Lactobacillus fermentum ME-3 – an antimicrobial and antioxidative probiotic

Figures & data

Table I.  Main endogenous factors determining the composition of the intestinal microbiota (IMB) [1], [2], [27–30].

1. Mucosal	2. Luminal
Specific	pH, gases
IgA	Secretion of digestive enzymes
Specificity of epithelial receptors	Peristalsis
Non-specific	Antagonism of microbiota
Complement	Lysozyme
Lysozyme
Interferon
Mucin
Dendritic cells/Toll-like receptors
Colonization resistance

Figure 1.  Prevalence (%) of lactobacilli among Estonian and Swedish children.

Table II.  Evidence-based probiotic lactobacilli according to randomized double-blind placebo-controlled studies (adapted from 68,69).

Strain affiliation	Licensing enterprise	Published clinical evidence (reference no.)
Lactobacillus acidophilus La5	Chr. Hansen, Denmark	Suppression of Helicobacter pylori infection [78]
L. acidophilus L1	Campina Melkunie, Holland	Blood cholesterol-lowering effect [79]
Lactobacillus johnsonii LA1	Nestle, Switzerland	Suppression of Helicobacter pylori colonization in children [80]
Lactobacillus gasseri OLI 27168	Meij Milk Products, Tokyo, Japan	Suppression of Helicobacter pylori infection [81]
Lactobacillus casei Shirota	Yakult, Japan	Reduction of constipation, reduced proteolytic activity of IMB [82], [83]
L. casei DN114001	Danone, France	Reduction of winter infections in elderly [84], diarrhea and respiratory infections in adults [85], [86].
Lactobacillus paracasei LP-33	Un-President Enterprise Corp., Tainan, Taiwan	Reduction of allergic rhinitis [87]
Lactobacillus rhamnosus GG ATCC 53103	Valio, Finland	Reduction of diarrhoeal infections and allergy [88–92]; increased expression of immune response genes [93]
L. rhamnosus GR-1 and L. reuteri RC-14	Chr. Hansen, Denmark	Reduction of diarrhoeal infections in children [94]
L. rhamnosus HN001	Danisco, Denmark	Enhanced immunity of consumers [95]
Lactobacillus plantarum 299V	Probi, Sweden	Reduction of infectious complications in transplantation patients; decrease of intestinal permeability [96]
Lactobacillus brevis CD2	VSL Pharmaceuticals, Inc., Fort Lauderdale, FL, USA	Reduction of Helicobacter pylori infection, reduction of levels of polyamines in gastric mucosa [97]
Lactobacillus reuteri ATCC 55730	BioGaia, Sweden	Reduction of diarrhoeal infections, enhancement of consumers’ immunity [98], [99]
Lactobacillus fermentum ME-3 DSM 14241	AS Tere, Estonia	Anti-atherogenic effect due to increase of antioxidative activity indices, decrease of GSSG/GSH ratio and oxLDL of sera [100–102]

IMB, intestinal microbiota; GSSG/GSH ratio, glutathione redox ratio; oxLDL, oxidized low-density lipoprotein.

Figure 2.  Lactobacillus fermentum ME-3 (DSM 14241) [118], [119]. (a) Light microscopy, Gram stain, magnification×1000. (b) Fluorescent in situ hybridization (FISH). Probe: Lab 158, Lactobacilli + enterococci according to Franks et al. [41].

Figure 3.  AP-PCR fingerprints for different L. fermentum strains. The two lanes of strain ME-3 have been generated using two DNA samples that were extracted with a time interval of 6 months. Lanes 2 and 3 contain DNA of L. fermentum strains isolated from the same person. Lane M contains a 100 bp DNA ladder.

Figure 4.  Phylogenetic tree based on 16S rRNA sequencing showing the relationship of L. fermentum ME-3 to the closest related lactobacilli. Analysis was performed with the ARB software package.

Figure 5.  Gas chromatography of polyamines of L. fermentum ME-3 in the decarboxylation medium with ornithine [123].

Table III.  Production of short chain fatty acids (SCFAs), ethanol, polyamines, conjugated linoleic acid (CLA) and nitric oxide (NO) by L. fermentum ME-3 DSM 14241 in comparison with Lactobacillus plantarum DSM 21380.

Indices*	ME-3	DSM 21380^1
Short chain fatty acids (mg/ml) 24–48 h incubation
Lactic acid	10.6–11.1	10.1–11.6
Acetic acid	0.8–0.9	0.08–0.1
Succinic acid	1.8–1.9	0.07–0.07
Ethanol	9.8–7.5	0
Polyamines (µg/ml) 96 h incubation
Decarboxylation of arginine
Putrescine	0	0
Cadaverine	0.6	0
Decarboxylation of glutamine
Putrescine	0.8	0
Cadaverine	0.5	0
Decarboxylation of ornithine
Putrescine	1.3	0.5
Cadaverine	0	0.6
Conjugated linoleic acid (mg/l)	12.4	39.9
NO production (µM)	1.2±0.2	2.6±0.8
H2O2 Production (µM)	484±200	196±129

*Microaerobic environment.

Figure 6.  Recovery of L. fermentum ME-3 in faecal samples of all volunteers after consumption of ME-3 fermented goat's milk.

Figure 7.  A net of prooxidants and the potency of antioxidant defence system normally balanced in the human body. (a) A summary effect of oxidative stressors and potency of antioxidant defence system of the human body are normally balanced. An imbalance leads to oxidative stress. PUFA, polyunsaturated fatty acids; SOD, superoxide dismutase; GSHPx, glutathione peroxidase; CAT, catalase; HO1, haem oxygenase; GSH, reduced glutathione. (b) Oxidative stress causes the production of oxidized LDL (oxLDL), which is a potent atherogenic and inflammatory agent. Strain ME-3 lowers the level of oxLDL. LDL, low-density lipoprotein; CVD, cardiovascular diseases.

Table IV.  Antioxidativity-related properties and effects of strain ME-3.

Property/effect	Experimental (ES), animal (AS), human (HS) study (reference nos)
Expression of Mn-SOD, prolonged survival time in presence of high H2O2, scavenging of superoxide and hydroxyl radicals	ES [156]
Characterized by high TAA and TAS values	ES [156], [157]
Containing of GSH and related antioxidative enzymes	ES [157], [158]
Working as natural antioxidant in soft cheese spreads with different fats	ES [159]
Maintaining its high TAA during production of probiotic cheese	ES [136]
Removal effect of metals (prooxidants) from environment	ES [160]
Elevation of blood TAS or TAA and TAA in the gut mucosa	HS, AS [100], [102], [157], [161], [162]
Elevation of oxyresistance of LDL	HS [100], [157], [162]
Lowering level of oxLDL	HS [100], [102], [162]
Lowering level of isoprostanes	HS [100], [162], [163]
Lowering the glutathione redox ratio in blood, in the gut mucosa, in skin	HS, AS [100], [102], [157], [161], [164]
Lowering lipid peroxidation in the gut mucosa	AS [161], [164]
Lowering level of BCD-LDL	HS [100], [162], [167]
Positive effects on post-prandial status of OxS, blood lipoprotein status and urine isoprostanes	HS [162], [163]

BCD-LDL, baseline diene conjugates in low-density lipoprotein; GSH, reduced glutathione; H₂O₂, hydrogen peroxide; LDL, low-density lipoprotein; Mn-SOD, manganese superoxide dismutase; oxLDL, oxidized low-density lipoprotein; OxS, oxidative stress; TAA, total antioxidative activity; TAS, total antioxidative status.

Figure 8.  The number of mice with viable Salmonella Typhimurium in ileum, blood and liver. Gr1, Salmonella Typhimurium (ST)-challenged mice; Gr2, ST treated with ofloxacin (OFX); Gr3, ST treated with strain ME-3; Gr4, ST treated with OFX + strain ME-3. ¹p=0.032 Gr1 vs Gr2 ST in ileum; ²p=0.002 Gr1 vs Gr3 and Gr4 ST in ileum; ³p=0.002 Gr1 vs Gr3 and Gr4 ST in liver.

Figure 9.  The number of mice with typhoid nodules in liver and spleen. ⁴p=0.027 Gr1 vs Gr3; ⁵p<0.001 Gr1 vs Gr4; ⁶p=0.023 Gr2 vs Gr4; ⁷p=0.002 Gr1 vs Gr2 and Gr4; ⁸p=0.048 Gr2 vs Gr3 and Gr3 vs Gr4.

Table V.  Indices of oxidative stress (with standard deviations) in the ileum mucosa in mice challenged with S. Typhimurium and treated with ofloxacin and/or the probiotic L. fermentum ME-3.

Experimental groups	LPO (pmol/mg protein)	GSSG/GSH
Salmonella Typhimurium challenged mice (Gr1)	338±46^1;^4	0.26±0.41^3;^5
ST treated with ofloxacin (OFX) (Gr2)	228±41^2	0.26±0.11
ST treated with strain ME-3 (Gr3)	169±11^1;^2	0.16±0.20^3
ST treated with OFX + strain ME-3 (Gr4)	161±27^1;^2	0.17±0.11^3
Control (PBS)	157±24^4	0.11±0.2 ^5

GSSG/GSH, glutathione redox ratio; LPO, lipid peroxides; OFX, ofloxacin; PBS, phosphate-buffered saline; ST, Salmonella Typhimurium. ¹p<0.001 Gr1 vs Gr3 and Gr4; ²p=0.002 Gr2 vs Gr3 and Gr 4; ³p=0.006 Gr1 vs Gr3 and Gr4; ⁴p<0.001 Gr1 vs control; ⁵p<0.003 Gr1 vs control.

Figure 10.  Increase of total faecal counts of lactobacilli in healthy volunteers consuming strain ME-3 in fermented goat milk and in the DBRP probiotic capsule efficacy trial [118]. *p < 0.005 difference from pretreatment values; ‡p < 0.01 difference between ME-3 and control treatments.

Table VI.  Improvement of OxS-related indices of blood sera in the synbiotic DBRP crossover study in healthy volunteers [167].

Blood indices	Baseline	Final synbiotic	Paired t test
TAA%	41±2	42±2	<0.001
oxLDL (ApoB-modified)	132.5±50.5	122.8±45.6	0.047
BDC-LDL (diene conjugates)	15.2±6.1	12.7±4.1	<0.001

BCD-LDL, baseline diene conjugates in low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; TAA, total antioxidant activity.

Figure 11.  Content of iron and diene conjugates in the skin in patients with atopic dermatitis (AD), regularly (3 months) consuming probiotic strain ME-3. *p<0.05 comparing the values before and after consumption.

Table VII.  Clinical and biochemical evaluations of stroke patients: parameters at the baseline (before) and after an application period (after) by means of SSS and FIM scale and biochemical indices (mean±SED) [162].

Parameter	ME-3 group		Placebo group
Clinical parameter	Before	After	Before	After
SSS	33±13	42±12^2	37±12	45±9^1
FIM	21±19	40±23^3	32±16	50±16, ^1,4
Biochemical indices
LDL-cholesterol (mmol/L)	3.9±2.2	3.8±1.9	3.2±0.8	3.2±1.1^4
oxLDL (U/L)	121±35	109±35^1	130±23	128±22^4
DC (µmol/L)	50±9	45±8^3	45±16	45±14
GSSG (µmol/L)	64±16	52±18^2	73±28	71±18^4
GSSG/GSH	0.07±0.01	0.05±0.01^1	0.07±0.02	0.06±0.01
TAA (%)	34±1	46±3^2	37±1	35±41^4

DC, diene conjugates; FIM, Functional Independence Measure; GSSG, oxidized glutathione; GSSG/GSH, glutathione redox ratio; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; SSS, Scandinavian Stroke Scale; TAA, total antioxidant activity. ¹p<0.05, ²p<0.01 and ³p<0.001 as compared to baseline value; ⁴p<0.05 as compared to the ‘After’ values in the ME-3 and placebo groups.

Figure 12.  Increase of oxiresistance of low-density lipoprotein (LDL) particles (minutes) and lowering oxidized-LDL level (absorbance units) after using strain ME-3. Oxidation of LDL is measured on the basis of conjugated dienes at 234 nm.

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