KR100887819B1 - Method of improving immune function in mammals using lactobacillus reuteri strains - Google Patents

Abstract

Relates to a method of improving the function - The invention immunity in a mammal using the product, this product containing Lactobacillus ruteri (Lactobacillus reuteri) The use of the strain, these Lactobacillus ruteri (Lactobacillus reuteri) strain as an immune enhancer. These selected strains show good toxin binding and neutralizing effects and good CD4 ⁺ cell recruitment.

Lactobacillus reuteri

Description

METHOD OF IMPROVING IMMUNE FUNCTION IN MAMMALS USING LACTOBACILLUS REUTERI STRAINS}

The present invention relates to the use of Lactobacillus reuteri strains as immunopotentiators and to improved methods for selecting strains most suitable for this purpose.

Due to the proliferation of antibiotic-resistant pathogens, probiotics containing various gastrointestinal microorganisms have been produced. Various Lactobacillus species, including L. reuteri , have been used to prepare probiotics. Lactobacillus reuteri occurs naturally in the gastrointestinal tract of animals and is commonly found in the gut of healthy animals. It is known to have antibiotic activity (US Patent No. 5,439,678, 5,458,875, 5,534,253, 5,837,238, 5,849,289). L. reuteri cells, when cultured in anaerobic conditions in the presence of glycerol, produce an antimicrobial substance known as lutein (β-hydroxy-propionaldehyde).

Immunomodulatory activity is also associated with L. reuteri (“Biotherapeutic effects of probiotic bacteria on candidiasis in immunodeficient mice”, Wagner RD, et al. Infect Immune 1997 Oct 65: 4165-72). However, since there is a difference in efficacy between strains, a method of selecting the most effective strains, for example, a method of selecting strains using the CD4 + cells presented in the present invention is required.

In addition, L. reuteri is known to be used as a beneficial probiotics as a whole, but the prior art is most effective for the Lactobacillus strain, which neutralizes the toxicity produced by pathogens already present in the gastrointestinal tract. The importance of the use was not recognized (“Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium”, El-Nezami HS, et al, Food Addit Contain 2002 Jul 19: 680). According to the present invention, such toxin neutralization, including binding, is important not only to mitigate the direct effects caused by these toxin-generating pathogens, but also to reduce the overall burden on the immune system.

Gastrointestinal problems caused by pathogenic microorganisms are caused by a number of different causes. For example, Helicobacter pylori (Helicobacteri pylori) is a gastric ulcer and duodenal ulcer and gastric mucosal lymphoid tissue lymphoma causes combined. Certain pathogenic Escherichia coli strains produce toxins such as Vero toxin (V) produced by E. coli 0157: H7, which neutralizes the effects of antibiotics.

For this reason, an object of the present invention is to provide a method for enhancing immune-function in mammals using Lactobacillus reuteri strains which have been found to be effective for both CD4 + cell recruitment and toxin neutralization, and such immuno-enhancing lactose To present a method for screening Bacillus lusteri ( L. reuteri ) strains, and to present a product containing such strains. Another object of the present invention is to provide a method of using the culture supernatant of L. reuteri strains effective for reducing the immune burden of these toxins.

Other objects and advantages are apparent from the following detailed description and the appended claims.

Summary of the Invention

Relates to a method of improving the function - The invention immunity in a mammal using the product, this product containing Lactobacillus ruteri (Lactobacillus reuteri) The use of the strain, these Lactobacillus ruteri (Lactobacillus reuteri) strain as an immune enhancer. These selected strains show good toxin binding and neutralizing effects and good CD4 ⁺ cell recruitment. Other objects and features of the present invention are more apparent from the following detailed description and the appended claims.

The present invention relates to the use of Lactobacillus reuteri strains used in the production of compositions that enhance immune-function in mammals and exhibit good toxin binding and neutralizing effects and good CD4 ⁺ cell recruitment. The present invention also relates to products containing such Lactobacillus reuteri strains and methods for enhancing immune-function in mammals using such products. In addition, the present invention relates to a method for screening such immune-enhancing Lactobacillus luteri ( L. reuteri ) strains.

Cytokines are immune system proteins that function as biological response regulators. They modulate T cell immune system interactions with antibodies and amplify immune responsiveness (10). Cytokines include monokines synthesized by macrophages and lymphokines produced by activated T lymphocytes and natural killer (NK) cells. In both humans and mice the CD4 + subset is based on cytokine production and main effector function. Th1 cells synthesize interferon-gamma (INF-γ), IL-2, tumor necrosis factor (TNF). They are primarily responsible for cellular immunity against intracellular microbes and inducing delayed hypersensitivity. They affect immunoglobulin G2a (IgG2a) synthesis and antibody dependent cell mediated cytotoxicity. The INF-γ they produce activates macrophages and consequently induces phagocytosis. Th2 cells are interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin- 13 (IL-13) is synthesized. These include IgE and IgG1 antibody reactions; Mucosal immunity by the synthesis of mast cells and eosinophil growth and differentiation factors; Induces IgA synthesis.

The present invention encompasses a method comprising administering L. reuteri strains, analyzing such strains, and including various typical steps to confirm the efficacy of such strains in both CD4 + cell recruitment and toxin neutralization. The data indicate, for example, that tablet formulations containing L. reuteri provide levels of gastrointestinal colonization similar to that of direct administration of cell culture. L. reuteri colonization is associated with the ingestion of L. reuteri and occurs when the washout period ( L. reuteri ) levels fall to pre-intake levels. Time) is at least 28 days after tablet administration, demonstrating that the present invention can be applied not only to L. reuteri cell cultures but also to products containing such L. reuteri cultures. .

Suitably, the present invention relates to the use of products containing L. reuteri strains as probiotics for the prophylactic improvement of immune-function in mammals. Such products may be various food products, such as dietary supplements, sweets or tablets, containing cells of the selected strains.

The L. reuteri strain according to the present invention can also be used in the preparation of a drug for the treatment of various microorganisms producing toxins, for example, Echericia coli . Thus, E. coli and VT toxins can be treated.

According to the invention, cells of L. reuteri strains and their cell cultures can be used for the production of prophylactic probiotic-type pharmaceutical compositions.

According to the present invention, L. reuteri strains showing good CD4 + cell recruitment and / or good toxin binding can be used. The presence of CD4 + cells can be examined, for example, using antibodies against CD4 by immunohistochemical methods (eg, Example 5) or immunofluorescent methods. Toxin binding can be confirmed by contacting L. reuteri cells or supernatants with toxins and examining differences in available toxins, as described in Example 1.

The prophylactic probiotic-type pharmaceutical product according to the present invention may contain additives and excipients suitable for nutritional or pharmaceutical use.

Features of the present invention are more apparent with reference to the following examples, but these examples do not limit the present invention.

Strains selected according to the present invention show good toxin binding and neutralizing effects and good CD4 ⁺ cell recruitment.

Example 1 Vero Toxin Study

Three lactic acid bacterial strains: L. reuteri ATCC 55730, L. bulgaricus strain LB12 (CHR, Horsholm, Denmark), L. casei strain 01 (CHR, Horsholm) , Denmark). Lactobacillus ruteri ( L. reuteri ) was inoculated in MRS liquid medium (+ 20 mM glucose) and then incubated for 24-48 hours at 37 ° C. under aerobic fixation conditions. In some cases, centrifugation at 2,500 rpm for 30 minutes after initial incubation, washing twice with phosphate buffer (PBS) to remove media components, suspended in 250 mM glycerol solution, 6 hours at 37 ° C. under aerobic fixation conditions. Incubated for L. bulgaricus and L. casei were incubated in MRS + 20 mM glucose (without glycerol) at 37 ° C. for 24-48 h under aerobic fixation conditions. Each test lactic acid bacterium was adjusted to 2 g / 30 ml (dry weight), centrifuged at 2,500 rpm for 30 minutes after incubation, the supernatant was recovered, and the pH was adjusted to 7.0 with NaOH (see below). Was used after filtration into a 2.0 μm filter. Glycerol solution and MRS liquid medium adjusted to pH 7.0 was used as a control after filtration in a 2.0 ㎛ filter.

Vero cells (African Green Monkey kidney cells, ATCC-CCL81) were minimally essential medium supplemented with 10% fetal bovine serum (FBS, Sigma) of 25 g / ml gentamycin (Sigma) in a 37 ° C., 10% C0 ₂ incubator. (MEM, Sigma) was incubated for 48 hours, and used after confirming monolayer formation.

Escherichia coli 0157: H7 (ATCC 43894), which secretes both VT1 and VT2, is inoculated in casein soybean digestive liquid medium (TSB), incubated for 24 hours with stirring in a stirred incubator at 37 ° C, 30 at 2,500 rpm Centrifuged for minutes and the culture supernatant was used after filtration.

500 Vero cells (2 × 10 ⁵ cells / ml) were inoculated into 96-well plates and incubated for 48 hours in a 37 ° C., 10% C0 ₂ incubator to confirm the formation of a monolayer. : A: VT alone (positive control); B: TSB (E. coli Escherichia coli 0157: H7 culture medium); C: MRS liquid medium (test lactobacillus bacteria); D: glycerol solution ( L. reuteri culture medium); E: VT + MRS liquid medium; F: VT + glycerol solution; G: supernatant of glycerol solution of VT + L. reuteri ; H: VT + MRS liquid medium culture supernatant of L. bulgaricus ; I: VT + L. bulgaricus culture supernatant; J: VT + Lactobacillus casei culture supernatant. Therapeutic agents B, C, and D determine whether each culture itself causes toxicity to Vero cells; E and F will determine if the culture medium of the test lactic acid bacteria has its own neutralizing capacity for VT. Each culture supernatant of the test lactic acid bacteria at G, H, I, J was diluted 2 × serially, and each diluted culture supernatant and VT of the test lactic acid bacteria were 400 ml + 100 ml, respectively; 300 ml + 200 ml; 200 ml + 300 ml; Combine 100 mL + 400 mL, and then incubate for 18 hours at 37 ℃, 10% C0 ₂ incubator to confirm whether the cytopathic effect (CPE) appears.

Culture of L. reuteri dissolved in 250 mM glycerol solution inhibited lutein production by addition of 300 mM neutralized semicalvazide hydrochloride (Sigma), which inhibits the production of lutein, and the upper layer as described above. The solution was collected. 100 μl Vero cells were seeded in 96-well plates and incubated for 24 hours in a 37 ° C., 10% C0 ₂ incubator to check for monolayer formation and then tested the following therapeutic substances: A: VT alone; B: VT + 25 mM glycerol solution; C: supernatant of glycerol solution of VT + L. reuteri ; D: Glycerol solution culture supernatant of L. reuteri inoculated after treatment with VT + lutein inhibitor. Each culture of L. reuteri treated with or without glycerol solution and lutein inhibitor at B, C, D was 20 + 80 μl each with VT; 30 + 70 μl; 40 + 60 μl; 50 + 50 μl; 60 + 40 μl; 70 + 30 μl; 80 + 20 μl was mixed and then incubated for 18 hours at 37 ° C. in a 10% C0 ₂ incubator to determine whether the cytopathic effect was observed. After microscopic observation, the cultures were stained with crystal violet and their OD (absorbance) values were read at 490 nm.

96-well plates were coated with Gb ₃ (globotriaosylceramide) (Sigma) blocked with 5% bovine serum albumin (BSA) and incubated in VT or VT + 250 mM glycerol solution ( L. reuteri). ) And the culture supernatant. The monoclonal antibody (mAb) against VT was then used as primary antibody, to which horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG was added, its absorbance (OD) being O-phenylene diamine After color development, the cells were read at 490 nm by ELISA reader. The experiment demonstrated the presence of a substance that inhibits the interaction between L. reuteri and Gb ₃ receptor.

From the results of the interaction between VT and 250 mM glycerol solution and the interaction between L. reuteri culture supernatants cultured in glycerol solution after coating with VT and Gb ₃ , VT and Lactobacillus luteri ( L. reuteri ) The combination of culture supernatants showed significantly lower OD values compared to VT alone as shown in FIG. 1. This means that the presence of a substance that inhibits the binding of the VT and Gb ₃ receptors in L. reuteri culture supernatant can be indirectly detected.

96-well plates were incubated in VT / NT + 250 mM glycerol solution, coated with L. reuteri culture supernatant blocked with 3% BSA and reacted with VT. The monoclonal antibody (mAb) against VT was then used as the primary antibody, to which horseradish peroxidase (HRP) -conjugated goat anti-mouse IgG (H + L) was added and its absorbance (OD) was After color development of O-phenylene diamine, it was read at 490 nm through an ELISA reader. The experiment demonstrated the presence of a substance that interacts with VT in L. reuteri supernatant.

Example 2 Investigation of the Neutralizing Effect of Lactic Acid Bacteria on Vero Toxin (VT) I and II Secreted by E. coli 0157: H7

When TSB, MRS liquid medium, and glycerol solution were added to Vero cells, no cytopathic effect was observed. In addition, when both VT and MRS liquid medium / glycerol solution were added to Vero cells, CPE was observed in Vero cells, demonstrating that the culture itself lacked neutralizing activity against VT.

When each cell culture supernatant of the test lactic acid bacteria was adjusted to pH 7.0, filtered and bound with VT, the results shown in Table 1 were observed. L. bulgaricus and L. casei showed CPE over the entire concentration range, while L. reuteri showed a much higher ratio of the tested lactic acid bacteria to VT of 4: 1. Although low CPE was seen, most of the glycerol supernatants did not show CPE. Thus, there was a recognizable neutralizing capacity for the VT of culture supernatants cultured in 250 mM glycerol solution. Incubation in MRS liquid medium (+20 mM glucose) showed CPE at all concentrations.

^* Culture supernatant obtained after incubating L. reuteri in 250 mM glycerol solution

^** Culture supernatant obtained after incubating L. reuteri in MRS liquid medium (+ 20 mM glucose)

^- CPE (cell deformation effects) No

⁺ CPE

^a slight CPE

The results of binding assays between competitive ELISA and VT and L. reuteri culture supernatants were performed by student validation using the Microcal Origin 6.1 (Microcal Software, Inc., Boston, Mass.) Program.

Example 3 Lactobacillus Luteri L. reuteri Subject administration

In this example, the subject is given two tablets of chewing tablet twice daily containing 1 × 10 ⁸ CFU (colony-forming units) of L. reuteri (SD2112: ATCC 55730) to give 4 × 10 O. ^A total daily dose of ⁸ CFU was provided with L. reuteri . All other excipients used in the tablets are known and meet international pharmacopoeia. The study consisted of two parts: an endoscopy examining the upper gastrointestinal tract and an ileoscopy examining the distal small intestine (see below). Exclusion criteria were as follows: taking antibiotics two weeks before and during the study; Taking probiotics three weeks before and during the study, followed by treatment with gastrointestinal related drugs; Serious organ disease (eg cancer) that requires regular treatment. The protocol for patient care was approved by the Danish Ethical Committee and complied with the Declaration of Helsinki. This study was conducted in Denmark.

Wilcoxon signed-rank test using (Wilcoxon signed-rank test) symptoms, blood levels, Lactobacillus histological differences before and after ingestion of fecal content ruteri, ruteri Lactobacillus (L. reuteri) of (L. reuteri) Compared. P <0.05 was considered significant.

All subjects completed the study. On gastroscopy, 10 healthy volunteers with a dietary habit of 18 years or older were examined. A gastroduodenal endoscopy was performed after fasting overnight on day 0 prior to ingestion of L. reuteri and on day 28 at the end of the study. For culture and histological examination of L. reuteri , biopsies were taken from the torso, the antrum of stomach and the third part of the duodenum. The average size of the biopsy was 33 mg. Biopsies were immediately placed in PBS (2: 1 Vol / wt sample), stored on ice and shipped to Biogaia AB Laboratories (Lund, Sweden) for analysis of L. reuteri total number. The time from tissue separation to analysis was within 36 hours.

In ileoscopy, nine ileal subjects who underwent colectomy due to ulcerative colitis (3 subjects) or Crohn's disease (6 subjects) were examined. In all subjects, the small intestine showed no signs of inflammation. The ileoscopy was performed on days 0 and 28. Biopsy samples were collected within distal 20㎝ of the small intestine, to the culture and histologic examination of Lactobacillus ruteri (L. reuteri) as other Lactobacillus bacteria (Lactobacillus), as described above.

Example 4: Microbial Analysis

Fecal samples were collected from each subject prior to ingestion of L. reuteri (day 0) and at the end of the study (day 28). In ileoscopy subjects (see below), feces were collected from the stoma. On day 42 (14 days after discontinuation of L. reuteri ingestion), fecal samples were collected from seven volunteers who had undergone gastroscopy and four volunteers who underwent ileoscopy. These samples (5 g or less) were collected in sterile containers and placed immediately in the refrigerator. Within 24 hours, 20 ml (1: 5 wt / vol) of 0.1% peptone water was added. Samples were homogenized and portions were distributed into cold-vials and frozen immediately at -70 ° C. Samples were frozen in dry ice and transferred to Biogaia AB laboratory for analysis of Lactobacillus and L. reuteri . Here, samples are thawed and diluted, in MRS-3 agar medium containing vancomycin (50 mg / l) for L. reuteri and LBS agar medium for total Lactobacillus total. Plates (KEBOLAB; AB, Lund, Sweden) were plated. MRS-3 is a modified MRS agar medium (KEBOLAB AB, Lund, Sweden) containing 2% sodium acetate (wt / vol). LBS agar medium was prepared by adding 1.32 mL cold acetic acid / L as recommended by the manufacturer. Agar plate was anaerobicly cultured for 48 hours at 37 ℃ using a BBL gas pack in an anaerobic bottle. DNA from isolates selected from this study was analyzed by PCR using Bacterial Barcodes repPROTM DNA Fingerprinting Kit (Bacterial BarCodes, Inc., Houston, TX), and DNA fingerprints were analyzed by Bionumerics software (Applied Maths BVBA, Sint-Martens-Latem, Belgium).

L. reuteri was not detected in the excretion of patients undergoing endoscopy before the start of the study and prior to administration of the study product (Table 2). Since Lactobacillus ruteri (L. reuteri) 28 days of intake, Lactobacillus ruteri (L. reuteri) in the feces of these subjects is 1.0 X 10 ² to 3.5 x 10 ⁵ CFU / g (colony-forming units / g feces) range It was present in an amount of, with an average of 4.0 × 10 ⁴ CFU / g, suggesting a significant increase in L. reuteri viable in excretion due to administration of the study product. Five subjects were selected to deliver fecal samples on days 42 and 56 (ie up to 28 days after administration of study product). In the feces of these subjects, L. reuteri persisted 4 weeks after ingestion (Table 2).

nd = not detected (<1.0 × 10 ² CFU / feces g); * = No sample provided by subject.

Excretion samples were collected on day 0 before supplementation with L. reuteri for 28 days.

Results are expressed in colony forming units (CFU) / g of wet weight excretion.

Example 5. Histological Evaluation

To assess any local immune response, quantitative changes in B-lymphocytes, T-lymphocytes, and macrophages were measured. Reference biopsies and D-28 biopsies were formalin-fixed and embedded in paraffin. 4 μm sections were then cut and stained histologically or immunohistochemically using standard techniques (Hematoxylin Eosin, Van Gieson, Periodic Acid Sciff-Alcain, Periodic Acid Sciff-diastase). A primary antibody obtained from DAKO (Glostrup, Denmark) are were as follows: CD20 (B- lymphocytes), CD3, CD4 +, CD8 (T- lymphocytes), CD68 (histiocytes), Helicobacter pylori (Helicobacter), Ki-67 (proliferation marker ). Immunohistochemical staining was performed on a DAKO TechMate ^™ 500 immunostainer to achieve uniform staining.

One pathologist evaluated the biopsy. Based on histochemical staining, tissue damage was graded according to Madsen et al. (Gastroenterol. 1999; 116: 1107-1114). These ratings represent the four criteria: ulcer formation, epithelial hyperplasia, the amount of monocyte cells, and the sum of the levels of neutrophil granulocytes in the mucosa. Based on immunohistochemical staining, the total number of CD20, CD3, CD4 +, CD8, CD68 positive cells in the mucosal lamina propria was assessed semiquantitatively. Three months later, the baseline biopsy and the D-28 biopsy were reevaluated blindly by the same pathology. The overall correlation between baseline biopsies and positive stained cells of D-28 biopsy was calculated and compared.

Significant single B-lymphocyte cells were observed in the stomach (body and eastern) of two subjects on day 0 and two subjects on day 28. One subject had cells dispersed in the stomach (east) on day 28. On day 0 four subjects had single cells in the duodenum and on day 28 eight subjects had significant single cells. On day 0, eight subjects had significant cells of CD3, CD4 +, and CD8 (T-lymphocytes) in the stomach (body and eastern), and on day 28, nine subjects showed significant single cells in the ventricles (body and eastern). Retained (Table 3). Scattered cells were observed in the duodenum of 8 subjects on day 0, and significantly dispersed T-lymphocytes were observed in the duodenum of 7 subjects on day 28.

Significant single histocytic cells were observed in the stomach (body and eastern) of 9 subjects on Day 0 and 5 subjects on Day 28. Significantly dispersed cells were observed in the duodenum of 6 subjects on day 0 and 6 (not identical) on day 28.

After the subject ingested L. reuteri , there was a statistically significant decrease in the total number of histocytes (CD68) in the biopsies obtained from the gastric body (p = 0.025) and the eastern (p = 0.046) and B in the duodenum. A significant increase (p = 0.046) was observed in the total number of cells (CD20) (Table 3). No significant changes in T-lymphocyte amounts due to L. reuteri ingestion were observed. The agreement between these two findings (first non-blind and second blind analysis of histological samples) was 76% (Table 3). In the upper torso and eastern histiocytes (CD68) cells, the concordance rates were 40% and 65%, respectively. The concordance rate of duodenal B-cells (CD20) was 60%. Therefore, there was some uncertainty in these findings. The agreement rate was much higher (up to 90%; Table 3) in the CD3, CD4 + and CD8 assays.

In President endoscopy, all biopsies were normal histologically H. pylori (Helicobacter) were negative. Ki-positive cells were normal in all biopsies. On day 0, significant single B-lymphocytes were observed in six subjects of cells and in two subjects of dispersed cells. On day 28 single cells were observed in all subjects. Significantly dispersed CD3 cells (B-lymphocytes) were observed in 7 subjects on both 0 and 28 days. Significantly dispersed CD4 + cells were observed in 7 subjects on day 0, and significantly adjacent cell populations were observed in 7 subjects on day 28. Significantly dispersed CD8 cells were observed in seven subjects on day 0 and in nine subjects on day 28 (Table 3). Significantly dispersed histiocyte cells were observed in 7 subjects on both 0 and 28 days.

After supplementation with L. reuteri for 28 days, the amount of CD4 + lymphocytes increased significantly (p = 0.046; Table 3). No significant change was observed in the amount of B-lymphocytes or histocytes due to L. reuteri administration. The agreement between the two findings was high overall for all cell types tested, and the matching rate for the CD4 + findings was 78%, suggesting significant reliability in these data (Table 3). All H. pylori (Helicobacter) staining was negative.

Biopsies were taken within 20 cm of the small intestine, fixed and cut into slices for histological examination using immunohistochemical staining of specific cell types.

“_” = No cells detected; “X” = single cell; “XX” = dispersed cell or cell population; “XXX” = contiguous multiple cell populations.

Histological score (1-10) to grade the extent of tissue damage.

Statistical analysis: Wilcoxon sign-rank test. Samples given nominal scores, X, XX and XXX, were set to 1, 2, 3 and 4, respectively, for statistical analysis.

Example 6 Biochemistry of Blood

Blood samples are taken on days 0 and 28 and hemoglobin, hematocrit, platelets, leukocytes, C-reactive protein, potassium, sodium, creatinine, b-urea, p-glucose, cholesterol, HDL (high density lipoprotein), LDL (Low density lipoprotein), VLDL (ultra low density lipoprotein), triglycerides, total bilirubin, urate, ALAT, alkaline phosphatase, lactate.

Most blood tests were normal before and after ingestion of L. reuteri . Although there were some singularities in blood parameters, no clinically significant abnormal and systemic changes were observed after treatment.

Example 7. DNA Fingerprinting

DNA fingerprinting assays were performed on L. reuteri isolates selected from this study. Thus, excreta were taken from three subjects who had ingested L. reuteri for 28 days, and one isolate from the duodenal biopsy and one from ileum biopsies on day 0 prior to L. reuteri administration. Isolates were harvested. All isolates were found to have 98% genetic similarity to each other. These isolates showed 97% similarity to SD2112, the strain incorporated into the purification.

Example 8 Preparation of Products for Improving Immune-Function in Humans

In this example, L. reuteri SD2112, ATCC 55730, which can be added to standard yoghurt, is selected using the method described above for toxin neutralization and CD4 + cell recruitment. The L. reuteri strain is grown and lyophilized using standard methods of growing Lactobacillus in the dairy industry. The culture is then added to the fermented milk using traditional yogurt culture at a level of 10E + 7 CFU / yogurt g, which is used as one method to improve human immune function.

Although the present invention has been described with reference to specific embodiments, many modifications are possible and all such modifications are considered to be within the spirit and scope of the invention.

Figure 1. Confirmation of inhibitory activity on the binding of vero toxin (VT) and Gb ₃ receptor in L. reuteri culture supernatant by competition ELISA. Each was reacted as follows on a plate coated with Gb ₃ and then ELISA was performed using the mAb for VT.

Control: VT + Casein Soy Digestion Liquid Medium (TSB).

VT + G: VT + 250 mM glycerol solution.

VT + LRS: L. reuteri culture supernatants incubated in VT + 250 mM glycerol solution.

Claims (7)

In a method of selecting a strain of lactic acid bacteria that enhances immune-function in a mammal, a) selecting Lactobacillus reuteri strain and culturing; b) obtaining culture supernatants of these strains; c) determining a decrease in cytotoxic activity in Vero-cells; d) i. Toxin binding and neutralizing effect; And ii. Selecting a Lactobacillus strain that exhibits all CD4 + cell recruitment measured by standard assays with antibodies on the appropriate cells. A composition containing cells of a strain of lactic acid bacteria selected by the method of claim 1, which is used in the manufacture of a medicament for improving immune-function in a mammal. A product containing the cells of the lactic acid bacteria strain obtained by the method of claim 1. 4. The product of claim 3, wherein the product is made into a tablet containing cells of the selected strain. 4. The product of claim 3, wherein the product is prepared from a dietary adjuvant containing cells of the selected strain. 4. The product of claim 3, wherein the product is made of a confectionary containing cells of the selected strain. The product according to claim 3, which is prepared as an oral medicament for improving the immune-function of a patient.

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