HK1162937B - Compositions comprising lactobacillus plantarum strains in combination with tannin and new lactobacillus plantarum strains - Google Patents

Description

Compositions containing lactobacillus plantarum strains in combination with tannin and novel lactobacillus plantarum strains

The present application is a divisional application of the "composition comprising lactobacillus plantarum strain in combination with tannin" and new lactobacillus plantarum strain "chinese patent application 200480015309.2, filed 4, 2/4/2004.

Technical Field

The present invention relates to compositions having anti-inflammatory properties and in vivo control of the intestinal microflora and in vitro preservative properties, containing optionally new tannase-producing strains of Lactobacillus plantarum (Lactobacillus plantarum) having a significant ability to adhere to the human intestinal mucosa.

Background

Tannins are defined as water-soluble phenolic products that precipitate proteins from aqueous solutions and are naturally occurring compounds. There are two classes of tannins, hydrolyzable tannins derived from gallic acid and ellagic acid, and condensed tannins, i.e., proanthocyanidins, which are oligomers and polymers of flavanols. Tannins inhibit the growth of a variety of microorganisms and resist microbial attack (Chung, K.T., et al., (1998), Tannins and human health: A review. Critical reviews in Food Science and Nutrition 38: 421-. Moulds and yeasts and some aerobic bacteria are generally best suited for degrading tannins but anaerobic degradation also occurs, as in the intestinal tract (Bhat, T.K., et al., (1998), Microbial degradation of tannins-A current perspective.Biodegradation 9: 343-.

Tannins are known to be antinutritional agents, i.e., they reduce the body's efficiency in converting digested nutrients into new body substances. However, there have also been reports of the beneficial health effects of tannins, such as anti-cancer effects, the ability to lower blood pressure and modulate immune responses. These effects may be due to the antioxidant properties of tannins (Chung et al, 1998). An effective antioxidant tannin reported to have anticancer properties is ellagic acid. Another type of tannin with very high antioxidant capacity is proanthocyanidins, such as found in grapes and olives. Thus, the different concentrations of tannins present in plant derived foods have a significant impact on human health. Ingestion of large amounts of tannins is not recommended because they may be involved in cancer formation and anti-nutritional activity, but by affecting metabolic enzymes, immune regulation or other functions, ingestion of small amounts of the correct species of tannins may be beneficial to health (Chung et al, 1998).

However, as produced in the gut, many anaerobic degradation products of tannins can produce compounds with beneficial health effects (Bhat et al, 1998). These degradation compounds are, for example, derivatives of phenylpropionic acid or phenylacetic acid (Bhat et al, 1998). These compounds have anti-inflammatory effects when absorbed in the gastrointestinal tract. These compounds, together with other degradation products of tannins, also have a broad antimicrobial effect in the gastrointestinal tract, inhibiting unwanted bacteria.

Most lactobacillus species are not able to degrade tannins, but strains of closely related lactobacillus plantarum, lactobacillus pentosus and l.paralantarum are able to have tannase activity, Osawa, r.a. (2000), Isolation of tannin-degrading lactobacillus from transformed foods, Applied and Environmental Microbiology 66: 3093-3097.

Some lactobacillus plantarum strains have the ability to specifically adhere to human epithelial cells by a mechanism that is blocked due to the presence of mannose, Adlerberth, i., et al, (1996), a mutation-specific additive mechanism binding to the human collagen cell line ht-29, Applied and Environmental Microbiology 62: 2244-2251.

Disclosure of Invention

It has been found that strains of Lactobacillus plantarum having the ability to adhere to human intestinal mucosa and produce tannase, when degrading tannin, produce compounds against harmful bacteria in the Gastrointestinal (GI) tract which have an anti-inflammatory effect when absorbed in the GI tract.

Drawings

The figure shows isolated DNA fragments obtained by cleaving chromosomal DNA of Lactobacillus plantarum strains HEAL9 (lane 2), HEAL 19 (lane 3), 299v (lane 4) and HEAL 99 (lane 5) with the restriction enzyme EcoRI. High molecular weight DNA marker (BRL) and DNA molecular weight marker VI (Roche) were used as standards (lane 1).

Detailed Description

The present invention relates to compositions comprising one or more tannase-producing strains of Lactobacillus plantarum or closely related Lactobacillus species having the ability to adhere to human intestinal mucosa in combination with tannin. The compositions will produce compounds with antimicrobial and anti-inflammatory effects in vivo and compounds with preservative effects in vitro.

The invention also relates to compositions comprising one or more tannase-producing lactobacillus strains in combination with tannin and a carrier.

Examples of carriers are oatmeal pastes, lactic acid fermented foods, resistant starches. Dietary fibre may be added to the composition in order to improve bacterial proliferation and increase production of anti-inflammatory or preservative derivatives. Dietary fibres such as fructooligosaccharides, galactooligosaccharides, lactulose, maltodextrin, beta-glucans and guar gum may also be used as carriers.

The invention especially relates to food compositions comprising tannase-producing strains of lactobacillus in combination with more or less pure tannin components such as ellagic acid, flavonoids such as proanthocyanidins and anthocyanidins, or lignans, or with tannin-rich food components such as oat, barley, red sorghum, flour made from the inner cortex of pine trees and juices or extracts of grapes, citrus, bilberry, blueberry, blackcurrant, cranberry, strawberry, raspberry and rosehip.

The invention also relates to pharmaceutical compositions containing a tannase-producing strain of lactobacillus plantarum, combined with a more or less pure tannin component, such as ellagic acid, flavonoids, such as proanthocyanidins or anthocyanidins, or lignans, or any other pharmaceutically acceptable tannin source.

In order to achieve a prophylactic or therapeutic effect of the composition of the present invention, the tannin content is preferably about 500-1000 mg/day. For example for rose hip powder this corresponds approximately to 100g, or 4 litres in the form of rose hip pulp.

Tannins are water-soluble phenolic products of varying molecular weights that precipitate proteins in aqueous solutions. There are two classes of tannins, hydrolyzable tannins derived from gallic acid and ellagic acid, and condensed tannins, i.e., proanthocyanidins, which are oligomers and polymers of flavanols.

So-called condensed or non-hydrolyzable tannins are more resistant to microbial degradation than hydrolyzable tannins. Tannins are commonly found in fruits and seeds such as grapes, apples, bananas, blackberries, cranberries, raspberries, strawberries, olives, beans, sorghum grains, barley and finger mills, cocoa, tea and coffee.

The composition of the invention may be a food composition wherein the carrier is a food product. In the pharmaceutical composition, the carrier should be a pharmaceutically acceptable carrier. The composition can be administered to the general consumer to improve health measures to ultimately prevent future diseases such as infections of gastrointestinal origin, diabetes, Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS), cancer and cardiovascular disease, or to alleviate these exemplified diseases.

The pharmaceutical compositions of the present invention may be formulated, for example, as suspensions, tablets, capsules, and powders, which may be administered orally. The formulation may also be administered as an enema.

The invention relates in particular to tannase-producing strains of Lactobacillus plantarum or a closely related Lactobacillus species, having the ability to adhere to human intestinal mucosa, characterized by tannase activity determined by known methods, wherein strains are excluded: lactobacillus plantarum 299, DSM 6595 and Lactobacillus plantarum 299v, DSM 9843, said methods being described by Osawa and Walsh in Applied and Environmental Microbiology, Vol.59, No.4, April1993, p 1251-1252.

Preferred tannase producing strains belong to the species Lactobacillus plantarum and have the ability to survive in the Gastrointestinal (GI) tract. Survival in this environment means that the strain is able to metabolize and expand (live) in the gastrointestinal tract for a period of time.

According to a preferred aspect, the present invention relates to new strains which have been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH at 11/28 in 2002 and which have been given deposit numbers, i.e.Lactobacillus plantarum HEAL9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, and Lactobacillus plantarum HEAL 99, DSM 15316, and variants thereof having essentially the same REA pattern.

The new strain has been isolated from the colonic mucosa of healthy adults and screened by culturing on Rogosa agar. The strain has subsequently been characterized by REA.

According to another aspect, the invention also relates to the use of a tannase-producing lactobacillus plantarum strain in combination with tannin for the preparation of a medicament for the prophylactic or therapeutic treatment of cardiovascular diseases, Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS), gastrointestinal infections, diabetes, cancer, alzheimer's disease or diseases of autoimmune origin. Examples of tannase-producing strains are the new strains HEAL9, HEAL 19 and HEAL 99, and the known strains lactobacillus plantarum 299, DSM 6595 and lactobacillus plantarum 299v, DSM 9843.

The amount of tannase-producing bacteria used in the composition of the present invention is preferably not less than 10⁹cfu/dose/day.

According to another aspect, the invention relates to the use of a tannase-producing strain of lactobacillus plantarum together with tannin in the preservation of food. Examples of tannase-producing strains are the new strains HEAL9, HEAL 19 and HEAL 99, and the known strains lactobacillus plantarum 299, DSM 6595 and lactobacillus plantarum 299v, DSM 9843. The strain will then produce preservatives in the food product directly after degradation of tannin. By supplementing the product with pure tannin components or by supplementing the product with natural, less defined, tannin-rich additives such as rosehips, red sorghum or flours made from the inner cortex of pine trees.

The mixture of tannin-capable lactobacillus strains and tannins can be administered for therapeutic purposes or as a health care effect, thereby reducing the risk factors for the following diseases: cardiovascular diseases, metabolic syndrome, diabetes, Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS), gastrointestinal tract infections or diseases associated with autoimmune origin.

The strains Lactobacillus plantarum HEAL9, HEAL 19 and HEAL 99 have a higher ability to adhere to human colonic mucosal cells compared to the strain Lactobacillus plantarum 299v, DSM 9843.

Test of

Isolation of bacterial strains

42 different, newly isolated Lactobacillus strains were tested and compared to the well known reference probiotic strain Lactobacillus plantarum 299v, DSM 9843 for their ability to produce tannase, i.e.to degrade tannin. The strains are listed in table 1 below.

Screening method

The method used to detect tannase activity was described earlier by Osawa and Walsh (1993). The detection principle is to measure the degradation of tannin and gallic acid methyl ester by the following procedures:

the test bacteria were grown anaerobically on MRS-agar (Merck, Darmstadt, Germany) at 37 ℃ for 2 days, after which the cells were harvested and suspended in 5ml of 0.9% (w/v) NaCl. The cell suspension was centrifuged and the cells were resuspended in 10ml of 0.9% NaCl and the absorbance at 620nm was measured (0.9% NaCl solution as standard). The cell suspension was diluted to an absorbance between 0.1 and 0.6 (spectrophotometer, Pharmacia LKB, Novaspec II). After centrifugation the cells were resuspended in 1ml of methyl gallate buffer (3.7g/l methyl gallate [ Aldrich chemical Company, Inc., Milwaukee, Wis., USA)]，4.5g/l NaH₂PO₄pH 5.0[ sterile filtration ]]) Neutralize and incubate the tubes at 37 ℃ for 24 hours. 1ml of NaHCO was added₃Buffer (42g/l NaHCO)₃pH 8.6) and the solution was incubated at room temperature for 1 hour, then absorbance (NaHCO) was measured at 440nm₃Buffer as standard). The color of the suspension was measured by visual observation.

The color that is graded as positive tannase activity should be brown or green. Absorbance of the cell suspension at the beginning of incubation with methyl gallate (A)₆₂₀(ii) a Cell amount) ratio of absorbance (A) after incubation with methyl gallate for 24 hours₄₄₀(ii) a Free gallic acid developed after exposure to oxygen in alkaline solution) to obtain a quantitative value for tannase activity.

Results

The results of screening lactobacillus strains having tannase activity are shown in table 1. Most of the test strains did not have any tannase activity. However, 11 strains were positive and are given in table 1.

TABLE 1 tannase activity in different Lactobacillus strains.

Positive tannase activity was shown green to brown. Free gallic acid in the cell suspension develops color after prolonged exposure to oxygen in alkaline solutions.

Tannase activity, expressed as the absorbance at 620nm of the cell suspension at the beginning of 24 h incubation with methyl gallate (A)₆₂₀) After the mixture is insulated with the gallic acid methyl ester (A)₄₄₀) Absorbance at 440nm (A)₄₄₀) The ratio of (a) to (b).

Three of the tannase positive strains of Lactobacillus plantarum showed higher tannase activity than the well known probiotic strains Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL9, Lactobacillus plantarum HEAL 19 and Lactobacillus plantarum HEAL 99. They have been isolated from healthy human intestinal mucosa.

Genotyping Using REA

According toM，Molin G，Persson A，AhrnéS &S, International Journal of Systematic Bacteriology, 40: 189, 193, 1990, and by Johansson, M-L, et al, International Journal of systematic Bacteriology, 45: 670-675, 1995, by means of the restriction enzyme analysis-REA-method, the chromosomal DNA cleavage pattern of the strain was examined. An exemplary REA can be described as follows: chromosomal DNA from the strain in this study was prepared and cleaved with restriction enzymes. 0.75. mu.g of each DNA was digested with 10 units of EcoRI and HindIII at 37 ℃ for 4 hours, respectively; each endonuclease was used separately. The cut DNA fragments were separated by size by gel electrophoresis using submerged horizontal agarose slab gels. The gel consisted of 150ml of 0.9% agarose (ultrapure DNA grade; low electro-endosmosis; BioRad Laboratories, Richmond, USA) and was made as a slab gel (150X 235 mm). Mu.g of high molecular weight DNA marker (Bethesda Res)Research Laboratories, MD, USA) with 0.5. mu.g of DNA molecular weight marker VI (Roche, Germany) was used as standard. Minimal band distortion and maximal sharpness were achieved by sample DNA in Ficoll loading buffer (2g Ficoll, 8ml water, 0.25% bromophenol blue).

The gel was electrophoresed at a constant pressure of 40V for 18 hours at about 6-8 ℃. Buffer during electrophoresis (89mM Tris, 23mM H₃PO₄2mM EDTA sodium salt, pH 8.3). The gel was then stained in ethidium bromide (2 μ g/ml) for 20 minutes and destained in distilled water, visualized at 302nm with a UV projector (UVP Inc., San Gabriel, USA) and photographed. For low to 1.2x10 molecular weights⁶This method of performing gel electrophoresis produces well-distributed and relatively well-separated bands.

The analysis result is shown in the figure.

Adherence to HT-29 cells

They were tested for their ability to adhere to intestinal epithelial cells of a human colon cancer cell line HT-29 with mannose-specific binding in a total of 32 Lactobacillus plantarum strains isolated from human mucosa (methods described in Wold, A, et al, Infection and Immunity, Oct.1988, p.2531-2537). Cells of human colon cancer cell line HT-29 were cultured in Eagle's medium (Sigmachemical Co., Saint Louis, Mo, USA) supplemented with 10% fetal bovine serum, 2mM L-glutamine and 50. mu.g/ml gentamicin. After several days when the cells reached confluence, the cells were detached with EDTA-containing buffer (0.54mM), washed and washed at 5X10⁶The/ml was resuspended in Hank's Balanced Salt Solution (HBSS). Bacteria were harvested, washed and washed at 5 × 10⁹Perml (2X optical density 1.5 at 597 nm) was resuspended in HBSS. Cells, bacteria and HBSS were mixed 1: 3 and incubated for 30 minutes in 4EC with end-to-end rotation. Cells were washed once with ice-cold PBS and washed with neutral buffered formalin (Histofix, Histolab,sweden) were fixed. The number of bacteria attached to each of at least 40 cells was measured using an interference phase contrast microscope (500x magnification)Several, Nicon optophor, with interferometric phase contrast devices,Instruments，sweden) determined and calculated the average number of bacteria per cell.

All strains, except the three HEAL strains, had values between 0.3 and 14 (adherence in saline solution; corresponding values of 0.5 and 2.4, respectively, in the presence of methyl mannoside). Most strains had values below 10. The results are given in table 2 below.

TABLE 2

Testing in laboratory mouse model

Method of producing a composite material

15 Balb/C mice were divided into 5 groups (3 mice/group) and fed different combinations of normal diet, rose hip powder (rich in tannin) and the tannase positive strain Lactobacillus plantarum 299 v. The ingredients are mixed with some water to give a paste-like density. Normal mice were fed with food in groups 1 and 2, normal food supplemented with rose hip powder (1.6 g/day) in group 3, and lactobacillus plantarum 299v (10) in group 4¹⁰Bacteria/dose), group 5 was given a normal diet supplemented with rose hip powder and lactobacillus plantarum 299v simultaneously. The mice were fed once daily for 6-8 days, and then induced ischemia/reperfusion injury. Lesions were made according to the following anatomical protocol: mice were anesthetized with 0.15ml ketamine/xylazine solution (7.85 mg/ml and 2.57mg/ml, respectively) administered subcutaneously. An incision was made at the midline of the abdomen and the superior mesenteric artery was closed with an atraumatic catheter ring and hemostats. For fluid resuscitation, 1.0ml PBS was injected into the peritoneal cavity. The artery was closed for 30 minutes, then the catheter loop and hemostat were removed and observedImmediate reperfusion of the tissue. The abdomen was then closed with vicryl 3-0 suture. The animals were waited for to recover from anesthesia and were returned to the cages by removing them from the warm pads. After 4 hours and 15 minutes, the animals were again anesthetized and tissue and fecal samples were obtained in the following order into pre-weighed tubes: liver tissue, ileal mesenteric tissue and cecal faeces were used for bacteriological sampling, and cecal and ileal tissue for colorimetric testing of lipid peroxidation, and cecal and ileal tissue for histological examination. Samples for bacteriological evaluation were weighed and placed in a freezing medium and immediately frozen at-70 ℃. Samples for colorimetric testing (LPO586) were rinsed in PBS, weighed, homogenized, aliquoted and immediately frozen at-70 ℃.

Analytical method

Anaerobic cultures (BBL Gas Pak Plus, Becton Dickinson and Company, Sparks, MD, USA) were carried out by culturing on Rogosa agar (Merck, Darmstadt, Germany) at 37 ℃ for 3 days, VRBD agar (Merck, Darmstadt, Germany) at 37 ℃ for 24 hours and brain heart perfusion agar (BHI, Oxoid, Basingstoke, Hampshire, England) at 37 ℃ for 3 days, and bacteriological evaluation was carried out by viable count. Viable count on BHI was also performed under aerobic conditions.

In spectrophotometer and assay kitLPO-586^TM(OxisResearch^TMOxygen Health Products, inc., Portland) for the colorimetric testing of lipid peroxidation. The analysis was performed according to the manufacturer's instructions.

Lipid peroxidation is a well-defined mechanism of cell 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 undergo decomposition to produce Malonadialdehyde (MDA) and 4-Hydroxylkenals (HAE). Measurement of MDA can be used as an indicator of lipid peroxidation. LPO-586^TMIs a colorimetric test designed for quantification of MDA and based on the chromogenic reagent N-methyl-2-Reaction of phenylindole with MDA at 45 ℃. One molecule of MDA reacts with two molecules of N-methyl-2-phenylindole to produce a stable chromogenic material with a maximum absorbance at 586 nm.

Results

Lipid peroxidation measured as malonadialdehyde (mda)/g colon tissue was measured in the differently treated mice and the results are shown in table 3. Ischemia/reperfusion increases MDA. Pretreatment of mice with rosehip powder in food (group 3) or lactobacillus plantarum 299v (group 4) reduced MDA compared to the positive control (group 2). However, the effect of the combined pretreatment of rose hip powder and lactobacillus plantarum 299v reduced MDA more significantly (group 5).

TABLE 3 lipid peroxidation after ischemia/reperfusion in mice

The results of viable count are shown in table 4. Ischemia/reperfusion injury increased viable count by a factor of 10 on BHI and Rogosa agar (compare group 1 and 2). Rose hip powder alone (group 3) resulted in a lower viable count compared to other feeding alternatives. The group given with lactobacillus plantarum 299v and rosehip powder simultaneously (group 5) showed the same viable bacterial count as the ischemia/reperfusion group without rosehip powder (groups 2 and 4), except for lower enterobacteriaceae. However, the viable count on the substrate (group 5) that allowed the growth of lactobacillus was mainly lactobacillus plantarum 299 v.

TABLE 4 bacterial communities in the cecum after ischemia/reperfusion in mice

Conclusion

Tannin in rose hip reduces the total bacterial load in the intestine of injured mice, but this reduction is alleviated when rose hip is given simultaneously with lactobacillus plantarum 299v in mice and the reduction induced by tannin is supplemented by lactobacillus plantarum 299 v. Thus, tannin supports the intestinal flora balance in favor of the probiotic strains. Administration of rose hip powder reduced lipid peroxidation but this effect was enhanced by the presence of lactobacillus plantarum 299v together with rose hip powder.

The strains lactobacillus plantarum HEAL9, HEAL 19 and HEAL 99 have higher tannase activity compared to lactobacillus plantarum 299v and additionally have a higher ability to adhere to human colonic mucosal cells than lactobacillus plantarum 299 v.

Claims (2)

1. Use of a strain of lactobacillus plantarum yielding a tannase enzyme, selected from the group comprising lactobacillus plantarum 299, DSM 6595 and lactobacillus plantarum 299v, DSM 9843, in combination with tannin for the preparation of a medicament for the prophylactic or therapeutic treatment of diabetes, Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS), gastrointestinal tract infections, cancer, alzheimer's disease or diseases of autoimmune origin, with the strain having the ability to adhere to the intestinal mucosa of humans.

2. Use of a strain of lactobacillus plantarum yielding tannase selected from the group comprising lactobacillus plantarum 299, DSM 6595 and lactobacillus plantarum 299v, DSM 9843, in combination with tannin, in food preservation, wherein the strain has the ability to adhere to human intestinal mucosa.