus, and Escherichia coli were used as indicator bacteria, and the Oxford cup method was employed for antibacterial testing. The bacterial suspension was centrifuged (8000×g) for 2 min, and the supernatant was filtered through a membrane and added to Oxford cups in solid culture dishes containing the indicator bacteria. After incubation at 37℃ for 24 h, the inhibitory effect on the indicator bacteria was detected. The size of the inhibition zone was observed and recorded, and the antibacterial ability of the strain was determined.

[0094] 3. Safety studies of the strain

[0095] 3.1 Drug sensitivity

[0096] The drug susceptibility of the strain was tested using the drug susceptibility test strip method according to WS / T 6639-2018. 150 μL of bacterial solution was added to a solid culture medium and spread evenly. Drug susceptibility test strips were then attached to the surface of the solid culture medium. The diameter of the inhibition zone was observed and recorded after 24 hours to determine the drug susceptibility.

[0097] 3.2 Hemolytic

[0098] The test strains were inoculated onto Columbia blood agar plates and cultured at 37°C for 24 hours. Staphylococcus aureus was set as a positive control. The presence of hemolytic zones around the colonies was observed to determine hemolysis.

[0099] 4. Data Analysis and Processing

[0100] Each experiment was repeated three times. After the experimental data were collected and organized, one-way ANOVA was used to analyze the data and generate graphs.

[0101] Screening of mycotoxin-degrading strains

[0102] like Figure 1 As shown, Enterococcus hirae (QHBMA068) can efficiently degrade mycotoxins. The comprehensive degradation rate of AFB1, DON, and ZEA mixed toxins by Enterococcus hirae QHBMA068 (the average degradation rate of AFB1, DON, and ZEA) is over 78%, and the degradation rates of AFB1, DON, and ZEA reach 73.60%, 77.02%, and 83.97%, respectively.

[0103] Example 2

[0104] Probiotic properties of Enterococcus hirae (QHBMA068)

[0105] 1. Determination of growth capacity and acid production capacity

[0106] like Figure 2 As shown in Figure A, the Enterococcus hirae QHBMA068 provided by this invention enters the logarithmic growth phase 2 hours after inoculation and reaches the plateau phase 8 hours later. Enterococcus hirae QHBMA068 exhibits strong growth and reproduction capabilities, reaching its peak at 20 hours, with an OD value of [missing value]. 600 nm is 1.86, OD at 48h 600 The nm value reached 1.83. For example... Figure 2 As shown in Figure B, lactic acid bacteria strain EK013 produces acid.

[0107] 2. The strain's resistance to acid, bile salts, and osmotic pressure.

[0108] like Figure 3As shown in Figure A, bile salts inhibit bacterial growth; at a bile salt concentration of 0.3%, bacterial growth essentially ceases. Figure 3 As shown in Figure B, the growth of the strain decreases with decreasing pH, and growth essentially ceases at pH 2.5. Strain QHBMA068 exhibits excellent growth under low pH conditions, demonstrating a certain degree of acid resistance. Figure 3 As shown in Figure C, the growth of strain QHBMA068 gradually decreased as the NaCl concentration increased from 0% to 8%, indicating that it has a certain ability to withstand osmotic pressure.

[0109] 4. Hydrophobicity, self-agglutination, and antibacterial properties of the strain

[0110] As shown in Table 1, strain QHBMA068 has a hydrophobicity of 95.43% and a self-agglutination rate of 89.06%. It also exhibits strong inhibitory effects against Escherichia coli and Staphylococcus aureus, as well as Salmonella.

[0111] Table 1. Hydrophobicity, autoagglutination rate, and inhibitory ability against pathogens of bacterial strains

[0112]

[0113] Note: (+) indicates an inhibition zone diameter of 9.0-12.99 mm; (++) indicates an inhibition zone diameter of 13.0-16.99 mm; (+++) indicates an inhibition zone diameter ≥ 17 mm.

[0114] 5. Strain safety

[0115] (1) Drug sensitivity

[0116] As shown in Table 2, *Enterococcus hesperidin* QHBMA068 was sensitive to tetracycline, ampicillin, and cefazolin; moderately sensitive to erythromycin and furazolidone; and insensitive to streptomycin, ofloxacin, tobramycin, ciprofloxacin, trimethoprim-sulfamethoxazole, and polymyxin B. Drug susceptibility testing is shown in [Table 2]. Figure 4 .

[0117] Table 2. Assessment of antibiotic susceptibility of strains

[0118]

[0119] Note: S indicates sensitive, R indicates insensitive, and I indicates moderately sensitive.

[0120] (2) Hemolytic

[0121] like Figure 5 The indicated pathogen Staphylococcus aureus showed β-hemolysis (complete hemolysis), while strain QHBMA068 showed γ-hemolysis, meaning it did not produce hemolysin.

[0122] The *Enterococcus hirae* QHBMA068 provided by this invention, with accession number CCTCCNO: M 20252987, was deposited at the China Center for Type Culture Collection on December 22, 2025. *Enterococcus hirae* QHBMA068 was isolated from the feces of wild yak and Tibetan wild ass in Qinghai Province, and was previously isolated and purified by the Key Laboratory of Conservation and Innovative Utilization of Plateau Livestock Genetic Resources in Qinghai Province.

[0123] The *Enterococcus helixeri* QHBMA068 provided by this invention can tolerate a certain degree of acidity, high bile salt content, and high osmotic pressure environments, exhibiting high hydrophobicity and autoaggregation rate. *Enterococcus helixeri* QHBMA068 is sensitive to tetracycline, ampicillin, and cefazolin; moderately sensitive to erythromycin and furazolidone; and insensitive to streptomycin, ofloxacin, tobramycin, ciprofloxacin, trimethoprim-sulfamethoxazole, and polymyxin B. *Enterococcus helixeri* QHBMA068 is non-hemolytic. It possesses good probiotic properties and safety, with no side effects on humans and animals, and can be used for experimental and production purposes.

[0124] Enterococcus hirae (QHBMA068) can efficiently degrade mycotoxins. The comprehensive degradation rate of AFB1, DON, and ZEA mixed toxins (the average degradation rate of AFB1, DON, and ZEA) by Enterococcus hirae QHBMA068 is over 78%, and the degradation rates of AFB1, DON, and ZEA reach 73.60%, 77.02%, and 83.97%, respectively.

[0125] Enterococcus haematobacterium QHBMA068 can efficiently degrade mycotoxins and has probiotic properties; it can be used as a feed additive or as a microbial inoculum for feed fermentation, while also improving feed utilization.

[0126] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0127] It should also be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0128] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A type of Enterococcus haematobacterium, characterized in that, The Enterococcus hirae mentioned is Enterococcus hirae QHBMA068, with accession number CCTCC NO: M 20252987, deposited at the China Center for Type Culture Collection.

2. A microbial preparation, characterized in that: The microbial preparation contains Enterococcus haematobacterium or a culture of the strain.

3. A composition, characterized in that: The composition comprises at least one of Enterococcus haematobacterium and a culture of the strain; The cultures of the strains include, but are not limited to, fermentation broth, fermentation precipitate, or lyophilized powder of Enterococcus hesperidin.

4. The composition according to claim 3, characterized in that: The composition is a microecological preparation or a pharmaceutical preparation.

5. The composition according to claim 4, characterized in that: The composition is a probiotic agent.

6. The composition according to claim 3, characterized in that: The composition also contains substances that help maintain the viability of Enterococcus hesperidin; the composition also contains other probiotics and / or prebiotics.

7. Use of the Enterococcus haematobacterium of claim 1, or the microbial preparation of claim 2, or the composition of any one of claims 3 to 6 in the preparation of a composition for degrading mycotoxins.

8. Use of the Enterococcus haematobacterium of claim 1, or the microbial preparation of claim 2, or the composition of any one of claims 3 to 6 in the preparation of probiotic preparations and / or probiotic preparation compositions.

9. Use of the Enterococcus haematobacterium of claim 1, or the microbial preparation of claim 2, or the composition of any one of claims 3 to 6 in the preparation of a composition for feed fermentation.

10. An animal feed, characterized in that: The animal feed contains Enterococcus helixeri and / or Enterococcus helixeri ferments according to claim 1 and / or the microbial preparation according to claim 2 and / or the composition according to any one of claims 3 to 6.

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