Lactococcus lactis ZJUIDS-C036, which promotes calcium absorption in pets, and its applications.

Abstract

This invention belongs to the field of microbial technology, specifically relating to *Pediococcus lactis* ZJUIDS-C036, which has the ability to accumulate calcium, and its applications. This invention discloses a *Pediococcus lactis* (… Pediococcus acidilactici ZJUIDS-C036, with accession number CGMCC NO. 34468, is also described in this invention. This invention also provides the application of the aforementioned *Pediococcus lactis* ZJUIDS-C036 in promoting calcium absorption / accumulation and in the preparation of drugs that promote calcium absorption. This strain has the potential to prevent osteoporosis and thus can improve calcium utilization in animals.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the calcium-accumulating Pediococcus lactis ZJUIDS-C036 and its applications. Background Technology

[0002] With the expansion of the pet market, canine and feline skeletal health has become a core concern for families. Calcium, a major component of bones (accounting for 99% of bone minerals), suffers from metabolic imbalances that lead to dental deformities in young pets and osteoporosis in adults. Traditional calcium supplementation methods suffer from drawbacks such as low absorption rates (inorganic calcium <30%), high costs (organic calcium is 3-5 times more expensive than inorganic calcium), and the risk of kidney stones. Under varying metal ion concentrations, bacteria can absorb various heavy metals. Lactobacilli, a common type of bacteria, can attach metal ions to their surface or transfer them into the body for storage, laying the foundation for the enrichment and utilization of trace elements. Current research shows that certain Bifidobacterium strains can significantly improve calcium bioavailability and enhance bone density. Some lactobacilli have also been shown to promote calcium absorption by regulating intestinal mucosa and immune function. The enrichment and conversion of calcium by beneficial microorganisms has become a research hotspot in this field, aiming to obtain probiotics with high calcium-accumulating capabilities. This provides a theoretical basis for the development of probiotic fermentation agents and products that use probiotics to supplement calcium and prevent osteoporosis. This provides a theoretical basis for the development of products that improve calcium utilization in dogs and cats, in particular.

[0003] CN119464163A, entitled "A Method for Preparing Lactobacillus rhamnosus Lr-Ca011, Postbiotics, Calcium Products and Their Nanoemulsification Homogenization for Promoting Calcium Absorption", discloses Lactobacillus rhamnosus Lr-Ca011 and the preparation of postbiotics and calcium products that promote calcium absorption using Lactobacillus rhamnosus Lr-Ca011.

[0004] CN119432643A, entitled "A strain of Lactobacillus plantarum NSL0125 and its application in the preparation of microbial preparations that promote calcium supplementation and growth and development," discloses Lactobacillus plantarum NSL0125 and the preparation of biological preparations using Lactobacillus plantarum NSL0125.

[0005] CN119464158A, titled "A type of Pediococcus lactis with acid resistance, bile salt resistance, and antibacterial activity and its application," discloses Pediococcus lactis TR19. Pediococcus lactis TR19 exhibits acid resistance, bile salt resistance, and antibacterial activity, while also demonstrating good degradation performance against nitrite and cholesterol.

[0006] CN119345237A, "Application of a strain of Pediococcus lactis," discloses Pediococcus lactis TG005, which was obtained through screening from feces of healthy individuals in China. This strain can lower cholesterol levels and prevent cardiovascular diseases; degrade uric acid precursors, reduce uric acid production, and prevent hyperuricemia and related diseases; it can also inhibit pathogenic bacteria such as Fusobacterium nucleatum and Bacteroides fragilis, and prevent colorectal cancer.

[0007] CN119286740A, entitled "A strain of Pediococcus lactis ZKPa1222, its Astragalus fermentation broth and its application in improving immunity", discloses Pediococcus lactis ZKPa1222. It describes the direct fermentation of Astragalus extract using Pediococcus lactis ZKPa1222, which simplifies the fermentation process and improves the fermentation efficiency. Summary of the Invention

[0008] The problem to be solved by the present invention is to provide a lactic acid cocci ZJUIDS-C036 that promotes calcium absorption in pets and its application.

[0009] To address the aforementioned technical problems, this invention provides a *Pediococcus acidilactici* strain ZJUIDS-C036, with accession number CGMCC NO. 34468.

[0010] This invention also provides the application of the above-mentioned Pyrococcus lactis ZJUIDS-C036 in promoting calcium absorption / calcium enrichment.

[0011] This invention also provides the application of the above-mentioned Pyrococcus lactis ZJUIDS-C036 in the preparation of drugs that promote calcium absorption.

[0012] An improvement to the application of this invention: used to promote calcium absorption in pets.

[0013] As a further improvement to the application of the present invention, *Pediococcus lactis* ZJUIDS-C036 possesses at least one of the following properties:

[0014] 1. It has a certain ability to accumulate calcium (promote intestinal calcium absorption);

[0015] 2. It has a certain acid-producing capacity;

[0016] 3. It can tolerate acid, bile salts, and artificial gastric and intestinal juices, is suitable for the gastrointestinal environment, and has a certain proliferative capacity;

[0017] 4. It has a certain degree of hydrophobicity and self-aggregating ability, which can promote intestinal colonization and enhance metabolic activity;

[0018] 5. It does not develop antibiotic resistance and has antibacterial activity.

[0019] 6. Strong antioxidant activity;

[0020] 7. It does not hemolyze on Columbia blood agar plates, indicating a certain degree of safety.

[0021] The preservation information of the strain ZJUIDS-C036 of this invention is as follows:

[0022] Preservation Name: Pediococcus acidilactici, Preservation Institution: China General Microbiological Culture Collection Center, Preservation Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Preservation Number: CGMCC NO. 34468, Preservation Date: May 9, 2025.

[0023] This invention screened a strain of *Pediococcus acidilactici* ZJUIDS-C036 from pet cats and identified the bacterium through bacterial morphology, physiology, culture characteristics, and 16S rDNA sequencing. The full-length 16S rDNA sequence of *Pediococcus acidilactici* ZJUIDS-C036 of this invention is shown in SEQ ID No: 1.

[0024] This invention first discovered a strain of *Pediococcus lactis* ZJUIDS-C036 with high calcium accumulation capacity and potential for preventing osteoporosis, thereby improving calcium utilization in animals. This invention then provides the application of the aforementioned *Pediococcus lactis* ZJUIDS-C036 in the preparation of products that promote intestinal calcium absorption. These products include bacterial powder, bacterial agents, milk powder, fermented yogurt, cat food, etc.

[0025] The technical advantages of this invention are:

[0026] (1) The present invention isolates a strain of Pyotrophic lactic acid bacteria ZJUIDS-C036 with calcium enrichment function, which is safe and non-pathogenic. At the same time, it has unique effects on calcium ion adsorption, transport and absorption promotion, thereby effectively promoting calcium absorption and growth and development of the body, filling the gap in the efficacy of probiotic products;

[0027] (2) This invention can provide a foundation for the development of foods and health products that promote the absorption of calcium in the intestines by beneficial bacteria, and has great application value and good market application prospects.

[0028] (3) Lactococcus lactis ZJUIDS-C036 has good probiotic properties in promoting calcium absorption in the intestines of dogs and cats.

[0029] In summary, this invention screened *Pediococcus lactis* strains with high calcium accumulation from probiotics isolated from pet dogs. This strain exhibits a high calcium accumulation capacity. It shows significant advantages over other probiotics in acid production and tolerance to artificial gastrointestinal fluids, demonstrating suitability for the intestinal environment and proliferative capacity. Furthermore, this strain possesses high hydrophobicity and strong self-aggregation ability, promoting intestinal colonization and enhancing metabolic activity. Simultaneously, this strain exhibits antibacterial activity, lacks antibiotic resistance, and does not hemolyze on blood agar plates, demonstrating a certain level of safety. The *Pediococcus lactis* strain of this invention can be widely used in developing probiotic products that prevent and improve calcium utilization in dogs and cats. Attached Figure Description

[0030] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] Figure 1 The colony morphology of ZJUIDS-C036 (left) and the cell morphology after Gram staining (right).

[0032] Figure 2 The calcium enrichment of ZJUIDS-C036.

[0033] Figure 3 This refers to the acid production capacity of ZJUIDS-C036.

[0034] Figure 4 The self-cohesive force of ZJUIDS-C036.

[0035] Figure 5 The growth curve of ZJUIDS-C036.

[0036] Figure 6 The hemolysis test for ZJUIDS-C036. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0038] Example 1: Screening and identification of Pediococcus lactis ZJUIDS-C036:

[0039] 1. Screening of Pediococcus lactis ZJUIDS-C036

[0040] 1.1 Sample Source

[0041] The strain used in this invention was isolated from the feces of pet cats in Zhejiang Province.

[0042] 1.2 Isolation and purification of strains

[0043] Samples were collected using rectal swabs. 1 mL of sterile saline was added to a sterile 1.5 mL EP tube beforehand. A sterile cotton swab was moistened with saline and then used to collect the rectal swab. The swab was then inserted into the EP tube and immersed in saline, with the wooden part removed. Each sample was collected in a sterile EP tube and immediately sent to the laboratory for bacterial isolation. The samples were vortexed and allowed to stand for 10 minutes. Then, 100 μL of enrichment solution was drawn up in a laminar flow hood and serially diluted tenfold with sterile saline. 10 μL of the diluted solution was selected for further analysis. -6 10 -7 10 -8 Three dilution gradients were used, with 100 μL of bacterial culture from each gradient plated onto MRS agar medium and incubated at 37°C for 48 h. After incubation, plates with 50–150 single colonies were selected from the agar medium, and typical colonies were picked and streaked multiple times on MRS agar plates for purification until the colonies on the entire plate had a uniform morphology. Single colonies were then picked and enlarged in MRS liquid medium. All obtained strains were stored frozen at -80°C in MRS liquid medium containing 40% glycerol.

[0044] Finally, a strain with strong calcium accumulation, good growth, and good genetic stability was selected and named ZJUIDS-C036.

[0045] 2. Identification of Pediococcus lactis ZJUIDS-C036

[0046] 2.1 Colony characteristics

[0047] After culturing the isolated and purified *Pediococcus lactis* ZJUIDS-C036 on MRS agar medium for 24 hours, the colonies were round, moist, smooth, with regular edges, and white in color. Figure 1 .

[0048] 2.2 Microscopic morphology:

[0049] Pediococcus lactis ZJUIDS-C036 colony smear: Gram-positive cocci, single, paired, or arranged in short chains, oval-shaped cocci, non-spore-forming, non-capsulated, see [see details]. Figure 1 .

[0050] 2.3 16S rDNA Identification

[0051] Genomic DNA of the target strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The extracted probiotic genomic DNA was used as a template for PCR amplification. PCR experiments of 16S rDNA were performed using universal bacterial primers 27F and 1492R. After the PCR reaction, the PCR product was sent to BGI Genomics Co., Ltd. for sequencing, and BLAST sequence alignment was performed on the NCBI website. The results showed that the sequence had more than 99% homology with the identified 16S rDNA sequence of Pediococcus lactis.

[0052] By combining the sequence alignment results and physiological and biochemical results of strain ZJUIDS24, the screened probiotic ZJUIDS-C036 was identified as Pediococcus acidilactici ZJUIDS-C036.

[0053] The preservation information of the strain ZJUIDS-C036 of this invention is as follows:

[0054] Preservation Name: Pediococcus acidilactici, Preservation Institution: China General Microbiological Culture Collection Center, Preservation Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Preservation Number: CGMCC NO. 34468, Preservation Date: May 9, 2025.

[0055] Example 2: Confirmation of calcium enrichment in Pediococcus lactis ZJUIDS-C036

[0056] 1. Preparation of fermentation broth for bacterial strains

[0057] The strain preserved in glycerol tubes was first streaked on MRS agar plates 2-3 times to activate it. Then, single colonies were picked and cultured in MRS liquid medium for expansion. The culture was incubated statically at 37°C for 18 hours. Then, 1% (v / v) of the culture was inoculated into MRS liquid medium and cultured aerobically at 37°C for 18 hours to obtain a bacterial suspension.

[0058] 2. Determination of in vitro calcium accumulation capacity

[0059] First, adjust the bacterial suspension to a bacterial cell concentration of 1×10⁻⁶. 8CFU / mL was added to MRS-CaCl2 liquid medium at an inoculum of 2% (V / V) (using CaCl2 as the calcium source, introducing a calcium ion concentration of 1.2 mg / mL into the MRS medium). After incubation at 37℃ and 200 r / min for 5 h with shaking, the medium was centrifuged at 8000 r / min for 20 min. The supernatant was collected, and the calcium ion content in the supernatant was determined by titration with disodium ethylenediaminetetraacetic acid (EDTA). The centrifuged bacterial cells were collected, freeze-dried for 48 h, and the bacterial mass was measured. The calcium enrichment was calculated using the following formula:

[0060] Calcium enrichment = (MRS-CaCl2 - Total calcium in liquid culture medium - Residual calcium in supernatant) / Bacterial weight × 100

[0061] The results are as follows Figure 2 The results showed that the calcium accumulation of *Pediococcus lactis* strain ZJUIDS-C036 was 54.17 mg / g, which was higher than that of the other two *Pediococcus lactis* strains (ZJUIDS-C033 and ZJUIDS-C004). After chelating free calcium with EDTA, probiotics need to achieve calcium accumulation through surface adsorption (such as charge binding) or active transport (such as calcium ion exchange). 2+ -ATPase) enriches calcium. This process mimics the mechanism of calcium uptake by active transport midgut epithelial cells, thus indicating that *Pediococcus lactis* ZJUIDS-C036 is a strain with similar capabilities. The aim is to obtain *Pediococcus lactis* strains with high calcium enrichment, further demonstrating that this strain has potential in promoting calcium absorption in the canine and feline intestines, improving calcium utilization in the body, and preventing the risk of diseases such as osteoporosis.

[0062] Example 3: Confirmation of the acid-producing ability of *Pediococcus lactis* ZJUIDS-C036 (conventional experimental method)

[0063] Activated *Pediococcus lactis* ZJUIDS-C036 was inoculated into MRS broth at a rate of 2% (V / V) and cultured at 37℃ for 24 hours. Samples were taken every 6 hours to determine the lactic acid content. 3 mL of fermentation broth was placed in an Erlenmeyer flask, and distilled water was added at a volume ratio of 1:3. Using phenolphthalein as an indicator, titration was performed with a standardized 0.1 mol / L sodium hydroxide solution to convert the total titratable acid into lactic acid content.

[0064] Lactic acid (%) = (N×V×K) / W ×100

[0065] In the formula:

[0066] N: Concentration of sodium hydroxide standard solution, in molybdenum per liter (mol / L);

[0067] V: The volume of sodium hydroxide standard solution consumed in the titration, in mL;

[0068] K: Lactic acid conversion factor, taken as 0.09;

[0069] W: Volume of fermentation broth, in mL.

[0070] The results are as follows Figure 3 The results showed that *Pediococcus lactis* strain ZJUIDS-C036 produced 0.6 ml, 1.1 ml, 1.4 ml, and 1.8 ml of acid at 6 h, 12 h, 18 h, and 24 h, respectively. The acid production of this strain gradually increased with prolonged culture time, and was higher than that of the other two *Pediococcus lactis* strains. Acid production capacity is an important indicator of probiotic quality. Excellent probiotics should possess both good growth and acid production capabilities, thereby improving the flavor and nutritional value of fermented products. In addition, the lactic acid produced by probiotic metabolism lowers the intestinal pH, creating an acidic environment. This acidification significantly increases the solubility of calcium, releasing it from poorly soluble complexes (such as calcium phosphate and calcium oxalate) as free calcium ions, which are then enriched by the bacteria, thus improving its utilization in the body and promoting intestinal calcium absorption.

[0071] Example 4: Confirmation of the acid resistance, bile salt resistance, and artificial intestinal fluid tolerance of *Pediococcus lactis* ZJUIDS-C036

[0072] 1. Acid resistance test

[0073] After three generations of continuous activation and liquid amplification culture of the test strain *Pediococcus lactis* ZJUIDS-C036, the bacterial suspension was inoculated into 0.05 M PBS buffer (pH=3) at a ratio of 1:9 (volume ratio). After mixing, the suspension was incubated at 37°C. Plate counts were performed at 0 and 3 hours. After 36 hours of static incubation at 37°C, the bacterial count was determined, and the survival rate was calculated using the following formula:

[0074] Acid tolerance rate (%) = A3 / A0 × 100;

[0075] In the above formula, A0 is the number of viable bacteria (CFU / mL) of the test strain at 0 h; A3 is the number of viable bacteria (CFU / mL) of the test strain at 3 h.

[0076] The results are shown in Table 1 below.

[0077] 2. Bile salt tolerance test

[0078] After three generations of continuous activation and liquid amplification culture of the test strain *Pediococcus lactis* ZJUIDS-C036, the bacterial suspension was inoculated into 0.30% PBS buffer (0.05 M, pH = 7.4) at a ratio of 1:9. After mixing, the suspension was incubated at 37℃. Plate counts were performed at 0 and 3 hours of incubation. After 36 hours of static incubation at 37℃, the bacterial count was determined, and the survival rate was calculated using the following formula:

[0079] Bile salt tolerance rate (%) = A3 / A0 × 100;

[0080] In the above formula, A0 represents the viable count (CFU / mL) of the test strain at 0 h; A3 represents the viable count (CFU / mL) of the test strain at 3 h. The results are shown in Table 1 below.

[0081] 3. Artificial intestinal fluid tolerance test

[0082] After three generations of continuous activation and liquid amplification culture of the test strain *Pediococcus lactis* ZJUIDS-C036, the bacterial suspension was inoculated into simulated gastric fluid at a ratio of 1:9, mixed thoroughly, and incubated at 37°C. Plate counts were performed at 0 and 3 hours. After static incubation at 37°C for 36 hours, the bacterial counts were performed to determine the survival rate. The survival rate was calculated using the following formula:

[0083] In the formula, A0 is the number of viable bacteria (CFU / mL) of the test strain at 0 h; A3 is the number of viable bacteria (CFU / mL) of the test strain at 3 h.

[0084] Artificial intestinal fluid tolerance rate (%) = A3 / A0 × 100;

[0085] The results are shown in Table 1 below.

[0086] As shown in Table 1, *Pediococcus lactis* ZJUIDS-C036 exhibited significantly better acid and bile salt tolerance (achieving growth within 3 hours) than the control strain ATCC53103, with a survival rate of 79.03 ± 0.85% in PBS at pH 3.0. Its survival rate in an environment containing 0.3% ox bile salts was as high as 38.41 ± 1.66%, indicating excellent bile salt tolerance. Even after 3 hours of treatment with artificial intestinal fluid, *Pediococcus lactis* maintained a high survival rate. These experiments demonstrate that *Pediococcus lactis* ZJUIDS-C036 possesses high gastrointestinal survival ability.

[0087] Table 1. Results of strains' tolerance to acid and bile salts and to artificial intestinal fluid.

[0088] Pediococcus acidilactici ZJUIDS-C036 79.03±0.85 38.41±1.66 65.8±4.09 Lactobacillus rhamnosus ATCC53103 62.12±0.18 1.03±0.23 39.46±0.01

[0089] Probiotics must be able to survive in the adverse environment of the gastrointestinal tract, including stomach acid and bile, to exert their beneficial effects. The *Pediococcus lactis* ZJUIDS-C036 provided by this invention can grow and proliferate under conditions of pH 3.0, 0.3% bovine bile salts, and simulated intestinal fluid for 3 hours. It can successfully pass through the acidic environment of the stomach to reach the small intestine, where it can survive and effectively improve the intestinal flora, thus exerting its beneficial effects.

[0090] Example 5: Confirmation of the hydrophobicity of Pediococcus lactis ZJUIDS-C036

[0091] ZJUIDS-C036 bacterial culture was inoculated into MRS liquid medium at a 5% (V / V) inoculum. After incubation at 37℃ and 200 r / min for 2 h with shaking, the culture was centrifuged (5000 r / min, 15 min) to collect the bacterial cells. The cells were resuspended in sterile PBS, and the OD 600 nm value was adjusted to 1. 3 mL of the bacterial culture was transferred, 1 mL of chloroform was added, and the culture was shaken. After separation, the lower aqueous phase was collected, and its absorbance at 600 nm (OD 600 nm value) was measured. The hydrophobicity of the strain was calculated using the following formula:

[0092] Hydrophobicity (%) = (1 - A2 / A0) × 100;

[0093] In the formula, A0 = absorbance of the probiotic strain at 0h; At = absorbance of the probiotic strain at 2h after treatment;

[0094] The results are shown in Table 2 below. The results indicate that *Pediococcus lactis* has a certain degree of hydrophobicity. This result suggests that the strain has strong adhesion ability; the higher the hydrophobicity, the easier it is for the bacteria to adhere to the intestinal epithelial cells, forming a stable biofilm or bacterial aggregation, improving the health of the intestinal flora, and continuing to promote calcium absorption.

[0095] Table 2. Results of hydrophobicity of strains

[0096] Pediococcus acidilactici ZJUIDS-C036 5.35±2.％ Lactobacillus rhamnosus ATCC53103 12.31±1.15％

[0097] Example 6: Confirmation of the self-aggregating ability of *Pediococcus lactis* ZJUIDS-C036

[0098] ZJUIDS-C036 bacterial culture was inoculated into MRS liquid medium at a 5% (V / V) inoculum and cultured at 37℃ and 200 rpm for 16 h with shaking. Afterwards, the culture was centrifuged (5000 rpm, 15 min) to collect the bacterial cells. The cells were resuspended in sterile PBS to achieve an OD 600 nm value of 0.8. The bacterial culture was then placed in test tubes and incubated at 37℃ for 12 h and 24 h. 1 mL of the supernatant was taken to measure the absorbance (OD 600 nm value) to calculate the self-aggregation ability of the strain. The calculation formula is as follows:

[0099] Self-agglomeration rate (%) = (A0 - At) / At × 100;

[0100] In the formula, A0 = absorbance of the probiotic strain at 0 h; At = absorbance of the probiotic strain at each treatment time.

[0101] The results are as follows Figure 4 The results showed that *Pediococcus lactis* ZJUIDS-C036 has a certain self-aggregation ability. Self-aggregation ability is positively correlated with its adhesion in the intestine; strains with high self-aggregation ability also have strong adhesion. Furthermore, self-aggregation ability can create steric hindrance for probiotics, hindering the colonization of pathogens in the intestine and continuously promoting calcium absorption.

[0102] Example 7: Confirmation of the growth curve determination of *Pediococcus lactis* ZJUIDS-C036

[0103] A single colony of ZJUIDS-C036 was inoculated into 30 mL of MRS medium and incubated statically at 37°C. The absorbance at OD 600 nm was measured every 2 hours, and the medium was zeroed using blank MRS medium. A bacterial growth curve was plotted with incubation time on the x-axis and absorbance at OD 600 nm on the y-axis.

[0104] The results are as follows Figure 5 The results showed that when the growth curve of single colony of *Pediococcus lactis* strain ZJUIDS-C036 was detected, the strain was in a lag phase within 4 hours; the OD value of ZJUIDS-C036 increased almost linearly from 4 to 18 hours, indicating a logarithmic growth phase; and ZJUIDS-C036 entered a plateau phase after 18 hours of culture.

[0105] Example 8: Confirmation of the inhibitory ability of Pyotrophic Lateral Sclerosis Lactococcus ZJUIDS-C036 against pathogens.

[0106] The antibacterial activity of probiotics was determined using the internationally accepted agar diffusion method. Four frozen indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) were activated 2-3 times on LB agar. Single colonies of each activated strain were picked and incubated in LB agar at 37 °C for 18 h. Bacterial cells were collected by centrifugation and resuspended in PBS buffer (pH 7.2-7.4) to achieve a concentration of 10⁻⁶. 8 CFU / mL. The indicator bacterial suspension was added at 1% (v / v) to sterilized LB solid medium cooled to approximately 55°C. After thorough mixing, the mixture was poured into petri dishes (15 mL / dish), and the pre-placed sterile Oxford cups were removed after cooling. Activated *Pediococcus lactis* ZJUIDS-C036 was inoculated into MRS medium at 1% (v / v). After 18 h of culture, the supernatant was collected by centrifugation (8000 rpm, 5 min, 4°C), and the bacterial precipitate was discarded. The fermentation supernatant was added to the wells (200 μL / well), with uninoculated MRS medium (pH 6.2) used as a blank control. The culture was incubated at 37°C, and the diameter of the inhibition zone was measured after 24 h. Strains with a clear inhibition zone around the well were selected, and the diameter of the inhibition zone was measured, with each measurement repeated three times.

[0107] The results are shown in Table 3. The metabolites of *Pediococcus lactis* ZJUIDS-C036 showed inhibitory effects against *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, and *Listeria monocytogenes*. These effects were superior to those of ATCC53103, indicating that the metabolites of this strain possess antibacterial properties.

[0108] Table 3. Antibacterial activity of the strains against four common pathogenic bacteria.

[0109]

[0110] Example 9: Confirmation of antibiotic susceptibility of Pediococcus lactis ZJUIDS-C036

[0111] According to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI), the disk diffusion method was used to determine the antibiotic susceptibility of bacterial strains. After centrifugation and washing of the bacterial cells, the concentration of the bacterial suspension was controlled to reach 10. 8 CFU / mL. Spread 100 μL of bacterial suspension evenly onto MRS solid medium. Then, using sterile forceps, gently press the antibiotic disc onto the plate surface and incubate at 37°C for 24 hours. Measure and record the diameter of the inhibition zone using calipers. The antibiotic sensitivity of the strain is determined based on the diameter of the inhibition zone. According to the CLSI Standards for Antimicrobial Susceptibility Testing (17th Edition), the antibiotic resistance of probiotics is determined, divided into three levels: sensitive (S), intermediate (I), and resistant (R).

[0112] Table 4 shows the diameters of the antibiotic resistance zones of *Pediococcus lactis* ZJUIDS-C036. It exhibited high sensitivity to florfenicol, imipenem, chloramphenicol, and tetracycline, and sensitivity to cephalothin. The experimental results indicate that *Pediococcus lactis* ZJUIDS-C036 is sensitive to common antibiotics and has a high safety profile.

[0113] Table 4. Antibiotic susceptibility results of Pediococcus lactis ZJUIDS-C036

[0114]

[0115] Example 10: Confirmation of the antioxidant capacity of *Pediococcus lactis* ZJUIDS-C036

[0116] 1. Sample preparation:

[0117] The ZJUIDS-C036 strain, preserved in glycerol tubes, was streaked onto MRS solid medium and incubated upside down at 37°C for 48 hours. A single colony was picked with an inoculation loop and inoculated into 30 mL of sterile MRS liquid medium, and incubated statically at 37°C for 18-24 hours to obtain the culture medium. The culture medium was adjusted to a probiotic cell concentration of 10% with distilled water. 8 CFU / mL, centrifuged (8000 rpm, 20 min, 4℃), and the supernatant was collected as the fermentation supernatant. The centrifuged bacterial pellet was resuspended and washed with 0.02 M PBS buffer (pH=7.4), and centrifuged (8000 rpm, 20 min, 4℃), repeated 3 times. The washed bacterial cells were resuspended in PBS buffer and the cell concentration was adjusted to 10cFU / mL. 8 CFU / mL yields the bacterial suspension.

[0118] The supernatant and bacterial cells of *Pediococcus lactis* ZJUIDS-C036 were used as sample solutions, respectively.

[0119] 2. DPPH removal rate

[0120] Mix 1 mL of sample solution with 1 mL of 0.2 mM DPPH anhydrous ethanol solution by shaking for 1 min, and react at room temperature in the dark for 30 min. Centrifuge and take the supernatant to measure the absorbance at 517 nm. Use 95% ethanol instead of DPPH as a control and PBS (pH 7.2-7.4) instead of sample solution as a blank.

[0121] DPPH clearance rate = 1 - (Ai - Aj) / Ac;

[0122] Explanation: Ai: Absorbance of sample solution + DPPH; Aj: Absorbance of sample solution + 95% ethanol; Ac: Absorbance of PBS + DPPH.

[0123] The results are shown in Table 5 below.

[0124] 3. Hydroxyl radical scavenging rate

[0125] (1) Take 1 mL of o-phenanthroline (2.5 mmol / L), add 1 mL of phosphate buffer (PBS concentration of 0.02 mol / L, pH=7.4) and 1 mL of distilled water in sequence, mix well, add 1 mL of ferrous sulfate (FeSO4 concentration of 2.5 mmol / L), mix well, add 1 mL of hydrogen peroxide (H2O2 mass fraction of 20 mmol / L), and measure its absorbance at 536 nm after incubating in a water bath at 37℃ for 1.5 h.

[0126] (2) Replace 1 mL of hydrogen peroxide in step 1) with 1 mL of distilled water, and keep the rest the same. The absorbance measured is Ab.

[0127] (3) Replace 1 mL of distilled water in step 1) with 1 mL of sample, and keep the rest the same. The absorbance is measured as As.

[0128] Hydroxyl radical scavenging rate = (As - Ap) / (Ab - Ap)

[0129] The results are shown in Table 5 below.

[0130] 4. Restoration ability

[0131] Experimental group: The reaction raw material solution was prepared by mixing 0.5 mL of sample solution, 0.5 mL of potassium ferricyanide (1% by mass), and 0.5 mL of phosphate buffer (PBS concentration of 0.2 mol / L, pH=6.6). The reaction raw material solution was heated in a water bath at 50℃ for 20 min. After the reaction, it was cooled to room temperature and 0.5 mL of trichloroacetic acid (TAC mass fraction of 10%) was added to precipitate the protein. After centrifugation, 1 mL of the supernatant was taken and reacted with 1 mL of ferric chloride (FeCl3 mass fraction of 0.1%). The absorbance was measured at 700 nm.

[0132] Blank control: Replace the sample solution in the experimental group with PBS or MRS liquid culture medium, otherwise the same as the experimental group.

[0133] Reduction capability = (As - Ab) / Ab

[0134] As: Absorbance of the experimental group; Ab: Absorbance of the blank group

[0135] The results are shown in Table 5 below.

[0136] Table 5 Antioxidant activity of *Pediococcus lactis* ZJUIDS-C036

[0137] DPPH removal rate 102.23±3.45 27.84±9.33 Restoration ability 123.00±20.82 3.72±0.64 Hydroxyl radical scavenging rate 1.13±0.36 32.82±0.68

[0138] As shown in Table 5, the fermentation supernatant of *Pediococcus lactis* ZJUIDS-C036 screened by this invention exhibits high DPPH scavenging and reducing abilities, while the bacterial cells show even higher DPPH scavenging and hydroxyl radical scavenging abilities. Overall, this strain and its metabolites possess good antioxidant capabilities.

[0139] Example 11: Confirmation of hemolytic activity of *Pediococcus lactis* ZJUIDS-C036

[0140] Prepare blood agar plates (use fresh, sterile, defibrinated blood. When preparing the culture medium, preheat the blood to about 40-50℃. Add the blood when the culture medium cools to about 50℃. When adding 10% whole blood to the culture medium, the temperature should not be too high. After adding the blood and mixing well, pour the agar plates immediately. The prepared plates should be about 15 mL-20 mL in size and should be refrigerated at about 2-8℃). Inoculate the calcium-rich bacterial solution onto Columbia blood agar medium using an inoculation loop. After incubating at 37℃ for 48 hours, observe whether there is a hemolysis zone around the colony.

[0141] When a grass-green ring forms around a colony due to incomplete rupture of red blood cells, it is α-hemolysis; when a clearly defined, completely transparent hemolytic ring forms around a colony due to complete rupture of red blood cells, it is β-hemolysis; when there is no change in the culture medium around a colony, it is non-hemolysis.

[0142] The results are as follows Figure 6 The pathogenicity of many bacteria is related to their hemolytic properties. The results showed that after culturing *Pediococcus lactis* ZJUIDS-C036 on Columbia blood agar plates, the surrounding culture medium remained unchanged, indicating that the strain was not hemolytic. Therefore, it was preliminarily determined that this strain was not pathogenic.

[0143] Example 12: Preparation of Probiotic Powder of *Pediococcus lactis* ZJUIDS-C036 to Promote Calcium Absorption in Pet Cats

[0144] 1. Preparation of Pediococcus lactis ZJUIDS-C036 mycelial sludge

[0145] A single colony of *Pediococcus lactis* ZJUIDS-C036 was inoculated into 50 mL of MRS liquid medium and incubated at 37°C for 18 h. It was then activated again by inoculating 5% of the colony into 250 mL of MRS liquid medium and incubated at 37°C for 24 h. Finally, the activated *Pediococcus lactis* ZJUIDS-C036 was inoculated at 5% in a 10 L fermenter and cultured at high density for 18 h at 37°C and pH 6.8. Afterward, the colony was centrifuged at 8000 rpm and 4°C for 15 min, the supernatant was discarded, and the bacterial precipitate was collected. The bacterial cells were washed twice with sterile PBS (pH 7.0). This yielded the *Pediococcus lactis* ZJUIDS-C036 bacterial sludge.

[0146] 2. Preparation of Protective Agent

[0147] The freeze-drying protectant contains 15% skim milk powder, 5% trehalose, 3% monosodium glutamate, 1% glycerol, 0.5% cysteine ​​hydrochloride, and the remainder water as a solvent. It is sterilized at 110°C for later use.

[0148] Preparation of Lactococcus lactis ZJUIDS-C036 probiotic powder to promote intestinal calcium absorption in pet cats

[0149] The prepared *Pediococcus lactis* ZJUIDS-C036 bacterial sludge was thoroughly mixed with a preservative solution at a ratio of 1:5. The mixture was pre-frozen at -40℃ for 5 hours to ensure uniform freezing onto the inner wall of the container. Then, it was freeze-dried under vacuum for 18–20 hours to obtain *Pediococcus lactis* ZJUIDS-C036 bacterial powder. After rehydration with physiological saline and washing twice, the viable count in the *Pediococcus lactis* ZJUIDS-C036 bacterial powder was determined to be 1.0 × 10⁻⁶. 11 ~1×10 12 CFU / g.

[0150] Example 13: Preparation of probiotic milk powder to promote calcium absorption in the intestinal tract of pet cats using Pediococcus lactis ZJUIDS-C036

[0151] A freeze-dried *Pediococcus lactis* ZJUIDS-C036 bacterial powder was prepared, with a viable count of 1.0 × 10⁻⁶. 11 ~1×10 12 CFU / g;

[0152] Adding ZJUIDS-C036 freeze-dried probiotic powder to regular pet milk powder ensures that the live bacteria remain at a concentration of 10... 8 CFU / g or higher.

[0153] Example 14: Preparation of probiotic canned food that promotes intestinal calcium absorption in pet cats using Pediococcus lactis ZJUIDS-C036

[0154] Adding ZJUIDS-C036 freeze-dried probiotic powder to regular pet food ensures a live bacteria count of 10. 9 CFU / g.

[0155] In addition, the present invention can also use Pediococcus lactis ZJUIDS-C036 to prepare probiotic fermented yogurt that promotes calcium absorption in the intestinal tract of pet cats, and use Pediococcus lactis ZJUIDS-C036 to prepare feed that promotes calcium absorption in the intestinal tract of pet cats.

[0156] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Pediococcus acidilactici ( Pediococcus acidilactici ZJUIDS-C036, characterized in that The accession number is CGMCC NO. 34468.

2. The application of the Lactococcus lactis ZJUIDS-C036 as described in claim 1 in promoting calcium absorption / calcium enrichment.

3. The use of Pleurotus ostreatus ZJUIDS-C036 as described in claim 1 in the preparation of drugs that promote calcium absorption.

4. The application according to claim 2 or 3, characterized in that: Used to promote calcium absorption in pets.

5. The application according to claim 2 or 3, characterized in that: Promotes intestinal calcium absorption.

6. The application according to any one of claims 1 to 5, characterized in that: Pediococcus lactis ZJUIDS-C036 possesses at least one of the following properties: It is resistant to acids and bile salts; produces acid; has hydrophobicity and self-polymerizing ability; has antioxidant properties; has antibacterial activity and no antibiotic resistance.

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