Probiotic supplement for metabolic health comprising lactobacillus genus

Abstract

Disclosed herein are strains of Lactobacillus genus that have beneficial effects on metabolic health in mammalian subjects. Further disclosed are compositions comprising such strains and their use for improving metabolic health or for reducing blood glucose levels in a mammalian subject.

Description

[0001] This application is a divisional application of the patent application with application number 201980070200.5 and invention name “Probiotic supplement for metabolic health containing Lactobacillus”.

[0002] References to deposited biological material

[0003] This application contains references to a deposit of biological material, which is incorporated herein by reference. For complete information, see the last paragraph of the specification. Technical Field

[0004] The present invention relates to bacterial strains, cellular components, metabolites and secreted molecules thereof having the ability to improve the metabolic health of mammalian subjects. The present application also relates to compositions comprising such bacterial strains, and the use of such compositions for improving the metabolic health of mammalian subjects. Background Art

[0005] In recent years, it has been shown repeatedly that bacteria have an important and significant impact on the health of mammals, especially humans, and it has been recognized that beneficial microbiota in the gastrointestinal tract are important for proper nutrition and health.

[0006] Ingestion of microbial cultures or strains (also known as probiotics) is now recognized as a way of using microbial cultures or strains to improve the health of an individual. In this regard, throughout the specification and claims, the terms probiotics, microbial cultures and microbial strains are understood in a generally accepted manner as "live microorganisms that confer a health benefit on the host when administered in adequate amounts" (http: / / www.nature.com / articles / nrgastro.2014.66?foxtrotcallback=true).

[0007] Microbial strains can be used both to improve an individual's poor health and to prevent the onset of poor health.

[0008] The beneficial properties of probiotics depend on the properties of the microbial strains that form the probiotics, and therefore it is important to provide new microbial strains with beneficial properties so that these strains can be used as probiotics. Summary of the invention

[0009] In a first aspect, the present invention relates to a Lactobacillus strain having the ability to improve the metabolic health of a mammalian subject, such as to alleviate prediabetes. In another or alternative aspect, the Lactobacillus strain of the present invention has the ability to reduce the blood sugar level of a mammalian subject. The Lactobacillus strain of the present invention can, for example, be a strain deposited in the DSMZ-"German Collection of Microorganisms and Cell Cultures" with the following accession numbers: DSM 17648, DSM 32851, DSM 32853, DSM 32910 and / or DSM 32911.

[0010] In a second aspect, the present invention relates to a composition comprising one or more Lactobacillus strains of the present invention, preferably formulated in a discrete dose formulation.

[0011] In a third aspect, the present invention relates to the use of one or more Lactobacillus strains of the present invention or a composition comprising one or more Lactobacillus strains of the present invention to improve the metabolic health of a mammalian subject, such as reducing prediabetes, maintaining normal blood sugar levels in an individual and / or reducing postprandial blood sugar response, preventing or reducing insulin resistance, or preventing or reducing weight gain in an individual on a high fat and / or high sugar diet.

[0012] Finally, in a fourth aspect, the present invention relates to a method for screening Lactobacillus strains having the ability to improve metabolic health, such as to alleviate pre-diabetes, in a mammalian subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shown is a bar graph representing the ratio of the anti-inflammatory cytokine interleukin 10 (IL10) to the pro-inflammatory cytokine interleukin 12 (IL12) measured by Luminex or ELISA in the supernatant of peripheral blood mononuclear cells (PBMC) incubated with Lactobacillus strains for 24h. Each column represents the mean of 3 experiments performed using healthy PBMC donors. For more details, see Example 2.

[0014] Figure 2 Shown is a bar graph representing the pro-inflammatory cytokine monocyte chemoattractant protein 1 (MCP-1) measured by Luminex in the supernatant of PBMCs stimulated with a pro-inflammatory stimulus (lipopolysaccharide) and incubated with Lactobacillus strains for 24 h. Each column represents the mean of 3 experiments performed using healthy PBMC donors. For more details, see Example 2.

[0015] Figure 3Shown is a bar graph representing pro-inflammatory osteopontin measured by Luminex in the supernatant of PBMCs stimulated with a pro-inflammatory stimulus (lipopolysaccharide) and incubated with Lactobacillus strains for 24 h. Each column represents the mean of 3 experiments performed using healthy PBMC donors. For more details, see Example 2.

[0016] Figure 4 Bar graphs showing cytokines secreted by dendritic cells (DCs) in a pro-inflammatory environment are shown. Each column represents the mean of n=1-3. A: MCP-1; B: osteopontin; C: IL12p70. Supernatants were harvested by centrifugation and used for cytokine analysis using multiplex assays. See Example 3 for more details.

[0017] Figure 5 Shown are supernatants of DC cultures incubated with Lactobacillus and a low-grade inflammatory cocktail consisting of TNFα+IFNγ+LPS (mixture) by BD TM Bar graph of the ratio of the anti-inflammatory cytokine interleukin 10 (IL10) to the pro-inflammatory cytokine interleukin 12p70 (IL12p70) measured by cytometric bead array (CBA). The data were normalized according to the mixture control from each donor. DCs were cultured with Lactobacillus for 4 hours, then the mixture was added and maintained for another 16 hours. The mixture control was DCs cultured with the mixture for 16 hours. Each column represents the ratio of 12 experiments performed with monocyte-derived DCs from individual healthy human PBMC donors.

[0018] Figure 6 Shown are supernatants of DC cultures incubated with Lactobacillus and a low-grade inflammatory cocktail consisting of TNFα+IFNγ+LPS (mixture) by BD TM Bar graph of the average concentration of monocyte chemoattractant protein-1 (MCP-1 / CCL2) measured by cytometric bead array (CBA). DCs were cultured with Lactobacillus for 4 hours, then the mixture was added and maintained for another 16 hours. The mixture control was DCs cultured with the mixture for 16 hours. Each column represents the mean of 12 experiments performed with monocyte-derived DCs from healthy human PBMC donors.

[0019] Figure 7Shown is a bar graph representing the average concentration of the cytokine interleukin 6 (IL6) measured by BD Cytometric Bead Array (CBA) in the supernatant of DC cultures incubated with Lactobacillus and a low-grade inflammatory mixture consisting of TNFα+IFNγ+LPS (mixture). DCs were cultured with Lactobacillus for 4 hours, then the mixture was added and maintained for another 16 hours. The mixture control was DC cultured with the mixture for 16 hours. Each column represents the mean of 12 experiments performed with monocyte-derived DCs from healthy human PBMC donors.

[0020] Figure 8 . The effect of Lactobacillus strains on the barrier strength of the epithelial cell line Caco2 after 24h of incubation at a multiplicity of infection (MOI) of 5 is shown. This effect was measured using the transepithelial electrical resistance (TEER) technique and is expressed as a TEER ratio. The TEER ratio is measured as A in the absence of pro-inflammatory stimuli and as B in the presence of pro-inflammatory stimuli. For more details, see Example 5.

[0021] Fig. 9 The effect of lactobacilli on the barrier strength of the epithelial cell line Caco2 is shown after 24 h of incubation in the presence and absence of Lactobacillus strains at an MOI of 5 and a pro-inflammatory cocktail consisting of TNF-alpha (TNFα) and INF-gamma (INFγ). A) TEER ratio of Lactobacillus HH02. Mean values ​​of 2-3 animals per group. B) TEER ratio of Lactobacillus HH04. Mean values ​​of 2-3 animals per group. C) TEER ratio of Lactobacillus HH05. Mean values ​​of 2-3 animals per group.

[0022] Fig.10 Shown are the transepithelial electrical resistance (TEER) of freeze-dried Lactobacillus strains HH02 and HH04, which were incubated with epithelial cells Caco2 at a multiplicity of infection (MOI) of 500 for 24 h and stored at -20°C, 4°C, 25°C, and 40°C for one month. A) TEER ratio of Lactobacillus strain HH02. Mean of 3-4 animals per group. B) TEER ratio of Lactobacillus strain HH04. Mean of 3-4 animals per group.

[0023] Fig.11Shown are the effects of Lactobacillus strains HH02 and HH04 at an MOI of 5 on the transepithelial electrical resistance (TEER) of the epithelial cell line Caco2 in an experimental setting simulating low sugar (solid dots) or high sugar uptake (7 mM fructose, solid inverted triangles) in the intestine, or simulating systemic high glucose (7 mM glucose, solid triangles) on the basolateral side, or a combination of both (solid squares). The medium control is depicted as an open circle. As depicted are continuous TEER values ​​over an incubation time of 24 h. A) TEER values ​​of Lactobacillus strain HH02. Mean values ​​of 1-3 per group. B) TEER values ​​of Lactobacillus strain HH04. Mean values ​​of 1-3 per group.

[0024] Fig.12 A bar graph representing the bile salt concentration consumed by lactic acid bacteria (LAB) in simulated bile salt juice is disclosed. Each column represents one experiment (n=1). For more details, see Example 7.

[0025] Fig.13 Shown is a bar graph representing the average concentration of glucagon-like peptide 1 (GLP-1) released by L cells (NCI-H716) incubated with cell-free growth broth of Lactobacillus strains cultured in YDA medium. Each test sample was diluted 10 times and 50 times and added to NCI-H716 cells for 2 hours. Each column represents the mean of 5 individual experiments. For more details, see Example 8.

[0026] Fig.14 Shown is a bar graph showing NCI-H716 viability (%) after 2 hours of incubation with cell-free culture growth broth (1:10 and 1:50 dilutions), fresh culture broth (1:10 and 1:50 dilutions) and 10 μM PMA positive control. Presto Blue solution was added to the cells and incubated for 25 minutes, and then Presto Blue fluorescence was measured at 567 nm (excitation) / 587 nm (emission). Each column represents the mean of 5 individual experiments.

[0027] Fig.15 .A line graph is shown representing the mean body weight (% of body weight on day -6) of each group of animals in a mouse acute inflammation (colitis) model. Each line corresponds to each treatment during the experiment (n=11-15). Lactobacillus strain treatment started on day -5. For more details, see Example 9.

[0028] Fig.16Bar graphs representing the Wallace score (raw colon injury score) for each treatment group in the mouse acute inflammation (colitis) model are shown. Each column represents the mean of n = 11-15. The inflammation of the colon was assessed, and the colon was macroscopically graded using an optical microscope to evaluate the visual appearance of the colon. This was performed by 2 blind observers based on the Wallace scoring method. The criteria for colon injury scoring are: 0-no injury; 1-congestion. No ulcers; 2-congestion and intestinal wall thickening. No ulcers; 3-one ulcer, no intestinal wall thickening; 4.-two or more than two ulcers or inflammation sites; 5.-two or more than two large sites of ulcers and inflammation, or one site of ulcer / inflammation extends >1cm along the length of the colon, and 6-10-if the injury covers >2cm along the length of the colon, the score increases by 1 for each additional centimeter. For more details, see Example 9.

[0029] Fig.17 Shown is a bar graph representing the mean colon wet weight (g) / length (cm) for each treatment group in a mouse acute inflammation (colitis) model. Each column represents the mean of n=11-15. See Example 9 for more details.

[0030] Fig.18 Shown is a bar graph representing cytokines in colon tissue in a mouse acute inflammation (colitis) model. Each column represents the mean of n=11-15. A: TNFα; B: IL1b; C: IL6; D: IL17A, E: IL25, F: IL12 and G: IL10. See Example 9 for more details.

[0031] Fig.19 PCA plots visualizing the effects of treatment on microbial composition (species level) are shown.

[0032] Fig. 20 The percentage differences associated with treatments are shown. The analysis was performed at the genus level.

[0033] Fig.21 A study overview of the mouse study described in Example 10 is shown.

[0034] Fig. 22The effects of HH04 and HH05 on weight development and body composition in a diet-induced obese mouse (DIO) model are shown. Means of 10-12 mice per group are shown: A) Weight after random assignment. B) Weight development of the experimental group after starting the experimental diet and tube feeding treatment. C) Fat mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points. D) As in C, but depicted as group means with connecting lines. E) Lean mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points. F) Body fluid mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points. More details can be found in Example 10.

[0035] Fig.23 The effects of Lactobacillus strains on glucose regulation and intestinal permeability are shown. A) 5h fasting blood glucose after 10 weeks of HFD feeding and daily gavage treatment. The bar graph represents the mean of 10-12 mice in each group. B) 5h fasting insulin after 10 weeks of HFD feeding and daily gavage treatment. The bar graph represents the mean of 10-12 mice in each group. C, E) Oral glucose tolerance test in 5h fasting mice after 10 weeks of HFD feeding and daily gavage treatment. C depicts blood glucose levels during stimulation (2μg / g lean mass), and E depicts plasma levels of endogenous insulin response to glucose stimulation. The mean of 10-12 mice in each group. D) Area under the curve of OGTT week 10. The bar graph represents the mean of 10-12 mice in each group. F) Area under the curve of GSIS week 10. The bar graph represents the mean of 10-12 mice in each group. G) Plasma sulfonic acid fluorescence units after oral challenge (150 μl / mouse, concentration: 10 mg / mL) in 5h fasted mice after 10 weeks of HFD feeding and daily gavage treatment. Mean values ​​of 10-12 mice per group. H) Cumulative feed intake per mouse during 12 weeks of HFD feeding and daily gavage treatment. Due to co-housing, the intake is the mean per mouse per cage, with n=4 in each group. The bar graph represents the mean. AG) ​​All mice were gavaged with the corresponding treatment 3 hours before oral challenge. More details about this study can be found in Example 10.

[0036] Fig.24 The effect of Lactobacillus strains on tissue weight or length in a diet-induced obesity mouse model is shown. The bar graph represents the mean of 10-12 mice per group: A) Liver weight at autopsy after 12 weeks of daily gavage. B) Colon length at autopsy after 12 weeks of daily gavage. The bar graph shows the mean of 10-12 mice per group. C) Weight of inguinal white adipose tissue (iWAT) at autopsy after 12 weeks of daily gavage. D) Weight of epididymal white adipose tissue (eWAT) at autopsy after 12 weeks of daily gavage. More details about this study can be found in Example 10.

[0037] Fig.25 The effect of Lactobacillus strains on tissue weights in a diet-induced obesity mouse model is shown. The bar graph represents the mean of 10-12 mice per group at autopsy after 12 weeks of daily gavage. A) Weight of retroperitoneal white adipose tissue (rpWAT). B) Weight of mesenchymal white adipose tissue (mWAT). C) Weight of brain. D) Weight of heart. More details about this study can be found in Example 10.

[0038] Fig.26 Biochemical analysis of the effects of Lactobacillus strains on plasma markers in a diet-induced obesity mouse model is shown. A) Alanine aminotransferase. B) Aspartate aminotransferase. C.1) Triglycerides, including all animals. C.2) Triglycerides, where two animals were excluded after evaluation exceeded the detection limit of a specific assay. D) Total cholesterol. Each bar graph represents the mean of 10-12 mice per group. More details about this study can be found in Example 10.

[0039] Fig. 27 Results are shown for the effect of Lactobacillus strains on plasma markers in a diet-induced obesity mouse model. A) Serum amyloid A. B) Adiponectin. C) Non-esterified fatty acids. D) Lipopolysaccharide. AE) Each bar graph represents the mean of 10-12 mice per group. More details on this study can be found in Example 10.

[0040] Fig.28 Results are shown for the effect of Lactobacillus strains on liver lipid accumulation in a diet-induced obesity mouse model. A) Triglyceride content in liver tissue (mg / liver), and B) Cholesterol in liver tissue (mg / liver). Each bar graph represents the mean of 10-12 mice per group. More details about this study can be found in Example 10.

[0041] Fig.29 Results of the effects of Lactobacillus strains on colonic cytokines in a diet-induced obesity mouse model are shown. A) Cytokine interferon gamma (IFNγ) levels in colonic tissue, and B) Cytokine interleukin-10 (IL-10) levels in colonic tissue. Each bar graph represents the mean of 10-12 mice per group. More details about this study can be found in Example 10.

[0042] Fig.30Results of the effects of Lactobacillus strains on intestinal microbiota composition in a diet-induced obesity mouse model are shown. A) DNA from HH04 Lactobacillus strains found in fecal samples. B) DNA from HH04 probiotic strains found in small intestinal contents. C) DNA from HH05 Lactobacillus strains found in fecal samples. D) DNA from HH05 Lactobacillus strains found in small intestinal contents. E) Principal coordinate analysis (PCoA) was performed using the weighted unifrac distances from the end of the study to cluster fecal microbiota composition in the specified group. Larger points indicate group means of 10-12 mice per group. F) Principal coordinate analysis (PCoA) was performed using the weighted unifrac distances from the end of the study to cluster small intestinal microbiota composition in the specified group. Larger points indicate group means of 10-12 mice per group. More details about this research can be found in Example 10.

[0043] Fig.31 The effects of HH04, HH05 and HH02 on weight development and body composition in the diet-induced obesity mouse (DIO) model are shown. Means of 10-12 mice per group are shown: A) Weight after random assignment. B) Weight development of individual groups after starting the experimental diet and tube feeding treatment. C) Fat mass obtained by magnetic resonance scan at the depicted time points (% of total body weight). D) Fat mass development (grams), which is depicted as group means with connecting lines. E) Lean mass (grams) obtained by magnetic resonance scan at the depicted time points. F) Body fluid mass (% of body weight) obtained by magnetic resonance scan at the depicted time points. More details can be found in Example 11.

[0044] Fig.32 Shown are the effects of Lactobacillus strains HH04, HH05 and HH02 on glucose regulation. A) 5h fasting blood glucose after 10 weeks of modified WD feeding and daily gavage. The bar graph represents the mean of 10-12 mice in each group. B) 5h fasting insulin levels after 10 weeks of modified WD feeding and daily gavage. C) Oral glucose tolerance test of 5h fasting mice during stimulation (1.5μg dextrose / g lean mass) after 10 weeks of modified WD feeding and daily gavage. D) As C, but showing the change of blood glucose value relative to fasting value during stimulation. E) Plasma levels of endogenous insulin response to glucose stimulation. F) C-peptide levels in plasma during stimulation. AF) Data represent the mean of 10-12 mice in each group. More details about this study can be found in Example 11.

[0045] Fig.33The effect of Lactobacillus strains HH04, HH05 and HH02 on tissue weight in a diet-induced obesity mouse model is shown. The bar graph represents the mean of 10-12 mice in each group: A) liver weight at autopsy after 12 weeks of daily gavage. B) weight of inguinal white adipose tissue (iWAT) at autopsy after 12 weeks of daily gavage. C) weight of retroperitoneal white adipose tissue (rpWAT) at autopsy after 12 weeks of daily gavage. D) weight of mesenchymal white adipose tissue (mWAT) at autopsy after 12 weeks of daily gavage. E) weight of intrascapular brown adipose tissue (BAT) at autopsy after 12 weeks of daily gavage. F) weight of gastrocnemius muscle at autopsy after 12 weeks of daily gavage. More details about this study can be found in Example 11.

[0046] Fig.34 Results are shown for the effect of Lactobacillus strains on plasma markers in a diet-induced obesity mouse model. A) Alanine aminotransferase. B) Aspartate aminotransferase. C) Triglycerides. D) Total cholesterol. AD) Each bar graph represents the mean of 10-12 mice per group. More details on this study can be found in Example 11.

[0047] Fig.35 Results of the effects of Lactobacillus strains on hepatic lipid accumulation in a diet-induced obesity mouse model are shown. A) Triglyceride content in liver tissue (μg / mg liver), B) Cholesterol in liver tissue (μg / mg liver). C) Ratio of phosphorylated (activated) Akt protein levels to total Akt protein levels in liver tissue of insulin-stimulated mice quantified by Western blot. AB) Each bar graph represents the mean of 10-12 mice per group. C) Each bar graph represents the mean of 3-4 mice per group. More details about this study can be found in Example 11.

[0048] Fig.36 Results are shown for the effect of Lactobacillus strains on inflammation of liver and colon tissues in a diet-induced obesity mouse model. A) Tumor necrosis factor alpha (TNFα) levels in liver tissue, and B) cytokine interferon gamma (IFNγ) levels in liver tissue. C) Tumor necrosis factor alpha (TNFα) levels in colon tissue. D) Cytokine interferon gamma (IFNγ) levels in colon tissue. AD) Each bar graph represents the mean of 10-12 mice per group. More details about this study can be found in Example 11.

[0049] Fig.37Results are shown for the effect of Lactobacillus strains on intestinal microbiota composition in a diet-induced obesity mouse model. Principal coordinate analysis (PCoA) was performed using weighted unifrac distances to cluster fecal microbiota composition from the end of the study in a given group. Larger points indicate group means for 10-12 mice per group. More details on this study can be found in Example 11.

[0050] Fig.38 The effects of HH04, HH05 and HH02 on weight development and body composition in two diet-induced obesity mouse (DIO) models are shown. Mean values ​​of 8-9 mice per group are shown: A) Weight of mice fed LFD or HFD after random assignment. B) Weight of mice fed LFD or modified WD after random assignment. C) Weight development of individual groups fed LFD or HFD after starting experimental diet and tube feeding treatment. D) As C, but in groups fed LFD or modified WD. E) Fat mass (% of total body weight) obtained by magnetic resonance scanning at the depicted time point for groups fed LFD or HFD. F) As E, but in groups fed LFD or modified WD. G) Fat mass development (grams) of groups fed LFD or HFD, which is depicted as group means with connecting lines. H) As G, but in groups fed LFD or modified WD. I) Lean mass (grams) obtained by magnetic resonance scanning at the depicted time point for groups fed LFD or HFD. J) As I, but in groups fed LFD or modified WD. AJ) More details can be found in Example 12.

[0051] Fig.39 The effects of Lactobacillus strains HH04, HH05 and HH02 on glucose regulation are shown. A) 5h fasting blood glucose after 10 weeks of feeding with modified LFD or HFD and daily gavage. B) As A, but in groups fed with LFD or modified WD. C) 5h fasting insulin levels after 10 weeks of feeding with LFD or HFD and daily gavage. D) As C, but fed with LFD or modified WD. E) Changes in blood glucose values ​​during oral glucose tolerance test relative to fasting values ​​after daily gavage and 10 weeks of feeding with LFD or HFD. F) As E, but depicting the LFD or modified WD feeding group. G) Plasma levels of endogenous insulin response to glucose stimulation in groups fed with LFD or HFD. H) As G, but depicting the groups fed with LFD or modified WD. I) C-peptide levels in plasma during stimulation in groups fed with LFD or HFD. J) As I, but in groups fed with LFD or modified WD. AJ) Bar graphs represent the mean values ​​of 8-9 mice per group. More details about this study can be found in Example 12. DETAILED DESCRIPTION

[0052] Lactobacillus strains

[0053] The present invention relates to Lactobacillus strains, cellular components, metabolites and / or any secreted molecules thereof having the ability to improve metabolic health in a mammalian subject, such as reducing pre-diabetes.

[0054] When used herein, the term "Lactobacillus strain" shall refer to one or more individual Lactobacillus strains, or a combination of a Lactobacillus strain and its cellular components, metabolites and / or any secreted molecules.

[0055] The term "prediabetes" is used for a condition in a subject where not all symptoms required for a diagnosis of diabetes are present, but, for example, blood glucose is abnormally high. As used herein, prediabetes includes conditions that cause symptoms such as, for example, insulin resistance, hyperglycemia, low-grade inflammation, and / or impaired intestinal barrier function, i.e., where the function of the intestinal barrier function is reduced.

[0056] In one aspect, the Lactobacillus strains of the invention survive in the gastrointestinal (GI) tract.

[0057] The term "survival in the gastrointestinal (GI) tract" is used herein to mean that the strain is active after passing through the GI tract. Survival in the intestine is determined, for example, by identifying the strain in a stool sample of a subject to whom the strain has been administered, such as by oral ingestion.

[0058] The Lactobacillus strains of the present invention may have the ability to improve one or more prediabetic symptoms such as insulin resistance, hyperglycemia, low-grade inflammation and / or intestinal barrier function. In one aspect, one or more Lactobacillus strains of the present invention or compositions comprising the same may improve a subject's postprandial glycemic response, and / or reduce a subject's insulin resistance.

[0059] The Lactobacillus strains of the invention may also or alternatively have the ability to improve one or more symptoms of type 2 diabetes such as insulin resistance, hyperglycemia, low-grade inflammation and / or intestinal barrier function. In one aspect, one or more Lactobacillus strains of the invention may improve a subject's postprandial glycemic response, and / or Mitigation Insulin resistance in a subject. Thus, in one embodiment, one or more Lactobacillus strains of the present invention or a composition comprising the same improves the postprandial glycemic response and / or reduces insulin resistance in a subject with type 2 diabetes.

[0060] The Lactobacillus strains of the present invention may also have the ability to lower blood glucose concentrations and / or reduce weight gain in individuals on a high fat and / or sugar diet, wherein one or more Lactobacillus strains of the present invention are present in the gastrointestinal tract. It should be understood that the Lactobacillus strains of the present invention have the ability to lower blood glucose levels in individuals whose blood glucose levels are higher than normal due to a particular diet. There is no doubt that the Lactobacillus strains of the present invention can lower blood glucose levels to below normal blood glucose levels. Therefore, when one or more Lactobacillus strains of the present invention are present in the gastrointestinal tract of an individual, they can reduce the weight gain of an individual who consumes a high fat diet, compared to the same individual who consumes the same diet without the one or more Lactobacillus strains of the present invention in the gastrointestinal tract.

[0061] The Lactobacillus strains of the present invention may also have the ability to improve microbial dysbiosis induced by a high fat or Western diet in the gastrointestinal tract of mammals, particularly humans. As a result, the Lactobacillus strains of the present invention, when present in the gastrointestinal microbiota, have the ability to protect the intestine from conditions and diseases that would normally cause severe disruptions in normal gastrointestinal function. For example, one or more Lactobacillus strains of the present invention may protect a subject from conditions such as colitis.

[0062] The Lactobacillus strains of the present invention can exert an anti-inflammatory effect on mammalian cells, for example, as indicated by a high IL10 / IL12 ratio in mammalian cells exposed to one or more Lactobacillus strains of the present invention in the presence of a pro-inflammatory stimulus. In one embodiment, the Lactobacillus strains of the present invention exert an anti-inflammatory effect on mammalian cells. In another embodiment, the one or more Lactobacillus strains of the present invention result in the production of a high IL10 / IL12 ratio in mammalian cells exposed to the Lactobacillus strains in the presence of a pro-inflammatory stimulus.

[0063] In one embodiment, the lactobacillus strain is selected from a lactobacillus strain, a Lactobacillus reuteri strain or a strain belonging to a Lactobacillus casei group. The strain belonging to the Lactobacillus casei group includes Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus; preferably Lactobacillus paracasei, Lactobacillus paracasei subsp. tolerans or Lactobacillus paracasei subsp paracasei.

[0064] Particularly preferred strains are selected from the group of Lactobacillus strains deposited with DSMZ - "German Collection of Microorganisms and Cell Cultures" under the following deposit numbers: DSM 17648, DSM 32851, DSM 32853, DSM 32910 or DSM 32911.

[0065] The Lactobacillus strains of the invention can be obtained in any manner known per se, such as by cultivation in a suitable culture medium, preferably in such a way and using such a culture medium that the microbial preparation thus obtained is suitable for administration to humans and / or animals and is in particular capable of colonizing the desired parts of the gastrointestinal tract.

[0066] As known in the art, the cultivation of the Lactobacillus strains of the present invention may be carried out in standard fermentation equipment suitable for fermenting Lactobacillus strains.

[0067] After cultivation, the Lactobacillus strain of the invention is recovered from the fermentation broth and converted into the composition of the invention using techniques known in the art, or alternatively the entire fermentation broth may be converted into the composition according to the invention.

[0068] filter

[0069] Lactobacillus strains having the ability to improve metabolic health in a mammalian subject, such as reducing pre-diabetes, can be selected from a library of candidate strains by one or more screening steps / methods for screening strains for desirable properties.

[0070] The one or more screening methods can be selected from screening methods that screen for properties such as immunomodulatory properties, cell adhesion, improvement in intestinal barrier function, reduction in lipid accumulation in adipocytes, minimum inhibitory concentration (MIC), and / or gastrointestinal survival (GI survival).

[0071] Preferred screening methods include, but are not limited to:

[0072] PBMC screening, wherein peripheral blood mononuclear cells are recovered from one or more donors, such as two or more than two, three or more than three, four or more than four or five or more than five donors, and incubated with a bacterial strain for a given period of time, after which the immunomodulatory characteristics of the strain are determined by analyzing the effect of this incubation on the levels of cytokine production, such as interferon gamma (IFNγ); interleukin 10 (IL10); IL12; IL13; IL17; IL1B; IL2; IL4; IL5; IL6; IL8; lipocalin; monocyte chemoattractant protein 1 (MCP-1): osteopontin and TNFα.

[0073] TEER measurement (transepithelial electrical resistance as described in B. Srinivasen et al. (2015). J Lab Autom 2015, 20: 107-126) with or without the addition of biologically active pro-inflammatory factors such as pro-inflammatory cytokines or lipopolysaccharide (LPS).

[0074] Dendritic cell (DC) assay, wherein DCs recovered from one or more donors, such as two or more than two, three or more than three, four or more than four, or five or more than five donors, are matured into inflammatory DCs by incubation with pro-inflammatory stimuli such as cytokines, LPS, TNFα, IFNγ, followed by incubation with a Lactobacillus strain for a given period of time, and measuring the cytokines. In a preferred embodiment, DCs may also be pre-incubated with a Lactobacillus strain, followed by the addition of the pro-inflammatory stimuli. Lactobacillus strains with the ability to improve metabolic health, such as to alleviate prediabetes, should have reduced IL12 levels, and the levels of anti-inflammatory cytokines (or the ratio between anti-inflammatory / pro-inflammatory cytokines) should be higher compared to strains that do not have this property.

[0075] Adipocyte assay (as described in Zhang et al. 2016, Scientific reports 6, 36083), in which 3T3-L1 cells were differentiated into mature adipocytes over 10 days, co-incubated with Lactobacillus strains at specific times for given periods of time, and lipid formation was measured using Oil Red O staining of intracellular accumulated lipids and gene expression of adipogenesis-related genes and pro-inflammatory marker genes by qPCR.

[0076] L cell assay, in which human L cell line NCI-H716 in growth medium containing 10% fetal bovine serum and 1% penicillin / streptomycin was seeded (passage 12-20 times) on pre-coated plates in 24-well plates (200.000 cells / well) in an incubator at 37°C / 5% CO2 and allowed to differentiate for 48 hours, then washed twice with Krebs Ringer Buffer containing bovine serum albumin (BSA, 0.2%) and finally incubated with diluted supernatant or medium control for 2h for glucagon-like peptide 1 (GLP-1) release assay in an incubator at 37°C / 5% CO2. Each test included phorbol 12-myristate 13-acetate (10 μM) and Triton X-100 (0.2%) as a GLP-1 positive control and for obtaining total release of GLP-1, respectively. All test samples contained 25 μM dipeptidyl peptidase-4 inhibitor. After incubation, all supernatants were removed and stored at -20°C until analyzed for GLP-1. Cell viability was analyzed. Supernatants were analyzed for GLP-1 using an active GLP-1 ELISA kit.

[0077] Bile salt hydrolase assay, in which Lactobacillus strains are incubated for 24 h at 37°C in MRS medium (De Man, Rogosa and Sharpe) in the presence of the following bile salts: glycocholate, taurocholate, glycochenodeoxycholate, taurochenodeoxycholate, glycodeoxycholate and taurodeoxycholate. The bile salts are present at the following concentrations: 1,56 mg / mL, 0,56 mg / mL, 1,51 mg / mL, 0,54 mg / mL, 1,51 mg / mL and 0,33 mg / mL, respectively. A reference containing bile salts in the absence of any bacteria is used. After incubation, the supernatant is harvested by centrifugation. The samples are extracted with acetic acid methanol and analyzed with high mass resolution on a reverse phase (RP) liquid chromatography mass spectrometry (LCMS). The method is established to quantify all six bile salts mentioned above. The conversion rate of conjugated bile salts (CBS) expressed as a percentage was calculated according to the formula CBS [%] = 100 [%] - (A * 100 [%] / B). A is the amount of bile salts after 24 hours of incubation in the presence of bacteria, and B is the amount of bile salts after 24 hours of incubation in the absence of any bacteria. The higher the CBS value, the more bile salts are converted.

[0078] MIC determination: MIC determination (as described by the EFSA panel on additives or products or substances for use in animal feed and approved for public consultation in May 2017), in which Lactobacillus and control strains were grown and distributed in the presence of different antibiotics in serial dilutions. Breakpoints for each antibiotic were calculated for 3 different replicates. In addition, a study of the absence of transferable resistance genes in the genome of the strains was performed using the Resfinder analysis (Zanktari et al., 2012, J. Antimicrob. Chemotherapy, PMID 22782487).

[0079] In a preferred embodiment, the Lactobacillus strain of the present invention is selected in a screening protocol comprising at least one screening method selected from the group consisting of: PBMC screening, TEER assay, DC assay, adipocyte assay, MIC assay, L cell assay and bile salt hydrolase assay.

[0080] In another preferred embodiment, the Lactobacillus strain of the present invention is selected in a screening protocol comprising at least two screening methods selected from the group consisting of: PBMC screening, TEER assay, DC assay, adipocyte assay, MIC assay, L cell assay and bile salt hydrolase assay.

[0081] In another preferred embodiment, the Lactobacillus strain of the present invention is selected in a screening protocol comprising the following steps: PBMC screening, TEER assay, DC assay, L cell assay, and bile salt hydrolase assay;

[0082] wherein the IL10 / IL12 ratio is higher than 10 in a PBMC assay, such as higher than 20, higher than 30 or higher than 50 when calculated as a mean in a PBMC assay using PBMCs from at least 3 healthy donors;

[0083] wherein the Lactobacillus strain is capable of increasing the TEER ratio in a TEER assay by a factor of at least 1.15, wherein the TEER ratio is calculated as the quotient of the TEER value of the Caco2 cells after 24 hours of incubation with the Lactobacillus strain and the TEER value after 0 hours relative to the TEER value of the medium control after 24 hours of incubation over the TEER value after 0 hours;

[0084] wherein the Lactobacillus strain is capable of reducing IL12 levels in a DC assay stimulated with a pro-inflammatory cocktail by at least 10%, such as at least 20%, compared to a pro-inflammatory stimulated control; and

[0085] • These strains performed at least at the same level as strain HH05 in the bile salts and L-cell assays.

[0086] In a particularly preferred embodiment, the Lactobacillus strain of the invention is selected in a screening protocol comprising the following steps: PBMC screening, TEER assay, DC assay and L cell assay.

[0087] In one embodiment, the TEER determination for the present invention is carried out with or without the addition of pro-inflammatory factors such as pro-inflammatory cytokines or LPS with biological activity. In another embodiment, the TEER determination for the present invention is carried out with the addition of pro-inflammatory factors such as pro-inflammatory cytokines or LPS with biological activity. In an alternative embodiment, the TEER determination for the present invention is carried out without the addition of pro-inflammatory factors such as pro-inflammatory cytokines or lipopolysaccharide (LPS) with biological activity. In another alternative embodiment, two TEER determinations are used in the screening scheme, wherein one TEER determination is carried out without the addition of pro-inflammatory factors such as pro-inflammatory cytokines or LPS with biological activity, and another TEER determination is carried out with the addition of pro-inflammatory factors such as pro-inflammatory cytokines or LPS with biological activity. In one embodiment, the TEER determination carried out with the addition of pro-inflammatory factors with biological activity is the TEER determination using pro-inflammatory cytokines.

[0088] Composition

[0089] The present invention further relates to a composition comprising one or more Lactobacillus strains of the present invention. 3 viable cells / gram of composition, preferably at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 In a preferred embodiment, the composition comprises 10 6 -10 14 viable cells / gram of composition, preferably 10 7 -10 13 viable cells / gram of composition.

[0090] One or more lactobacillus strains can be dried, such as freeze drying or spray drying. When the microorganism is dried, such as freeze drying, it is important to use a method that ensures a satisfactory high viable cell count and retains the activity of the selected strain. Such technology is known in the art, and these methods known in the art also may be applicable to the present invention.

[0091] The dried, such as freeze-dried, preparation may contain suitable adjuvants known per se, for example cryoprotectants, such as nutritional sugars, maltose or prebiotics (eg galacto-oligosaccharides). It is preferred to use cryoprotectants that have no or low energy value or have a very low glycemic index.

[0092] The composition may further comprise other ingredients known in the art of preparing foods and food supplements, for example selected from fillers, nutrients, preservatives, stabilizers, flavoring agents and coloring agents.

[0093] The composition may be in the form of a powder, paste or gel and filled into sachets, tablets or capsules. For ease of administration, the composition is preferably formulated into discrete dosage forms such as sachets, tablets or capsules.

[0094] Composition can be a food supplement, or is the form of food or food composition.Alternatively, composition can be the form of pharmaceutical composition.On the one hand, composition is a food, food composition or food supplement comprising one or more lactobacillus strains and optionally comprising one or more acceptable excipients.On the other hand, composition is a pharmaceutical composition comprising one or more lactobacillus strains and one or more pharmaceutically acceptable excipients.On the one hand, pharmaceutical composition is used together with medicine such as metformin.In another embodiment, pharmaceutical composition further comprises medicine such as metformin.

[0095] When the preparation of the present invention is in the form of a food supplement, it may be in the form of a single administration, such as a capsule, tablet, powder or the like, which preferably contains a unit dose of the microorganism, containing 10 7 -10 13 cells / dose, most preferably 10 10 -10 11 cells / dose.

[0096] The food supplement may also be in powder form or similar form, which is added to or mixed with a suitable food (composition) or a suitable liquid or solid carrier to prepare a food ready for consumption.

[0097] For example, the food supplement may be in the form of a lyophilized powder that can be reconstituted using a suitable liquid, such as water, an oral rehydration solution, milk, juice or a similar drinkable liquid. The food supplement may also be in the form of a powder that is mixed with a solid food or a food with a high water content, such as a fermented milk product, e.g. yogurt.

[0098] The composition of the invention may also be in the form of a food product ready for consumption. Such a food product may be prepared, for example, by adding the above-described supplement of the invention to a food product or food matrix known per se; adding the microorganisms (alone or in a mixture) to a food product or food matrix known per se in the amount desired for administration; or culturing the desired bacteria in a food culture medium until a food product containing the amount of bacteria desired for administration is obtained. The food culture medium is preferably such that it already forms part of the food product or will form part of the food product after fermentation.

[0099] In this regard, the food or food substrate may be fermented or non-fermented.

[0100] The composition of the present invention may be a food for oral consumption, such as a whole food or an infant formula.

[0101] The composition may further contain prebiotic compounds, in particular fibers that produce butyrate / butyric acid or propionate / propionic acid when fermented; macronutrients containing amino acids, such as proteins; and specific vitamins, minerals and / or trace elements. Regarding the latter, and as can be seen from the examples, increased or moderately high amounts of vitamins A, K, B 12 The presence of Mg, Cr and Zn may be advantageous, as may the presence of folate and ω3 fatty acids.

[0102] The food supplement may further comprise fiber, for example in an amount of at least 0,5 g fiber per 100 g total preparation.

[0103] As fiber, the preparation preferably contains resistant starch or another butyrate generator, and a suitable propionate generator, such as gum or soy polysaccharide, in the amounts indicated above. Short chain fatty acids, such as butyric acid and propionic acid, may also be used as such, preferably in a suitable encapsulated form, or in the form of their physiological equivalents, such as sodium propionate, in an amount of at least 0.1 g / 100 g of the total composition.

[0104] Amino acids / peptides, vitamins, minerals and trace elements may also be included, for example in the form of yeast extract.

[0105] The composition, in particular when it is in the form of a whole food, may also contain peptides and / or proteins, in particular proteins rich in glutamate and glutamine; lipids; carbohydrates; vitamins; minerals and trace elements. Preferably, glutamine / glutamate precursors are used in an amount corresponding to 0.6-3 g glutamine / 100 g product, as well as small polypeptides with a high glutamine content. Alternatively, glutamine-rich proteins such as milk proteins, wheat proteins or hydrolysates thereof may be added.

[0106] The compositions of the present invention may be lactose-free, halal, vegetarian and / or kosher compositions.

[0107] use

[0108] Preferably 10 7 -10 13 cells / day, preferably 10 10 -10 11 One or more Lactobacillus strains of the invention or a composition comprising one or more Lactobacillus strains of the invention are administered in an amount of 10 cells / day. For both adults and children, the cells can be administered in a single administration, or the cells can be divided into 2 or up to 10 doses per day.

[0109] The compositions are administered orally, typically with a meal.

[0110] One or more Lactobacillus strains or compositions of the invention should be administered daily, as long as the individual receiving it has a need for it, or as long as the individual receiving it wishes to adapt to the properties of the Lactobacillus strains of the invention, for example, as long as the individual wishes to stop, reduce or limit weight gain caused by consuming an overly rich diet, and / or improve their postprandial blood sugar response and / or reduce their insulin resistance.

[0111] The term "insulin resistance" is the reduced ability of cells to respond to the action of insulin when transporting glucose (sugar) from the bloodstream into cells. Insulin resistance can be measured, for example, by an oral glucose tolerance test and / or glucose-stimulated insulin secretion or by measuring fasting insulin and blood glucose.

[0112] The term "reducing insulin resistance" is used to describe the outcome of insulin resistance in a subject who has received one or more strains of Lactobacillus compared to the insulin resistance of the same subject who has not received a strain of Lactobacillus.

[0113] Thus, in a preferred embodiment, one or more Lactobacillus strains or compositions of the invention are administered daily for an unspecified period of time, which in principle could continue for several years.

[0114] The Lactobacillus strains of the invention have a positive effect on the gastrointestinal tract. For this purpose, the composition comprising one or more Lactobacillus strains may further contain health-improving compounds known per se, such as drugs, etc. In particular, the composition may contain compounds having a beneficial effect on the gastrointestinal tract, such as glutamine / glutamate or its precursors.

[0115] One or more Lactobacillus strains or compositions of the invention can be used to reduce prediabetes, reduce or improve type 2 diabetes, improve insulin resistance, improve liver health, reduce intestinal inflammation, improve intestinal barrier function, lower blood sugar levels and / or prevent or reduce weight gain caused by a high fat diet in a mammalian subject.

[0116] The term "liver health" is used herein to evaluate liver status (compared to untreated disease or healthy controls) by assessing the expression of cytokines, triglycerides, cholesterol and / or liver-related enzymes ALT and / or AKT in liver tissue, and / or measuring liver-related enzymes ALT and / or AKT in plasma, serum and / or blood samples, and / or measuring liver weight, and / or evaluating liver status based on histological examination of liver tissue.

[0117] The term "intestinal inflammation" is used herein for situations when inflammatory factors such as proinflammatory cytokines are produced in the intestine and / or when intestinal barrier function is damaged causing intestinal leakage. Intestinal inflammation and intestinal leakage can be assessed by measuring cytokine concentrations in intestinal samples and / or plasma samples and / or by measuring LPS in plasma.

[0118] The term "intestinal barrier" is used herein to refer to the intestinal mucosa to ensure that undesirable luminal substances are adequately contained in the intestine while maintaining the ability to absorb nutrients. Intestinal barrier function can be assessed in vitro by measuring the TEER across the intestinal epithelial cell layer, and in vivo by measuring LPS levels in plasma samples and / or by in vivo sulfonic acid determination.

[0119] The term "blood glucose level" is used herein for the concentration of glucose in the blood, as measured, for example, by an enzyme-based assay. The body maintains blood glucose concentrations within a narrow range. This tight regulation is called glucose homeostasis. Insulin, which lowers blood glucose, or GLP-1, which enhances insulin secretion, is one of the best-known hormones involved in blood glucose regulation.

[0120] One or more Lactobacillus strains or compositions of the present invention can be used to improve the metabolic health of individuals, such as reducing prediabetes and / or type 2 diabetes. In one aspect of the present invention, one or more Lactobacillus strains or compositions are used to improve or reduce one or more prediabetes and / or type 2 diabetes symptoms such as the following, such as insulin resistance, hyperglycemia, low-grade inflammation and / or intestinal barrier function. On the other hand or in another aspect, one or more Lactobacillus strains or compositions are used to improve the postprandial blood glucose response of the subject, and / or reduce the insulin resistance of the subject. On another hand or in another aspect, one or more Lactobacillus strains or compositions are used to prevent or reduce colitis. On still another hand or in another aspect, one or more Lactobacillus strains or compositions are used to reduce the weight gain of individuals who take in high fat and / or high sugar. In a specific aspect of the present invention, one or more Lactobacillus strains or compositions are used to improve or reduce two or more than two prediabetes and / or type 2 diabetes symptoms selected from the group consisting of: insulin resistance, hyperglycemia, low-grade inflammation and intestinal barrier function. In another specific aspect of the present invention, one or more Lactobacillus strains or compositions are used to improve or alleviate three or more prediabetes and / or type 2 diabetes symptoms selected from the group consisting of: insulin resistance, hyperglycemia, low-grade inflammation, and intestinal barrier function. In yet another specific aspect of the present invention, one or more Lactobacillus strains or compositions are used to improve or alleviate the following prediabetes and / or type 2 diabetes symptoms: insulin resistance, hyperglycemia, low-grade inflammation, and intestinal barrier function.

[0121] Preferred Embodiments

[0122] The present invention can also be described by the following numbered embodiments:

[0123] Example 1. A Lactobacillus strain having the ability to improve metabolic health, such as reducing pre-diabetes, or having the ability to reduce blood glucose levels in a mammalian subject.

[0124] Embodiment 2. The Lactobacillus strain according to embodiment 1, wherein the strain has the ability to alleviate prediabetes.

[0125] Embodiment 3. The Lactobacillus strain according to embodiment 1 or 2, wherein the strain has the ability to improve or alleviate one or more pre-diabetic symptoms selected from the group consisting of: insulin resistance, hyperglycemia, low-grade inflammation and intestinal barrier function.

[0126] Embodiment 4. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain has the ability to prevent and / or alleviate type 2 diabetes.

[0127] Embodiment 5. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain has the ability to prevent and / or alleviate one or more symptoms of type 2 diabetes selected from the group consisting of: insulin resistance, hyperglycemia, low-grade inflammation and intestinal barrier function.

[0128] Embodiment 6. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain has the ability to improve a subject's postprandial blood sugar response and / or reduce a subject's insulin resistance.

[0129] Embodiment 7. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain survives in the gastrointestinal (GI) tract.

[0130] Embodiment 8. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain is selected in a screening protocol comprising at least one screening method selected from the following: PBMC screening, TEER assay, DC assay, adipocyte assay, L cell assay, bile salt hydrolase assay and MIC assay.

[0131] Embodiment 9. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain is selected in a screening protocol comprising at least two screening methods selected from the following: PBMC screening, TEER assay, DC assay, adipocyte assay, L cell assay, bile salt hydrolase assay and MIC assay.

[0132] Embodiment 10. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain is selected in a screening protocol comprising PBMC screening, TEER assay, TEER assay with addition of pro-inflammatory cytokines, and DC assay.

[0133] Embodiment 11. The Lactobacillus strain according to any one of the preceding embodiments, wherein the IL10 / IL12 ratio in the PBMC assay is higher than 10, such as higher than 20, such as higher than 30, such as higher than 50, calculated as a mean using PBMCs from at least 3 healthy donors.

[0134] Embodiment 12. The Lactobacillus strain according to any of the preceding embodiments, wherein the strain is capable of increasing the TEER ratio in a TEER assay by a factor of at least 1.15, wherein the TEER ratio is calculated as the quotient of the TEER value of the PBMCs after 24 hours of incubation with the Lactobacillus strain and the TEER value after 0 hours, relative to the TEER value of the culture medium control after 24 hours of incubation over the TEER value after 0 hours.

[0135] Embodiment 13. The Lactobacillus strain according to any of the preceding embodiments, wherein the strain is capable of reducing IL12 levels in a DC assay stimulated with a pro-inflammatory cocktail by at least 10%, preferably at least 20% compared to a pro-inflammatory stimulated control.

[0136] Embodiment 14. The Lactobacillus strain according to any one of the preceding embodiments, wherein the Lactobacillus strain is a Lactobacillus reuteri strain or a Lactobacillus brevis strain or belongs to the Lactobacillus casei group.

[0137] Embodiment 15. The Lactobacillus strain according to any one of the preceding embodiments, wherein the Lactobacillus strain is a Lactobacillus reuteri strain or belongs to the Lactobacillus casei group.

[0138] Embodiment 16. The Lactobacillus strain according to any one of the preceding embodiments, wherein the Lactobacillus strain is a Lactobacillus reuteri strain, a Lactobacillus casei strain, a Lactobacillus paracasei strain and / or a Lactobacillus rhamnosus strain.

[0139] Embodiment 17. The Lactobacillus strain according to embodiment 14 or 15, wherein the Lactobacillus strain is of the Lactobacillus casei group and is selected from the group consisting of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus.

[0140] Embodiment 18. The Lactobacillus paracasei strain according to embodiment 17, wherein the Lactobacillus paracasei strain is selected from the group consisting of Lactobacillus paracasei subsp. firmus and Lactobacillus paracasei subsp. paracasei strains.

[0141] Embodiment 19. The Lactobacillus strain according to any one of the preceding embodiments, wherein the strain is selected from the strains deposited as DSM 17648, DSM 32910, DSM 32911, DSM 32851 and DSM 32853 and mutants thereof.

[0142] Embodiment 20. The Lactobacillus strain according to embodiment 19, wherein the mutants are derived from natural evolution.

[0143] Embodiment 21. The Lactobacillus strain according to embodiment 19, wherein the mutants comprise 1 to 50 amino acid substitutions, deletions and / or additions, such as 1 to 40, 1 to 30, 1 to 20 or 1 to 10 amino acid substitutions, deletions and / or additions to the Lactobacillus strain deposited as DSM 17648, DSM 32910, DSM 32911, DSM 32851 or DSM 32853.

[0144] Embodiment 22. A composition comprising one or more Lactobacillus strains according to any one of embodiments 1-21.

[0145] Embodiment 23. The composition of embodiment 22, comprising two or more Lactobacillus strains according to any one of embodiments 1 to 21, such as three or more, four or more, or five or more Lactobacillus strains according to any one of embodiments 1 to 21.

[0146] Embodiment 24. The composition according to any one of embodiments 22 to 23, wherein the composition comprises the Lactobacillus strain deposited as DSM 17648 and one or more Lactobacillus strains selected from the strains deposited as DSM 32910, DSM 32911, DSM 32851 and DSM 32853.

[0147] Embodiment 25. The composition according to any one of embodiments 22 to 23, wherein the composition comprises the Lactobacillus strain deposited as DSM 32910 and one or more Lactobacillus strains selected from the strains deposited as DSM 17648, DSM 32911, DSM 32851 and DSM 32853.

[0148] Embodiment 26. The composition according to any one of embodiments 22 to 23, wherein the composition comprises the Lactobacillus strain deposited as DSM 32911 and one or more Lactobacillus strains selected from the strains deposited as DSM 17648, DSM 32910, DSM 32851 and DSM 32853.

[0149] Embodiment 27. The composition according to any one of embodiments 22 to 23, wherein the composition comprises the Lactobacillus strain deposited as DSM 32851 and one or more Lactobacillus strains selected from the strains deposited as DSM 17648, DSM 32910, DSM 32911 and DSM 32853.

[0150] Embodiment 28. The composition of any one of embodiments 22 to 23, wherein the composition comprises a Lactobacillus strain deposited as DSM 32853 and one or more Lactobacillus strains selected from the group consisting of strains deposited as DSM 17648, DSM 32910, DSM 32911 and DSM 32851.

[0151] Embodiment 29. The composition according to embodiments 22 to 28, comprising up to five Lactobacillus strains according to any one of embodiments 1 to 21, such as up to four, three or two Lactobacillus strains according to any one of embodiments 1 to 21.

[0152] Embodiment 30. The composition of any one of embodiments 22 to 29, wherein the composition further comprises a microbial strain from a different species.

[0153] Embodiment 31. The composition according to embodiment 30, wherein the further microbial strain is selected from the group consisting of a Lactobacillus rhamnosus strain and a strain belonging to the species of Akkermansia.

[0154] Embodiment 32. The composition of any one of embodiments 22 to 31, wherein the composition further comprises cellular components, metabolites and / or secreted molecules thereof.

[0155] Embodiment 33. The composition of embodiment 32, wherein the cellular components, metabolites and / or secreted molecules are from the retentate of the culture after fermentation and centrifugation.

[0156] Embodiment 34. The composition according to any one of embodiments 22 to 33, wherein the strains are dried, such as freeze-dried or spray-dried.

[0157] Embodiment 35. The composition of any one of embodiments 22 to 33, wherein the strains are freeze-dried.

[0158] Embodiment 36. A composition according to any one of embodiments 22 to 35, wherein the composition comprises 10 6 -10 14 colony forming units (CFU) / g of the composition, preferably comprising 10 7 -10 13 , preferably 10 8 -10 12 CFU / g.

[0159] Embodiment 37. The composition according to any one of embodiments 22 to 36, wherein the composition is formulated into unit dose formulations, each of which contains 10 6 -10 14 Colony forming units (CFU) / unit, preferably 10 7 -10 13 , preferably 10 8 -10 12 CFU / unit.

[0160] Embodiment 38. A composition according to any one of embodiments 22 to 37, wherein the composition comprises 10 6 with 10 14 Between, such as 10 6 with 10 13 cells between 10 8 with 1012 cells between 10 9 with 10 11 The total number refers to the total amount of living cells and dead cells.

[0161] Embodiment 39. A composition according to any one of embodiments 22 to 37, wherein the composition comprises 10 6 with 10 14 Between, such as 10 6 with 10 13 cells between 10 8 with 10 12 cells between 10 9 with 10 11 The total number refers to the total number of living cells.

[0162] Embodiment 40. A composition according to any one of embodiments 22 to 37, wherein the composition comprises 10 6 with 10 14 Between, such as 10 6 with 10 13 cells between 10 8 with 10 12 cells between 10 9 with 10 11 The total number refers to the total number of dead cells.

[0163] Embodiment 41. The composition according to any one of embodiments 22 to 40, wherein the composition is a pharmaceutical composition comprising a Lactobacillus strain and one or more pharmaceutically acceptable excipients.

[0164] Embodiment 42. The composition of any one of embodiments 22 to 40, wherein the composition is a food, a food supplement, or a food composition.

[0165] Embodiment 43. The composition of embodiment 42, comprising one or more acceptable excipients.

[0166] Embodiment 44. The composition of any one of embodiments 42 to 43, wherein the composition is ready for consumption.

[0167] Embodiment 45. The composition according to any one of embodiments 42 to 44, comprising an additional food supplement.

[0168] Embodiment 46. The composition according to embodiment 45, wherein the other food supplements are selected from one or more prebiotics, enzymes, vitamins and minerals.

[0169] Embodiment 47. The composition of any one of embodiments 22 to 46, wherein the composition further comprises cellular components, metabolites and / or secreted molecules thereof.

[0170] Embodiment 48. A dietary supplement comprising one or more Lactobacillus strains according to any one of embodiments 1 to 21.

[0171] Embodiment 49. The dietary supplement of embodiment 48, further comprising one or more narratively beneficial ingredients, such as prebiotics, enzymes, vitamins and / or minerals.

[0172] Embodiment 50. Use of one or more Lactobacillus strains according to any one of embodiments 1 to 21 or a composition according to any one of embodiments 22 to 47 for improving metabolic health in a mammalian subject, such as reducing prediabetes.

[0173] Embodiment 51. Use of one or more Lactobacillus strains according to any one of embodiments 1 to 21 or a composition according to any one of embodiments 22 to 47 for reducing prediabetes, preventing or reducing type 2 diabetes, reducing insulin resistance, improving liver health, reducing intestinal inflammation, improving intestinal barrier function, lowering blood glucose levels and / or preventing or reducing weight gain caused by a high fat diet in a mammalian subject.

[0174] Embodiment 52. The use according to embodiment 50 or 51, wherein the use is for reducing prediabetes, preventing or reducing type 2 diabetes, reducing insulin resistance, improving liver health, reducing intestinal inflammation and / or improving intestinal barrier function in a mammalian subject.

[0175] Embodiment 53. The use according to any one of embodiments 50 to 52, wherein the use is for reducing insulin resistance, improving liver health, reducing intestinal inflammation and / or improving intestinal barrier function in a mammalian subject.

[0176] Embodiment 54. Use of one or more Lactobacillus strains according to any one of embodiments 1 to 21 or the composition according to any one of embodiments 22 to 47 for lowering blood glucose levels in a mammalian subject.

[0177] Embodiment 55. Use of one or more Lactobacillus strains according to any one of embodiments 1 to 21 or a composition according to any one of embodiments 22 to 47 for preventing or reducing weight gain in an individual on a high fat diet.

[0178] Embodiment 56. The use according to any one of embodiments 50 to 55, wherein the mammal is a human.

[0179] Embodiment 57. The use according to any one of embodiments 50 to 56, wherein 10 6 -10 14 Colony forming units (CFU), preferably 10 7 -10 13 , preferably 10 8 -10 12 .

[0180] Embodiment 58. A method for screening a Lactobacillus strain according to any one of embodiments 1 to 21, using a screening protocol comprising at least one screening method selected from the following: PBMC screening, TEER assay, DC assay, adipocyte assay, L cell assay, bile salt hydrolase assay and MIC assay.

[0181] Embodiment 59. The method according to embodiment 58 uses a screening protocol comprising at least two screening methods selected from the group consisting of: PBMC screening, TEER assay, DC assay, and adipocyte assay.

[0182] Embodiment 60. The method according to embodiment 58 or 59, which uses a screening protocol comprising the following screening methods: PBMC screening, TEER assay, TEER assay performed with the addition of pro-inflammatory cytokines, and DC assay.

[0183] Embodiment 61. The method according to any one of embodiments 58 to 60, which uses a screening protocol comprising the following screening methods: PBMC screening, TEER assay, DC assay and L cell assay.

[0184] Examples

[0185] Materials and Methods

[0186] Lactobacillus, Enterococcus, Micrococcus and Bifidobacterium strains are cultured using standard culture media, e.g., culture media providing 15-20 g of peptone or tryptone and about 5 g of yeast extract per liter. For the preparation of the culture media, standard procedures (component concentration, sterilization, inoculation) are applied, and the microorganisms are grown for a sufficient time (e.g., 8-48 hours) at an elevated temperature (e.g., 35-42°C).

[0187] Example 1. Screening of suitable Lactobacillus strains

[0188] Using the screening protocol described below, Lactobacillus strains were discovered that had beneficial effects on metabolic health, such as alleviating prediabetes.

[0189] 1. Pre-screening: 640 lactobacilli were screened by analyzing the IL10 / IL12 ratio as an indicator of the immunomodulatory potential of the strains in a PBMC screening assay:

[0190] For peripheral blood mononuclear cell (PBMC) assay, PBMC from one donor were incubated with Lactobacillus strains for 24 h after acclimatization for 24 h in the absence of other pro-inflammatory stimuli. After incubation, appropriate dilutions of supernatants containing cytokines were measured by Luminex assay (Invitrogen) and / or ELISA according to the manufacturer's instructions, and the IL10 / IL12 ratio was determined.

[0191] In the pre-screening, 180 strains with IL10 / IL12 ratios >3 were selected.

[0192] 2. Screening for immunomodulation and intestinal integrity

[0193] The 180 strains selected in the prescreen were subjected to the same PBMC confirmation screen as the prescreen, but the strains were tested on PBMCs from three different donors to confirm the beneficial IL10 / IL12 ratio. In addition to the IL10 / IL12 ratio, 14 other cytokines were measured.

[0194] The strains were further subjected to TEER assessment (transepithelial electrical resistance) (as described in B. Srinivasen et al. (2015). J Lab Autom [Journal of Laboratory Automation] 2015, 20: 107-126) to evaluate the effect of Lactobacillus strains on intestinal integrity without the addition of any pro-inflammatory stimuli such as TNFα. TEER measurements were performed after 0 and 24 hours.

[0195] Twenty strains that confirmed an IL10 / IL12 ratio >3 and increased TEER values ​​in the PBMC confirmation screening were selected for subsequent screening methods.

[0196] 3. Hit confirmation in the presence of pro-inflammatory triggers

[0197] In this confirmation step, TEER assessment with the addition of TNFα was also performed for the 20 strains selected from the TEER assessment performed in the absence of TNFα in step 2.

[0198] A Lactobacillus strain is selected which causes an increase in the TEER value compared to a control without the strain.

[0199] These strains were further screened in the DC assay. For this test, DCs were generated from PBMCs derived from 3 donors. DCs were matured into inflammatory DCs by treatment with 0.1 μg / mL LPS+50ng / ml TNF-α+20ng / ml IFNγ and incubated for 4 hours. Aliquots of mature DCs were incubated with 4 different doses of each Lactobacillus strain, and cytokine levels were determined using Luminex afterwards.

[0200] A Lactobacillus strain is selected that reduces IL12 induction compared to a control composition without the strain.

[0201] HH01

[0202] Species: Lactobacillus reuteri

[0203] Confirmed by 16S and WGS

[0204] Accession number: DSM 17648.

[0205] HH02

[0206] Species: Lactobacillus reuteri

[0207] Confirmed by 16S and WGS

[0208] Accession number: DSM 32910.

[0209] HH03

[0210] Species: Lactobacillus rhamnosus

[0211] Confirmed by 16S and WGS

[0212] Accession number: DSM 32911

[0213] HH04

[0214] Species: Lactobacillus paracasei subsp. tenacious

[0215] Confirmed by 16S and WGS

[0216] Accession number: DSM 32851

[0217] HH05

[0218] Species: Lactobacillus paracasei subsp. paracasei

[0219] Confirmed by 16S and WGS

[0220] Accession number: DSM 32853

[0221] Example 2. Induction of cytokines in PBMCs

[0222] The strains HH01-HH05 selected in Example 1 were further characterized in vitro.

[0223] 1x10 isolated from healthy donors 6 PBMC / mL with vehicle or containing 1x10 6 The medium of each Lactobacillus strain HH01-HH05 / mL was incubated together for 24 hours, and the total volume was 1ml. According to the manufacturer's instructions, Luminex was used to measure cytokines IL10, IL12, MCP-1 and osteopontin. Phosphate buffered saline (PBS) pH 7,4 (Cat. No. 10010-023, Gibco) without MgCl was used as a medium.

[0224] result:

[0225] Compared to vehicle controls, strains HH01-HH05 induced increased IL10 / IL12 ratios, confirming that the selected strains possess anti-inflammatory properties ( Figure 1 ).

[0226] Figure 2 Shown is the level of MCP-1 in the supernatant of PBMCs stimulated with the pro-inflammatory stimulus lipopolysaccharide (LPS). The results show that while significant production of MCP-1 was seen with vehicle alone, strains HH01-HH05 significantly reduced MCP-1 induction, supporting the anti-inflammatory properties of these strains.

[0227] Figure 3 The content of osteopontin in the supernatant of PBMCs stimulated with a pro-inflammatory stimulus (lipopolysaccharide) is shown. The results show that a large production of osteopontin was seen with vehicle alone, while strains HH01-HH05 significantly reduced osteopontin induction, confirming the anti-inflammatory properties of these strains.

[0228] Example 3. Induction of cytokines in dendritic cells

[0229] In the first set of assays, dendritic cells (DCs) were recovered from PBMCs from 3 healthy donors and aliquots were incubated for 24 h with vehicle or vehicle containing Lactobacillus strains HH01, HH03, or HH05. The mixtures were centrifuged, the supernatants were collected, and MCP-1, osteopontin, and IL12p70 were measured using Luminex (Invitrogen) according to the manufacturer's instructions.

[0230] In the second set of assays, DCs were recovered from PBMCs from 12 healthy donors and aliquots were incubated for 24 h with vehicle or vehicle containing Lactobacillus strains HH02, HH04, or HH05. The mixtures were centrifuged, the supernatants were collected, and IL10, IL12p70, MCP-1 were measured using Luminex (Invitrogen) according to the manufacturer's instructions.

[0231] result:

[0232] In the first set of DC assays, the results showed that the concentrations of pro-inflammatory cytokines MCP-1, IL12, and osteopontin were lowest when DCs were co-incubated with Lactobacillus strain HH03 compared to strains HH01 and HH05 ( Figure 4 ).

[0233] In the second set of DC assays, results showed that Lactobacillus strains HH01, HH04 and HH05 stimulated with the low-grade inflammatory mixture all induced an increased IL10 / IL12p70 ratio compared to controls stimulated with the low-grade inflammatory mixture alone. In addition, DCs stimulated with the low-grade inflammatory mixture produced MCP-1, but at a reduced level compared to the mixture control, confirming that the selected strains have anti-inflammatory properties.

[0234] Example 4. Induction of cytokines in DC cultures

[0235] The strains HH02, HH04 and HH05 selected in Example 1 were further characterized in a co-incubation assay with DCs. Selected cytokines were measured after stimulation with a pro-inflammatory cocktail.

[0236] Human PBMCs were isolated from buffy coats (N=12) and CD14+ monocytes were purified by positive selection (CD14 microbeads, Miltenyi Biotech). Monocytes were cultured with IL4 (20 ng / mL) and GM-CSF (20 ng / mL) for 6 days to differentiate into immature DCs (imDCs). To differentiate into mature DCs, 1×10 5 cells and the selected Lactobacillus strain HH02 (10 6Lactobacillus strains / mL) and a low-grade inflammatory mixture consisting of TNFα (10ng / mL), IFNγ (10ng / mL) and LPS (100ng / mL) were cultured together. ImDCs were stimulated with Lactobacillus strains for 4 hours, and then the low-grade inflammatory mixture was added for another 16 hours. DC maturation was evaluated after in vitro stimulation with BD Cytometric BeadArray (CBA) to measure IL10, IL12p70, MCP-1 and IL6 produced by DCs. The effects of strains HH02, HH04, and HH05 on these cytokines were compared with controls without strains stimulated only with the pro-inflammatory mixture.

[0237] result

[0238] The results showed that DCs stimulated by the low-grade inflammation mixture produced IL10, and the presence of strains HH02, HH04, and HH05 increased the concentration of IL10 compared with stimulation with the mixture alone, DCs stimulated by the low-grade inflammation mixture produced IL12p70, and the presence of strains HH02, HH04, and HH05 increased the concentration of IL12p70 compared with stimulation with the mixture alone, and Lactobacillus strains HH01, HH04, and HH05 all induced an increase in the IL10 / IL12p70 ratio compared with the mixture control ( Figure 5 ), thus confirming that the selected strains possess anti-inflammatory properties.

[0239] Furthermore, it was shown that DCs stimulated by the low-grade inflammatory mixture produced MCP-1, and the presence of Lactobacillus strains HH02, HH04, and HH05 all reduced the MCP-1 concentration during the culture period ( Figure 6 In addition, the results showed that DCs stimulated by the low-grade inflammatory mixture produced high levels of IL6, and the presence of the Lactobacillus strain HH04 maintained the IL6 concentration at the level of the mixture control, while strains HH02 and HH05 increased the IL6 concentration ( Figure 7 ).

[0240] Example 5. TEER assay using Lactobacillus strains HH01-HH05

[0241] The TEER determination was performed according to B. Srinivasen et al. (2015). J Lab Autom [Journal of Laboratory Automation] 2015, 20: 107-126. For this determination, CaCo-2 cells with the number ACC 169 purchased from DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkultur GmbH) were used.

[0242] Inoculate 2x10 5 CaCo-2 cells / mL and differentiated for 3 weeks. In the absence of pro-inflammatory stimuli, these cells were incubated with vehicle or 1x10 6 The cells were incubated with the medium containing Lactobacillus strains HH01-HH05 at a concentration of 10 cells / mL for 24 hours, and then the TEER ratio was determined. Phosphate buffered saline pH 7.4 without MgCl2 (Catalog No. 10010-023, Gibraltar) was used as a medium. A TEER ratio>1 indicates that the Lactobacillus strain is able to enhance the integrity of the intestinal barrier function.

[0243] In the presence of pro-inflammatory stimuli in the form of 100 ng / mL TNFα, CaCo-2 cells were incubated with vehicle or vehicle containing Lactobacillus strains HH01-HH05 for 24 h, and then the TEER ratio was determined. PBS pH 7,4 without MgCl2 (Cat. No. 10010-023, Gibraltar) was used as a vehicle. A TEER ratio>1 indicates that the Lactobacillus strain is able to enhance the intestinal barrier function.

[0244] result:

[0245] TEER data showed that Lactobacillus strains HH04 and HH03 were not present in the presence of Figure 8 A) or exists ( Figure 8 B) Pro-inflammatory factors can best enhance the intestinal epithelial barrier. In the presence of pro-inflammatory factors, strains HH01, HH02 and HH05 are also able to increase TEER compared to the disease control alone (medium with pro-inflammatory factors).

[0246] Example 6. TEER determination

[0247] Effects of Lactobacillus strains HH02, HH04 and HH05 on the barrier integrity of the epithelial cell line Caco2 in the presence of an inflammatory cocktail:

[0248] The effect of Lactobacillus strains HH02, HH04 and HH05 on the barrier integrity of the epithelial cell line Caco2 in the presence of an inflammatory cocktail was determined using the TEER EVOM2 assay performed in the same manner as described in "Example 5. TEER assay" with the following modifications. At the beginning of the co-incubation, an inflammatory cocktail containing TNFα-alpha (100 ng / mL) and INFγ-gamma (10 ng / mL) was added on the basolateral side.

[0249] result:

[0250] The results showed that the inflammatory mixture reduced the TEER ratio, an indicator of a weakened Caco2 barrier integrity. This effect was complemented by the tested strains HH02, HH04 and HH05, with the strongest complementation being by HH04 ( Fig. 9 ).

[0251] TEER enhancement after one month of storage:

[0252] The formulated products containing freeze-dried Lactobacillus strains HH02 or HH04 were tested for their TEER enhancing ability after storage for one month at -20°C, 4°C, 25°C and 40°C. The effect of the formulated products on the barrier integrity of the epithelial cell line Caco2 in the presence of the formulated products was determined using the TEER EVOM2 assay performed in the same manner as described in "Example 5. TEER assay", with the following modifications. The formulated products containing five times the amount of Lactobacillus strains (infection multiplicity (MOI) of 500) as the inoculated Caco2 cells were dissolved in 500 μL of culture medium in the apical culture medium.

[0253] result:

[0254] The results showed that the TEER enhancement ability of the HH02 and HH04 formulations was stable for at least 1 month at storage temperatures of -20°C, 4°C, and 25°C ( Fig.10 ). For products stored at 40°C for more than one month, lower TEER activity was seen.

[0255] Screening of the epithelial cell line Caco2 by Lactobacillus strains HH02 and HH04 in the presence / absence of fructose or glucose The impact of fault integrity :

[0256] The effect of Lactobacillus strains HH02 and HH04 on the barrier integrity of the epithelial cell line Caco2 was determined using a TEER cellZscope assay performed in the same manner as described in "Example 5. TEER assay" with the following modifications. On the apical side, medium alone or medium containing 7 mM fructose was added. On the basolateral side, medium alone or medium containing 7 mM glucose was added.

[0257] result:

[0258] The results show that HH02 minimally increases TEER, with different concentrations having little effect. The presence of high concentrations of glucose on the basolateral side allows HH04 to increase TEER more rapidly than when the strain is tested in standard medium on the basolateral side. This result suggests that the effect of HH04 is increased in subjects with high blood glucose values ​​compared to the effect in healthy individuals. A faster increase in TEER was also observed with HH04 in the presence of high fructose concentrations on the apical side compared to medium alone. This result suggests that the effect is increased in subjects with a high fructose load in the intestine compared to the effect in fasting subjects ( Fig.11 ).

[0259] Example 7. Conversion of bile salts

[0260] Strains HH01, HH02, HH04 and HH05 were incubated at 37°C in MRS medium (DeMan, Rogosa and Sharpe) for 24 hours in the presence of the following bile salts: glycocholate, taurocholate, glycochenodeoxycholate, taurochenodeoxycholate, glycodeoxycholate and taurodeoxycholate. Bile salts were present at the following concentrations: 1,56 mg / mL, 0,56 mg / mL, 1,51 mg / mL, 0,54 mg / mL, 1,51 mg / mL and 0,33 mg / mL, respectively. References containing bile salts in the absence of any bacteria were used. After incubation, the supernatant was harvested by centrifugation. The samples were extracted with acetic acid methanol and analyzed by high mass resolution on a reverse phase (RP) liquid chromatography mass spectrometer (LCMS). The method was established to quantify all six bile salts mentioned above. The conversion rate of conjugated bile salts (CBS) expressed as a percentage was calculated according to the formula CBS [%] = 100 [%] - (A * 100 [%] / B). A is the amount of bile salts after 24 hours of incubation in the presence of bacteria, and B is the amount of bile salts after 24 hours of incubation in the absence of any bacteria. The higher the CBS value, the more bile salts are converted.

[0261] The results are as follows Fig.12 , and showed that strains HH01 and HH02 were able to partially convert all bile salts, indicating an effect on reducing fat absorption, while strains HH04 and HH05 were able to partially convert taurodeoxycholate, indicating a positive effect on metabolism.

[0262] Example 8. L-cell assay for measuring lactobacillus-stimulated glucagon-like peptide 1 (GLP-1) release

[0263] L cell assays were performed as described in Gagnon and Brubaker (2015) NCI-H716 Cells. pp. 221-228, Verhoeckx K. et al. (eds.) The Impact of Food Bioactives on Health. Springer, Cham. The human L cell line NCI-H716 was grown in RPMI-1640 (Roswell Park Memorial Institute) medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cells (passages 12-20) were seeded in 24-well plates (200.000 cells / well) and allowed to differentiate for 48 hours. The 24-well plates were pre-coated with 0.5 g Matrigel / well prior to cell seeding.

[0264] For the glucagon-like peptide 1 (GLP-1) release assay, plates containing human L cells were washed twice with Krebs Ringer buffer containing bovine serum albumin (BSA, 0.2%), and then diluted samples of Lactobacillus supernatants from HH02 and HH04 or medium controls were added for 2 hours in an incubator at 37°C / 5% CO2. Phorbol 12-myristate 13-acetate (PMA) (10 μM) and Triton X-100 (0.2%) were included in each test as a GLP-1 positive control and for obtaining total release of GLP-1, respectively. All test samples contained 25 μM dipeptidin A (Sigma-Aldrich), a dipeptidyl peptidase-4 inhibitor. After incubation, all supernatants were removed and stored at -20°C until analysis of GLP-1. Cell viability was analyzed using Presto Blue viability staining (Invitrogen). The supernatants were analyzed for GLP-1 using an active GLP-1 ELISA kit (EGLP-35K, Millipore-Merck). Broth for Lactobacillus culture (yeast dextrose agar broth (YDA)) and broth for control bacteria Staphylococcus epidermidis ATCC 35984 (SE) culture (glucose-containing medium 17 (GM17)) were included as additional controls in the absence of strains.

[0265] result:

[0266] The results showed that if the cell-free broth of strains HH02 and HH04 was diluted 10-fold, it stimulated the release of GLP-1 from L cells. The release from the cell-free broth of these strains was higher than that from the YDA medium control diluted 10-fold. If the cell-free growth broth was tested at a 50-fold dilution, no release above background levels was observed ( Fig.13 The cell-free broth obtained from strains HH02 and HH04 did not affect the viability of NCI-H716 during the 2-h incubation period, demonstrating that GLP-1 release was not due to L cell proliferation ( Fig.14 ).

[0267] Example 9. Prophylactic treatment with Lactobacillus in experimental colitis using a mouse model

[0268] In this study, the trinitrobenzene sulfonic acid (TNBS)-induced acute colitis model was used (Foligne et al., 2007 doi: 10.3748 / wjg.v13.i2.236). Colitis was induced in mice by a single intrarectal administration of TNBS, after which the mice were observed for 2 days. The Lactobacillus strains HH01-HH05 described in Example 1 were used for this study.

[0269] Research Plan

[0270] Seven groups of 15 Balb / c mice were used for the experiment. The mice were transferred to cages on day -19 to acclimate to the environment and had free access to feed and sterile water.

[0271] On day -5 to day 1, mice received 100 μL oral gavage each containing vehicle, Lactobacillus strains as specified in the following protocol. PBS pH 7,4 without MgCl2 (Cat. No. 10010-023, Gibraltar) was used as vehicle.

[0272] On day 0, mice in the TNBS and TNBS+Lactobacillus strain groups, respectively, each received intrarectal administration of ethanol containing 100 mg / kg 2,4,6-TNBS to induce colitis.

[0273]

[0274] Mice were sacrificed on day 2.

[0275] sampling

[0276] Monitor clinical signs on Days -6 to 2:

[0277] Weight (see Fig.15 )

[0278] • Wallace scoring of colon (Foligne et al., 2007 doi: 10.3748 / wjg.v13.i2.236).

[0279] At the end of the study, colon wet weight and colon length were determined (see Fig.16 ), and collect the following samples:

[0280] Tail blood plasma samples

[0281] · Rectal quick-frozen samples

[0282] Colon samples (see Fig.17 )

[0283] Cecal contents samples

[0284] Feces collected on days -6 and 0

[0285] result:

[0286] The tested strains showed good anti-inflammatory effects as reflected by the Wallace score.

[0287] As expected, the vehicle control group did not show any colonic lesions, while the mean lesion score of the disease control group (TNBS group) was between 2 and 3. The groups treated with Lactobacillus strains HH01-HH05 strains showed reduced colonic lesions as reflected by a mean lesion score of about 1 compared to the TNBS (disease control) group.

[0288] In addition, the levels of pro-inflammatory cytokines IL12, TNF-α, IL1b, IL25, IL6, and IL17A, as well as the regulatory cytokine IL10, in the Lactobacillus strain group were lower than those in the disease controls (see Fig.18 ), which confirmed a reduction in inflammation. Thus, this experiment showed that Lactobacillus strains are able to reduce colon inflammation.

[0289] Thus, Lactobacillus strains HH01-HH05 improved the overall clinical condition.

[0290] Example 10. Effect of Lactobacillus strains in a diet-induced mouse model fed a classic high-fat diet

[0291] The study aimed to demonstrate that two Lactobacillus strains tested (HH04 and HH05) achieved metabolic health improvements, such as reduction of prediabetes, in a diet-induced obesity (DIO) mouse model. The study adopted a preventive approach to DIO, where the strains were administered by daily oral gavage for 12 weeks while on a high-fat diet (HFD) (for a study overview, see Fig.21 ).

[0292] Research Overview:

[0293] C57BL / 6J mice (Jackson Lab.Bar Harbour, Maine USA) were recruited in 4 batches, 12 mice per batch (3 mice per group), and treatment was started weekly (for 4 weeks). At the beginning of the experiment (week 0), all mice were 8 weeks old. Two weeks before the experiment began, mice were transferred to a cage to acclimate. During the acclimatization period, mice had free access to a low-fat diet (LFD) and water.

[0294] The experiment started at week 0 when mice were transferred to the experimental diet and received daily oral gavages in a total volume of 100 μL according to the following regimen. The Lactobacillus strains used in this example were strain HH04 and strain HH05 described in Lactobacillus Example 1.

[0295]

[0296] The high-fat / sucrose diet (HFD) contained 45% of energy from fat (87% lard, 13% soybean oil), 35% of energy from carbohydrates (mainly sucrose and maltodextrin), and 20% of energy from protein (casein) (D12451, Research Diet Inc., NJ, USA)

[0297] The low-fat reference diet (D12450H, Research Diet Inc., NJ, USA) contained 10% of energy from fat. Both diets contained 17% of the total mass from sucrose.

[0298] Sampling and Measurement

[0299] Body composition of individual mice was assessed by the mean of three measurements on magnetic resonance (MR) scans (Bruker, calibrated daily when used) at weeks 0, 4, 8, and 12 of the experimental protocol.

[0300] Fresh feces were collected at the beginning of the light cycle (9 AM ± 1 hour) at the same time as the MR scans before daily gavage. The samples were immediately frozen on dry ice and stored at -80°C until further processing.

[0301] Body weight was measured weekly between 8-9 AM.

[0302] Weight development and body composition Fig. 22Shown in. Mean values ​​of 10-12 mice per group are shown: A) Weight after random assignment. B) Weight development of individual groups after starting experimental diet and gavage treatment. C) Fat mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points. D) Fat mass (grams), which is depicted as group means with connecting lines. E) Lean mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points. F) Body fluid mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points.

[0303] At (9AM ± 1 hour) mice were transferred to new clean cages with enrichment materials but without nesting materials. Feed was measured at the time of transfer and again 24 hours later, when mice were transferred to clean standard cages (including nesting materials). Feces were carefully collected from the litter for 24 hours and stored at -80°C until further processing.

[0304] Glucose tolerance test, glucose-stimulated insulin secretion, and intestinal permeability test

[0305] Oral glucose tolerance test was carried out in the 10th week of the experimental scheme. Mice were fasted for 5h at 8AM and gavaged at 10AM. Before gavage with 4 μL 50% dextrose solution / g lean mass and 150 μL sulfonic acid solution, 5h fasting blood glucose measurement (OneTouch Vario Flex, LifeScan company) and blood sampling from tail vein were performed. Sulfonic acid solution was composed of 1.5mg fluorescein-5 (6)-sulfonic acid (Invitrogen company (Invitrogen)) dissolved in a distilled water suspension of 150 μL 0,5% carboxymethylcellulose sodium salt (CMC) (Sigma company (Sigma)). Blood glucose from tail vein puncture was measured at time points 0, 15, 30, 60, 90 and 120 min after dextrose challenge, and blood samples for insulin and intestinal permeability were placed in ethylenediaminetetraacetic acid (EDTA) treated capillaries (Sarstedt) at time points 0, 15, 30, 60 and 120 min after challenge.

[0306] After this procedure, mice received 0.5mL normal saline (Hospira company) to make mice rehydration.Blood sample was centrifuged at 4 ℃ with 1000rcf for 10min.For insulin measurement, the first 5 μL plasma was transferred to 96-well PCR plates on dry ice and remained at-80 ℃ until downstream processing.The next 5 μL plasma for intestinal permeability test was transferred to black 96-well optical button plates (Nunc company (Nunc)), and remained on wet ice until adding 150 μL of distilled water containing 0,5% CMC and fully mixing.Read plate under excitation / emission 485 / 528nm wavelength on Synergy HT microplate reader (BioTek, U.S.A.).

[0307] Insulin levels were measured by mouse ultrasensitive insulin ELISA (Alpco) following the manufacturer's protocol and quantified on an EnSpire 2300 multilabel reader (PerkinElmer).

[0308] Effects of Lactobacillus strains on glucose regulation and intestinal permeability Fig.23 Shown in: A) 5h fasting blood glucose after 10 weeks of HFD feeding and daily gavage treatment. The bar graph represents the mean of 10-12 mice in each group. B) 5h fasting insulin levels after 10 weeks of HFD feeding and daily gavage treatment. The bar graph represents the mean of 10-12 mice in each group. C, E) Oral glucose tolerance test in 5h fasting mice after 10 weeks of HFD feeding and daily gavage treatment. C depicts blood glucose levels during stimulation (2μg / g lean mass), and E depicts plasma levels of endogenous insulin response to glucose stimulation. The mean of 10-12 mice in each group. D) Area under the curve of OGTT week 10. The bar graph represents the mean of 10-12 mice in each group. F) Area under the curve of GSIS week 10. The bar graph represents the mean of 10-12 mice in each group. G) Plasma sulfonic acid fluorescence units after oral challenge (150 μL / mouse, concentration: 10 mg / mL) in 5h fasted mice after 10 weeks of HFD feeding and daily gavage treatment. Mean values ​​of 10-12 mice per group. H) Cumulative feed intake per mouse during 12 weeks of HFD feeding and daily gavage treatment. Due to co-housing, intake is the mean per mouse per cage, with n=4 in each group. Bar graphs represent means. AG) ​​All mice were gavaged with the corresponding treatment 3 hours before oral challenge.

[0309] Autopsy

[0310] Autopsy was performed at the 12th week of the experimental scheme. Mice were fasted from 7AM and gavaged at 10AM. Euthanasia was performed in an alternating order, and one mouse was taken per cage. The euthanasia of batch 1 started from group B, then C, etc. Batch 2 started from group C, and the same applies to the remaining batches, starting from groups D and A, respectively. Mice were anesthetized with isoflurane (Fresenius Kabi). Cardiac puncture was performed using a 25G needle and 1mL syringe coated with EDTA (Sigma-Aldrich). Blood was transferred to an Eppendorf tube containing 1 μL dipeptidyl peptidase-IV (DPP-IV) inhibitor (Millipore) and 1 μL protease inhibitor mixture (Sigma). Samples were centrifuged for 10min at 1000rcf, and plasma was aliquoted in triplicate, placed on dry ice and transferred to -80°C for storage until further processing.

[0311] Tissue harvesting

[0312] The weights of liver, pancreas, epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), retroperitoneal white adipose tissue (rpWAT), mesenchymal white adipose tissue (mWAT), heart, quadriceps, gastrocnemius, brain, and colon were measured, and the tissues were immediately frozen in liquid nitrogen and stored at -80°C. The tissues were dissected by the same operator and taken in the same order for all mice. The brain was frozen in liquid nitrogen for <60 sec after death.

[0313] Effect of Lactobacillus strains on tissue weight Fig.24 As shown, where the bar graphs represent the mean of 10-12 mice per group: A) Liver weight at necropsy after 12 weeks of daily gavage. B) Colon length at necropsy after 12 weeks of daily gavage. The bar graphs show the mean of 10-12 mice per group. C) iWAT weight at necropsy after 12 weeks of daily gavage. D) eWAT weight at necropsy after 12 weeks of daily gavage; and Fig.25 Figure 2, where the bar graphs represent the mean of 10-12 mice per group at necropsy after 12 weeks of daily gavage. A) rpWAT weight. B) mWAT weight. C) Brain weight. D) Heart weight.

[0314] The length of the small intestine (from the stomach to the cecum) and colon (from the cecum to the rectum) was measured and kept on a Plexiglas plate cooled by wet ice below during the entire processing time. The duodenum was considered to be the first 5 cm of the small intestine, and the remaining small intestinal tissue was divided into 3 parts of equal length. The first 3 cm was discarded, and the remaining part of the proximal 2 / 3 of the small intestine was classified as the jejunum. The first 3 cm of the distal 1 / 3 of the small intestine was discarded, and the remaining tissue was classified as the ileum. Before clearing the intestinal tissue, the most proximal cm of the duodenum, jejunum, ileum, and colon was stored in Carnoy's solution for histology (to maintain the integrity of the mucosal layer).

[0315] The contents of the small intestine, cecum, and colon were separated by mechanical pressure, frozen on dry ice, and then stored at -80 ° C. Tissues from the duodenum, jejunum, ileum, colon, and cecum were quickly frozen in liquid nitrogen and stored at -80 ° C. In batch 1, tissues from the liver, pancreas, eWAT, iWAT, rpWAT, mWAT, heart, quadriceps, and gastrocnemius were stored in 4% paraformaldehyde solution for 72 hours and then stored in 70% ethanol. In batches 2-4, tissues from the liver, eWAT, iWAT, and mWAT were stored in 4% paraformaldehyde solution for 72 hours and then stored in 70% ethanol. For all batches, liver tissue was additionally stored in OCT compound (Sakura Finetek) to achieve histological oil red O lipid staining.

[0316] Plasma biochemical analysis

[0317] Effects of Lactobacillus strains on plasma biomarkers of liver damage and lipid levels Fig.26 As shown in, where the bar graphs represent the mean of 10-12 mice per group: A) alanine aminotransferase (ALT), B) aspartate aminotransferase (AST), C.1) triglycerides, including all data points, C.2) as C.1, but excluding two data points after evaluating the detection limit of the assay used. D) total cholesterol. These analyses were performed by the medical biochemistry department at the Heart and Lung Institute of Laval University according to locally developed procedures that meet the highest standards for the processing and analysis of human and animal samples.

[0318] Effects of Lactobacillus strains on other plasma markers such as Fig. 27Shown in the figure, where the bar graphs represent the mean values ​​of 10-12 mice per group: A) serum amyloid A (SAA), B) adiponectin, C) non-esterified fatty acids (NEFA), D) LPS measurements were performed according to the ELISA kit manufacturer's protocol.

[0319] Lipid accumulation in the liver

[0320] The liver of each mouse was homogenized in liquid nitrogen by cryo-grinding the tissue with a mortar and pestle while keeping it frozen and transferred to a fresh tube. Lipids were extracted from 50 mg of powdered tissue. The effect of Lactobacillus strains on hepatic lipid accumulation was measured by calorimetry and as Fig.28 Shown in Figure , where the bar graphs represent the mean of 10-12 mice per group: A) triglycerides and B) cholesterol.

[0321] Intestinal cytokine quantification

[0322] Cytokine levels were measured in homogenized colonic tissue using the Bio-Plex 200 Luminex system according to the manufacturer's instructions. Fig.29 Shown in Figure 1 are: A) interferon-γ (IFNy) and B) IL-10, where the bars represent the mean of 10-12 mice per group.

[0323] Intestinal microbiota analysis

[0324] Bacterial DNA was extracted from frozen stool samples, and the hypervariable V3-V4 16S rRNA region was amplified and sequenced using the Illumina MiSeq platform.

[0325] Sequence data were preprocessed, tags were identified and trimmed, paired-end reads were merged, truncated with a quality score of 4, and at least 100 bp overlap was required, with the merged read length between 300 and 600 bp in length. Sequences were strictly deduplicated, and clusters with less than 5 sequences were discarded. Sequences were clustered at 97% sequence similarity by using the most abundant strictly deduplicated reads as centroids and discarding suspicious chimeras based on internal comparisons. Taxonomic assignment of OTUs was performed using the database in the Ribosomal Database Project.

[0326] The amount of probiotic DNA present in samples from intestinal contents was quantified using quantitative PCR with primers specific for HH04 or HH05 strains. Relative amounts expressed in arbitrary units are as follows Fig.30, where the bar graphs indicate the means of 10-12 samples per group, including: A) HH04 content in stool samples from the indicated groups, B) HH04 amount in small intestinal contents, C) HH05 in stool samples, and D) HH05 in small intestinal samples.

[0327] Effects of probiotics on the composition of intestinal microbiota Fig.30 , where E) is the principal coordinate analysis (PCoA) of the weighted UniFrac distance of the fecal microbiota composition, where the large dots indicate the mean of each group and the small dots indicate individual samples. F) As E, but for the microbiota composition of the small intestine contents.

[0328] Results - Weight Development

[0329] The weight distribution in all groups was similar, and then at week 0, in the HFD experimental group, the adaptation diet was changed from LFD to HFD. Starting from week 6 and continuing, daily oral gavage with HH04 in group C significantly reduced the weight gain on the high fat diet compared to group B. Group D, which was gavaged with HH05, now did show the same effect on weight gain. During the entire study period, starting from week 2, the weight gain of group A on the low fat diet was significantly lower than that of group B.

[0330] There was no difference in fat mass at week 0 between these groups.At the 4th, 8th and 12th weeks, the fat percentage using high fat diet in group C was lower with HH04 tube feeding compared with group B. The results show that lower weight gain is caused by lower fat mass increase.Compared with group B, group A similarly showed that fat percentage was significantly lower at the 4th, 8th and 12th weeks.At any time point during the entire research phase, the fat percentage of group D and group B did not show differences.Compared with group B, group A was at the 4th, 8th and 12th weeks, and the absolute fat grams of group C at the 8th and 12th weeks were significantly lower.At all measurement time points, group D had similar absolute fat mass with group B.

[0331] At week 0, the lean mass (percentage of total body weight) was equal between the groups. Compared with group B, the lean mass percentage of group C and group A increased significantly at weeks 4, 8, and 12. These results further support that the lower weight gain is specific to the lower fat mass gain. These results support the conclusion that HH04 has a significant effect on body composition and that daily administration of this strain can reduce fat content.

[0332] Results - Effects of Lactobacillus strains on glucose regulation and intestinal permeability.

[0333] 5h fasting blood glucose was significantly lower in Group A and Group C compared to Control Group B. Insulin levels after 5h fasting were significantly lower in Group A and Group C compared to Group B. Group D showed no difference in fasting blood glucose or insulin levels compared to Group B.

[0334] Throughout the oral glucose tolerance test, blood glucose levels in Group A were lower than those in Group B. At 30, 60, and 90 min after glucose challenge, Group C showed lower blood glucose levels than Group B, indicating better glucose tolerance. The area under the curve of the oral glucose tolerance test similarly showed that glucose tolerance was generally improved in Group A and Group C. Group D showed neither improved blood glucose values ​​nor a lower area under the curve during the oral glucose test.

[0335] Compared with group B, group C has lower insulin secretion of glucose stimulation in the first 15min after glucose challenge, which shows that insulin sensitivity improves after oral gavage HH04. On the contrary, compared with group B, the insulin level of group A after oral glucose challenge does not show statistical difference. In the first hour of test, after glucose challenge, the insulin secretion of group A is less than that of group B, and group D is no different from group B in the whole test. The area under the curve of insulin level during oral glucose tolerance test shows that there is a significant difference between group A and group B, while the remaining groups are no different.

[0336] Results - Effect of Lactobacillus strains on tissue weight

[0337] The liver weights of Group A and Group C were lower than those of Group B, respectively.

[0338] The iWAT mass, ie, subcutaneous fat stores, was lower in groups A and C compared with group B, whereas there was no difference in group D.

[0339] The mWAT mass, i.e., visceral fat stores, was significantly lower in Groups A and C compared with Group B. No differences were observed between Groups B and D.

[0340] There were no differences in brain and heart weights between any of the groups, independent of diet or Lactobacillus strain administration.

[0341] Results - Effects of Lactobacillus strains on lipid profiles and biomarkers of liver health

[0342] Alanine aminotransferase (ALT), a common biomarker of liver damage, was lower in group C treated with HH04 compared to the disease control group B.

[0343] Aspartate aminotransferase (AST), a surrogate liver disease biomarker, varied less between the groups compared with ALT levels but trended toward being lower in HH04 (Group C) compared with the disease control group B.

[0344] There were no differences in circulating triglyceride levels between the groups.

[0345] Total circulating cholesterol levels were elevated in disease controls (Groups B and C) and decreased by HH04 or HH05 treatment (Groups C and D) compared to healthy controls.

[0346] Results - Effects of Lactobacillus strains on hepatic lipid accumulation

[0347] The accumulation of triglycerides and cholesterol in liver tissue is an indicator of obesity and non-alcoholic fatty liver disease (NAFLD), as observed in the increased lipid levels found in Group B compared to Group A. Both triglyceride and cholesterol levels were reduced in Group C compared to Group B, while Group D had similar lipid accumulation.

[0348] Results - Effects of Lactobacillus strains on intestinal inflammation

[0349] The classical pro-inflammatory cytokine interferon gamma (IFNγ) was increased in group B compared with group A. IFNγ levels were decreased in group C compared with group B. The anti-inflammatory cytokine IL-10 was increased in group B compared with group A.

[0350] Results - Effects of Lactobacillus strains on the composition of the intestinal microbiota

[0351] HH04 DNA was only found in mice treated with this Lactobacillus strain, and the amount in feces and small intestinal samples was similar. A small amount of HH05 DNA was found in fecal samples of mice treated with vehicle control and HH04, but it was much more abundant in mice treated with HH05. In the small intestinal contents, HH05 was found only in mice treated with this Lactobacillus strain. The overall fecal microbiota composition is greatly affected by diet. The fecal microbiota composition of mice treated with HH05 is clustered between HFD-fed mice and LFD-fed mice treated with vehicle, and the mice treated with HH04 have fecal microorganisms similar to the control. Diet also greatly affects the microbiota composition of the small intestine, and the mice treated with HH04 and HH05 are all clustered away from the control and therefore have different microbial compositions. Indicator Lactobacillus strain treatment can moderately regulate the intestinal microbiota composition.

[0352] in conclusion

[0353] When a classic high-fat diet was used, Lactobacillus strain HH04 reduced the gain in body weight and fat mass, while increasing the percentage of lean mass. In addition, HH04 reduced 5-h fasting blood glucose and insulin levels and increased glucose tolerance and insulin sensitivity compared with high-fat controls. Lactobacillus strain HH04 also reduced the mass of subcutaneous fat storage iWAT and visceral fat storage mWAT as well as liver lipid accumulation, and shifted intestinal inflammation toward a more anti-inflammatory profile.

[0354] When combined with a classic high-fat diet, HH05 reduced circulating cholesterol levels and altered the intestinal microbiota.

[0355] Example 11 Effect of Lactobacillus strains in a diet-induced mouse model fed a modified high-fat diet

[0356] This study was designed to demonstrate that metabolic health, such as pre-diabetes improvements, was achieved with three Lactobacillus strains tested (HH02, HH04, and HH05) in a diet-induced obesity (DIO) mouse model using a modified Western diet designed to induce inflammation similar to the human obesity phenotype. This study was identical to the study shown in Example 10, except that the diet and Lactobacillus strain HH02 were added. Study Overview Fig.21 as shown in .

[0357] Research Overview:

[0358] The study was designed and conducted as described in Example 10, except that Group E was subsequently added and the diet was altered to induce an obese phenotype characterized by enhanced inflammation:

[0359]

[0360] A modified Western diet (HFD) contains 43% of energy from fat (milk fat), 42% of energy from carbohydrates (mainly sucrose and fructose), and 15% of energy from protein (mixed sources) (D12079 mod*, Ssniff Special Diets, Germany (Ssniff Germany)

[0361] A low-fat reference diet matched with modified WD (Ssniff Special Diet, Germany) Germany)) contains 14% of its energy from fat.

[0362] Sampling and Measurement:

[0363] Weight development and body composition Fig.31 Shown in. Mean values ​​of 9-12 mice per group are shown: A) Body weight after random assignment and before starting the experimental diet and treatment. B) Weight development of individual groups after starting the experimental diet and gavage treatment. C) Fat mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points. D) Fat mass (grams), which is depicted as group means with connecting lines. E) Lean mass (grams) obtained by magnetic resonance scan at the depicted time points. F) Body fluid mass (% of total body weight) obtained by magnetic resonance scan at the depicted time points.

[0364] Glucose tolerance test and glucose-stimulated insulin secretion

[0365] Oral glucose tolerance test was carried out in the 10th week of experimental scheme.Mice were fasted for 5h at 8AM and gavaged at 11AM.Before oral gavage with 1.5 μg dextrose / g lean mass, 5h fasting blood glucose measurement (OneTouch VarioFlex, LifeScan company) and blood sampling from tail vein were performed.The blood glucose from tail vein puncture was measured at the time point 0,15,30,60,90 and 120min after dextrose excitation, and the blood sample for insulin and c-peptide was placed in the capillary (Sarstedt company (Sarstedt)) processed by EDTA at the time point 0,15,30,60 and 120min after excitation.Mice received 0.5mL normal saline (Hospira company) to make mice rehydration after this program.

[0366] Effects of Lactobacillus strains on glucose regulation and intestinal permeability Fig.32 Shown in: A) 5h fasting blood glucose after 10 weeks of WD feeding and daily gavage treatment. The bar graph shows the mean of 9-12 mice per group. B) 5h fasting insulin levels after 10 weeks of WD feeding and daily gavage treatment. The bar graph shows the mean of 9-12 mice per group. C) Oral glucose tolerance after 10 weeks of WD feeding and daily gavage treatment, which shows absolute blood glucose values. D) As C, but depicting the change in blood glucose relative to baseline during the challenge. E) Plasma levels of endogenous insulin response to glucose tolerance test. F) C-peptide levels during glucose tolerance test.

[0367] Necropsy and tissue harvest

[0368] Autopsy was performed after 12 weeks of the experimental protocol. Mice were fasted from 8AM and gavaged at 11AM. Euthanasia was performed in alternating order, and one mouse was taken and cardiac puncture was performed using a 25G needle and 1mL syringe coated with EDTA (Sigma-Aldrich). The weight was measured, and the tissue was immediately frozen in liquid nitrogen and stored at -80°C. The tissue was dissected by the same operator and all mice were taken in the same order.

[0369] Effect of Lactobacillus strains on tissue weight Fig.33 As shown in the figure, where the bar graph represents the mean of 9-12 mice in each group: A) liver weight (g), B) inguinal white adipose tissue (iWAT) weight, C) retroperitoneal white adipose tissue (rpWAT) weight, D) mesenchymal white adipose tissue (mWAT) weight, E) intrascapular brown adipose tissue (BAT) weight, and F) gastrocnemius muscle (Gastroc) weight.

[0370] Plasma biochemical analysis

[0371] Effects of Lactobacillus strains on plasma biomarkers and lipid levels of liver injury Fig.34 As shown in, where the bar graph represents the mean of 9-12 mice per group. These analyses were performed by the medical biochemistry department at the Heart and Lung Institute of Laval University according to locally developed procedures that met the highest standards for processing and analyzing human and animal samples. A) Alanine aminotransferase (ALT) biomarker of liver damage, B) Aspartate aminotransferase (AST) biomarker of liver damage, C) plasma triglyceride levels, D) plasma total cholesterol levels.

[0372] Hepatic lipid accumulation and activation of insulin signaling pathways in liver tissue

[0373] The liver of each mouse was homogenized in liquid nitrogen by cryo-grinding the tissue with a mortar and pestle while keeping it frozen and transferred to a fresh tube. Lipids were extracted from 50 mg of powdered tissue. The effect of Lactobacillus strains on hepatic lipid accumulation was measured by calorimetry and as Fig.35 The bar graphs show the mean values ​​of 9-12 mice in each group. A) The total amount of triglycerides in liver tissue, and B) The total amount of cholesterol in liver tissue.

[0374] The protein levels of phosphorylated (activated) Akt and total Akt in liver tissues of 3-4 insulin-stimulated mice per group (Groups A, B, C, E) were quantified by Western blotting using pS473 Akt and pan-Akt antibodies (Cell Signaling) and quantified using BioRad software. Effect of Lactobacillus strains on sensitivity to insulin stimulation in Akt pathway activation Fig.35 Shown: C) Ratio of activated Akt (pS473 Akt) to total Akt protein levels in liver tissue expressed in arbitrary units.

[0375] Tissue cytokine quantification

[0376] Cytokine levels were measured in homogenized liver and colon tissues using the Bio-Plex 200 Luminex system according to the manufacturer's instructions. Fig.36As shown in the figure, the bar graphs represent the mean values ​​of 9-12 mice in each group. A) Tumor necrosis factor alpha (TNFa) level in liver tissue, B) Interferon gamma (IFNy) level in liver tissue, C) Tumor necrosis factor alpha (TNFa) level in colon tissue, D) Interferon gamma (IFNy) level in colon tissue.

[0377] Intestinal microbiota analysis

[0378] Bacterial DNA was extracted from frozen stool samples, and the hypervariable V3-V4 16S rRNA region was amplified and sequenced using the Illumina MiSeq platform.

[0379] Sequence data were preprocessed, tags were identified and trimmed, paired-end reads were merged, truncated with a quality score of 4, and at least 100 bp overlap was required, with the merged read length between 300 and 600 bp in length. Sequences were strictly deduplicated, and clusters with less than 5 sequences were discarded. Sequences were clustered at 97% sequence similarity by using the most abundant strictly deduplicated reads as centroids and discarding suspicious chimeras based on internal comparisons. Taxonomic assignment of OTUs was performed using the database in the Ribosomal Database Project.

[0380] Effects of Lactobacillus strains on the composition of the intestinal microbiota Fig.37 As shown in , the figure shows the principal coordinates analysis (PCoA) of the weighted UniFrac distances of the fecal microbiota composition, where the large dots indicate the mean of each group and the small dots indicate individual samples.

[0381] Results - Effect of Lactobacillus strains on weight development

[0382] At the beginning of the study, the groups had similar body weights before starting daily treatment and experimental diets by oral gavage. During the study period, the groups treated with HH04 or HH05 gained slightly less weight when fed the modified WD, while the group treated with HH02 gained less weight during the study.

[0383] At the beginning of the study period, all groups had similar % fat, and feeding the modified WD increased % fat. During weeks 4, 8, and 12 of the study, groups treated with HH04, HH05, or HH02 had slightly lower % fat, while lean mass and fluid mass did not change in the control group.

[0384] Results - Effects of Lactobacillus strains on glucose regulation

[0385] When mice were fed the modified WD for 10 weeks without Lactobacillus strain treatment, blood glucose levels increased after 5 hours of fasting. However, the corresponding 5h fasting insulin levels were slightly reduced in mice treated with HH04 and significantly reduced in mice treated with HH05 or HH02, indicating that glucose homeostasis was improved by these treatments.

[0386] During the oral glucose challenge, there were small improvements in the groups treated with Lactobacillus strains, most notably in the group treated with HH02. The corresponding glucose-stimulated insulin secretion (GSIS) was greatly reduced in mice treated with HH02 or HH05, indicating improved insulin sensitivity, while it was slightly reduced in the HH04 treated group. C-peptide levels further supported the reduced insulin production during the glucose challenge, particularly by strains HH02 and HH05, as well as some effects from strain HH04.

[0387] Results - Effect of Lactobacillus strains on tissue weight

[0388] Liver weight was greatly increased by feeding modified WD, which was reduced by treatment with Lactobacillus strains HH02, HH04 or HH05. Treatment with Lactobacillus strains tended to reduce various white adipose tissues, which was most obvious in mice treated with HH02. Brown adipose tissue tended to decrease in the group treated with HH04, while muscle mass did not differ by treatment with any of the Lactobacillus strains.

[0389] Results - Effects of Lactobacillus strains on lipid profiles and biomarkers of liver health

[0390] ALT, a marker of liver damage, was greatly increased by feeding modified WD. This was improved by treatment with Lactobacillus strains, most significantly with HH02. Similar observations were made for another marker of liver damage, AST. In mice treated with HH05 or HH02, respectively, the levels of circulating triglycerides and cholesterol were reduced. Treatment with HH02, HH04 or HH05 reduced the accumulation of triglycerides and cholesterol in liver tissue observed in mice fed with modified WD. In the liver tissue of mice fed with modified WD, the activation of the Akt pathway observed by the increase in the ratio of phosphorylated Akt to total Akt protein levels was reduced. As observed by increased Akt activation, HH04 or HH02 treatment offset this indication of insulin resistance in the liver.

[0391] Results - Effects of Lactobacillus strains on inflammation in liver and intestinal tissue

[0392] In the absence of Lactobacillus strain treatment, mice fed the modified WD had increased levels of proinflammatory cytokines TNFa and IFNy in liver tissue. Mice treated with HH05 or HH02 had reduced cytokine levels in colon tissue.

[0393] Results - Effects of Lactobacillus strains on the composition of the intestinal microbiota

[0394] The overall fecal microbiota composition was greatly affected by diet. The fecal microbiota composition of mice treated with HH02, HH04 or HH05 clustered and separated from controls, but this effect was smaller than the distance of LFD and WD control groups. This indicates that these Lactobacillus strain treatments induce a moderate effect on the intestinal microbiota composition.

[0395] in conclusion

[0396] This study indicates that diet affects the efficacy of Lactobacillus strains. In the current study where mice were fed a modified WD that promotes inflammation, mice treated with HH04 showed little effect on body and liver weight loss, however, HH04 treated mice secreted less insulin during a glucose tolerance test, indicating improved insulin sensitivity. This was further supported by a reduction in liver damage markers measured in plasma, reduced liver lipid and cholesterol accumulation, and enhanced insulin signaling in liver tissue.

[0397] Treatment with HH05 significantly reduced fasting and glucose-stimulated insulin, indicating improved insulin sensitivity. In addition, HH05 treatment reduced liver weight, liver damage markers, liver triglyceride and cholesterol levels. HH05 treatment also reduced circulating cholesterol levels and reduced colon inflammation.

[0398] During the study, HH02-treated mice showed reduced weight gain. During the glucose tolerance test, mice treated with HH02 recovered their baseline blood glucose faster than the control group, and fasting and glucose-stimulated insulin levels were greatly reduced, indicating greatly improved insulin sensitivity. In addition to reducing the levels of circulating cholesterol and triglycerides, HH02 treatment also reduced liver and mesenchymal adipose tissue weight. Markers of liver damage were also greatly reduced and were associated with increased insulin sensitivity in liver tissue. In colon tissue, pro-inflammatory cytokine levels were reduced.

[0399] Example 12 - Reproducibility of probiotic effects in two different diet-induced mouse models

[0400] This study aimed to demonstrate the reproducibility of three probiotic test strains (HH02, HH04 and HH05) in improving metabolic health, such as prediabetes, in two different diet-induced obesity (DIO) mouse models using a classic HFD and a modified Western diet. This study was identical to the studies presented in Examples 10 and 11, which aimed to reproduce the observed findings. In addition, the performance of HH02 in a classic HFD model was tested for the first time. Fig.21 as shown in .

[0401] Research Overview:

[0402] The study was designed and conducted as described in Examples 10 and 11, involving the same probiotic treatments in mice simultaneously fed either of two high-fat diets:

[0403]

[0404] The high-fat / sucrose diet (HFD) contained 45% of energy from fat (87% lard, 13% soybean oil), 35% of energy from carbohydrates (mainly sucrose and maltodextrin), and 20% of energy from protein (casein) (D12451, Ssniff Special Diets, Germany). Germany)

[0405] The modified Western diet (modified WD) contained 43% of energy from fat (milk fat), 42% of energy from carbohydrates (mainly sucrose and fructose), and 15% of energy from protein (mixed sources) (D12079 mod*, Ssniff Special Diets, Germany (Ssniff Germany)

[0406] A low-fat reference diet matched with modified WD (Ssniff Special Diet, Germany) Germany)) contains 14% of its energy from fat.

[0407] Sampling and Measurement:

[0408] Weight development and body composition Fig.38Shown in. Shows the mean of 8-9 mice per group: A) During the study (AE), the body weight of the group fed LF or HFD diet after random assignment and before the start of the experimental diet and treatment. B) During the study (A, FI), the body weight of the group fed LF or modified WD diet after random assignment and before the start of the experimental diet and treatment. C) After the start of the experimental diet and tube feeding treatment, the weight development of the individual groups (AE) fed LFD or HFD. D) After the start of the experimental diet and tube feeding treatment, the weight development of the individual groups (A, FI) fed LFD or modified WD. E) The fat mass (% of total body weight) obtained by magnetic resonance scanning at the depicted time point for the group (AE) fed LFD or HFD. E) The fat mass (% of total body weight) obtained by magnetic resonance scanning for the group (A, FI) fed LFD or modified WD at the depicted time point. G) The fat mass (grams) of the group (AE) fed LFD or HFD, which is depicted as the group mean with connecting lines. H) Fat mass (g) of the groups fed LFD or modified WD (A, FI) depicted as group means with connecting lines. I) Lean mass (g) obtained from magnetic resonance scans of the groups fed LFD or HFD (AE) at the depicted time points. J) Lean mass (g) obtained from magnetic resonance scans of the groups fed LFD or modified WD (A, FI) at the depicted time points. Glucose tolerance test and glucose-stimulated insulin secretion

[0409] Oral glucose tolerance test was carried out in the 10th week of experimental scheme.Mice were fasted for 5h at 8AM and gavaged at 11AM.Before oral gavage with 1.5 μg dextrose / g lean mass, 5h fasting blood glucose measurement (OneTouch VarioFlex, LifeScan company) and blood sampling from tail vein were performed.The blood glucose from tail vein puncture was measured at the time point 0,15,30,60,90 and 120min after dextrose excitation, and the blood sample for insulin and c-peptide was placed in the capillary (Sarstedt company (Sarstedt)) processed by EDTA at the time point 0,15,30,60 and 120min after excitation.Mice received 0.5mL normal saline (Hospira company) to make mice rehydration after this program.

[0410] Effects of probiotics on glucose regulation and intestinal permeability Fig.39Shown in: A) 5h fasting blood glucose after 10 weeks of HFD feeding and daily gavage treatment. The bar graph shows the mean of 9-12 mice in each group (AE) fed with LFD or HFD. B) 5h fasting blood glucose after 10 weeks of HFD feeding and daily gavage treatment. The bar graph shows the mean of 9-12 mice in each group (A, FI) fed with LFD or modified WD. C) 5h fasting insulin levels after 10 weeks of HFD feeding and daily gavage treatment. The bar graph shows the mean of 9-12 mice in each group (AE) fed with LFD or HFD. D) 5h fasting insulin levels after 10 weeks of HFD feeding and daily gavage treatment. The bar graph shows the mean of 9-12 mice in each group (A, FI) fed with LFD or modified WD. E) Oral glucose tolerance after 10 weeks of HFD feeding and daily gavage treatment, showing the change in blood glucose relative to baseline during the challenge period in the groups fed LFD or HFD (AE). F) Oral glucose tolerance after 10 weeks of HFD feeding and daily gavage treatment, showing the change in blood glucose relative to baseline during the challenge period in the groups fed LFD or modified WD (A, FI). G) Plasma levels of endogenous insulin response to glucose tolerance test in the groups fed LFD or HFD (AE). H) Plasma levels of endogenous insulin response to glucose tolerance test in the groups fed LFD or modified WD (A, FI). I) C-peptide levels during the first 30 minutes of glucose tolerance test in the groups fed LFD or HFD (AE). J) C-peptide levels during the first 30 minutes of glucose tolerance test in the groups fed LFD or modified WD (A, FI).

[0411] Results - Effect of Probiotics on Weight Development

[0412] At the beginning of the study, the groups were similar in body weight before starting daily treatment and experimental diets by oral gavage. During the study, the group fed HFD (as in Example 10) and treated with HH04 gained less weight. The groups fed modified WD did not reduce weight gain regardless of the probiotic treatment. As observed in Example 10, the groups fed HFD and treated with HH04 reduced fat mass gain. When the mice were fed modified WD, no probiotic treatment reduced the gain of fat mass. Probiotic treatment did not affect lean mass gain regardless of diet.

[0413] Results - Effects of probiotics on glucose regulation

[0414] Mice fed HFD or modified WD had increased blood glucose levels after 5 hours of fasting, and this was not affected by probiotic treatment. When mice were fed HFD, insulin levels after 5 hours of fasting increased, while in mice treated with HH04, they decreased. Mice fed modified WD already showed increased fasting insulin levels. In addition, mice treated with HH05 had significantly reduced fasting insulin levels, with mice treated with HH02 having greatly reduced fasting insulin values.

[0415] After 10 weeks of feeding HFD, during the oral glucose tolerance test, similar to the findings in Example 10, mice treated with HH04 had reduced blood glucose values ​​during the test, which showed improved glucose tolerance. Mice fed modified WD performed poorly in the glucose tolerance test, but this could be reduced by treatment with HH05 or HH02. In the case of feeding HFD, in mice fed HH04, glucose-stimulated insulin secretion observed by insulin and c-peptide levels during the test was reduced compared to their control group. When the modified WD was fed to mice treated with HH02, the levels of both insulin and c-peptide were reduced, indicating that the insulin sensitivity of these mice was significantly improved. Mice fed modified WD and treated with HH05 showed reduced c-peptide levels compared to controls.

[0416] in conclusion:

[0417] The beneficial effects of different probiotic strains depend on the diet fed to the mice. The effects of each probiotic strain on body weight and glucose regulation observed in Examples 10 and 11 using two experimental diets were generally reproduced in this study. In summary, strain HH04 showed a body weight and fat mass reduction effect on a normal HFD, which translated into improved glucose regulation. When mice were fed a modified WD that promoted inflammation, strain HH05 showed beneficial effects on different glucose tolerance test readings. When mice were fed a modified WD that promoted inflammation, strain HH02 showed a great reduction in insulin resistance, thereby improving glucose regulation.

[0418] Biomaterial Deposit

[0419] The following biological material has been deposited under the terms of the Budapest Treaty with the Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany) and has been given the following accession numbers:

[0420] Lactobacillus reuteri (HH01); Accession number: DSM 17648; Deposit date: 12-10-2005; Country of origin: Germany.

[0421] Lactobacillus reuteri (HH02); Accession number: DSM 32910; Deposit date: 04-09-2018; Country of origin: Germany.

[0422] Lactobacillus rhamnosus (HH03); Accession number: DSM 32911; Deposit date: 04-09-2018; Country of origin: Germany.

[0423] Lactobacillus paracasei subsp. firmus (HH04); Accession number: DSM 32851; Deposit date 04-07-2018; Country of origin: Germany.

[0424] Lactobacillus paracasei subsp. paracasei (HH05); Accession number: DSM 32853; Deposit date: 04-07-2018; Country of origin: Germany.

[0425] These strains are deposited under the following conditions, which ensure that the right to use the culture can be obtained during the indefinite period of authorization determined by foreign patent laws. The deposit represents the substantially pure culture of the deposited strain. It is necessary to provide the deposit by the foreign patent laws of some countries, and submit a copy of the subject application in these countries, or its subsequent text. However, it should be understood that the availability of the deposit does not constitute a license for the practice of the subject of the present invention for the patent right approved by government action.

Claims

1. A Lactobacillus strain having the ability to improve metabolic health, wherein the strain is a strain deposited under DSM 32910.

2. The Lactobacillus strain according to claim 1, wherein the strain has the ability to alleviate prediabetic symptoms.

3. The Lactobacillus strain according to claim 1 or 2, wherein the strain has the ability to improve or alleviate one or more pre-diabetic symptoms selected from the group consisting of: insulin resistance, hyperglycemia, low-grade inflammation and intestinal barrier function.

4. The Lactobacillus strain according to claim 1 or 2, wherein the strain has the ability to prevent and / or alleviate type 2 diabetes.

5. The Lactobacillus strain according to claim 1 or 2, wherein the strain has the ability to prevent and / or alleviate one or more symptoms of type 2 diabetes selected from the group consisting of: insulin resistance, hyperglycemia, low-grade inflammation and intestinal barrier function.

6. The Lactobacillus strain according to claim 1 or 2, wherein the strain has the ability to improve a subject's postprandial blood sugar response and / or reduce a subject's insulin resistance.

7. The Lactobacillus strain of claim 1 or 2, wherein the strain survives in the gastrointestinal (GI) tract.

8. The Lactobacillus strain according to claim 1 or 2, wherein the strain is selected in a screening protocol comprising at least one screening method selected from the group consisting of: PBMC screening, TEER assay, dendritic cell assay, adipocyte assay, L cell assay, bile salt hydrolase assay and MIC assay.

9. The Lactobacillus strain according to claim 1 or 2, wherein the strain is selected in a screening protocol comprising at least two screening methods selected from the group consisting of: PBMC screening, TEER assay, DC assay, adipocyte assay, L cell assay, bile salt hydrolase assay and MIC assay.

10. The Lactobacillus strain according to claim 1 or 2, wherein the strain is selected in a screening protocol comprising a screening method selected from the group consisting of: PBMC screening, TEER assay, TEER assay with addition of pro-inflammatory cytokines, and DC assay.

11. The Lactobacillus strain according to claim 1 or 2, wherein the IL10 / IL12 ratio is higher than 20 in a PBMC assay.

12. The Lactobacillus strain according to claim 1 or 2, wherein the IL10 / IL12 ratio is higher than 30 in a PBMC assay.

13. The Lactobacillus strain according to claim 1 or 2, wherein the IL10 / IL12 ratio is higher than 50 in a PBMC assay.

14. The Lactobacillus strain according to claim 1 or 2, wherein the strain is capable of reducing IL12 levels in a DC assay stimulated with a pro-inflammatory cocktail by at least 20% compared to a pro-inflammatory stimulated control.

15. The Lactobacillus strain according to claim 1 or 2, which is a Lactobacillus reuteri strain.

16. A composition comprising one or more Lactobacillus strains according to any one of claims 1 to 15.

17. A composition according to claim 16, wherein the composition comprises the Lactobacillus strain deposited as DSM 32910 and one or more Lactobacillus strains selected from the group consisting of strains deposited as DSM 17648, DSM 32911 and DSM 32853.

18. The composition according to claim 16, wherein the further comprised microbial strain is selected from a strain of the genus Lactobacillus rhamnosus and a strain belonging to the species of the genus Akkermansia.

19. The composition of claim 16, wherein the composition further comprises cellular components, metabolites and / or secreted molecules thereof.

20. The composition of claim 19, wherein the cellular components, metabolites and / or secreted molecules are derived from the retentate of the culture after fermentation and centrifugation.

21. The composition of claim 16, wherein the strains are dried.

22. The composition of claim 16, wherein the strains are spray dried.

23. The composition of claim 16, wherein the strains are freeze-dried.

24. The composition according to claim 16, wherein the composition comprises 10 6 -10 14 Colony forming units (CFU) per gram of the composition.

25. The composition of claim 16, wherein the composition is formulated into unit dose formulations, each of which comprises 10 6 -10 14 Colony forming units (CFU) / unit.

26. The composition according to claim 16, wherein the composition comprises 10 6 with 10 14 The total number refers to the total amount of living cells and dead cells.

27. The composition according to claim 16, wherein the composition comprises 10 6 with 10 14 The total number refers to the total number of living cells.

28. The composition according to claim 16, wherein the composition comprises 10 6 with 10 14 The total number refers to the total number of dead cells.

29. The composition of claim 16, wherein the composition is a pharmaceutical composition comprising a Lactobacillus strain and one or more pharmaceutically acceptable excipients.

30. Use of a Lactobacillus strain according to any one of claims 1 to 15 or a composition according to any one of claims 16 to 29 for the preparation of a food, food supplement or food composition for preventing or reducing weight gain in an individual on a high fat diet.

Images