Compositions comprising Lactobacillus plantarum strains in combination with tannin and new Lactobacillus plantarum strains

Abstract

The invention refers to a composition comprising one or more tannase-producing strains of Lactobacillus having the ability to adhere to the human intestinal mucosa in combination with tannin. New tannase-producing strains of Lactobacillus plantarum are a.

Description

This is brake break down tannins. The strains are listed in Table 1 below.

Screening Method

The applied method to detect tannase activity has earlier been described by Osawa and Walsh (1993). The detecting principle is that the breakdown of the tannin, methylgallate, is measured by the following procedure:

The test bacterium is cultured anaerobically on MRSagar (Merck, Darmstadt, Germany) for 2 d at 37° C. and then the cells are harvested and suspended in 5 ml 0.9% (w/v) NaCl. The cell-suspension is centrifuged and the cells re-suspended in 10 ml 0.9% NaCl and the absorbance is measured at 620 nm (0.9% NaCl solution as standard). The cell-suspension is diluted until the absorbance is between 0.1 and 0.6 (spectrophotometer, Pharmacia LKB, Novaspec II). After centrifugation, the cells are re-suspended in 1 ml methylgallate-buffer (3.7 g/l methylgallate [Aldrich Chemical Company, Inc., Milwaukee, Wis., USA], 4.5 g/l NaH₂PO₄, pH=5.0 [sterile filtered]) and the tube is incubated at 37° C. for 24 h. One ml of NaHCO₃-buffer (42 g NaHCO₃ per litre, pH=8.6) is added and the solution is incubated for 1 h at room temperature, before measurement of the absorbance at 440 nm (NaHCO₃-buffer as standard). The colour of the suspension is measured by visual determination.

The colour should be brown or green to be graded as positive tannase activity. A quantitative value of the tannase activity was obtained by the ratio between the absorbance of the cell-suspension (A₆₂₀; amount of cells) at the start of the incubation with methylgallate versus the absorbance after the 24 h incubation with methylgallate (A₄₄₀; coloration of free gallic acids after exposure to oxygen in an alkaline condition).

Results

The result of the screening for Lactobacillus strains possessing tannase activity is shown in Table 1. A majority of the tested strains did not have any tannase activity. However, 11 strains were positive and are presented in Table 1.

TABLE 1
Tannase activity in different Lactobacillus strains.
		Tannase activity*	Quantitative
		(positive or	tannase activity**
Organism	Strain	negative)	(A440/A620)
Lactobacillus	299v	+	6.2
plantarum	DSM 9843
Lactobacillus	LP2	+	4.9
plantarum
Lactobacillus	LP5	+	3.3
plantarum
Lactobacillus	4LF:1	+	6.1
plantarum
Lactobacillus	17LF:1	+	5.4
plantarum
Lactobacillus	HEAL 9	+	6.4
plantarum	DSM 15312
Lactobacillus	HEAL 19	+	7.4
plantarum	DSM 15313
Lactobacillus	HEAL 99	+	6.8
plantarum	DSM 15316
*Positive tannase activity is shown as a green to brown coloration of free gallic acid in the cell-suspension after prolonged exposure to oxygen in an alkaline condition.
**The tannase activity expressed as the ratio between the absorbance of the cell-suspension at 620 nm (A620) at the start of the 24 h incubation with methylgallate versus the absorbance at 440 nm (A440) after the incubation with methylgallate (A440).

Three of the tannase positive L. plantarum strains had a higher tannase activity than the well known probiotic strain Lactobacillus plantarum 299v, DSM 9843, i.e. L. plantarum HEAL 9, L. plantarum HEAl 19 and L. plantarum HEAL 99. They have been isolated from healthy, human intestinal mucosa.

Genotypic Identification by REA

The strains were examined as to the cleavage pattern of the chromosomal DNA, through restriction-endonuclease analysis—REA—method according to Ståhl M, Molin G, Persson A, Ahrné S & Ståhl S, International Journal of Systematic Bacteriology, 40:189-193, 1990, and further developed by Johansson, M-L, et al., International Journal of Systematic Bacteriology 45:670-675, 1995. Schematically REA can be described as follows: Chromosomal DNA from the strains involved in the study were prepared and cleaved by restriction endonucleases. 0.75 μg of each DNA was separately digested at 37° C. for 4 h with 10 units of EcoRI and Hind III; each endonuclease was used separately. The cleaved DNA fragments are separated as to size by gel electrophoresis using submerged horizontal agarose slab gels. The gels consisted of 150 ml of 0.9% agarose (ultrapure DNA grade; low electro-endo osmosis; BioRad Laboratories, Richmond, USA) and were cast as slab gels (150 by 235 mm). 0.2 μg of the High Molecular Weight DNA marker (Bethesda Research Laboratories, MD, USA) together with 0.5 μg of a DNA molecular weight marker VI (Roche, Germany) were used as standards. Minimal band distortion and maximal sharpness were achieved by applying the sample DNA in Ficoll loading buffer (2 g of Ficoll, 8 ml of water, 0.25% bromphenol).

Gels were run at a constant voltage of 40V for 18 h at about 6-8° C. The buffer (89 mM Tris, 23 mM H₃PO₄, 2 mM sodium EDTA, pH 8.3) was recirculated during the running period. Thereafter, the gels were stained for 20 minutes in ethidium bromide (2 μg/ml) and destained in distilled water, visualized at 302 nm with a UV transilluminator (UVP Inc., San Gabriel, USA) and photographed. This way of running the gel electrophoresis gave well distributed and relatively well-separated band down to a molecular weight of 1.2×10⁶.

The results of the analysis are presented in the FIGURE.

Adhesion to HT-29 Cells

In total 32 L. plantarum strains isolated from human mucosa were tested as to adherence to intestinal epithelial cells of human colonic carcinoma cell-line HT-29 with a mannose-specific binding (method as described by Wold, A, et al, Infection and Immunity, October 1988, p. 2531-2537). Cells of the human adenocarcinoma cell line HT-29 were cultured in Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and 50 ig/ml of gentamicin (Sigma Chemical Co., Saint Louis, Mo., USA). A few days after the cells had reached confluence they were detached with EDTA-containing buffer (0.54 mM), washed and suspended in Hank's balanced salt solution (HBSS) at 5×10⁶/ml. The bacteria were harvested, washed and suspended in HBSS at 5×10⁹/ml (2× an optical density of 1.5 at 597 nm). Cells, bacteria and HBSS were mixed in the ratio 1:1:3 and incubated with end-over-end rotation for 30 minutes at 4EC. The cells were washed once with ice cold PBS and fixed with neutral buffered formalin (Histofix, Histolab, Götebrog, Sweden). The number of bacteria attached to each of at least 40 cells was determined using interference contrast microscopy (500× magnification, Nicoll Optophot, with interference contrast equipment, Bergström Instruments, Göteborg, Sweden) and the mean number of bacteria per cell was calculated.

All strains except the three HEAL-strains had values between 0.3-14 (adhesion in salt solution; corresponding values in the presence of methyl-mannoside were 0.5 and 2.4, respectively). Most strains had a value lower than 10. The results are given in Table 2 below.

TABLE 2
		Adhesion to HT-29 cells
		(number of bacteria per cell)
		In salt	In presence of
Organism	Strain	solution	methyl-mannoside
Lactobacillus plantarum	299v	11.7	3.4
	DSM 9843
Lactobacillus plantarum	HEAL 9	20	2.1
Lactobacillus plantarum	HEAL 99	20	2.0
Lactobacillus plantarum	HEAL 19	23	5.0
Lactobacillus plantarum	ATCC 14917T	5.2	2.2
Lactobacillus plantarum	78B	0.3	0.5

Test in Experimental Mouse Model
Method

Fifteen Balb/C mice were divided into five groups (3 mice per group) and fed different combinations of normal food, rose hip powder (rich in tannins) and the tannase positive strain Lactobacillus plantarum 299v. The constituents were mixed with some water to get a mushy consistency. Groups 1 and 2 were given normal mouse food, Group 3 got the normal food supplemented with rose hip powder (1.6 g per day), Group 4 got normal food supplemented with L. plantarum 299v (10¹⁰ bacteria per dose) and Group 5 got normal food supplemented with both the rose hip powder and L. plantarum 299v. The mice were fed once a day for 6-8 days before inducing an ischemia/reperfusion injury. The injury was done according to the following dissection protocol: Mice were given 0.15 ml of Ketamine/Xylazine solution (7.85 mg/ml and 2.57 mg/ml, respectively) subcutaneously for anesthesia. A midline abdominal incision was made and the superior mesenteric artery was occluded using atraumatic vessel loops and hemostat. 1.0 ml PBS was injected into the peritoneal cavity for fluid resuscitation. The artery was occluded for 30 min before the vessel loop and hemostat were removed and the tissue was observed for immediate reperfusion. The abdomen was then closed using a running vicryl 3-0 suture. The animal was allowed to awake from anesthesia and was removed from the warming pad and placed back into the cage. After 4 h and 15 min, the animal was given anesthesia again and tissue and stool samples were obtained in the following order and placed in preweighed tubes: liver tissue, ilium mesentery tissue and cecum stool for bacteriological sampling, and cecum and ilium tissue for colorimetric assay for lipid peroxidaton, and cecum and ilium tissue for histological examination. The samples for bacteriological evaluation were weighed and placed in freezing media and frozen immediately at −70° C. Samples for colorimetric assay (LPO586) were rinsed in PBS, weighed, homogenized, aliquoted and then frozen immediately at −70° C.

Analysis Methods

Bacteriological evaluation was performed by viable count by anaerobic incubation (BBL Gas Pak Plus, Becton Dickinson and Company, Sparks, Md., USA) on Rogosa-agar (Merck, Darmstadt, Germany) at 37° C. for 3 d, VRBD-agar (Merck, Darmstadt, Germany) at 37° C. for 24 h and Brain heart infusion agar (BHI; Oxoid, Basingstoke, Hampshire, England) at 37° C. for 3 d. Viable count on BHI was also done aerobically.

Colorimetric assay for lipid peroxidation was done with the aid of a spectrophotometer and the analysing kit Bioxytech® LPO-586™ (Oxis Research™, Oxis Health Products, Inc., Portland). The analysis was performed in accordance with the description of the manufacturer.

Lipid peroxidation is a well-established mechanism of cellular injury and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydoxyalkenals (HAE) upon decomposition. Measurement of MDA can be used as indicator of lipid peroxidation. LPO-586™ is a colorimetric assay designed to quantify MDA and is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole with MDA at 45° C. One molecule of MDA reacts with two molecules of N-methyl-2-phenylindole to yield a stable chromophore with maximal absorbance at 586 nm.

Results

The lipid peroxidation measured as malondialdehyde (MDA) per g colonic tissue was measured in the differently treated mice and the results are presented in Table 3. The ischemia/reperfusion increased the MDA. Pre-treatment of mice with rose hip powder (Group 3) or L. plantarum 299v (Group 4) in the food decreased the MDA compared to the positive control (Group 2). However, the effect of combined pre-treatment with rose hip powder and L. plantarum 299v decreased the MDA much more pronounced (Group 5).

TABLE 3
Lipid peroxidation after ischemia/reperfusion injury in mice.
	Malondialdehyde (MDA)
	per g colonic tissue
Mouse group	[median-value]
G1.	Control A; uninjured (no	4.3
	ischemia/reperfusion); normal food
G2.	Control B; normal food	6.3
G3.	Normal food + rose hip powder (RHP)	5.1
G4.	Normal food + L. plantarum 299v	5.8
G5.	Normal food + RHP + L. plantarum	3.6
	299v

The results of the viable count are presented in Table 4. The iscemia/reperfusion injury increased the viable counts on BHI and Rogosa agar with a factor of 10 (compare Group 1 and Group 2). Rose hip powder alone (Group 3) resulted in a lower viable count than the other feeding alternatives. The group that was given both L. plantarum 299v and rose hip powder (Group 5) showed the same viable count as the ischemia/reperfusion injury groups without rose hip powder (Groups 2 and 4) except for Enterobacteriacea that was lower. However, the viable count on the substrate allowing growth of lactobacilli was now (in Group 5) dominated by L. plantarum 299v.

TABLE 4
Bacterial flora in caecum after ischemia/reperfusion injury in mice.
	Median of viable count
	(CFU per g caecal content)
	Total	Total	Lacto-	Enterobac-
Mouse	anaerobes	aerobes	bacilli	teriaceae
G1.	Control A; uninjured	2 × 108	1 × 108	5 × 108	3 × 103
	(no ischemia/reper-
	fusion); normal food
G2.	Control B; normal food	3 × 109	1 × 109	1 × 109	4 × 103
G3.	Normal food + rose	1 × 108	4 × 108	1 × 108	<102
	hip powder (RHP)
G4.	Normal food +	3 × 109	4 × 109	2 × 109	3 × 103
	L. plantarum 299v
G5.	Normal food + RHP +	4 × 109	2 × 109	3 × 109	<102
	L. plantarum 299v

CONCLUSION

The tannins in the rose hip decreased the total load of bacteria in the intestine of the injured mice, but when the mice were administrated L. plantarum 299v simultaneously with rose hip the decrease was mitigated and the tannin-induced reduction was filled up by the L. plantarum 299v. Thus, the tannins supported the balance of the intestinal flora in favour of the probiotic strain. The lipid peroxidation was mitigated by administration of rose hip powder but this effect was enhanced by the presence of L. plantarum 299v together with the rose hip powder.

The strains L. plantarum HEAL 9, HEAL 19 and HEAL 99 have higher tannase activity than L. plantarum 299v and in addition the capacity to adhere to human, colonic mucosa cells are higher than for L. plantarum 299v.

Claims

1. A composition comprising:

(a) one or more isolated tannase-producing strains of Lactobacillus plantarum; and

(b) tannin;

wherein said one or more isolated tannase-producing strains of Lactobacillus plantarum are selected from the group consisting of: Lactobacillus plantarum strain HEAL 9, which is deposited as DSM 15312; Lactobacillus plantarum strain HEAL 19, which is deposited as DSM 15313; Lactobacillus plantarum strain HEAL 99, which is deposited as DSM 15316; and combinations thereof.

2. The composition according to claim 1, wherein said composition further comprises a carrier.

3. The composition according to claim 1 which is a food composition.

4. The composition according to claim 1 which is a pharmaceutical composition.

5. A isolated tannase-producing strain of Lactobacillus plantarum, wherein said isolated tannase-producing strain of Lactobacillus plantarum is selected from the group consisting of: Lactobacillus plantarum strain HEAL 9, which is deposited as DSM 15312; Lactobacilus plantarum strain HEAL 19, which is deposited as DSM 15313; and Lactobacillus plantarum strain HEAL 99, which is deposited as DSM 15316.

6. The isolated tannase-producing strain according to claim 5, wherein said isolated tannase-producing strain is Lactobacillus plantarum strain HEAL 9, which is deposited as DSM 15312.

7. The isolated tannase-producing strain according to claim 5, wherein said isolated tannase-producing strain is Lactobacillus plantarum strain HEAL 19, which is deposited as DSM 15313.

8. The isolated tannase-producing strain according to claim 5, wherein said isolated tannase-producing strain is Lactobacillus plantarum strain HEAL 99, which is deposited as DSM 15316.

9. A medicament for prophylactic or curative treatment of cardiovascular diseases, diabetes, inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), gastrointestinal infections, cancer, Alzheimer's disease or diseases with an autoimmune origin, wherein said medicament comprises:

(a) one or more isolated tannase-producing strains of Lactobacillus plantarum having the ability to adhere to the human intestinal mucosa; and

(b) tannin;

wherein said one or more isolated tannase-producing strains of Lactobacillus plantarum are selected from the group consisting of: Lactobacillus plantarum strain HEAL 9, which is deposited as DSM 15312; Lactobacillus plantarum strain HEAL 19, which is deposited as DSM 15313; Lactobacillus plantarum strain HEAL 99, which is deposited as DSM 15316; and combinations thereof.

10. A composition for the preservation of food comprising:

(a) one or more isolated tannase-producing strains of Lactobacillus plantarum having the ability to adhere to the human intestinal mucosa; and

(b) tannin;

wherein said one or more isolated tannase-producing strains of Lactobacillus plantarum are selected from the group consisting of: Lactobacillus plantarum strain HEAL 9, which is deposited as DSM 15312; Lactobacillus plantarum strain HEAL 19, which is deposited as DSM 15313; Lactobacillus plantarum strain HEAL 99, which is deposited as DSM 15316; and combinations thereof.

11. A food product comprising:

(a) one or more isolated tannase-producing strains of Lactobacillus plantarum having the ability to adhere to the human intestinal mucosa; and

(b) tannin;

wherein said one or more isolated tannase-producing strains of Lactobacillus plantarum are selected from the group consisting of: Lactobacillus plantarum strain HEAL 9, which is deposited as DSM 15312; Lactobacillus plantarum strain HEAL 19, which is deposited as DSM 15313; Lactobacillus plantarum strain HEAL 99, which is deposited as DSM 15316; and combinations thereof.

12. The composition according to claim 2 which is a food composition.

13. The composition according to claim 2 which is a pharmaceutical composition.

14. A method for prophylactic or curative treatment of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or gastrointestinal infection comprising administering to an individual with an inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or a gastrointestinal infection, in combination with a tannin, a tannase-producing strain of Lactobacillus plantarum having the ability to adhere to the human intestinal mucosa, wherein the tannase-producing strain of Lactobacillus plantarum is selected from the group consisting of Lactobacillus plantarum HEAL 9, which is deposited as DSM 15312; Lactobacillus plantarum HEAL 19, which is deposited as DSM 15313; Lactobacillus plantarum HEAL 99, which is deposited as DSM 15316; and combinations thereof.