Lactobacillus paracasei KQNIU, microbial preparation and application of lactobacillus paracasei KQNIU

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a lactobacillus paracasei KQNIU, a microbial preparation and application of the lactobacillus paracasei KQNIU. The Lactobacillus paracasei KQNIU is preserved in the China General Microbiological Culture Collection Center on August 25, 2025, the preservation number is CGMCC No.35739, and the Lactobacillus paracasei KQNIU is classified and named as Lactobacillus paracasei, and the Lactobacillus paracasei KQNIU is named as Lactobacillus paracasei KQNIU. The Lactobacillus paracasei KQNIU is named as Lactobacillus paracasei KQNIU. The lactobacillus paracasei KQNIU provided by the invention has good gastrointestinal tract tolerance, has an inhibition effect on pathogenic bacteria such as escherichia coli, salmonella and the like, can be prepared into a microbial preparation or a feed additive for preventing and treating calf diarrhea, replaces antibiotics and realizes green and healthy breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of paracasei KQNIU, microbial preparation and application thereof. BACKGROUND

[0002] Healthy breeding of beef calves is the basis for high-quality beef fattening and improving meat quality, and is also the key to improving the breeding efficiency, economic benefits and market competitiveness of the beef industry. However, calf diarrhea has become a key problem restricting the breeding of beef and dairy cattle, which not only reduces the growth efficiency of beef calves, but also restricts the economic benefits of the industry. At present, the main cause of calf diarrhea is the overgrowth of intestinal pathogenic bacteria such as E. coli and Salmonella or viral infection leading to intestinal flora imbalance, and traditional antibiotic treatment has problems such as drug residues and increased bacterial resistance, which does not meet the green development direction of livestock breeding. Therefore, it is of great significance to explore an antibiotic substitute to alleviate calf diarrhea for the livestock breeding industry. SUMMARY

[0003] In order to explore an antibiotic substitute to alleviate calf diarrhea, the present application provides a strain of paracasei KQNIU, microbial preparation and application thereof. The paracasei KQNIU provided by the present application has good gastrointestinal tolerance and inhibitory effect on pathogenic bacteria such as E. coli and Salmonella, and can be made into a microbial preparation or a feed additive for preventing and treating calf diarrhea, replacing antibiotics and realizing green and healthy breeding.

[0004] The present application provides a strain of paracasei KQNIU, Lacticaseibacillus paracasei The paracasei KQNIU was deposited with the China General Microbiological Culture Collection Center on August 25, 2025, and the deposit number is CGMCC No. 35739.

[0005] The paracasei KQNIU provided by the present application has good gastrointestinal tolerance and inhibitory effect on pathogenic bacteria such as E. coli and Salmonella, and can replace antibiotics to realize green and healthy breeding.

[0006] The present application further provides a microbial preparation containing the paracasei KQNIU.

[0007] Further, the microbial preparation further comprises a plant extract.

[0008] Further, the plant extract is a radix anemarrhena asphodeloides extract, a glycyrrhizic flavone, a white asparagus root alcohol, a hippophae rhamnoides flavone, a radix isatidis extract or a mulberry leaf flavone.

[0009] Further, in the microbial preparation, the mass of the plant extract is 2 times the mass of the paracasei KQNIU.

[0010] The application also provides application of the Paracaseicillus casei KQNIU or microbial preparation in preparation of a product for preventing or treating cow diarrhea.

[0011] Further, the product is a feed additive.

[0012] Further, the feed additive is used for inhibiting cow intestinal pathogens.

[0013] Further, the intestinal pathogens are any one or several of Escherichia coli, Salmonella and Staphylococcus aureus.

[0014] Compared with the prior art, the application has the beneficial effects that: The Paracaseicillus casei KQNIU provided by the application has the following significant beneficial effects: the survival rate is 63.84% in an environment with a pH of 3.0 for 8 hours, and the survival rate is more than 73% when the bile salt is 0.3%, which indicates that the Paracaseicillus casei KQNIU has strong acid and bile salt resistance and can smoothly pass through the gastrointestinal tract of a calf; the surface hydrophobicity is 56.65%, which indicates that the Paracaseicillus casei KQNIU has high adhesion and can be efficiently colonized in the intestinal tract; the Paracaseicillus casei KQNIU can effectively inhibit calf diarrhea pathogenic bacteria such as Escherichia coli and Salmonella, and the diameter of the inhibition zone is 13mm to 14.5mm; the co-aggregation rate with common pathogenic bacteria is more than 66%; the Paracaseicillus casei KQNIU has good antioxidant property, and the hydroxyl radical clearance rate is 56.62%; and the Paracaseicillus casei KQNIU is sensitive to various antibiotics, which indicates that the Paracaseicillus casei KQNIU has no drug resistance risk and is high in safety.

[0015] The Paracaseicillus casei KQNIU provided by the application is fermented after being compounded with plant extracts such as mulberry leaf flavonoids and Pulsatilla, so that the growth performance, antibacterial ability and antioxidant ability can be significantly improved. After being compounded with Pulsatilla extract, the Paracaseicillus casei KQNIU has the largest Staphylococcus aureus inhibition zone of 17.76mm, the Salmonella inhibition zone of 14.96mm and the Escherichia coli inhibition zone of 13.99mm, and the DPPH clearance rate is 54.39%. After being compounded with mulberry leaf flavonoids, the Paracaseicillus casei KQNIU has the Staphylococcus aureus inhibition zone of 14.71mm, the Salmonella inhibition zone of 13.50mm and the Escherichia coli inhibition zone of 14.66mm, and the highest DPPH clearance rate is 55.32%.

[0016] The Paracaseicillus casei KQNIU provided by the application has wide application scenarios and can be made into various forms of feed additives such as living cells and heat-inactivated cells, or be used for probiotic preparation and fermented feed preparation, so that the calf diarrhea rate can be effectively reduced, the breeding efficiency can be improved, antibiotics can be replaced to avoid drug residues, the development of green animal husbandry can be promoted, and the value in the field of animal husbandry is outstanding.

[0017] Biological material preservation information description: KQNIU, referred to in this application as *Lactobacillus paracasei* KQNIU, was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35739. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Its classification name is *Lactobacillus paracasei*. Lacticaseibacillus paracasei . Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The figures show the isolation, microscopic examination, and electrophoresis results of the KQNIU strain; in the figure, A is a single colony photograph of the KQNIU strain; B is the Gram staining microscopic examination result of the KQNIU strain; and C is the 16S rDNA electrophoresis result of KQNIU.

[0020] Figure 2 A phylogenetic tree constructed based on the 16S rDNA gene sequence.

[0021] Figure 3 The figures show the growth curve and acid production curve of strain KQNIU; in the figure, A is the growth curve of strain KQNIU; and B is the acid production curve of strain KQNIU.

[0022] Figure 4 The acid resistance of the KQNIU strain.

[0023] Figure 5 This refers to the bile salt tolerance of the KQNIU strain.

[0024] Figure 6 The figure shows the adhesion ability of KQNIU strain and its co-aggregation ability with pathogens; in the figure, A represents the adhesion ability of KQNIU strain; B represents the co-aggregation ability of KQNIU strain with pathogens.

[0025] Figure 7 The figure shows the antioxidant capacity of the KQNIU strain; in the figure, A represents the DPPH scavenging rate of the KQNIU strain; and B represents the hydroxyl radical scavenging rate of the KQNIU strain. Detailed Implementation

[0026] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0027] Example: A strain of paracasein lactobacillus KQNIU, microbial preparation and its application.

[0028] I. Materials and methods 1. Sample collection The test samples were collected from the beef cattle breeding base of the Agricultural and Pastoral Science and Technology Park of Inner Mongolia University for Nationalities. Fresh rectal feces were collected at 7 days of age. Whitehead extract, glycyrrhizin flavone, white asparagus alcohol, sea buckthorn flavone, radix isatidis extract and mulberry leaf flavone were purchased from Xi'an Jiayun Biological Feed Company, with serial numbers JT20250915, JT20250715, JT20250720, JT20250615, JT20250625 and JT20250725.

[0029] 2. Main reagents and culture medium MRS liquid medium, MRS agar medium, LB medium, gram staining kit, Columbia blood agar medium, pepsin, trypsin, bovine bile salt, antibiotic sensitivity tablet and oxford cup were purchased from Tianjin Sikai Biological Technology Company; indicator bacteria Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus and Salmonella Salmonella were provided by the Inner Mongolia Autonomous Region Beef Disease Prevention and Control Engineering Technology Research Center Laboratory strain library and activated; bacterial DNA extraction kit was purchased from Shengong Biological Company; penicillin, streptomycin, gentamicin, kanamycin, ampicillin and ofloxacin were purchased from Bikeman Biological Reagent Factory.

[0030] 3. Strain screening In the clean bench, 5g of fresh calf feces sample was taken and placed in a 95mL sterile physiological saline conical flask, and mixed thoroughly to make the original bacterial suspension. Then using ten-fold gradient dilution method, 1mL of bacterial solution was taken in a centrifuge tube, 100µL was taken and added to 900µL of sterile physiological saline, vortexed and shaken, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7Gradient dilution; take 100 μL of each gradient dilution and spread on the surface of MRS agar medium, incubate in a 37°C incubator for 48h. After colony formation, select typical colonies with white round shape, smooth and full edge, purify single colonies by three times of streaking, inoculate the obtained single colonies into MRS liquid medium and incubate at 37°C for 24h to obtain the test strain for subsequent strain identification.

[0031] 4. Strain identification 4.1. Colony and bacteria identification Perform Gram staining on the test strain and observe its morphological characteristics under an oil lens. Select colonies with purple staining and morphological characteristics consistent with lactic acid bacteria, and take single colonies for hydrogen peroxide test. The presence of bubbles is positive, and the absence of bubbles is negative.

[0032] 4.2. Biochemical identification of strains Use bacterial biochemical identification tubes to analyze the biochemical characteristics of the strain. Sterilized rings are dipped into the test strain and added to each biochemical identification tube. Immediately after inoculation, seal the biochemical identification tube with a sealing film and incubate in a 37°C incubator for 12h. Observe the color change of the biochemical identification tube. A color change is positive, and no color change is negative. Extract the DNA of KQNIU according to the bacterial DNA extraction kit method, and use bacterial 16S rDNA gene universal primers 27F and 1492R for PCR amplification. The PCR amplification reaction program is as follows: initial denaturation at 95°C for 5min; denaturation at 94°C for 30s, annealing at 57°C for 30s, 35 cycles; extension at 72°C for 90s; total extension at 72°C for 10min; and storage at 4°C. The amplified products are detected by 1.5% agarose gel electrophoresis. The samples meeting the requirements are sent to Shengong Bioengineering Co., Ltd. for sequencing. According to the sequencing results, the N-J method in MEGA software is used to construct a phylogenetic tree.

[0033] 5. Determination of biological characteristics of strains Inoculate the test strain into MRS liquid medium at a 2% inoculation amount and incubate at 37°C for 36h. Take samples at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 28h, and measure the absorbance at a wavelength of 600nm. Repeat 3 times. Plot the growth curve with incubation time as the abscissa and absorbance value as the ordinate. Inoculate the bacterial liquid into 10mL MRS liquid medium with pH 6.5 at a 2% inoculation amount, and incubate in a 37°C constant temperature environment. Measure the pH every 2h using a portable pH meter. Plot the pH change curve of the strain in different fermentation stages with incubation time as the abscissa and pH as the ordinate to show the change trend of the acid production rate of the strain.

[0034] 5.1. Determination of acid tolerance of strains The pH of the MRS liquid medium was adjusted with 1 mol / L hydrochloric acid to set five gradients of 2.0, 3.0, 4.0, 5.0 and 6.0, while the medium with pH 6.0 was kept as a blank control. The test strain was inoculated into the above-mentioned MRS medium with different pH at a ratio of 1%, and placed in a 37°C constant temperature incubator for 8h. The OD600 of each test group was measured, and the survival rate was calculated.

[0035] Survival rate (%) = 100 x test group OD600 / control group OD600.

[0036] 5.2, Determination of the bile salt tolerance of the strain Bovine bile salt was added to the MRS liquid medium to prepare MRS liquid medium with bile salt concentrations of 0%, 0.15%, 0.3% and 0.6%, with 0% as a blank control and each concentration repeated three times. The test strain was inoculated into the above-mentioned medium with different bile salt concentrations at a ratio of 1%, and incubated at 37°C in a constant temperature environment for 8h. The OD600 of each test group was measured, and the survival rate was calculated to analyze the effect of different bile salt concentrations on the growth of the strain.

[0037] 5.3, Determination of the adhesion of the strain The hydrophobicity of the test strain was determined by xylene extraction method. After the test strain was cultured at 37°C for 24h, it was centrifuged at 4°C and 3000r / min for 10min, and the supernatant was discarded. The precipitate was washed twice with PBS and then resuspended in PBS, so that the OD600 of the resuspension was fixed at 0.8, which was A0. 2mL of the resuspension was mixed with 1mL of xylene and incubated at 37°C for 2h. The organic phase was removed, and the OD600 of the aqueous phase was measured as At. The hydrophobicity was calculated according to the following formula: Hydrophobicity (%) = [(A0-At) / A0] x 100.

[0038] The test strain was inoculated into MRS liquid medium at a ratio of 1% and incubated at constant temperature for 24h. It was then centrifuged at 4°C and 5000r / min for 10min, and the bacterial precipitate was collected. The precipitate was washed twice with PBS and then resuspended in PBS to obtain a resuspension, and the OD600 of the resuspension was measured as A0. The resuspension was incubated at 37°C for 2h, and the natural precipitate was collected, and the OD600 of the supernatant was measured again as A1. The self-aggregation rate was calculated according to the following formula: Self-aggregation rate (%) = [(A0-A1) / A0] x 100.

[0039] 5.4, Determination of the pathogenic bacteria co-aggregation ability of the strain The test bacteria liquid was centrifuged at 4 DEG C and 5000 r / min for 10 min, the bacterial bodies were collected, the bacteria liquid was washed twice with PBS, and then was resuspended in PBS, the OD600 was adjusted to 0.5, 200 μL of the bacterial suspension was mixed with E. coli, S. aureus and Salmonella respectively, and was placed at room temperature for 18 h, the supernatant was taken to measure OD600 as A2, and the coagulation rate was calculated according to the following formula: Coagulation rate (%) = [(0.5-A2) / 0.5]x100.

[0040] 5.5, Antibiotic sensitivity determination of the strain In order to evaluate the sensitivity of the strain to antibiotics, the paper disc diffusion method was used to determine the antibiotic sensitivity of the test strain. The test strain was prepared into a bacterial suspension with a concentration of 1x10 8 CFU / mL, 100 μL of which was spread on MRS solid medium, and was placed for 10 min to absorb the bacteria liquid. Penicillin, streptomycin, gentamicin, ampicillin, kanamycin and ofloxacin were placed on the MRS agar medium by tweezers, and was incubated at 37 DEG C for 24 h, and the diameter of the inhibition zone was recorded.

[0041] 5.6, Determination of antioxidant capacity of the strain Preparation of bacterial suspension: the test strain was incubated at 37 DEG C for 24 h, and was centrifuged at 4 DEG C and 8000 r / min for 15 min, the precipitate was washed with PBS for 3 times, and then was resuspended in PBS, the OD600 was adjusted to 1.0±0.05, and the bacterial suspension was obtained.

[0042] Preparation of sterile broken material: the bacterial suspension was broken by ultrasonic at 200 W and 20 Hz, the working time was 9 s and the rest time was 12 s, and the total time was 10 min; after breaking, the supernatant was obtained by centrifuging at 4 DEG C and 9500 r / min for 15 min.

[0043] Determination of the clearance rate of the strain to DPPH free radical and hydroxyl free radical: 1 mL of the bacterial suspension was taken and mixed with 1 mL of 0.2 mmol / L DPPH solution, and was reacted at room temperature for 0.5 h in the dark, the OD517 was measured, and the blank was anhydrous ethanol. The DPPH clearance rate of the bacterial suspension was calculated according to the following formula: DPPH clearance rate (%) = [1-(A1-A2) / A0]x100%; In the formula, A0 is the OD517 of 1 mL of DPPH+1 mL of anhydrous ethanol; A1 is the OD517 of 1 mL of DPPH+1 mL of the bacterial suspension; and A2 is the OD517 of 1 mL of anhydrous ethanol+1 mL of the bacterial suspension.

[0044] The DPPH clearance rate of the sterile broken material was detected according to the above steps.

[0045] Respectively take 1 mL of 2.5 mmol / L phenanthroline, PBS and distilled water, mix well, then add 1 mL of 2.5 mmol / L ferrous sulfate solution, 20 mmol / L H2O2 solution, mix well, 37℃ water bath for 90 min, 4℃, 5000r / min centrifugal 6 min, measure OD536. Measure OD563 of PBS as A0; use 1 mL of distilled water instead of 1 mL of H2O2, measure OD536 as A1; use 1 mL of bacterial suspension instead of 1 mL of distilled water, measure OD536 as A2. Calculate the hydroxyl radical scavenging rate of bacterial suspension according to the following formula: Hydroxyl radical scavenging rate (%) = [(A2-A0) / (A1-A0)]x100%.

[0046] Hydroxyl radical scavenging rate detection of sterile body broken product is the same as the above steps.

[0047] 5.7, determination of antibacterial performance of strains After the test strain was cultured in MRS liquid medium at 37℃ for 18h, it was centrifuged at 8000r / min for 12min, the supernatant was discarded, the precipitate was washed with PBS, and the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL for standby. The indicator strains Staphylococcus aureus, Escherichia coli and Salmonella were cultured in LB liquid medium at 37℃ for 24h, and the concentration of the indicator strains was adjusted to 1×10 8 CFU / mL for standby. Respectively take 200μL of the indicator strains and coat on the LB solid medium, after the bacterial solution is absorbed, use sterile forceps to put it into the Oxford cup, gently press to ensure that there is no gap between the Oxford cup and the medium, add 200μL of the test strain to the Oxford cup, stand for 1h, then invert and incubate in an anaerobic incubator at 37℃ for 24h, 3 replicates for each treatment. Measure the diameter of the inhibition zone with a vernier caliper, and the results are expressed as mean ± standard deviation. The size of the inhibition zone is used to judge the ability of the strain to inhibit the pathogenic indicator strains.

[0048] 6, preparation and effect of fermentation composition Respectively mix each extract with water at a ratio of 8g:80mL, sterilize at 121℃ for 15min, obtain each plant extract solution; mix 200μL of the bacterial solution of the test strain with 400μL of each plant extract solution into 10mL of MRS liquid medium, ferment at 37℃ for 24h, then sample the fermentation broth, and detect OD600 value, pH value, antioxidant performance and antibacterial ability. Antioxidant performance detection is the same as 5.6; antibacterial ability detection is the same as 5.7.

[0049] 7, feeding test Test site: Inner Mongolia Kexin Beef Cattle Breeding Co., Ltd., test animals: 30 beef calves with similar body conditions (body weight 46±5 kg) that were naturally delivered were selected as test animals.

[0050] Test design: 30 7-day-old calves were selected for the single-factor randomized test design and divided into 3 groups, namely the control group, the mulberry flavone group, and the anemone extract group. Each group had 10 male calves, and the test period was 14 days, with continuous gavage for 7 days. The feces of the calves were observed for 7 days and 14 days. The control group was not added with feed additives in the starter feed. The KQNIU mulberry flavone group was gavaged with 0.184 g of bacteria powder and 0.368 g of mulberry flavone powder per calf. The anemone group was gavaged with 0.184 g of bacteria powder and 0.368 g of anemone extract per calf. The calves were supplemented with starter feed at 0.2% of their body weight. The KQNIU freeze-dried powder was prepared by Inner Mongolia Hanen Biological Company according to the tested strain, with a viable bacterial count of 2.8×10 11 CFU / g. The plant extracts included mulberry flavone and anemone extract. The calf feces morphology was observed and recorded, and the evaluation criteria are shown in Table 1. A score of 3 or 4 was diarrhea, and the diarrhea rate was calculated: Diarrhea rate (%) =∑[(number of diarrhea calves × diarrhea days) / (total number of calves × recording days)]×100.

[0051] Table 1: Evaluation criteria for calf feces morphology

[0052] II. Test results 1. Strain isolation and identification Four rod-shaped, gram-positive, and hydrogen peroxide-negative single strains were obtained through isolation and purification in this test. A strain of Lactobacillus was finally selected based on its colony morphology characteristics, gram staining, and genomic DNA electrophoresis results, as shown in Figure 1 .

[0053] 2. Biochemical identification of the strain The biochemical identification results are shown in Table 2. The strain was negative in the hydrogen peroxide test, could decompose sucrose, glucose, maltose, mannitol, raffinose, fructose, xylose, lactose, esculin, galactose, sorbose, cellobiose, melibiose, salicylic acid, and rhamnose, and could not ferment arabinose. The 16S rDNA sequencing results had a high homology of 99.74% with the sequence of Paracaseolus casei in the database, confirming that the strain was Paracaseolus casei. The phylogenetic tree was constructed using the N-J method of MEGA 11 software, and the results are shown in Figure 2 . The strain was closely related to Paracaseolus casei Lacticaseibacillus paracasei ATCC 25302, Lacticaseibacillus paracaseiNBRC 15889 is in the same branch. Combined with physiological and biochemical identification, the strain is identified as Paracaseolyticum, which is named as Paracaseolyticum Lacticaseibacillus paracasei , which is referred to as KQNIU in the present application.

[0054] Table 2 Biochemical identification results of strains

[0055] 3. Biological characteristics of the strain 3.1 Growth curve and acid production performance of the strain As shown in FIG. A, the growth curve of KQNIU reveals the growth dynamics of KQNIU at different culture stages: slow growth before 6h, logarithmic growth phase after 6h, and growth speed increases; stable growth phase after 18h, and growth tends to be stable. Figure 3 As shown in FIG. B, the acid production capacity of KQNIU is weak from 0h to 6h, the metabolism is relatively slow, and it is in the adaptation period; the acid production capacity is strong from 6h to 16h, the metabolism is accelerated; the acid production capacity tends to be stable from 18h to 28h, and the pH is stable at 3.7 to 4.0. Figure 3

[0056] 3.2 Acid tolerance of the strain The acid tolerance of the strain is one of the important indicators for evaluating its probiotic potential. The acid tolerance test results of KQNIU are shown in FIG. C. With pH 6.0 as the control group, the viable cell count of KQNIU decreases significantly with the decrease of pH, and the survival rate is as high as 92.43% at pH 5.0; the survival rate decreases significantly to 78.49% at pH 4.0; the survival rate decreases significantly to 63.84% at pH 3.0; the growth of KQNIU is seriously affected at pH 2.0, and the survival rate decreases to 44.82%. The results show that KQNIU has strong acid tolerance, which indicates that it has stronger tolerance and survival ability in the intestinal tract. Figure 4 3.3 Cholate tolerance of the strain

[0057] The results of the cholate tolerance of KQNIU are shown in FIG. D. The data show that the survival rate of KQNIU decreases with the increase of cholate concentration, and the survival rate can reach 89.16% when the cholate concentration is 0.15%, and the survival rate is still above 72.43% when the concentration increases to 0.3%, and the survival rate decreases significantly when the concentration reaches 0.6%, but it is maintained at 56.91%. The prerequisite for probiotics to enter the intestinal tract and play a probiotic function is the tolerance to gastric acid and cholate environment. The growth of the test strain under the condition of 0.3% cholate and good survival rate can be used as probiotics for subsequent application. Figure 5

[0058] ​​3.4, Adhesion ability of the strain The hydrophobic properties of KQNIU were as shown in Table 2A, and the self-aggregation ability was 39.70%, and the surface hydrophobicity was as high as 56.65%, belonging to a highly hydrophobic strain, indicating that KQNIU had good intestinal epithelial cell adhesion properties, which helped to inhibit the colonization of pathogenic bacteria in the intestinal tract. Figure 6

[0059] 3.5, Co-aggregation ability of the strain The co-aggregation results of KQNIU with pathogenic bacteria were as shown in Table 2B, and the co-aggregation rates of KQNIU with E. coli, Salmonella, and Staphylococcus aureus were 66.33%, 67.87%, and 69.60%, respectively, showing significant differences. The high co-aggregation ability of KQNIU with pathogenic bacteria helped to inhibit the colonization of pathogenic bacteria in the intestinal tract. Figure 6

[0060] 3.6, Antibiotic sensitivity of the strain The sensitivity of probiotics to antibiotics is directly related to their safety in application and potential impact on human health. The antibiotic sensitivity results of KQNIU are shown in Table 3.

[0061] Table 3 Antibiotic sensitivity results of KQNIU

[0062] Note: I in the table indicates moderate sensitivity, and S indicates sensitivity.

[0063] KQNIU showed different degrees of sensitivity to 9 antibiotics, including gentamicin, streptomycin, kanamycin, ampicillin, ofloxacin, doxycycline, tetracycline, lincomycin, and erythromycin. Among them, KQNIU showed sensitivity to gentamicin and doxycycline, and moderate sensitivity to streptomycin, kanamycin, ampicillin, ofloxacin, tetracycline, lincomycin, and erythromycin.

[0064] 3.7, Antioxidant properties of the strain The DPPH free radical scavenging rate results of KQNIU are shown in Table 4A, and the DPPH scavenging rate of the sterile body broken material was 37.56%, and the DPPH scavenging rate of the bacterial suspension was 28.50%; the hydroxyl radical scavenging rate results are shown in Table 4B, and the hydroxyl radical scavenging rate of the sterile body broken material was 56.62%; the hydroxyl radical scavenging rate of the bacterial suspension was 36.67%. In the evaluation of the antioxidant capacity of the strain, the DPPH free radical scavenging rate and the hydroxyl radical scavenging rate are two important indicators, and the higher the free radical scavenging rate, the stronger the antioxidant activity. The results of this test showed that KQNIU had high antioxidant capacity, could scavenge intestinal oxidative stress, protect intestinal mucosa, and relieve stress. Figure 7 Figure 7

[0065] ​​​​3.8, Bacteriostatic performance of the strain To evaluate the bacteriostatic activity of KQNIU, the common indicator pathogenic bacteria of calf diarrhea, Escherichia coli, Salmonella and Staphylococcus aureus were selected for bacteriostatic test, and the results are shown in Table 4. KQNIU has inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus, and the inhibitory capacity is different, and the inhibitory capacity order is: Staphylococcus aureus > Salmonella > Escherichia coli.

[0066] Table 4 Bacteriostatic performance of KQNIU

[0067] 4, Growth, acid production, antioxidant and bacteriostatic performance of the strain and extract after fermentation The results are shown in Tables 5 and 6. After fermentation of KQNIU with mulberry leaf flavonoids and anemonae extract, the growth trend is good, the acid production performance and antioxidant performance are strong, and the bacteriostatic effect on Salmonella, Escherichia coli and Staphylococcus aureus is high (the diameter of bacteriostatic circle is significantly larger than that of single KQNIU control), which can be used as an important indicator for screening extract.

[0068] Table 5 Growth, acid production and antioxidant performance of KQNIU and extract after fermentation

[0069] Table 6 Bacteriostatic performance of KQNIU and extract after fermentation

[0070] 5, Feeding test After 7 days of feeding test and 14 days of feces observation, the results are shown in Table 7. KQNIU mulberry leaf flavonoids group and KQNIU anemonae extract group are better than the control group in feces score and diarrhea index, which shows that KQNIU combined with mulberry leaf flavonoids and anemonae extract has a positive effect on improving the feces condition of calf and reducing the diarrhea rate.

[0071] Table 7 Results of KQNIU and extract feeding test

[0072] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept.

[0073] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application belong to the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei KQNIU, characterized in that, The *Lactobacillus paracasei* KQNIU was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35739.

2. A microbial preparation containing *Lactobacillus paracasei* KQNIU as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation also contains plant extracts.

4. The microbial preparation according to claim 3, characterized in that, The plant extracts are Pulsatilla chinensis extract, glycyrrhiza flavonoids, resveratrol, Hippophae rhamnoides flavonoids, Isatis indigotica extract, or mulberry leaf flavonoids.

5. The microbial preparation according to claim 3, characterized in that, In the microbial preparation, the mass of the plant extract is twice the mass of the *Lactobacillus paracasei* KQNIU.

6. The use of the *Lactobacillus paracasei* KQNIU as described in claim 1 or the microbial preparation as described in any one of claims 2 to 5 in the preparation of products for the prevention or treatment of bovine diarrhea.

7. The application according to claim 6, characterized in that, The product is a feed additive.

8. The application according to claim 7, characterized in that, The feed additive is used to inhibit bovine intestinal pathogens.

9. The application according to claim 7, characterized in that, The intestinal pathogen is any one or more of Escherichia coli, Salmonella, and Staphylococcus aureus.

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