Use of <i>Lactobacillus paracasei </i>subsp. <i>paracasei </i>K56 capable of regulating gastrointestinal flora balance

Abstract

A new use of Lactobacillus paracasei subsp. paracasei K56 capable of regulating the gastrointestinal flora balance is described. The deposit number of the Lactobacillus paracasei subsp. paracasei is DSM27447. This strain alone has the ability to significantly promote the growth of intestinal Bifidobacterium and Lactobacillus, suppress Desulfovibrio and/or Enterobacteria in the intestine, suppress Helicobacter and/or Escherichia-Shigella, and can endure a simulated in vitro gastrointestinal fluid stress environment. Experiments in mice show that this strain has no acute oral toxicity, no antibiotic resistance, and may be safely used in food processing.

Claims

1. A method for regulating gastrointestinal flora, comprising administering a composition comprising Lactobacillus paracasei subsp. paracasei to a subject, wherein the deposit number of the Lactobacillus paracasei subsp. paracasei is DSM27447, wherein the composition increases the amount of Bifidobacterium, Lactobacillus or both in the intestine, and suppresses the amount of Desulfovibrio, Enterobacter or both in the intestine, and suppresses the amount of Helicobacter, Escherichia-Shigella or both, and wherein the amount of the Lactobacillus paracasei subsp. paracasei present in the composition is 1.0×10.sup.3 CFU to 5.0×10.sup.8 CFU/kg body weight/day.

2. The method according to claim 1, wherein the composition is in the form of a solid or liquid bacterial preparation.

3. The method according to claim 1, wherein the composition includes a food composition, a feed composition, or a pharmaceutical composition.

4. The method according to claim 1, wherein the composition is a food composition, and wherein the food is a fermented dairy product, cheese, a dairy beverage, a solid beverage, or milk powder.

5. A method for regulating gastrointestinal flora, comprising administering an effective amount of Lactobacillus paracasei subsp. paracasei to a subject, wherein the deposit number of the Lactobacillus paracasei subsp. paracasei is DSM27447, wherein the Lactobacillus paracasei subsp. paracasei increases the amount of Bifidobacterium, Lactobacillus or both in the intestine, and suppresses the amount of Desulfovibrio, Enterobacter or both in the intestine, and suppresses the amount of Helicobacter, Escherichia-Shigella or both, and wherein the Lactobacillus paracasei subsp. paracasei is administered to the subject in an amount of 1.0×10.sup.3 CFU to 5.0×10.sup.8 CFU/kg body weight/day.

6. The method according to claim 5, wherein the Lactobacillus paracasei subsp. paracasei is administered to the subject in an amount of 1.0×10.sup.4 CFU to 5.0×10.sup.8 CFU/kg body weight/day.

7. The method of claim 1, wherein the amount is 1.0×10.sup.4 CFU to 5.0×10.sup.8 CFU/kg body weight/day.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1A and FIG. 1B show a schematic microscopic photograph of intestinal adhesion of Lactobacillus paracasei subsp. paracasei K56.

(2) FIG. 2 shows the results of the intestinal adhesion comparison test of Lactobacillus paracasei subsp. paracasei K56.

(3) FIG. 3 shows the test results of Lactobacillus paracasei subsp. paracasei K56 in regulating intestinal flora.

DESCRIPTION OF EMBODIMENTS

(4) For better understanding of the technical features, the purpose, and advantageous effects of the present invention, the technical solutions of the present invention are now described in detail in connection with specific examples. It should be understood that these examples are only used to illustrate the present invention, but not to limit the scope of the present invention. In the examples, the starting reagents and materials are commercially available, and the experimental methods without specified conditions are conventional methods and conventional conditions well known in the art, or in accordance with the conditions recommended by the instrument manufacturer.

(5) Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the relevant art commonly understand. Unless otherwise specified, all numbers used in the present invention indicating the amounts of ingredients, cell culture, treatment conditions and the like should be understood as being modified by the term “about” under all circumstances. Therefore, unless otherwise stated, the numerical parameters are approximate values and may vary according to the desirable characteristics intended to be obtained by the present invention. Unless otherwise stated, the term “at least” preceding a series of elements should be understood to refer to each element in the series.

(6) In each example of the present invention, unless otherwise specified, the experimental data is expressed as Mean±S.E.M. The data is calculated by PRISM version 5.0 (GraphPad, San Diego, Calif., USA). Differences between groups are calculated by one-way ANOVA followed by Tukery's multiple comparison test. A significant statistical difference is present at P<0.05.

EXAMPLE 1: GASTRIC ACID RESISTANCE TEST

(7) The MRS culture medium was adjusted to pH 2.0, pH 2.5, and pH 3.0 respectively with a 0.1N HCl solution, and 100 μL (109 CFU/ml) of the activated bacteria solution was inoculated in 10 mL of test solutions at different pH. The viable bacteria having an initial concentration of approximately 107 CFU/mL was placed at 37° C. for 1 hour and sampled to measure the number of the remaining bacteria. 1 mL of the bacteria solution was taken, serially diluted with a 0.85% saline solution, applied on MRS agar, and incubated at 37° C. for 24 to 48 hours to calculate the number of colonies generated. In addition, 100 μL of a Lactobacillus solution (109 CFU/ml) was added to an MRS culture medium without pH adjustment (pH 6.8) as control.

(8) The acid resistance of the strain (%)=(the number of remaining bacteria in the test solution at pH 2.0 (or pH 2.5 or pH 3.0)/the number of bacteria in the MRS culture solution without pH adjustment)×100%

(9) Acid resistance is considered as one of the necessary characteristics for Lactobacillus to survive in the acidic environment in the stomach. The results of the acid resistance of K56 in different acidic environments are shown in Table 1 below. The results show that the number of the initial viable bacteria was 2.96×107 CFU/mL. After 1 hour in an acidic environment, the strain K56 survived in the tolerant conditions of pH 2.5 and pH 3.0, though it was almost completely killed after 1 hour of pH 2.0 treatment (0%). The survival rate was 6.39% at pH 2.5, and the strain was less sensitive to an acidic environment at pH 3 and maintained a survival rate of 84.31%.

(10) TABLE-US-00001 TABLE 1 K56 acid resistance test results Number of bacteria in Survival rate under different control (CFU/mL) acidic environments (%) Strain MRS (pH6.8) pH2.0 pH2.5 pH3.0 K56 2.96E+07 0.00% 6.39% 84.31%

EXAMPLE 2. BILE SALT RESISTASNCE TEST

(11) MRS culture media containing test solutions having oxgall bile at different concentrations of 0.1%, 0.5% and 1%, respectively, were prepared. 100 μL (109 CFU/ml) of activated bacteria solution was taken and inoculated in 10 mL of the test solutions containing oxgall bile at different concentrations. The viable bacteria having an initial concentration of about 107 CFU/mL was placed at 37° C. for 1 hour and sampled to measure the number of the remaining bacteria. 1 mL of the bacteria solution was taken, serially diluted with PBS (0.1 M, pH 6.2), applied on MRS agar, incubated at 37° C. for 24 to 48 hours to calculate the number of colonies generated. In addition, 100 μL of a Lactobacillus solution (109 CFU/ml) was added to an MRS culture medium (pH 6.8) without oxgall bile as control.

(12) Bile salt resistance of the strain (%)=(the number of remaining bacteria in the test solution containing 0.1% (or 0.5% or 1%) oxgall bile/the number of bacteria in the test solution without oxgall bile)×100%

(13) Bile salt resistance is considered as one of the necessary characteristics for Lactobacillus to survive in the small bowel. The results of the resistance of the strain K56 to different concentrations of bile salt for 1 hour are shown in Table 2. It can be seen that as the bile salt concentration increases, the sensitivity of the strain to the bile salt increases, which results in the increase in the mortality rate. the initial number of viable bacteria was 4.34×107 CFU/mL, and the strain K56 had 89.60% resistance to 0.1% bile salt after 1 hour incubation, 82.73% resistance to 0.5% bile salt after 1 hour incubation, and a 69.44% survival rate at 1% bile salt concentration. In summary, it shows that K56 is highly resistant to an acidic environment and a bile salt-containing environment.

(14) TABLE-US-00002 TABLE 2 K56 bile salt resistance test results Number of Survival rate under bacteria in environments with oxgall control bile at different (CFU/mL) concentrations (%) Strain MRS 0.1% Oxgall 0.5% Oxgall 1% Oxgall K56 4.34E+07 89.60% 82.73% 69.44%

EXAMPLE 3: INTESTINAL CELL ADHESION EFFECT

(15) Caco-2 cells were cultured in a culture flask. A DMEM cell culture medium containing 10% heat-inactivated fetal bovine serum and double antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) was added into the flask and placed and cultured in an incubator at 37° C., 5% CO2, with the culture medium changed every 2 days. After the cells grew into an adhering single cell layer (5 to 7 days), they were digested and passaged with 0.25% trypsin, and stained with a 0.4% trypan blue staining solution. A hemocytometer was used to determine the number and activity of the cells under a microscope in order to ensure a cell activity of above 95%.

(16) The adhesion of the strain to Caco-2 cells was tested in an adhesion experiment. The results are shown in FIG. 1 (panel B is an enlarged view of panel A). FIG. 2 shows that K56 has an intestinal adhesion ability similar to LcS and LcA and better than LGG and NCFM.

EXAMPLE 4: INTESTINAL FLORA REGULATION EFFECT

(17) In this example, it is intended to verify the effect of Lactobacillus paracasei subsp. paracasei according the present invention in intestinal regulation. Reference can be made to “Technical Specifications for Health Food Examination and Evaluation: Standards for Intestinal Flora Function Regulation” for the principles and procedures.

(18) Forty-two healthy SPF BABL/c mice weighing 18-22 g (supplied by Beijing Huafukang Biotechnology Co., Ltd.) were taken. After 3 days of adaptive feeding, they were randomly divided into 3 groups, each with 14 animals, i.e., a blank control group and a sample group. Each group of animals was gavaged with sterile water having dissolved Bifidobacterium lactis K56 powder (gavage volume 0.2 mL/10 g), and the blank control group was gavaged with sterile water of the same volume. The feeding or gavage was done once a day for 14 days consecutively. Gavage volume: 1.3×10.sup.7 CFU/ml (converted in accordance with an amount of 2×10.sup.9 CFU/d as needed by human, with a conversion factor between human and mouse of 0.0026). After the adaptive feeding, mouse feces was collected under aseptic conditions into numbered sterile centrifuge tubes, with 2-3 pellets of about 100 mg from each mouse, and transferred to an aseptic operation room under low temperature conditions for flora measurement. At the end of the experiment, mouse feces were collected again. The mice were grouped and numbered with picric acid, weighed on the 8th and 14th days of administration of the test substance, and the gavage volume of the mice was calculated. The mice were weighed once at the end of the experiment. Colony counting: selective media were prepared according to the strain to be identified. The strain to be tested and the corresponding medium are shown in Table 3. Sterilization was carried out followed by uniform shaking, cooling to 45° C.-50° C., and pouring into a plate before use.

(19) TABLE-US-00003 TABLE 3 Test strains and corresponding selective medium Strains to be tested Selective medium Enterobacter Eosin Methylene Blue (EMB) Agar Enterococcus Sodium Azide-Crystal Violet-Aescin Agar Bifidobacterium BBL Agar Lactobacillus LBS Agar Clostridium perfringens Tryptone-Sulfite-Cycloserine (TSC) Agar

(20) The collected mouse feces were placed in a sterile tube containing 0.5 mL of normal saline, prepared into a bacterial suspension, and shaken for 1 min before use. 0.1 mL of the bacteria suspension was taken with a 0.1 mL micropipette, slowly injected into 0.9 mL of sterile saline, shaken or repeatedly pipetted to mix well to make a 1:10 bacteria suspension. A 10-fold gradient dilution was conducted in the same way to 10 to 7 g/ml by using another 0.1 mL micropipette tip. According to the number of viable bacteria to be identified, two consecutive appropriate dilutions were selected. For each dilution, 10 μL of bacterial suspension was taken by a 10 μL micropipette, surface coated on a plate with the selective agar, and cultured according to the culture conditions shown in Table 2. For the colony counting method, reference can be made to “GB 4789.2-2010 National Food Safety Standard, Food Microbiological Examination: Aerobic Plate Count”.

(21) TABLE-US-00004 TABLE 4 Intestinal flora test medium and identification method Items Medium Culture condition Enterobacter Eosin Methylene Blue Agar 24 h culture, 36° C. ± 1° C. Enterococcus Sodium Azide-Crystal 48 h culture, 36° C. ± 1° C. Violet-Aescin Agar Bifidobacterium BBL Agar 48 h anaerobic culture, 36° C. ± 1° C. Lactobacillus LBs Agar 48 h culture, 36° C. ± 1° C. Clostridium TSC Agar 24 h anaerobic culture, perfringens 36° C. ± 1° C.

(22) SPSS17.0 was used for data statistics. The changes of Bifidobacterium, Lactobacillus, Enterococcus, and Enterobacteria before and after the experiment and between the groups were compared. For the test group, the change before and after the experiment was significant, and the animal test result of the test sample could be determined as positive if any of the following conditions was met: (i) there was a significant increase in Bifidobacterium or Lactobacillus in feces, a decrease or no significant change in Clostridium, no significant change in Enterococcus or Enterobacter; (ii) there was a significant increase in Bifidobacterium or Lactobacillus in feces, a decrease or no significant change in Clostridium, and a significant increase in Enterococcus and Enterobacter with the increase being lower than the increase in Bifidobacterium or Lactobacillus.

(23) The results of the body weight changes of animals during the experiment are shown in Table 5. During the experiment, the animals showed normal characteristics, and no adverse reaction occurred after the administration of the test substance. Over the experiment period, there was no significant difference in body weight between the two groups of animals. From Table 6 to Table 10, it can be seen that Lactobacillus paracasei subsp. paracasei K56 can significantly promote the growth of Bifidobacterium and Lactobacillus, while having no significant effect on Enterobacter, Enterococcus, and Clostridium perfringens. According to the “Technical Specifications for Health Food Examination and Evaluation: Standards for Intestinal Flora Function Regulation”, it can be concluded that the Lactobacillus paracasei subsp. paracasei K56 in this study has the effect of regulating intestinal flora (See FIG. 3).

(24) TABLE-US-00005 TABLE 5 Animal body weight changes Number of Initial weight Mid-term Final Group animals (g) weight (g) weight (g) Control 14 21.89 ± 1.25 22.14 ± 0.87 21.24 ± 0.87 K56 14 22.11 ± 1.08 21.79 ± 1.17 20.95 ± 0.22

(25) TABLE-US-00006 TABLE 6 Changes in animal intestinal Bifidobacterium before and after the test (LgCFU/g) p value for in-group p value for Number comparison before comparison with of Before After and after control after Group animals intervention intervention intervention intervention Control 14 8.66 ± 0.57 8.94 ± 0.46   0.196 K56 14 8.87 ± 0.59 9.80 ± 0.63** 0.009 0.002

(26) TABLE-US-00007 TABLE 7 Changes in animal intestinal Lactobacillus before and after the test (LgCFU/g) p value for in-group p value for Number comparison comparison with of Before After before and control after Group animals intervention intervention after intervention intervention Control 14 8.54 ± 0.57 8.45 ± 0.18   0.566 K56 14 8.76 ± 0.56 9.72 ± 0.57** 0.000 0.000

(27) TABLE-US-00008 TABLE 8 Changes in animal intestinal Enterobacter before and after the test (LgCFU/g) p value for in-group p value for Number comparison comparison with of Before After before and control after Group animals intervention intervention after intervention intervention Control 14 6.48 ± 0.32 6.98 ± 0.74 0.123 K56 14 7.00 ± 0.38 7.30 ± 0.13 0.001** 0.055

(28) TABLE-US-00009 TABLE 9 Changes of animal intestinal Enterococcus before and after the test (LgCFU/g) p value for in-group p value for Number comparison comparison with of Before After before and control after Group animals intervention intervention after intervention intervention Control 14 6.62 ± 0.27 6.78 ± 0.61 0.467 K56 14 6.97 ± 0.30 7.21 ± 0.41 0.058 0.052

(29) TABLE-US-00010 TABLE 10 Changes in animal intestinal Clostridium perfringens before and after the test (LgCFU/g) p value for in-group p value for Number comparison comparison with of Before After before and after control after Group animals intervention intervention intervention intervention Control 14 8.71 ± 0.17 9.10 ± 0.49 0.060 K56 14 8.60 ± 0.43 8.74 ± 0.57 0.465 0.091

EXAMPLE 5: COMPARISON OF THE INTESTINAL FLORA REGULATING EFFECTS OF K56 AT DIFFERENT DOSES

(30) In this example, the intestinal flora regulating effect of K56 at different doses was tested.

(31) Viable bacteria sample: according to the sample specification, 1 g of K56 viable bacteria sample was weighted and suspended in a PBS solution to 40 ml; namely, the concentration of the viable bacteria was 2.5×10.sup.9 CFU/ml.

(32) High-dose group: the gavage dose for a 20 g mouse was 0.4 ml as calculated according to a gavage amount of 0.2 ml/10 g in mice, and the gavage dose for the mice in the high-dose group was 10.sup.9 CFU/20 g.

(33) Medium-dose group: 5 ml of the high-dose suspension was taken and added to PBS to a volume of 50 ml; the gavage dose for a 20 g mouse was 0.4 ml as calculated according to a gavage amount of 0.2 ml/10 g in mice, and the gavage dose for the mice in the medium-dose group was 10.sup.8 CFU/20 g.

(34) Low-dose group: 5 ml of the medium-dose suspension was taken and added to PBS to a volume of 50 ml; the gavage dose for a 20 g mouse was 0.4 ml as calculated according to a gavage amount of 0.2 ml/10 g in mice, and the gavage dose for the mice in the low-dose group was 10.sup.7 CFU/20 g.

(35) Six-week-old BABL/c mice were raised in a clean grade animal housing at a temperature of 22° C. and humidity of 10-60%, with 12 hour-lighting alternating between light and darkness, and provided with standard feed and free drinking water. The mice was adaptively fed for 5 days and randomly divided into groups with 14 mice in each group. The groups are shown in Table 11.

(36) TABLE-US-00011 TABLE 11 Groups in the experiment of intestinal flora regulation Number Gavage amount Test of (Calculated as CFU/d in terms Groups substance animals of daily intake by human) Control PBS 14 — Low-dose PBS + K56 14 3.88 × 10.sup.9 Medium-dose PBS + K56 14 3.88 × 10.sup.10 High-dose PBS + K56 14 3.88 × 10.sup.11

(37) Before the gavage, the feces of each mouse was collected under aseptic conditions, labeled, and stored at −20° C., and the intestinal flora was examined. In the experiment, each test substance was administered according to a gavage amount of 0.2 ml/10 g, and PBS was given to the control group on Day 1 to Day 14. The experimental groups were given the corresponding dose of test substance by gavage according to Table 11. The mice were weighed once a week, and the gavage volume was adjusted according to the body weight. After 14 days, the feces of each mouse were collected under aseptic conditions, labeled, and stored at −20° C., and the intestinal flora was examined.

(38) Before and after the experiment, there was no significant difference in body weight among the mice in each group. At the phylum level, after supplementing of different doses of probiotics, the relative abundance of Firmicutes in the mouse intestinal flora increased while the relative abundance of Bacteroidetes and Proteobacteria decreased. Studies have shown that the ratio of Firmicutes to Bacteroides was closely correlated with intestinal diseases in human, and patients with obesity tend to have a lower ratio. However, patients with enteritis and intestinal stress syndrome tend to have a higher abundance of Proteobacteria.

(39) The effect of K56 on the intestinal flora at the genus level is shown in Table 12.

(40) TABLE-US-00012 TABLE 12 The effect of K56 on the intestinal flora Genus Control Low-dose Medium-dose High-dose Bacteroides 9.8061 ± 2.094  6.0503 ± 1.6172 7.4157 ± 2.3149 6.5026 ± 1.553  Lactobacillus 3.0166 ± 0.4635 0.5798 ± 0.5605 4.0343 ± 0.5534  2.158 ± 1.2288 Desulfovibrio 2.1391 ± 0.5097  0.853 ± 0.3645 1.5655 ± 0.9228 0.8367 ± 0.4632 Enterobacter 0.3447 ± 0.0971  0.254 ± 0.1668 0.2834 ± 0.1117 0.2134 ± 0.0873

(41) At the genus level, in the probiotic family of the intestinal flora, the K56 medium-dose group can significantly increase the relative abundance of Lactobacillus in the mouse intestine as compared to the control group. The K56 low-dose group and high-dose group have a significant suppressive effect on Desulfovibrio.

(42) The inhibitory effect of K56 on the pathogenic bacteria Helicobacter and Escherichia-Shigella is shown in Table 13.

(43) TABLE-US-00013 TABLE 13 The inhibitory effect of K56 on pathogenic bacteria Genus Control Low-dose Medium-dose High-dose Helicobacter 0.1254 ± 0.0492 0.059 ± 0.028 0.0978 ± 0.0468 0.0459 ± 0.0325 Escherichia-Shigella 0.0281 ± 0.0054 0.0104 ± 0.0061 0.0236 ± 0.0061 0.0027 ± 0.0031

(44) The results of the analysis of pathogenic bacteria show that the K56 low-dose group had a significant inhibitory effect on Escherichia-Shigella, and all the groups have a significant inhibitory effect on Helicobacter.

(45) The above experiments demonstrate that K56 can regulate the intestinal flora balance, promote the growth of beneficial bacteria, and inhibit harmful bacteria and even pathogenic bacteria.