AU2023226655B1 - Breast Milk-Derived Lactobacillus Plantarum HM-P2 and Use Thereof - Google Patents

Abstract

The present application relates to the field of microorganisms, particularly to a breast milk-derived Lactobacillus plantarum HM-P2, a bacterial agent and composition comprising the same, and a use thereof The breast milk-derived Lactobacillus plantarum HM-P2 provided by the present application is screened from healthy breast milk, has an extremely high bile salt resistance ability, can well resist a high bile salt environment (such as small intestine) in a human body, and has a strong bacteriostatic effect on pathogenic Escherichia coli and/or Staphylococcus aureus, and meanwhile has a certain safety on organisms. Therefore, the breast milk-derived Lactobacillus plantarum HM-P2 of the present application is expected to be developed and applied to food industry. 17

Description

Description

Breast Milk-Derived Lactobacillus Plantarum HM-P2 and Use Thereof

CROSS-REFERENCE The present application claims priority to Chinese patent application with an application number 202211142331.X filed on September 20, 2022, entitled "BREAST MILK-DERIVED LA CTOBA CILL US PLANTAR UM HM-P2 AND USE THEREOF", the entire disclosure of which is incorporated herein by reference.

Technical Field The present application relates to the field of microorganisms, particularly to a breast milk-derived Lactobacillusplantarum HM-P2, a bacterial agent and composition comprising the same, and a use thereof

Background Art Breast milk, as an essential source of nutrition for infants in the early stage of growth, plays a crucial role in the growth and development of infants. Breast milk has been confirmed to contain viable bacteria. Microorganisms having probiotic properties in breast milk are foundations for infants to initially build their immune systems and resist diseases, which replace pathogenic bacteria that occupy infant's intestines and are colonized through competitive rejection, so as to improve infant's intestinal environment by exerting their probiotic properties. At present, researches have shown that lactic acid bacteria have multiple drug resistance and carry drug resistance genes. Meanwhile, the drug resistance genes can be horizontally transferred to pathogenic bacteria, thereby leading to drug resistance of pathogenic bacteria and causing harm to a human body. Lactiplantibacillusplantarum is one of lactic acid bacteria in breast milk microorganisms, which is widely used in food, medicine, agriculture and other fields. The information disclosed in the part of the background art is only intended to increase an understanding of the overall background of the present application, and should not be regarded as acknowledging or implying in any form that the information constitutes prior art that is already known to persons of ordinary skill in the art.

Summary of the Invention The objective of the invention

In order to solve the above technical problem, the objective of the present application is to provide a breast milk-derived Lactobacillus plantarum HM-P2, a bacterial agent and composition comprising the same, and a use thereof. Solution In order to achieve the objective of the present application, the present application adopts the following technical solution. In a first aspect, the present application provides a breast milk-derived Lactobacillus plantarum HM-P2, the breast milk-derived Lactobacillus plantarum being classified and named as Lactobacillusplantarum with a preservation number CGMCC NO.23651. The above-mentioned breast milk-derived Lactobacillusplantarum HM-P2 has been preserved in China General Microbiological Culture Collection Center with a preservation number CGMCC NO.23651 on October 25, 2021. The address of the collection unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. In a potential embodiment for the above-mentioned breast milk-derived Lactobacillusplantarum, the breast milk-derived Lactobacillus plantarum has a survival rate of 118.2 % or more after surviving for 1 h under a condition of 0.3 % bile salt, and/or, a survival rate of 172.4 % or more after surviving for 3 h under a condition of 0.3 % bile salt. It indicates that the breast milk-derived Lactobacillus plantarum has an extremely high bile salt resistance ability which is obviously superior to that of existing commercial bacteria, and can well resist a high bile salt environment in a human body. In a potential embodiment for the above-mentioned breast milk-derived Lactobacillusplantarum, the breast milk-derived Lactobacillus plantarum has a good bacteriostatic effect on pathogenic Escherichia coli and/or Staphylococcus aureus, and has a bacteriostatic ability obviously superior to that of commercial bacteria; in a specific embodiment, a diameter of an bacteriostatic zone of the breast milk-derived Lactobacillus plantarum for pathogenic Escherichia coli is 26.21 ±0.52 cm, and a diameter of an bacteriostatic zone of the breast milk-derived Lactobacillus plantarum for Staphylococcus aureus is 24.30 ±0.52 cm. In a potential embodiment for the above-mentioned breast milk-derived Lactobacillusplantarum, the breast milk-derived Lactobacillusplantarum is confirmed to have the following properties: 1) no tryptophan decomposition ability; 2) no amino acid dehydroxylase activity; 3) no nitroreductase activity; and 4) no annotation of toxic virulence factors in virulence factor database search. For the above-mentioned Lactobacillusplantarum, in a specific embodiment, an acute toxicity test is performed by taking a high dose of the bacterial solution of the above-mentioned Lactobacillus plantarum HM-P2. The results show that the behavior of mice is abnormal, but no death phenomenon occurs, so that it is concluded that the LD5 o of the breast milk-derived Lactobacillus plantarum for mice is more than 10 g/kg. For the above-mentioned Lactobacillusplantarum, in a specific embodiment, it is found through 28-day gavage of the bacterial solution of the above-mentioned Lactobacillusplantarum HM-P2 for mice that there is no significant difference between an experimental group and a control group for body weight change (P>0.05); there is no significant difference between an experimental group and a control group for organ index (P >0.05); in addition, no colony growth is found after the organs of mice are coated onto an MRS culture medium for culture, that is to say, no bacterial translocation phenomenon occurs; after the gavage, it is found by detecting the blood and serum biochemical parameters of mice that blood and serum biochemical parameters of all dose groups of Lactiplantibacillus plantarum HM-P2 are kept normal; it can be seen that the breast milk-derived Lactiplantibacillusplantarum HM-P2 of the present application has a certain safety. The present application further provides a method for preparing the above-mentioned breast milk-derived Lactiplantibacillusplantarum, comprising the following steps: Selecting a fresh breast milk sample, performing gradient dilution by using sterilized PBS, coating the diluted sample onto an MRS culture medium containing 1 % CaCO3 to be cultured in an anaerobic incubator at 37 °C for 48 h, picking out white or milky white single colonies with obvious transparent zones and good growth for Gram staining and microscopic inspection, selecting rod-shaped strains that are Gram-positive bacteria to undergo enrichment culture in an anaerobic incubator at 37 °C; mixing the bacterial solution with sterilized 30 % glycerol in a ratio of 1:1 and then storing the obtained mixture in a refrigerator at -80 °C for later use, wherein compared with other types of lactic acid bacteria, lactobacillus has relatively more advantageous probiotic properties, and therefore the screening target of the present application is lactobacillus in lactic acid bacteria; performing bile salt resistance and bacteriostatic activity tests on the screened strains to obtain a Lactiplantibacillusplantarum having optimal bile salt resistance along with good acid resistance and bacteriostatic performance. In a second aspect, the present application provides a bacterial agent, the bacterial agent comprising an active ingredient comprising the breast milk-derived Lactobacillus plantarum HM-P2 as described in the first aspect. In a third aspect, the present application provides a composition, the composition comprising the breast milk-derived Lactobacillusplantarum HM-P2 as described in the first aspect, or comprising the bacterial agent as described in the second aspect. In a fourth aspect, the present application provides a use of the breast milk-derived Lactobacillus plantarum HM-P2 as described in the first aspect, or the bacterial agent as described in the second aspect, or the composition as described in the third aspect in preparing dairy products. In a preferred embodiment, the dairy products are selected from milk powders and fermented dairy products. In a preferred embodiment, the fermented dairy products are selected from yogurt, Kefir, fermented buttermilk, yogurt wine, milk wine and lactobacillus beverages. Beneficial effects The breast milk-derived Lactiplantibacillusplantarum HM-P2 provided by the present application is screened from healthy human milk; the Lactiplantibacillusplantarum HM-P2 has extremely high bile salt resistance ability, and can well resist a high bile salt environment (such as small intestine) in a human body. At the same time, the Lactiplantibacillusplantarum HM-P2 also has good acid resistance (the 1-hour survival rate and the 3-hour survival rate in a pH 3.0 environment are 166 % and 139 %, respectively), a strong bacteriostatic effect on pathogenic Escherichia coli and/or Staphylococcus aureus, and a certain safety to organisms, and therefore is expected to be developed and applied to food industry.

Brief Description of the Drawings Exemplary illustrations are made to one or more examples by pictures in accompanying drawings. These exemplary illustrations do not constitute limitations to the examples. The specialized word "exemplary" here means "as an example, an embodiment, or an illustration". Any examples

explained here as "exemplary" need not be interpreted as superior to or better than other examples. FIG. 1 shows bile salt resistance experiment results of breast milk-derived Lactobacillus plantarum HM-P2 described in Example 1 of the present application; FIG. 2 is an identification result graph of breast milk-derived Lactobacillus plantarum HM-P2 described in Example 2 of the present application; FIG. 3 is an acid resistance determination result graph of breast milk-derived Lactobacillus plantarum HM-P2 described in Example 3 of the present application; and FIG. 4 is a bacteriostatic ability determination result graph of breast milk-derived Lactobacillus plantarum HM-P2 described in Example 4 of the present application. The breast milk-derived Lactobacillus plantarum HM-P2 provided by the present application is classified and named as Lactobacillus plantarum, preservation date: October 25, 2021, preservation unit: China General Microbiological Culture Collection Center, preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, preservation number: CGMCC NO.23651.

Detailed Description of the Invention To make the objective, technical solutions and advantages of the examples of the present application more clear, the technical solutions of the examples of the present application will be clearly and completely described. Obviously, the described examples are some examples of the present application but not all the examples. Based on the examples of the present application, other examples obtained by persons of ordinary skill in the art without creative efforts all fall within the scope of protection of the present application. Unless otherwise explicitly stated, in the entire description and claims, the term "comprise" or its transformations such as "comprising" or "comprises" will be understood to include the stated elements or components, without excluding other elements or components. In addition, in order to better illustrate the present application, numerous specific details are provided in the specific implementation methods below. Those skilled in the art should understand that the present application can also be implemented without certain specific details. In some examples, there are no detailed descriptions of raw materials, components, methods, means, etc. that are familiar to those skilled in the art, in order to highlight the main purpose of the present application. In the following examples, An MRS solid culture medium was purchased from Beijing Land Bridge Technology Co., Ltd., with a product number CM188; An MRS broth culture medium was purchased from Beijing Land Bridge Technology Co., Ltd., with a product number CM187; Nutrient agar was purchased from Beijing Land Bridge Technology Co., Ltd., with a product number CM107; Kovacs's indole matrix reagent was purchased from Qingdao Haibo Biotechnology; Control strains L. plantarum 299253, L. reuteri 254476 and L. gasseri 339385 were all purchased from Beina Chuanglian Biotechnology Co., Ltd, L. reuteri DSM17938 was purchased from Bayer, and L. rhamnosus LGG was purchased from Kangcuile. Example 1: Screening of breast milk-derived LactobacillusplantarumHM-P2 A Fresh breast milk sample was selected and subjected to gradient dilution by using sterilized PBS (Purchased from Guangzhou Jiete Biofiltration Co., Ltd), then the diluted sample was coated onto an MRS solid culture medium containing 1 % CaCO 3 to be cultured in an anaerobic incubator at 37 °C for 48 h. White or milky white single colonies with obvious transparent zones and good growth were picked for Gram staining and microscopic inspection, and strains with good growth were selected from many rod-shaped strains that were Gram-positive bacteria to undergo enrichment culture in an anaerobic incubator at 37 °C. The screened strains were subjected to a bile salt resistance test to obtain a strain of Lactobacillus plantarum with optimal bile salt resistance, i.e., the Lactobacillus plantarum HM-P2 of the present application; the bacterial solution was mixed with sterilized 30 % glycerol in a ratio of 1:1 and then stored in a refrigerator at -80 °C for later use. The bile salt resistance test was carried out by using the following method: Under a sterile condition, strains to be tested were subcultured for 3 generations in an MRS liquid culture medium for activation, and the bacterial solution was diluted with an empty culture medium until the number of viable bacteria was 108 CFU/mL; the activated bacterial solution was transferred to a sterilized centrifugal tube to be centrifuged for 10 min at 3000 rpm so as to collect third-generation thalli, then the thalli were resuspended in an MRS liquid culture medium containing 0.3 % bile salt, and the concentration of the thalli was adjusted to 108 CFU/mL; the thalli were subjected to anaerobic culture at 37 °C, samples were taken at 0 h, 1 h and 3 h respectively and then subjected to 10-fold gradient dilution with sterilized PBSA buffer solutions, and 100 pL of samples with 3 proper dilution degrees were selected and coated onto an MRS solid culture medium plate and cultured for 48 h at 37 °C, followed by counting. The test results are shown in FIG. 1. It can be seen from FIG. 1 that the survival rates of the breast milk-derived Lactobacillusplantarum HM-P2 after surviving for 1h and 3 h under a condition of 0.3 % bile salt respectively reach 118.2 % and 172.4 % which are significantly higher than those of commercial strains. Example 2: Identification of breast milk-derived Lactobacillusplantarum HM-P2 In this example, whole genome sequencing analysis was performed on breast milk-derived Lactobacillusplantarum HM-P2 screened in Example 1. Specifically, whole genome sequencing analysis was performed on breast milk-derived Lactobacillusplantarum HM-P2 by using Pacbio (10Kb SMRT Bell library) and Illumina PE150 (350bp small fragment library), and then the sequencing results were subjected to NR (Non-Redundant Protein Database) database annotation (for message authentication of species and as species classification). The results are shown in FIG. 2. It can be seen from FIG. 2 that 86.68 % of genes in Lactobacillus plantarum HM-P2 meet Lactiplantibacillus plantarum, and HM-P2 is identified as Lactiplantibacillus plantarum in combination with morphological characteristics (round-ended and straight rod-shaped bacteria, short-chained, and Gram-positive bacteria) of HM-P2. Example 3: Acid resistance determination of breast milk-derived Lactobacillus plantarum HM-P2 Under a sterile condition, strains to be tested (including Lactiplantibacillusplantarum HM-P2 and L. plantarum BNCC299253, L. reuteri BNCC254476, L. gasseri BNCC339385, L. reuteri DSM

17938 and L. rhamnosus LGG as controls in the present application) were subcultured for 3 generations in an MRS liquid medium, and each bacterial solution was diluted with an empty culture medium until the number of viable bacteria was 108 CFU/mL. The activated bacterial solution was transferred to a sterilized centrifugal tube to be centrifuged for 10 min at 3000 rpm to collect thalli, then the thalli were washed twice with an MRS liquid culture medium, resuspended in an MRS liquid culture medium at pH 3.0 and subjected to anaerobic culture at 37 °C. Samples were taken at 0 h, 1 h and 3 h respectively and then subjected to 10-fold gradient dilution with sterilized PBSA buffer solutions, and 100 pL of samples with proper dilution degrees were coated onto an MRS plate (2 parallel for each dilution degree) and cultured for 48 h at 37 °C, followed by counting. The test results are shown in FIG. 3. It can be seen from FIG. 3 that the survival rates of the breast milk-derived Lactobacillus plantarum HM-P2 after surviving for 1 h and 3 h at pH 3.0 respectively reach 165.66 % and 139.4 % which are significantly higher than those of control strains, especially its homologous strain L.plantarum BNCC299253. Example 4: Bacteriostatic ability determination of breast milk-derived Lactobacillus plantarum HM-P2 1) Indicator bacteria Escherichiacoli ATCC 25922 and Staphylococcus aureus ATCC 25923 2) Method: After strains to be tested (including Lactiplantibacillus plantarum HM-P2 in the present application and L. plantarum BNCC299253, L. reuteri BNCC254476, L. gaseriBNCC339385, L. reuteri DSM 17938, L. rhamnosus LGG as control) were cultured and activated for three generations, then the third-generation bacterial solutions were centrifuged for 20 min at 4 °C at 5000 rpm, and the supernatants were stored at 4 °C for later use. The indicator bacteria were inoculated into a nutritional broth culture medium in an inoculation amount of 2 % to be cultured for 24 h at 37 °C and continuously activated for three generations. The concentration of thalli was adjusted to 106 CFU/mL (about 10 3 gradient), and then 200 pL of thalli were sucked and coated in a nutritional agar plate. The cover of the plate was opened, and the plate was put in aseptic air for 0.5 h so that a certain amount of water in the plate culture medium was lost, which benefits for diffusion of bacteriostatic substances. Refer to an Oxford cup agar diffusion method for bacteriostatic experiments. Specifically, the oxford cups (8 mm in diameter) were gently placed in Petri dishes by using sterile tweezers and gently pressurized. Four oxford cups were placed at equal intervals on each dish; 10 ml of nutrient agar (45 C) was added to fix the Oxford cups, and the Oxford cups were taken after coagulation; 200 pL of fermented supernatant was added into three of the Oxford cups, and an equal amount of

MRS liquid culture medium was added to the other Oxford cup as a negative control; the dishes were distributed at room temperature for 3 hours and incubated at 37 °C for 12 hours. The appearance of a bacteriostatic zone was observed and the diameter of the bacteriostatic zone was recorded. The results are expressed as mean standard deviation. The measurement results are shown in FIG. 4. It can be seen from FIG. 4 that the diameter of the bacteriostatic zone of Lactobacillusplantarum HM-P2 for Escherichia coli is 26.21 ±0.52 cm, and the diameter of the bacteriostatic zone of Lactobacillusplantarum HM-P2 for Staphylococcus aureus is 24.30± 0.52 cm. The bacteriostatic performances of the Lactobacillus plantarum HM-P2 are significantly superior to those of other commercial bacteria, especially its homologous strain L.plantarum BNCC299253. Example 5: Prediction of virulence factor of breast milk-derived Lactobacillus plantarum HM-P2 The genome sequence of Lactobacillusplantarum HM-P2 was compared with a virulence factor database VFDB (Virulence Factors of Pathogenic Bacteria), and a similarity rate of 70 % or more was regarded as a credible prediction. The results are shown in Table 1 below. Table 1 Predicted virulence factors in genome of Lactobacillusplantarum HM-P2

Stra Virulenc Pseudoge Identity Annotation Strain in e factor ne or not value

UTP-glucose-I-lymphoid Streptococcus galU No 75.70% tissue pyogenes ATP-dependent Clp protease clpP No 70.40% L. monocytogenes protein hydrolysis subunit

Streptococcus HM eno No 72% Phosphopyruvate hydratase pneumoniae -P2 Two-component reaction Listeria lisR No 76.80% regulator monocytogenes Mycoplasma Translation elongation factor tuf No 71.20% mycoides subsp. Tu Mycoides

It can be seen from Table 1 that any truly toxic virulence factor of Lactiplantibacillusplantarum HM-P2 is not annotated of in VFDB database search. Example 6: In-vitro toxicity research of breast milk-derived Lactobacillusplantarum HM-P2 1) Amino acid decarboxylase experiment

An experimental principle: bacteria with amino acid decarboxylase can decompose amino acids so that the amino acids are decarboxylated to generate amines (lysine - cadaverine, ornithine putrescine, and arginine -> spermine) and carbon dioxide, a culture medium turns alkaline and an indicator (bromocresol purple) turns purple, indicating a positive result; if no decarboxylation reaction occurs, the bacteria of enterobacteriaceae can decompose glucose to produce acids so that the indicator bromocresol purple turns yellow, indicating a negative result. An experimental process: Lactiplantibacillusplantarum HM-P2 was activated twice in an MRS culture medium (i.e., subculture for 2 generations); then, the activated Lactiplantibacillus plantarum HM-P2 was subcultured for 5 times in an MRS culture medium containing 0.1

% precursor amino acid (lysine, arginine and ornithine each being 0.1 %) and 0.005 % pyridoxal -phosphatemonohydrate (so as to induce decarboxylase); afterwards, the Lactiplantibacillus plantarum HM-P2 was cultured for 4 days in a decarboxylase culture medium containing an indicator (bromocresol purple). The color of the culture medium was observed and the pH value of the culture medium was measured. The preparation method of the used decarboxylase culture medium was: 5 g of tryptone, 5 g of yeast powder, 5 g of beef extract, 2 g of sodium chloride, 0.5 g of glucose, 1 g of Tween 80, 0.5 g of MnSO 4, 0.2 g of MgSO 4 , 0.04 g of FeSO 4, 0.01 g of thiamine, 2 g of K2 HPO4 , 0.1 g of CaCO 3 , 0.06 g of bromocresol pruple, 0.05 g of pyridoxal '-phosphate, and precursor amino acids (lysine, arginine, ornithine, each being 5g) were added with deionized water to 1 L. The experimental results are shown in Table 2 below. The results show that the Escherichia coli ATCC 25922 culture medium as a positive control is purple (i.e., positive), while the Lactiplantibacillusplantarum HM-P2 culture medium is yellow (i.e., negative), indicating that Lactiplantibacillusplantarum HM-P2 has no amino acid decarboxylase activity. Table 2 Experimental results of amino acid decarboxylase

Strain HM-P2 ATCC 25922

Lysine +

Ornithine +

Arginine +

Note: "+" is positive, and "-" is negative

2) Indole experiment: An experimental principle: some bacteria can decompose tryptophan in peptone to produce indole. The presence of indole can be demonstrated by a color development reaction. Indole was combined with p-dimethylaminobenzaldehyde to form rose indole which is a red compound. An experiment process: the activated Lactiplantibacillusplantarum HM-P2 and quality control bacteria ATCC 25922 (as a positive control) were respectively inoculated into an indole test culture medium at an inoculation amount of 3 %, and cultured for 72 h at 37 °C. To extract and concentrate indole, a small amount of ether was added to the culture medium, shaken and mixed evenly. After the ether layer floats on the surface of the culture medium, 8-10 drops of Kovacs's indole reagent were slowly added. If a red zone appears, the indole reaction was positive. The results are shown in Table 3 below. The results show that after the Kovacs's indole reagent is dropwise added, a rosy zone appears in a detection tube of quality control bacteria Escherichia coli ATCC 25922 (as a positive control), that is, the indole reaction is positive, while the zone does not appear in a detection tube of Lactiplantibacillusplantarum HM-P2, that is, the indole reaction is negative. These results indicate that Lactiplantibacillusplantarum HM-P2 has no tryptophan decomposition ability. Table 3 Indole experimental results

Strain HM-P2 ATCC 25922

Results

+ Note: "+" is positive, and "-" is negative

3) Nitroreductase experiment An experimental principle: the nitroreductase of bacteria can activate nitro drugs, so that cytotoxicity is produced. An experiment process: the activated Lactiplantibacillusplantarum HM-P2 and quality control bacteria ATCC 25922 (as a positive control) were respectively inoculated into a nitroreductase test culture medium in an inoculation amount of 3 %, and cultured for 72 hours at 37 °C; then a mixture of an a-naphthylamine solution and a p-aminobenzylsulfonic acid solution was sequentially added into the culture medium. If the culture medium turned red, it indicated a positive result for nitroreductase detection, otherwise it indicated a negative result. The results are shown in Table 4 below. The results show that the quality control strain Escherichia coli ATCC 25922 is nitroreductase-positive, and the Lactiplantibacillusplantarum HM-P2 culture solution does not turn red, that is, it is nitroreductase-negative. It can be seen that Lactiplantibacillusplantarum HM-P2 in the present application has no nitroreductase activity. Table 4 Nitroreductase detection results

Strain HM-P2 ATCC 25922

Results +

Note: "+" is positive, and "-" is negative

Example 7: In-vivo safety research of breast milk-derived Lactobacillusplantarum HM-P2 1) Preparation of gastric lavage bacterial solution: Lactiplantibacillusplantarum HM-P2 was taken from an ultra-low temperature refrigerator at -80 °C and inoculated into an MRS liquid culture medium for subculture and activation for three generations; the activated third-generation bacterial solution was taken and inoculated into an MRS medium in an inoculation amount of 2 %, and cultured for 12 hours at 37 °C; after 12 h, the bacterial solution was diluted with a series gradient of 10-_107, 100 tL of bacterial solution with a proper dilution gradient was taken and coated onto an MRS solid culture medium and cultured for 48 h at 37 °C; finally, a plate with a calculated bacterial count being between 30 and 300 was selected. The number of viable bacteria was calculated by using the following formula: CFU/mL = (average number of colonies * dilution ratio)/coating amount 2) Adaptive feeding of test mice: SPF grade C57BL/6JNifdc mice were put in plastic cages of 37 * 26 * 17 cm 3, with 5-6 mice in each plastic cage for eating and drinking freely; the temperature of the feeding room was set at (22 ±2) °C, the relative humidity was set as 50 %-60 %, and the light and darkness lasted for both 12 hours. The test was conducted after 7 days of feeding. 3) Acute oral toxicity test: With reference to a method of a maximum limit in GB15193.3-2014 "Acute Oral Toxicity Test", mice were randomly selected from 7 days of adaptive feeding and divided into an experimental group and a control group, with 5-6 mice in each group. The control group was infused with 0.85 % normal saline/day, and the experimental group was infused with a bacterial solution to be tested/day, which has a high concentration of 1010 cfu/mL, with an intragastric volume of 0.2 mL/mouse. During the period, sufficient food and water were ensured, and the mice were gavaged for 7 consecutive days. During the gavage, the physiological status and death of mice were observed and recorded to deduce median lethal dose LDo. 4) 28d oral toxicity test With reference to GB15193.22-2014 "28d oral toxicity test", toxicity studies were conducted. Specifically, mice were randomly selected from 7 days of adaptive feeding and divided into 3 experimental groups and 1 control group, with 5-6 mice in each group. The experimental groups are shown in Table 5 below. The control group was infused with 0.85 % normal saline, while the experimental groups were respectively infused with HM-P2 bacterial solutions having a high concentration (1010 CFU/mL), a medium concentration (109 CFU/mL) and a low concentration (10" CFU/mL) in a gavage dose of 0.2 mL/mouse. During this period, sufficient food and water were ensured, and the mice were fed continuously for 28 days. During this period, the weights of the mice were recorded weekly to study the trend of weight changes during the gavage. The mice were fed on the evening of day 28, and executed at 8:00 am on day 29. Table 5 28d gavage feeding grouping and gavage dose

Group Gavage strain Concentration of gavage bacterial solution (cfu/mL)

HM-P2-G HM-P2 1010

HM-P2-M HM-P2 10 9 HM-P2-L HM-P2 108 Control Normal saline 0.85%

) Organ indexes of mice On day 29, the mice were executed and then dissected to observe the color of their hearts, livers, spleens, lungs and kidneys, as well as whether there were any significant changes; specifically, the heart, liver, spleen, lung and kidney were taken with a sterile scalpel and put on a sterile plate. These organs were rinsed with sterile normal saline and dried with filter paper. Each organ was weighed and organ-to-body ratios of mice were calculated according to the following formula. Organ-to-body ratio = organ mass (g)/mouse body weight mass (g) 6) Determination of hematology and serum biochemical indexes of mice: After 28 days of feeding, the mice fasted overnight and bloods were collected from their eyeballs on the next day for blood and serum biochemical analysis. Specifically, 150 pL of whole bloods of each mouse were taken and placed in centrifuge tubes with an anticoagulant and then sent to a detection institution at 4 °C to detect the blood parameters of the mice with a full-automatic hematology analyzer. The remaining bloods were coagulated at 4 °C and then centrifuged at 3000 rpm for 10 minutes at 4 °C to obtain serums, the serums were then sent to a detection institution at -80 °C to detect the livers, kidneys, blood sugar, blood lipid and cholesterol levels of the mice by using a fully automated biochemical analyzer. The results are expressed as mean standard deviation. 7) Bacterial translocation experiment Each organ of experimental mice was coated onto an MRS culture medium for culture, and the growth of sterile colonies was observed.

The experimental results of the above-mentioned experiments were as follows: Acute oral toxicity test results: After continuous gavage for 7 days, various daily indicators of the mice were observed, and it was found that the mice were not poisoned or died. Therefore, LD5 0 was calculated to be greater than g/kg. The observation results of items such as behaviors and actions of mice are shown in Table 6 below. Table 6 Observation items for poisoning manifestations of mice

Observation and Organ system General manifestation inspection items

Central nervous system and body Behavior No abnormal movements or postures, no movements abnormal calls

Motion Normal motion without tremors

Stimulus response Normal stress reaction

Pupil size Normal, no zoom in/out Autonomic nervous system Secretion No lacrimation

nostril No rhinorrhea Respiratory system Respiratory rate Normal respiration

Abdominal shape Normal, no diarrhea or constipation found Gastrointestinal system Fecal hardness and color Fecal formation, normal color

Vulva, mammary gland No expansion Reproductive Urinary system Perineal region No pollution

Color, tension Normal, no looseness, no rash Skin and fur Integrity No vertical hair phenomenon

Eyeball Not prominent

Eye Transparency Not cloudy

Eyelid Normal

Organ index results of mice

After 28 days of feeding, the mice were subjected to gross dissection and each organ was observed.

The results show that none of the organs has any organic lesions, normal color, soft texture, and no

lumps; at the same time, various organs were collected and weighed to calculate their

corresponding organ indexes. The results are shown in Table 7 below.

The heart-to-body ratios, liver-to-body ratios, spleen-to-body ratios and lung-to-body ratios of

mice in different concentration groups of test strains were calculated and compared with those in

the control group. The results show that there is no significant difference between the test strain

groups and the control group (P>0.05), indicating that the gavage of Lactiplantibacillusplantarum

HM-P2 has no effect on the organs of mice.

Table 7 Organ indexes of mice

Heart-to-body Liver-to-body Spleen-to-body Lung-to-body Kidney-to-body Group ratio % ratio % ratio % ratio % ratio %

Control 0.508 +0.061 3.67+0.36 0.290 +0.036 0.586 +0.063 1.067 +0.085 group

R2-L 0.507+0.038 4.00+0.31 0.313+0.038 0.595+0.050 1.069+0.041

R2-M 0.524+0.061 3.82+0.16 0.285+0.018 0.617+0.089 1.117+0.055

R2-G 0.488 ±0.063 3.62±0.23 0.298 ±0.070 0.581 +0.029 1.087 ±0.055

Note: P>0.05 Blood routine and serum biochemical parameter detection results of mice: The blood parameter detection results of mice are shown in Table 8 below. It can be seen from Table 8 that all blood parameters of mice are increased or decreased, but there is no significant difference compared to the control group (P>0.05), indicating that the gavage of Lactiplantibacillusplantarum HM-P2 has no adverse effect on the blood parameters of mice. Table 8 Effect of test strains on blood parameters of mice

Group WBC RBC HGB HCT MCV MCH PLT

(10 9 /L) (10 12/L) (g/L) (L/L) (fl) (pg) (10 9/L)

Control 10.30 + 164.6 + 0.33 + 39.44 + 20.00 + 1559.40

+ group 1.50 8.26±0.76 0.89 0.04 0.81 1.96 42.40

HM-P2-L 156.4 ± 0.32 ± 39.52 ± 19.48 ± 1389.80 ±

9.29±0.86 8.01+0.65 12.54 0.03 0.76 1.43 68.34

HM-P2-M 162.2 ± 0.33 ± 39.38 ± 19.16 ± 1501.80 ±

9.37± 1.38 8.46±0.57 3.77 0.02 0.36 1.14 139.21

HM-P2-G 10.45 ± 158.2 ± 0.32 ± 39.06 ± 19.48 ± 1588.00 ± 8.12±0.54 1.56 3.35 0.02 0.47 1.09 148.63

Note: P>0.05 The serum parameter detection results of mice are shown in Table 9 below. It can be seen from Table 9 that there is no significant difference between the mice and the control group in various serum biochemical parameters (P>0.05), indicating that the gavage of Lactiplantibacillus plantarum HM-P2 has no adverse effect on serum biochemical parameters of mice. Table 9 Effect of test strains on serum biochemical parameters of mice

Group ALT( TC(mm TG(mm TBIL(p DBIL(p BUN(m UA(pm CREA(p GLU(m

U/L) ol/L) ol/L) mol/L) mol/L) mol/L) ol/L) mol/L) mol/L)

Contr 39.83 2.73 ± 0.27 ± 2.41 ± 2.29 ± 9.30 ± 265.67 10.00 ± 4.60 ± ol ±2.88 0.12 0.04 0.25 0.09 0.95 ± 11.06 0.14 1.20 group

HM-P 37.23 2.65 ± 0.25 ± 2.25 ± 2.21 ± 9.52 ± 284.00 9.79 ± 4.96 ±

2-L ±4.88 0.03 0.05 0.06 0.09 0.51 ± 12.95 1.38 0.78

HM-P 33.80 2.72 ± 0.27 ± 2.51 ± 2.41 ± 9.26 ± 276.33 10.33 ± 4.77 ±

2-M ±4.23 0.11 0.06 0.59 0.67 0.27 ±11.21 0.58 0.55

HM-P 38.90 2.79 ± 0.32 ± 2.26 ± 2.14 ± 10.12 ± 268.33 10.38 ± 4.49 ±

2-G ±1.22 0.16 0.04 0.09 0.08 0.99 ±6.66 0.15 0.82

Note: P>0.05

Bacterial translocation experiment results:

After all organs were coated onto the MRS culture medium for culture, no colony growth was

observed, indicating that no translocation of Lactiplantibacillusplantarum HM-P2 occurred in

mice, which did not bring corresponding safety problems to the organism.

The above in-vivo experiment results show that the Lactiplantibacillusplantarum HM-P2 of the

present application has extremely high safety in organisms.

Finally, it should be noted that the above embodiments are only used to illustrate, but not limit, the

technical solution of the present application; although the present application has been described

in detail with reference to the aforementioned examples, persons of ordinary skill in the art should

understand that amendments are still made to the technical solutions described in various

examples, or equal replacements are made to a part of technical features therein; whereas these

amendments or replacements do not allow corresponding technical solutions to depart from the

spirit and scope of the technical solutions in each example of the present application.

Industrial applicability The breast milk-derived Lactobacillus plantarum HM-P2 of the present application has an

extremely high bile salt resistance ability, can well resist a high bile salt environment (such as

small intestine) in a human body, has a strong bacteriostatic effect on pathogenic Escherichiacoli

and/or Staphylococcus aureus, also has a certain safety on organisms, and therefore is expected to

be developed and applied to food industry.

The reference in this specification to any prior publication (or information derived from it), or to

any matter which is known, is not, and should not be taken as an acknowledgment or admission or

any form of suggestion that that prior publication (or information derived from it) or known matter

forms part of the common general knowledge in the field of endeavour to which this specification

relates.

Claims (9)

Claims

1. A breast milk-derived Lactobacillus plantarum HM-P2, wherein the breast milk-derived Lactobacillusplantarum is preserved in China General Microbiological Culture Collection Center on October 25, 2021 with a preservation number CGMCC NO.23651.

2. The breast milk-derived Lactobacillus plantarum HM-P2 according to claim 1, wherein the survival rate of the breast milk-derived Lactobacillus plantarum after surviving for 1 h under a condition of 0.3 % bile salt is 118.2 % or more, and/or, the survival rate of the breast milk-derived Lactobacillusplantarum after surviving for 3 h under a condition of 0.3 % bile salt is 172.4% or more.

3. The breast milk-derived Lactobacillus plantarum HM-P2 according to claim 1, wherein the breast milk-derived Lactobacillus plantarum has an inhibitory effect on pathogenic Escherichia coli and/or Staphylococcus aureus.

4. The breast milk-derived Lactobacillus plantarum HM-P2 according to claim 1, wherein the breast milk-derived Lactobacillus plantarum has no tryptophan decomposition ability, no amino acid dehydroxylase activity and no nitroreductase activity, and no annotation of toxic virulence factors in virulence factor database search.

5. A bacterial agent, comprising an active ingredient comprising the breast milk-derived Lactobacillusplantarum HM-P2 according to any one of claims 1-4.

6. A composition, comprising the breast milk-derived Lactobacillusplantarum HM-P2 according to any one of claims 1-4, or comprising the bacterial agent according to claim 5.

7. A use of the breast milk-derived Lactobacillus plantarum HM-P2 according to any one of claims 1-4 or comprising the bacterial agent according to claim 5 or the composition according to claim 6 in preparing dairy products.

8. The use according to claim 7, wherein the dairy products are selected from milk powders and fermented dairy products.

9. The use according to claim 8, wherein the fermented dairy products are selected from yogurt, Kefir, fermented buttermilk, yogurt wine, milk wine and lactobacillus beverages.