<i>Lactobacillus paracasei </i>ET-22 and use thereof

Abstract

The present application relates to a Lactobacillus paracasei ET-22 strain and use thereof. The present application provides a Lactobacillus paracasei ET-22 strain with a deposit number of CGMCC No. 15077, a composition containing the strain and a method of treating a subject using the strain. The present application also relates to a method for treating a subject, including administering an effective amount of the Lactobacillus paracasei ET-22 strain to a subject in need thereof for 1) whitening teeth and/or inhibiting oral pathogens; 2) adjusting balance of flora in the subject; 3) promoting growth of bifidobacteria and/or lactic acid bacteria; and/or 4) enhancing immunity of the subject.

Claims

1. A method for treating a subject, comprising administering an effective amount of Lactobacillus paracasei ET-22 strain to a subject in need thereof for 1) whitening teeth and/or inhibiting oral pathogens; 2) adjusting balance of flora in the subject; 3) promoting growth of bifidobacteria and/or lactic acid bacteria; and/or 4) enhancing immunity of the subject.

2. The method of claim 1, wherein the ET-22 strain is in a form of a composition which comprises the ET-22 strain, an excipient, a diluent and/or a carrier.

3. The method of claim 1, wherein the ET-22 strain is an active strain and/or a deactivated strain.

4. The method of claim 1, wherein the ET-22 strain is administered to a mammalian subject via a gastrointestinal route.

5. The method of claim 1, wherein the ET-22 strain is administered to the subject in an amount of equal to or more than 10.sup.6 CFU.

6. The method of claim 1, wherein the effective amount of the Lactobacillus paracasei ET-22 strain is administered to the subject further for enhancing humoral immunity and/or cellular immunity of the subject, increasing the number of antibody-producing cells, increasing half hemolysis concentration HC.sub.50, and/or increasing activity of NK cells.

7. The method of claim 1, wherein the ET-22 strain is administered to the subject in an amount of equal to or more than 10.sup.7 CFU.

8. The method of claim 1, wherein the ET-22 strain is administered to the subject in an amount of equal to or more than 10.sup.8 CFU.

9. The method of claim 1, wherein the ET-22 strain is administered to the subject in an amount of equal to or more than 10.sup.10 CFU.

10. The method of claim 1, wherein the ET-22 strain is administered to the subject in an amount of equal to or more than 10.sup.11 CFU.

11. The method of claim 2, wherein the composition is a food composition, a pharmaceutical composition, or an oral cleaning composition.

12. The method of claim 11, wherein the composition is a food composition, and wherein the excipient, diluent and/or carrier is dairy drinks, tea, coffee, chewing gum and dentifrice, jerky for pet food or a combination thereof.

13. The method of claim 11, wherein the composition is an oral cleaning composition, and wherein the excipient, diluent and/or carrier is toothpaste, tooth powder, mouthwash, breath freshener spray, fluorine-coating agent, denture cleaner, pet tooth cleaning gum or pet laxatone, tooth brush, interdental brush, dental floss, oral swab or pet tooth cleaning bone.

14. The method of claim 11, wherein the composition is a pharmaceutical composition, which 1) is formulated an oral dosage form; 2) includes a therapeutic and/or prophylactic composition; 3) includes a nutritional composition; 4) is a dry or wet preparation; or 5) is formulated into a gel, cream, spray, aerosol, ointment, emulsion, suspension, patch, buccal tablet or sublingual tablet.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Evaluation of the tolerance of L. paracasei ET-22 in a simulated intestinal environment, a gastric acid and bile salt environment.

(2) FIG. 2. Evaluation of the intestinal adhesion of L. paracasei ET-22.

(3) FIG. 3. Regulation of intestinal flora by L. paracasei ET-22.

(4) FIG. 4. Comparison of the regulation of intestinal flora by L. paracasei ET-22 and Lactobacillus casei LC-01.

(5) FIG. 5. The number of antibody-producing cells by L. paracasei ET-22.

(6) FIG. 6. The serum hemolysin half hemolysis concentration (HC.sub.50) by L. paracasei ET-22.

(7) FIG. 7. The activity of NK cell by L. paracasei ET-22.

(8) FIG. 8. Test results of the production of hydrogen peroxide by L. paracasei ET-22.

(9) FIG. 9. Test results of inhibiting oral pathogens by L. paracasei ET-22.

(10) FIG. 10. Test results of inhibiting oral pathogens by deactivated L. paracasei ET-22 strain.

(11) [Preservation of Biomaterials]

(12) The Lactobacillus paracasei ET-22 strain of the present disclosure is deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit date of Dec. 18, 2017, and the deposit number CGMCC No. 15077; the address of the depositary unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichenxi Road, Chaoyang District, Beijing, China.

(13) The Lactobacillus paracasei K56 strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit date of Dec. 29, 2017, and the deposit number CGMCC No. 15139; the address of the depositary unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichenxi Road, Chaoyang District, Beijing, China.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(14) Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the relevant field.

(15) The freeze-dried culture of the lactic acid bacteria strain according to the present disclosure has been deposited in China Center for Type Culture Collection and the China General Microbiological Culture Collection Center. The details of the deposit are shown in the table below:

(16) TABLE-US-00001 TABLE 1 Deposit information of the lactic acid bacteria strain Strain name Classification Deposit number Deposit date ET-22 Lactobacillus CGMCC 15077 Dec. 18, 2017 paracasei

(17) Morphology and General Properties of the Lactic Acid Bacteria Strain

(18) The taxonomic characteristics of the strain are confirmed by 16S rDNA sequence analysis and API bacterial identification system analysis results. The morphological and general characteristics of the above strain are detailed in the table below:

(19) TABLE-US-00002 TABLE 2 Morphology and general characteristics of the lactic acid bacteria strain Strain name Morphological characteristics Lactobacillus 1. When cultured in MRS medium, the strain is paracasei short and medium rod-shaped, with rounded ET-22 strain ends, usually chain-shaped and occasionally appear in pairs. 2. Gram-positive bacilli, does not produce spores, has no catalase, oxidase and motility, can grow in both aerobic and anaerobic environments, the most suitable growth temperature being 37° ± 1° C., facultative heterogeneous fermentation, does not produce gas during glucose metabolism.

(20) The fermentation conditions of this strain are as follows:

(21) MRS liquid medium: peptone, 10.0 g; beef extract, 10.0 g; yeast extract powder, 5.0 g; glucose, 20.0 g; dipotassium phosphate, 5.0 g; diammonium citrate, 2.0 g; sodium acetate, 5.0 g; magnesium sulfate heptahydrate, 0.5 g; manganese sulfate tetrahydrate, 0.2 g; Tween 80, 1.0 g; agar 15.0 g; distilled water 1000 mL, with a pH between 6.2 and 6.4 and sterilize at 121° C. for 15 minutes.

(22) L. paracasei ET-22 is a micro-aerobic bacteria, which has better growth in a facultative anaerobic environment, produces lactic acid, has acid resistance, can survive in an acid environment of pH 2.5 and an environment of 0.4% bile salt for 4 hours, a mesophilic bacteria, with a growth temperature ranging of 15-45° C., and the optimal growth temperature of about 37° C.

Example 1: Tolerance to Artificial Gastric Fluid, Intestinal Fluid, Artificial Bile

(23) The L. paracasei ET-22 strain was activated three times and divided into 8 tubes. One tube was used for colonies counting to calculate the number of initial viable bacteria. The remaining seven tubes were centrifuged at 4000 rpm for 10 minutes, and after removing the supernatant, the cells were cultured in pH 2.5 medium at 37° C. for 1 to 3 hours. Every hour, one tube was centrifuged at 4000 rpm for 10 minutes, and after removing the supernatant, the pellet was washed twice with 5 mL RO water to calculate the number of viable bacteria. The remaining 4 tubes were centrifuged at 4000 rpm for 10 minutes after 3 hours, and after removing the supernatant (pH 2.5 MRS broth), the 4 tubes were mixed well with MRS broth with 1.5% bovine bile, then evenly distributed into 4 tubes, and incubated at 37° C. for 1-4 hour. Every hour, one tube was centrifuged at 4000 rpm for 10 minutes, and after removing the supernatant, the pellet was washed twice with 5 mL RO water to calculate the number of viable bacteria. The change of the numbers of viable bacteria measured at different times was compared and analyzed. The survival rate of the strains is shown in FIG. 1.

(24) The results showed that, after continuous processing through acidic medium and bile salt environment, i.e., after 7 hours of continuous gastric acid and cholate treatment in a simulated digestive environment, L. paracasei ET-22 maintains the total number of bacteria to the 5th power, which proves that L. paracasei ET-22 can pass the strict test of the human digestive system environment.

Example 2. Intestinal Cell Adhesion

(25) The cover glass was picked up with tweezers, sterilized on an alcohol lamp, and then placed in a 6-well plate for use. Caco-2 cells were taken out from the preservation tube, and after adjustment, were transferred to the 6-well plate for culture until confluence, and then used in the test. During the test, the liquid in the plate was removed, and after washing twice with PBS buffer, 1.5 mL of bacterial suspension (1×10.sup.9 CFU/mL) and 1.5 mL of cell culture medium (containing 10% PBS and 1% penicilin-streptomycin) per well were added and mixed well. After culturing in a constant temperature incubator (37° C., 5% CO.sub.2) for 4 hours, the plate was washed twice with sterile PBS, the cells were fixed with methanol, Gram stained, and the number of viable bacteria was counted under a microscope.

(26) The adhesion of the strain to Caco-2 cells was tested by an adhesion test. The results are shown in FIG. 2, which shows that L. paracasei ET-22 has strong adhesion to Caco-2 cells.

Example 3: Intestinal Flora Regulation Effect

(27) This example is intended to confirm the effect of the Lactobacillus paracasei of the present disclosure for intestinal flora regulation.

(28) 36 healthy SPF BABL/c mice weighing 18-22 g (provided by Beijing Huafukang Biotechnology Co. Ltd.) were adaptively reared for 3 days, and then were randomly divided into 3 groups, namely the blank control group and the test groups, each with 12 mice. Animals in each test group were administered by gavage with sterile water dissolved with Bifidobacterium lactis BL-99 powder (gavage volume 0.2 mL/10 g), once a day for continuous 14 days, and the blank control group was given the same volume of sterile water. Intragastric dose: 1.3×10.sup.7 CFU/ml (calculated according to the human body dose of 2×10.sup.9 CFU/day, with a conversion factor between human and mouse of 0.0026). Centrifuge tubes were sterilized, numbered, and filled with feces of mice collected under aseptic conditions after adaptive feeding, 2-3 pellets, about 100 mg, per tube. The tubes were then transferred to the sterile operation room at a low temperature for intestinal flora detection. At the end of the experiment, mouse feces were collected again. After grouping and numbering the mice with picric acid, the mice were weighed on the 8th and 14th days of administration of the test substance, respectively, and the gavage amount of the mice was calculated. The mice were weighed once at the end of the experiment. Colonies counting: Selective medium was prepared according to the strain to be identified. The strain to be tested and the corresponding medium are shown in the table below. The culture medium was sterilized, shaken well, cooled to 45° C.-50° C., poured into the plate for use.

(29) TABLE-US-00003 TABLE 3 Strains to be tested and corresponding selective media Strains to be tested Selective media Enterobacter Eosin Methylene Blue (EMB) Agar Bifidobacterium BBL agar Lactobacillus LBS agar

(30) The collected mouse feces were placed in a sterile tube containing 0.5 mL of physiological saline to prepare a bacterial suspension. The suspension was shaken for 1 min before use. 0.1 mL of the bacterial suspension was pipetted with a 0.1 mL micropipette and slowly injected into 0.9 mL of sterile physiological saline, shaken or repeatedly pipetted to mix well to prepare a 1:10 bacterial suspension, which was then serially diluted 10 times in a similar manner with another 0.1 mL micropipette, until 10.sup.−7 g/ml concentration. According to the number of viable bacteria to be identified, two consecutive appropriate dilutions were selected. 10 μL of bacterial suspension of each dilution was pipetted with a 0.1 mL micropipette and surface-coated on selective agar plates, and cultured under the culture conditions indicated in the below table. The colonies counting was carried out with reference to the “GB 4789.2-2010 National Food Safety Standard Food Microbiology Examination: Aerobic Plate Count”

(31) TABLE-US-00004 TABLE 4 Culture media and identification methods for intestinal flora Item Media Culture condition Enterobacter Eosin Methylene 36° C. ± 1° C. Blue Agar for 24 h Bifidobacterium BBL agar 36° C. ± 1° C. for 48 h, anaerobic Lactobacillus LBS agar 36° C. ± 1° C. for 48 h

(32) SPSS17.0 was used for data statistics. The changes in bifidobacteria, lactic acid bacteria, and enterobacteria within and between groups before and after the experiment were compared.

(33) The results of animal weight changes during the experiment are shown in Table 5 below. During the experimental period, the animals were characterized as normal, and no adverse reactions occurred after the administration of the test substance. During the experimental period, there was no significant difference in the weight between the two groups of animals. It can be seen from Tables 6-8 that Lactobacillus paracasei ET-22 can significantly promote the growth of Bifidobacterium and Lactobacillus, and has no significant effect on Enterobacter. According to the comparison before and after the intervention within the group, the effect of ET-22 is significantly higher than that of the well-known strain Lactobacillus casei LC-01 (DSM 19465), and thus has an excellent effect.

(34) Therefore, in this study, Lactobacillus paracasei ET-22 has the effect of regulating intestinal flora (FIG. 3).

(35) TABLE-US-00005 TABLE 5 change of animal weight number Initial Mid-term Final of weight weight weight Group animals (g) (g) (g) Control group 14 21.89 ± 1.25 22.14 ± 0.87 21.24 ± 0.87 L. casei LC-01 14 22.46 ± 1.12 22.83 ± 1.04 22.03 ± 1.73 L. paracasei ET-22 14 21.90 ± 1.50 21.42 ± 1.08 19.38 ± 1.66

(36) TABLE-US-00006 TABLE 5 change of numbers of animal intestinal bifidobacteria (LgCFU/g) before and after the test P value P value after before intervention and compared before after after with the No. of inter- inter- inter- control Group animals vention vention vention group Control 14 8.66 ± 8.94 ± 0.196 group 0.57 0.46 L. casei 14 8.94 ± 9.70 ± 0.001 0.003 LC-01 0.50 0.56** L. paracasei 14 8.67 ± 9.91 ± 0.000 0.000 ET-22 0.59 0.46**

(37) The results showed that the increase rate of the ET-22 group after the intervention was significantly higher than that of the well-known strain LC-01: the number of bifidobacteria was increased by 1.24 orders of magnitude for the ET-22 group, and 0.76 orders of magnitude for the LC-01 group (FIG. 4). ET-22 belongs to a Lactobacillus genus, but it can significantly increase the number of Bifidobacterium genus by more than one order of magnitude, higher than that of LC-01, showing that ET-22 has extremely strong ability to increase intestinal bacteria.

(38) TABLE-US-00007 TABLE 7 change of numbers of animal Lactobacillus (LgCFU/g) before and after the test P value after P value inver- before vention No. and compared of before after after with the Group ani- inter- inter- inter- control mals vention vention vention group Control 14 8.54 ± 8.45 ± 0.566 group 0.57 0.18 L. casei 14 8.96 ± 9.57 ± 0.000 0.000 LC-01 0.62 0.57** L. paracasei 14 9.17 ± 9.94 ± 0.000 0.000 ET-22 0.91 0.77**

(39) The results showed that the increase rate of the ET-22 group after the intervention was significantly higher than that of the well-known strain LC-01: the number of Lactobacillus was increased by 0.77 orders of magnitude for the ET-22 group, and 0.61 orders of magnitude for the LC-01 group (FIG. 4).

(40) TABLE-US-00008 TABLE 8 Numbers of animal Enterobacter (LgCFU/g) before and after the test P value P value compared with before after before the control No. of inter- inter- and after group after Group animals vention vention intervention intervention Control 14 6.48 ± 6.98 ± 0.123 group 0.32 0.74 L. casei 14 6.71 ± 6.97 ± 0.071 0.423 LC-01 0.68 0.40 L. paracasei 14 6.84 ± 6.98 ± 0.119 0.239 ET-22 0.66 0.52

Example 4: Intestinal Flora Regulation Effect (Sequencing Result)

(41) This example is intended to confirm the effect of Lactobacillus paracasei of the present disclosure in intestinal flora regulation. For the principles and procedures, please refer to “Technical Specifications for Inspection and Evaluation of Health Foods-Judgment Criteria for Regulating Intestinal Flora Function”. The difference between this Example and Example 3 is that this experiment uses 16S rDNA sequencing results.

(42) Experimental samples: Bacterial powder samples (see packaging specifications for the number of live bacteria) were provided by Inner Mongolia Dairy Technology Research Institute Co., Ltd.

(43) Experimental material: 182 healthy adult 6-week-old BABL/c mice weighing approximately 18-20 g.

(44) Experimental Procedure

(45) 1. Sample Preparation

(46) Live bacteria samples (ET-22): According to the sample specifications, 1 g of each live bacteria sample was weighed and suspended to 40 ml in PBS solution, to obtain a concentration of live bacteria of 2.5×10.sup.9 CFU/ml.

(47) High-dose group: Based on the gavage amount of 0.2 ml/10 g mice, the amount for a mouse weighting 20 g was 0.4 ml. The gavage dose for mice in the high-dose group was 10.sup.9 CFU/20 g.

(48) Medium-dose group: 5 ml of the high-dose suspension was added to PBS to make up to 50 ml. Based on the gavage amount of 0.2 ml/10 g mice, the amount for a mouse weighting 20 g was 0.4 ml. The gavage dose for mice in the medium-dose group was 10.sup.8 CFU/20 g.

(49) Low-dose group: 5 ml of the medium-dose suspension was added to PBS to make up to 50 ml. Based on the gavage amount of 0.2 ml/10 g mice, the amount for a mouse weighting 20 g was 0.4 ml. The gavage dose for mice in the medium-dose group was 10.sup.7 CFU/20 g.

(50) 2. Regulation of Intestinal Flora

(51) (1) 6 week-old BABL/c mice were housed in a clean-grade animal room with a temperature of 22° C., a humidity of 10-60%, alternating 12 hours of light and dark, standard feed feeding, and free access to water.

(52) (2) After adaptive feeding for 5 days, 182 mice were randomly divided into 13 groups, with 14 mice in each group. See Table 9 for grouping conditions.

(53) TABLE-US-00009 TABLE 9 Experimental grouping for regulating intestinal flora gavage dose (calculated by number of human daily Group Samples animals (n) intake, cfu/d) Control PBS 14 — group ET-22 ET-22 live 14 3.88 × 10.sup.9  low- bacteria dose group samples ET-22 ET-22 live 14 3.88 × 10.sup.10 medium- bacteria dose group samples ET-22 ET-22 live 14 3.88 × 10.sup.11 high- bacteria dose group samples

(54) (3) Before starting the gavage, the feces of each mouse were collected under aseptic conditions, labeled, and stored at −20° C. for detection of intestinal flora.

(55) (4) In the experiment, each test substance was given by gavage administration of 0.2 ml/10 g. The control group was given PBS on days 1-14, and the test groups were given the corresponding dose of test substance by gavage according to Table 9 respectively. Mice were weighed once a week, and the amount of gavage was adjusted according to the body weight.

(56) (5) After 14 days, the feces of each mouse were collected under aseptic conditions, labeled, and stored at −20° C. for detection of intestinal flora.

(57) 3. Changes of Microbial Diversity in the Intestine of Mice

(58) Microbial diversity was determined based on the Illumina HiSeq sequencing platform, using the paired-end sequencing (Paired-End) method to construct a library of small fragments for sequencing. Species composition of the sample can be revealed by splicing and filtering of Reads, OTUs (Operational Taxonomic Units) clustering, and species annotation and abundance analysis; the optimized sequence was clustered, divided into OTU, and the species classification was obtained according to the sequence composition of OTU. Based on the OTU analysis results, taxonomic analysis was performed on the samples at various classification levels to obtain the community structure of each sample at the genus level.

(59) Alpha diversity analysis was used to study the species diversity within a single sample. The Ace, Chao1, Shannon, and Simpson indexes of each sample at the 97% similarity level were counted, and the sample dilution curve and grade abundance curve were drawn; Alpha Diversity, Beta Diversity, and significant species difference analysis were further carried out to discover differences between samples. Comparison of intestinal flora alpha diversity index: Chao1 and ACE index measure species abundance, that is, the number of species; Shannon and Simpson index are used to measure species diversity, and are affected by species abundance and species uniformity in the sample community. Under the same species abundance, the greater species uniformity in the community, the greater the diversity of the community. The larger the Shannon index value and the smaller the Simpson index value, the higher the species diversity of the sample. Compared with the control group in the genus level, the ET-22 intervention can significantly increase the relative abundance in the intestine of mice. The OTU, ACE, and Chao1 indexes of the low-dose group increased significantly, showing that the low-dose ET-22 can most significantly increase the intestinal microbial diversity.

(60) TABLE-US-00010 TABLE 10 Intestinal microbial diversity of mice Before intervention After intervention Group OTU ACE Chaol Simpson Shannon OTU ACE Chaol Simpson Shannon Control group 294.50 ± 311.92 ± 316.19 ± 0.08 ± 3.46 ± 303.67 ± 323.29 ± 369.52 ± 0.04 ± 3.43 ± 21.52 20.16 20.04 0.03 0.18 24.71 23.3 24.96 0.01 0.19 ET22 low-dose 300.00 ± 319.73 ± 322.82 ± 0.06 ± 3.65 ± 349.83 ± 370.05 ± 374.87 ± 0.05 ± 3.96 ± group 25.75 23.81 25.23 0.02 0.29 22.63 19.97 22.53 0.01 0.22 ET22 286.25 ± 309.35 ± 314.09 ± 0.1 ± 3.26 ± 328.58 ± 353.35 ± 362.58 ± 0.07 ± 3.72 ± medium-dose 32.90 30.25 31.48 0.04 0.37 43.62 40.35 39.74 0.06 0.48 group ET22 high-dose 292.16 ± 315.04 ± 317.05 ± 0.09 ± 3.41 ± 319.83 ± 337.6 ± 343.34 ± 0.06 ± 3.78 ± group 39.34 38.49 39.67 0.06 0.55 45.74 41.48 42.91 0.04 0.4

(61) TABLE-US-00011 TABLE 11 Test results at genus levels after ET-22 intervention (%) ET22 low- ET22 medium- ET22 higjh- Control dose dose dose Genus group group group group Desulfovibrio 2.1391 ± 0.5097 1.3084 ± 0.4245 1.1237 ± 0.5559 1.6218 ± 0.8902 Enterorhabdus 0.3447 ± 0.0971 0.2563 ± 0.1022 0.3013 ± 0.1801 0.2298 ± 0.1687 Helicobacter 0.1254 ± 0.0492 0.2174 ± 0.6786 0.0748 ± 0.0267  0.06 ± 0.0288 Coprococcus_1 0.0795 ± 0.0126 0.0785 ± 0.0507 0.1009 ± 0.0734 0.0802 ± 0.0649 Escherichia- 0.0281 ± 0.0054 0.0045 ± 0.0025 0.0036 ± 0.0022 0.0011 ± 0.0010 Shigella Enterococcus 0.0158 ± 0.0067  0.005 ± 0.0018  0.001 ± 0.0011 0.0004 ± 0.0009 Akkermansia 0.0009 ± 0.0022 2.1671 ± 4.8421 2.5336 ± 8.4747  0.001 ± 0.0026 underlined: statistically significant difference compared to the control group (p < 0.05).

(62) At the level of the pathogenic genus, compared with the control group, Desulfovibrio in the intestinal tract of mice in both the low-dose and the medium-dose ET-22 groups was significantly reduced; the numbers of Helicobacter in the middle-dose and high-dose groups were significantly reduced; Escherichia-Shigella of the three groups of low-dose and middle-dose decreased significantly. BL-99 can significantly increase the relative abundance of Lactobacillus in the intestine of mice, and the BL-99 low-dose group leads to the most significant increase in Lactobacillus.

(63) Therefore, supplementing with different doses of ET-22 can adjust the balance of intestinal flora, inhibit the number of harmful bacteria and even pathogenic bacteria, and play a potential health effect.

Example 5: Analysis of Immunomodulatory Activity

(64) 5.1 Humoral Immunity Test

(65) 5.1.1 Antibody-Producing Cell Detection Experiment

(66) The spleen cell suspension of mice immunized with sheep red blood cells (SRBC) was mixed with a certain amount of SRBC. With the participation of complement, the SRBCs around the antibody-producing spleen cells were dissolved to form plaques visible to the naked eye. The number of hemolytic plaques can reflect the number of antibody-producing cells.

(67) The PBS suspension was used to adjust the concentration of live bacterial samples according to different gavage amounts before the animals were gavaged (the dosage was about 10.sup.7-10.sup.9 CFU/day). After 28 consecutive days of administering to the animals, each mouse was immunized by intraperitoneal injection of 0.2 mL of SRBC. The mice were sacrificed 4 days after SRBC immunization, and the spleen was collected to prepare a cell suspension of 5×10.sup.6 cells/mL. After heating to dissolve, agarose was mixed with an equal amount of double Hank's solution, and divided into small test tubes, 0.5 mL per tube. Then 50 μL of 20% (V/V, packed cell volume, in physiological saline) SRBC, and 200 μL of spleen cell suspension were added to the tube, mixed quickly, and poured onto a six-well plate with a thin layer of agarose. After solidification of the agar, the plate was placed in a carbon dioxide incubator and incubated for 1 h, then complement diluted in SA buffer (1:10) was added, incubated for 2 h, and then the number of hemolytic plaques was counted.

(68) The results of the number of antibody-producing cells are shown in FIG. 5. Compared with the control group, L. paracasei ET-22 and L. paracasei K56 (CGMCC 15139) significantly increased the number of antibody-producing cells (p<0.05).

(69) 5.1.2 Determination of Serum Hemolysin Half Hemolysis Concentration (HC.sub.50)

(70) After 28 consecutive days of administration to the animals, each mouse was immunized by intraperitoneal injection of 0.2 mL of SRBC. After 4 days, the eyeballs were removed and blood was collected in a 1.5 mL centrifuge tube, and placed at 4° C. for about 1 hour to allow serum full released. The tube was centrifuged at 2000 r/min for 10 minutes to collect the serum. The serum was diluted 100 times with SA buffer. The diluted serum was added to a 96-well plate, 100 μL per well, and then 50 μL of 10% (v/v) SRBC, and 100 μL of complement (diluted 1:8 with SA solution) were added sequentially. The plate was placed in a constant temperature water bath at 37° C. for 30 min, and centrifuged at 1500 r/min for 10 min. 50 μL of supernatant from each sample well and blank control well was added to another 96-well culture plate, and 150 μL of Venchi's reagent was added. In addition, a half hemolysis well was set up, into which 12.5 μL of 10% (v/v) SRBC and then 2000 μL of Venchi's reagent were added. The plate was thoroughly mixed in the shaking period, and after placing for 10 minutes, the optical density value of each well was measured with an automatic microplate reader at 540 nm.

(71) The amount of hemolysin is expressed as the half hemolysis concentration (HC.sub.50), calculated according to the following formula:
HC.sub.50 of the sample=Optical density of the sample/Optical density at SRBC half hemolysis×Dilution factor

(72) It can be seen from FIG. 6 that the half hemolysis concentration HC.sub.50 of the L. paracasei ET-22 group is significantly higher (p<0.05) that of the control group, and higher than that of the L. paracasei K56.

(73) 5.2 NK Cell Activity Test

(74) After 28 consecutive days of administration to the animals, the target cells YAC-1 were subcultured 24 hours before the start of the experiment, washed twice with Hank's solution, and the cell concentration was adjusted before use to 1×10.sup.5 cells/ml (target cells) with RPMI 1640 complete culture medium containing 10% calf serum. The mice were sacrificed by cervical dislocation, and the spleen was removed aseptically to prepare a spleen cell suspension. The suspension was washed twice with Hank's solution and centrifuged at 1000 rpm for 10 min. The pellet was resuspended in 2 mL of RPMI 1640 complete medium containing 10% calf serum, stained with trypan blue for counting (the number of live cells should be above 95%), and the cell concentration was adjusted to 1×10.sup.7 cells/mL (effector cells), so that the effector-target ratio was 100:1. 100 μL of target cells and 100 μL of effector cells were added to the U-shaped bottom 96-well culture plate, 100 μL of target cells and 100 μL of culture medium were added to the natural release wells of target cells, and 100 μL of target cells and 100 μL of 1% NP40 were added to the maximum release wells of target cells, all of the above were provided with three parallel wells. The 96-well culture plate was incubated in a 37° C., 5% CO.sub.2 incubator for 4 hours, and centrifuged at 1500 rpm for 5 minutes. 100 μL of the supernatant from each well was pipetted into a flat-bottomed 96-well culture plate, and 100 μL of LDH matrix solution was added to react for 3 minutes, and then 30 μL of 1 mol/L HCL solution was added to each well to stop the reaction. The OD value was measured at 490 nm on a microplate reader and the NK activity was calculated as follows:
NK cell activity=(reaction well OD−natural release well OD)/(maximum release well OD-natural release well OD)×100%

(75) It can be seen from FIG. 7 that L. paracasei ET-22 has higher ability to activate NK cell activity than L. paracaset K56, and the difference from the control group is significant (p<0.05).

(76) It can be seen from the experimental results that, since the L. paracasei ET-22 antibody-producing cell test and the half hemolysis concentration HC.sub.50 result were positive, the humoral immunity of the sample L. paracasei ET-22 is determined to be positive; since the NK cell activity test result was positive, it can be determined that the NK cell activity test of the sample L. paracasei ET-22 is positive. In summary, L. paracasei ET-22 can be considered to have the effect of enhancing immunity.

(77) L. paracasei ET-22 Inhibits Oral Pathogens

(78) At present, it is known that some strains are effective in inhibiting caries bacteria and periodontal bacteria. However, the literature indicates that most studies were conducted on the efficacy of individual strains in the oral cavity, and not all test results prove that lactic acid bacteria are helpful for oral health, and there are individual differences in the efficacy of strains (Anna Haukioja, European Journal of Dentistry 2010 (4): 348-355). For example, Vuotto C et al. (International Journal of Oral Science 2014 (6): 189-194) analyzed the inhibitory ability of probiotics against pathogenic bacteria, and pointed out that the result for any one probiotic strain will not be the same, and even for different probiotic strains of the same species, the test results are sometimes opposite. This result emphasizes that the efficacy of probiotics in inhibiting oral pathogens and whitening teeth is indeed a strain-specific phenomenon.

(79) In fact, as can be seen from the following test results of the present disclosure, most Lactobacillus strains do not have the effect of inhibiting oral pathogens and whitening teeth. The current domestic and foreign literature reports on the research of probiotics in maintaining oral health-related functions have only recently increased. In the early days, it was thought that the genus of lactic acid bacteria in probiotics may coexist with oral pathogens due to the acid-generating properties, and they may also cause corrosion of enamel and caries. However, in fact, experiments have shown that lactic acid bacteria can not only inhibit dental caries and periodontal bacteria, but also compete with them for the living space of oral mucosa and nutritional source, and cause the aggregation of oral pathogens and thus easy to be excluded. But these characteristics must be confirmed through experiments, and not all strains have the same characteristics and experimental results.

Example 6: Analysis of the Characteristics of the Strain to Produce Hydrogen Peroxide to Confirm the Function of Teeth Whitening

(80) The ability of the lactic acid bacteria strain of the present disclosure—Lactobacillus paracasei ET-22 strain to produce hydrogen peroxide was examined to verify the ability of the lactic acid bacteria strain of the present disclosure to whiten teeth. The experimental steps were as follows:

(81) 1. A plate for screening hydrogen peroxide producing probiotics was prepared;

(82) 2. 0.25 mg/mL of Trimethylborane TMB and 0.01 mg/mL of Horseradish Peroxidase HRP were added to the plate′

(83) 3. The plate was divided into four partitions for cultivating target strains;

(84) 4. After incubation for two days, if there was a lactic acid bacteria strain that produced hydrogen peroxide, the surrounding area of the colonies would appear blue;

(85) 5. The concentration of hydrogen peroxide in the lactic acid bacteria and secondary metabolites was determined using hydrogen peroxide test paper;

(86) 6. The lactic acid bacteria liquid was cultured and centrifuged at 4500 rpm for 5 minutes;

(87) 7. A fraction of the bacterial cells was dissolved in 4.9 ml of 100 mM Piperazine-1,4-bisethanesulfonicacid (PIPES); and

(88) 8. The culture was incubated at 37° C. for 5 hours and centrifuged at 220 rpm. 10 microliters (μL) of the supernatant or sedimented bacterial cells were dropped in a hydrogen peroxide test paper (Merck) and reacted for 10 seconds. The color change was observed and compared with a colorimetric card to record the concentration.

(89) The experimental results are shown in FIG. 8. FIG. 8 shows the results of the ability of the Lactobacillus paracasei ET-22 strain to secrete hydrogen peroxide on the plate after culturing on the hydrogen peroxide detection plate.

(90) It can be seen from the test results in FIG. 8 that after two days of cultivation, the Lactobacillus paracasei ET-22 strain of the present disclosure secreted hydrogen peroxide well during the growth of the strain. It should also be noted that other lactic acid bacteria strains of the same species other than the strain of the present disclosure, such as Lactobacillus paracasei strain 9 (BCRC16093), have no ability of secreting hydrogen peroxide, as shown in FIG. 8.

Example 7: Inhibition of Oral Pathogens by Active Lactic Acid Bacteria for Maintaining Oral Health

(91) In order to exert the oral health function of lactic acid bacteria, the resistance of oral pathogens is the most important. In addition to the well-known Streptococcus mutans, oral pathogens also include Fusobacterium nucleatum subsp. polymorphum, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis, etc. These strains may cause several symptoms, for example, Streptococcus mutans causes caries; Fusobacterium nucleatum subsp. polymorphum mainly causes periodontal disease, halitosis and colon cancer; Aggregatibacter actinomycetemcomitans is the main bacterial species causing periodontitis, oral inflammation and pneumonia; and Porphyromonas gingivalis is the main cause of adult periodontitis and halitosis. In addition, for oral odor, in addition to Porphyromonas gingivalis as the main bacteria causing halitosis, Fusobacterium nucleatum subsp. polymorphum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis all produce sulfide, and are the main bacterial species that affect oral odor.

(92) Therefore, the present disclosure conducted a pathogenic bacteria inhibition test to evaluate the ability of the active Lactobacillus paracasei ET-22 strain of the present disclosure to inhibit oral pathogenic bacteria, thereby achieving the effect of maintaining oral health. The experimental steps were as follows:

(93) 1. The active lactic acid bacteria was applied to the center of the MRS plate with a diameter of two centimeters and cultured for two days;

(94) 2. The culture medium for oral pathogens was poured on the plate where the lactic acid bacteria had grown, and after solidification, the high concentration of oral pathogens were evenly spread on the pathogen culture plate;

(95) 3. The plate was incubated at 37° C. for 2-4 days; and

(96) 4. The inhibition diameter was measured if the growth of pathogenic bacteria was inhibited in the central part where the lower medium was coated with lactic acid bacteria.

(97) The experimental results are shown in FIG. 9, which shows the analysis of the ability of the lactic acid bacteria strain of the present disclosure to inhibit oral pathogens.

(98) FIG. 9 shows the results of the Lactobacillus paracasei ET-22 strain of the present disclosure in inhibiting the survival of oral pathogens. Compared with another strain of Lactobacillus paracasei 9, the inhibitory effect of ET-22 is more significant.

Example 8: Inhibition of Oral Pathogens by Deactivated Lactic Acid Bacteria for Maintaining Oral Health

(99) In addition to the function of active lactic acid bacteria to inhibit oral pathogenic bacteria, it is unclear whether the deactivated lactic acid bacteria have the function of inhibiting oral pathogenic bacteria. Therefore, a pathogen inhibition test was conducted to evaluate the ability of the inactivated Lactobacillus paracasei ET-22 strain of the present disclosure to inhibit oral pathogens, thereby achieving the effect of maintaining oral health. The experimental steps were as follows:

(100) 1. Oral pathogens were activated;

(101) 2. The number of heat-killed lactic acid bacteria was adjusted and the bacteria were added to co-culture with the pathogenic bacteria according to the ratio of one billion bacteria per milliliter;

(102) 3. The bacteria were incubated in an anaerobic manner at 150 rpm in a 37° C. environment for 2-4 days; and

(103) 4. The number of pathogenic bacteria was counted.

(104) The experimental results are shown in FIG. 10, which is an analysis of the ability of the deactivated lactic acid bacteria strain of the present disclosure to inhibit oral pathogens; the histograms are all expressed in Mean±SD and compared with another strain of Lactobacillus paracasei 9.

(105) FIG. 10 shows the results of the deactivated Lactobacillus paracasei ET-22 strain of the present disclosure in inhibiting the survival of oral pathogens. Compared with the inhibitory effect of the Lactobacillus paracasei 9 strain, ET-22 is more remarkable.

(106) Based on the above test results, the food composition and pharmaceutical composition containing the lactic acid bacteria strain of the present disclosure can inhibit the growth of oral pathogenic bacteria, and can be used for reducing human caries, periodontal disease and breath odor. Preferably, the strain of lactic acid bacteria that can secrete hydrogen peroxide also has the ability to whiten teeth. The present disclosure found lactic acid bacteria that have no side effects on the human body and are beneficial to maintaining oral health as a new option for maintaining oral health.

(107) Unless otherwise stated, all numbers used in this specification (including claims) to indicate the amount of components, cell culture, processing conditions, etc. should be understood to be modified by the term “about” under all conditions. Therefore, unless otherwise stated to the contrary, the numerical parameters are approximate and may vary according to the desired characteristics sought to be obtained by the present disclosure. Unless otherwise stated, the term “at least” before a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize or be able to determine with no more than routine experimentation many equivalents of the specific embodiments of the invention described herein. The appended claims are intended to cover such equivalents.

(108) Those skilled in the art will understand that many modifications and variations of the present disclosure can be made without departing from the spirit and scope thereof. The specific embodiments described herein are provided by way of example only, and are not meant to be limiting in any way. The true scope and spirit of the present disclosure are shown by the appended claims, and the description and embodiments are merely exemplary.

(109) The above-mentioned examples are only to illustrate the technical ideas and features of the present disclosure, and their purpose is to enable those skilled in the art to understand the content of the present disclosure and implement it accordingly, but not to limit the patent scope of the present disclosure, that is, changes or modifications made according to the spirit of the present disclosure should still be covered by the patent scope of the present disclosure.