WO2006048457A1

WO2006048457A1 - Probiotica and polyphenol

Info

Publication number: WO2006048457A1
Application number: PCT/EP2005/055787
Authority: WO
Inventors: Karl Anders Olof Henriksson
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2004-11-05
Filing date: 2005-11-07
Publication date: 2006-05-11
Anticipated expiration: 2007-05-05

Abstract

The present invention relates to the field of infection caused by undesirable bacteria and associated diseases, and in particular to compositions comprising probiotica and polyphenols, which have the capacity to prevent and/or treat these infections and that may also be used as a preservative.

Description

PROBIOTICA AND POLYPHENOL

Field of the invention

The present invention relates to the field of infection caused among others by Gram positive Bacteria and associated diseases, and in particular to compositions comprising probiotica and polyphenol, which have the capacity to prevent and/or treat these infections and that may also be used as a preservative.

Background of the invention

Microbial pathogens remain a serious threat to human health despite the availability of traditional anti-microbial therapies. Such pathogens are known to be both Gram positive and Gram negative bacteria. Staphylococci are a group of Gram positive bacteria that are known to cause numerous infections, including neonatal infections, mastitis, post-operative infections, furuncles and carbuncles, pneumonia, bacteremia, endocarditis and enterocolitis (1). Pathogenic streptococci, including Streptococcus pyrogenes are commonly associated with the skin and nasopharynx where they may cause disease. Streptococcus pneumoniae is another human pathogen that causes infection in the lower respiratory tract (2). Other streptococci, such as Streptococcus mutans, are involved in plaque formation on teeth and therefore are of great concern in relation to dental health. Gram positive bacteria such as staphylococci and to some extent streptococci often carry multiple antibiotic resistances, which makes successful treatment difficult and in some cases impossible. Certain Gram negative bacteria are also known to cause prolonged and even chronic infections due to a lack of effective treatments. Helicobacter pylori is an example on such an pathogen. Gastric infection by this pathogen would mostly lead to peptic ulcer, in some cases (1%) developing to gastric cancer. Gastric infection with H. pylori is often treated with a combination therapy. However, emerging strains with resistance to such therapies are a problem (3). Due to the demand for new effective treatments, alternative treatment methods have been explored. Such methods include the use of probiotics for treatment of infections of the gastrointestinal tract (4-6) the nasopharynx (7) and the skin (8). Epigallocatechin gallate (EGCG ) is a compound that provides some protection against infection by Helicobacter pylori (9) and inhibits many other pathogenic bacteria in vitro. High dosage levels of EGCG may be required to achieve desired inhibition pathogens in vivo.

In addition to a direct effect on growth of pathogens, it is known that on one side probiotics and on the other side EGCG have immune modulatory properties (10, 11), which also may contribute to the treatment of infections by certain pathogens.

For a probiotic culture to be active, it needs to compete with other microorganisms, in the targeted area. Therefore, prebiotic compounds have been useful in stimulating growth and activity of probiotics in specific targeted areas to maximize the beneficial effect of probiotic cultures. The drawback with such prebiotics is that they may also stimulate growth and activity of certain undesirable bacteria.

There is still a need for improved treatments of infections caused by bacteria, which do not have the drawback of the known treatments (among other emergence of resistant strains).

Detailed description of the invention

The invention relates to a composition comprising a probiotic and a polyphenol.

A probiotic is defined herein as a live microbial strain, which beneficially affects the human host cell by improving its microbial balance. Preferably, the probiotic is selected from the group consisting of a Lactobacillus, a Bifidobacterium species and mixture thereof.

According to a more preferred embodiment, the Lactobacillus is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus fermentum and mixtures thereof and the Bifidobacterium is selected from the group consisting of

Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis and mixture thereof. Even more preferably, the probiotica is selected from the group consisting of the Lactobacillus acidophilus CBS 116411, the Lactobacillus casei CBS 116412, and the Bifidobacterium lactis B94 CBS 118529 all three protected by the trademark LAFTI™ (DSM, The Netherlands), a Bifidobacterium species. According to a preferred embodiment, the probiotic used in the composition of the invention comprises a biologically pure culture of said deposited strain. According to a more preferred embodiment, the probiotic used in the composition of the invention comprises a biologically pure culture of said deposited strain in combination with any other valuable probiotic strain.

The polyphenol present in the composition of the invention derives preferably from a tea extract or is preferably a tea extract. According to a more preferred embodiment, the tea extract is a green tea extract. According to a preferred embodiment, the polyphenol is or comprises catechins. The major catechins present in green tea extract are epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC). Preferably, the green tea extract comprises EGCG. EGCG is a polyphenol derived from the green tea plant Camellia sinensis. EGCG preparations are already known. A process for preparing EGCG preparations has already been described for example in EP 1 ,077,211. In this process, EGCG is highly enriched starting from any green tea extract, e.g. green tea leaves are typically extracted with hot or cold water to form a solution containing tea catechins and caffeine. This green tea solution can be further concentrated to form either a concentrated extract solution or a dry powder. Tea extract powders are commercially available e.g. from Guizhou Highyin Biological Product Co., Guiyang, P. R. China, or Zhejang Zhongke Plant Technical Co. Ltd., Hangzhou, Zhejang, P. R. China. More preferably, the green tea extract comprises as major compound EGCG. Major compound means that the green tea extract comprises at least 90% w/w, preferably at least 93 %, more preferably at least 95 % EGCG. Most preferably, the green tea extract is Teavigo™ (DSM Nutritional products Ltd., Kaiseraugst, Switzerland) or is EGCG.

According to a most preferred embodiment, the composition comprises EGCG and a probiotica is selected from the group consisting of the Lactobacillus acidophilus CBS 116411, the Lactobacillus casei CBS 116412, and the Bifidobacterium lactis B94 CBS 118529

Green tea extract is a powerful antioxidant and is known to have some anti¬ microbial activities, against undesirable bacteria such as Gram positive and Gram negative bacteria. Gram positive bacteria appear to be particularly sensitive to polyphenol. Surprisingly, we demonstrate that several probiotic strains (Lactobacilli and Bifidobacteria strains), which are Gram positive bacteria are relatively resistant to the anti-microbial activity of green tea extract. These strains grow at concentrations that are close to 10 times higher than the minimum concentrations that inhibit other Gram positive bacteria and some Gram negative bacteria. Therefore, the composition of the invention could be used in any types of applications wherein the undesirable agent is among others a Gram positive bacteria: the green tea extract would act as an anti-microbial agent which would allow the probiotic to colonize the treated human, animal or product. Increased colonization capacity and activity of the probiotic will improve the performance and efficacy of the probiotic culture. It has been surprisingly found that the combination of these two components together (polyphenol and probiotic) works synergistically such that the combination is more effective against undesirable bacteria than each agent taken separately. Surprisingly, the composition of the invention has a synergistic action on the modulation of the immune system. The composition of the invention has the capacity to kill an undesirable bacterium or to inhibit its growth, or to prevent the germination of its spores in vitro in a given medium and/or in vivo in a given food product matrix, crop protection application, pharmaceutical or cosmetic product. Undesirable bacteria are defined as being either a Gram positive or a Gram-negative bacterium. Preferably, undesirable bacteria are bacteria, which are not defined as probiotic. Undesirable Gram-negative bacteria that are selectively targeted by the composition of the invention are a.o. Helicobacter pylori. Preferably, undesirable Gram-positive bacteria that are susceptible and are targeted by the composition of the invention are selected from the group consisting of a Streptococcus, Staphylococcus species and mixture thereof. The effect of killing undesirabie bacteria or inhibiting their growth is preferably measured using the following in vitro assay: 10⁷ of

probiotic Lactobacillus acidophilus L10 CBS 116411 and/or Lactobacillus casei L26 iTM i ,:™ TM serve as controls: (1 ) 10⁷ cfu of the chosen undesirable bacteria as defined above are

above and (2) 10 cfu of the chosen undesirable bacteria is

TM

-3, in sis, is monitored for at feast 4h, I he number of cfu is

mϊcroaerophϊlic conditions. According to a preferred embodiment, the composition is said to have the capacity to kill undesirable bacteria if the reduction that is at least 20% greater

1 additional iog10 unit of the undesirable bacteria have been killed, more preferably at ieasf 2 log 10 units, even more preferably at least 3 log 10 units, even more preferably at ieast 4 log 10 units, even more preferably at least 5 log 10 units, even more preferably at

achieved in the control experiments 1 or 2.

According to another preferred embodiment, the composition is said to have to prevent the growth of the undesirabie bacteria if in the /n v/fro test d« undesirabie bacteria at the end of the assays is the same

10% ϊess than the number of undesirable bacteria compared to control experiment 1 or 2, more preferably at least 1 iog10 unit less, even more preferably at least 2 log 10 unit less, even more preferably at least 50% less and most preferabϊy at ieast 7 Ϊog10 units less.

Depending on the type of applications and/or administration, the composition of the

at least 10⁶ cfu probϊotic, or at least 10⁷ cfu probϊotic, at ieast 10⁸ cfu probϊotic or

associated

at least 100μg/mi polyphenol or EGCG, or at ieast 200 μg/ml polyphenol or EGCG, or at least 300 μg/ml poiyphenoi or EGCG, or at least 50 mg/ml poϊyphenoϊ or or EGCG or at ϊeast 300 rng/rni polyphenol or E mg polyphenol or EGCG/capsuie at least 100 m, ΞGCG/capsule or fabiet or a polyphenol or E¹ mg polyphenol or EGCG/capsuie

The preferred probiotic strains have been defined earlier in the description. The two

Iy t

, In

^•, the two components, may be first 1 as a smqϊe to the human or to the product to

iater on In the description.

According to another preferred embodiment, the composition of the invention comprises as probiotic any of the strains as defined earlier, preferably the strain Lactobacillus acidophilus CBS 116411 and/or the strain Lactobacillus casei CBS 116412 and/or Bifidobacterium lactis CBS 118529 and mixtures thereof with polyphenol or EGCG or preferably Teavigo™ in combination with at least one of the following ingredients in order to get a synergistic combination on the human being treated. Preferred ingredients are: vitamin A, vitamin C, vitamin D3, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, nicotinamide, folic acid, beta carotene, lutein, Zinc, Copper, selenium, and prebiotic.

According to a more preferred embodiment, the composition of the invention comprises a probiotic, a polyphenol and a prebiotic. It has been surprisingly found that the combination of these three components together (polyphenol, probiotic and prebiotic) works synergistically such that the combination is more effective against undesirable bacteria than each agent taken separately. Surprisingly, the composition of the invention has a synergistic action on the modulation of the immune system.

Prebiotic is defined as a component, which has a favourable influence on the growth of certain beneficial intestinal bacteria, preferably probiotic. Preferably the prebiotic is selected from the group consisting of pentosans (or arabinoxylans), b glucans, pectins, and pectic polysaccharides, mannans, arabinans, galactans, fructo- oligosaccharides, resistant starch and mixture thereof. Prebiotic have been fully described in US 6,241 ,983. Most preferred fructo-oligosaccharides are selected among the group consisting of inulin, banana fiber, and soy fructo-oligosaccharides or soy oligosaccharides. Most preferred mannans are selected among the group consisting of guar gum, locust bean gum, konjac and ad xanthan gum. Resistant starch as used in the context of this invention includes tiose forms defined as RS1 , RS2, RS3, and RS4 as defined in Brown et al (Brown, Mc Knaught and Moloney (1995) Food Australia 47:272- 275). Resistant starch and modified resistant starch have been both extensively described in US 6,348,452.

The Composition of the invention is preferably for use as a medicament. According to a further aspect, the invention relates to the use of the composition of the invention for the preparation of a medicament for preventing and/or treating infections caused by undesirable bacteria and associated diseases in a human being in a need thereof. The medicament, which comprises the composition of the invention is preferably used for preventing and/or treating infections caused by undesirable bacteria and associated diseases in a human being in a need thereof. Preferably, the undesirable bacterium is among other a Gram positive bacteria. In the context of this invention, a human being in the need of the composition of the invention is preferably defined as follows: human subjects that experience some form of fatigue and/or stress, bed sores, gingivitis, nose, throat infection and peptic ulcer. Stress and fatigue can have any origin but could have a professional origin: shiftwork, international travel, academic stress in students, over-training in sport (athletes). Alternatively and/or in combination with stress having a professional origin, stress could also have a domestic origin: family stress, stress caused by children. Alternatively and/or in combination with previous mentioned possible origins of stress, stress is caused by mid-life crisis and/or diet-related stress. More preferably, the stress and/or fatigue cause impaired physical performance in human over-training in sport such as athletes. Humans, In a need of the composition of the invention are preferably individuals that have been subjected to physical stress, e.g. afhietes or other individuals that are susceptible to infections caused by among other

ilng to a preferred embodii undesirable bacteria in human means that after daϋy oral administration of at Ieast 10 cfu (colony forming units) of probiotic associated with at least 10mg polyphenol per day during at least 1 day, a normal level of at least one of the following biomarkers is defected using a specific given assay. According to a preferred embodiment, the biomarker Is nitric oxide (NO), which is measured by a standard method known to the skilled person such as the Griess reaction (14), A norma! level of NO is 15G-2GGμM using this reaction. According to another preferred embodiment, the biomarker Is the ievei of presence of an undesirable bacterium for exarnpϊe in the nose. The undesirable bacterium Is preferably a Streptococcus or a Staphylococcus species. The undesirable bacterium is defected by Nutrient agar. According to another preferred embodiment, the biomarker is the Cross Reactive Protein (CRP), which is measured as described by (15). A norma! level of CRP is iess than 7mg/L blood as measured by this assay. More

even more preferably at least 60 days, even more preferably at least four month and most preferably during at least one year.

In particular, the composition of the invention can be used to prevent and/or treat gingivitis (mouthwash), skin infection (skin cream), nose and throat infections (tablet, capsule, spray), Helicobacter infections (tablet, capsule). The skilled person will understand that depending on the type of infections treated and targeted area, the type of formulation and possibly the quantity of each constituent of the composition of the invention would have possibly to be adjusted to obtain the most efficient formulation.

The composition of invention may be in any galenic form that is suitable for administrating to the animal body including the human body, especially in any form that is conventional for oral administration, e.g. in solid form such as (additives/supplements for) food or feed, food or feed premix, fortified food or feed, tablets, pills, granules, dragees, capsules, and effervescent formulations such as powders and tablets, or in liquid form such as solutions, emulsions or suspensions as e.g. beverages, pastes and oily suspensions. The pastes may be filled into hard or soft shell capsules, whereby the capsules feature e.g. a matrix of plant proteins or ligninsulfonate for example. Examples for other application forms are forms for topical, transdermal, parenteral or injectable administration. The composition of the invention may be in the form of controlled (delayed) release formulations. Preferably, for topical applications (skin) or mouthwash, the composition comprises: - at least 100 μg/ml polyphenol or EGCG or 200 μg/ml polyphenol or EGCG or at least 300 μg/ml polyphenol or EGCG associated with at least 10⁶ cfti/ml probiotic or at least 10⁷ cfti/ml probiotic or at least 10⁸ cfu/ml probiotic. Preferably, for oral administration, the preparation comprises:

- at least 10 mg polyphenol or EGCG as the daily dose, or at least 200 mg polyphenol or EGCG as the daily dose or at least 500mg polyphenol or EGCG as the daily dose, or at least 1000 mg polyphenol or EGCG as the daily dose associated with - at least 10⁸ cfu probiotic or at least 10¹ ° cfu probiotic at least 10^{1 1} cfu probiotic.

Preferably, for both types of applications, the polyphenol is as defined earlier a green tea extract or derived thereof or ECGC or TEAVIGO™. Preferably, the probiotic is one of the probiotic strain defined earlier on.

The invention further relates to a product comprising the composition of the invention. The product is preferably selected from the group consisting of a medicament, a feed product, a food product, a cosmetic, an oral hygiene product and an agricultural product.

In a preferred embodiment, the composition of the invention is a medicament or for use in the preparation of a medicament or is a food product or part thereof. According to a preferred embodiment, the composition of the invention is used for the manufacture of said medicament or food product. The medicament of the invention may comprise any physiologically or pharmacologically acceptable excipient and/or diluent. If the composition of the invention is part of a food product, the composition of the invention can be present as a dry formulation, e.g. granule, powder, tablet or the like. Alternatively, the composition of the invention may be formulated in a liquid form. The food product of the invention comprising the composition of the invention may comprise any physiologically or acceptable excipient and/or diluent. Preferred food products are cultured milk, yoghurt, cheese, milk drink, milk powder, sport drink, coveratures (defined as mixtures of oil, sugar(s) and milk protein (whey)), infant formulas and fermented meat products. Since the composition of the invention may be incorporated into a (food) product, which already contains endogenous probiotic and/or polyphenol, all amounts of probiotic and/or polyphenol given in the description relates to added amounts of these components. Alternatively and according to another preferred aspect of the invention, a Lactobacillus strain as defined before, preferably the Lactobacillus acidophilus strain CBS 116411 and/or the Lactobacillus casei CBS 116412 and/or Bifidobacterium lactis CBS 118529 and mixtures thereof and a polyphenol, preferably EGCG as defined before are used in the preparation of a food product for preventing and/or combating infections associated with undesirable bacteria and associated diseases in a human being in a need thereof. Preferably, the undesirable bacteria is a Gram positive bacteria as defined before.

The product (medicament or food product) of the invention comprises a probiotic, preferably a Lactobacillus strain as defined earlier, preferably the Lactobacillus acidophilus strain CBS 116411 and/or the Lactobacillus casei CBS 116412 and/or Bifidobacterium lactis CBS 118529 and a polyphenol, preferably EGCG as defined before in an effective amount to prevent and or treat infections associated with undesirable bacteria and associated diseases. According to a preferred embodiment, the food product or medicament comprises at least 10⁶ to 10⁸ cfu probiotic per gram product and at least 2 to 5 mg polyphenol or EGCG per gram product. According to a more preferred embodiment, the food product or medicament comprises at least 10⁶ to 10⁸ cfu probiotic and at least 2 to 5 mg TEAVIGO™ or EGCG per gram product. According to another preferred embodiment, the composition and the product of the invention (medicament and food product) is in the form of tablet, capsule, powder or granule, orally liquid administered liquid preparations, or suppositories, or dry preparations. More preferably, the probiotic present in the composition and the product of the invention is in the form of an enteric tablet, capsule, powder or granule, meaning it will survive the stomach and arrive intact in the intestine. All these forms can be prepared by known means, using food grade, respectively pharmaceutically acceptable carriers, excipients, solvents or adjuvants. For the preparation of the medicament, standard ingredients and method of preparation as already described in Remington: The science and practice of pharmacy, 1995, Mack Publishing, Co Easton, PA 18042, USA can be used, which content is herewith incorporated by reference.

The invention therefore also relates to a method wherein the composition of the invention, comprising a probiotic and a polyphenol preferably EGCG and/or preferably a Lactobacillus strain as defined earlier, more preferably the Lactobacillus acidophilus strain CBS 116411 and/or the Lactobacillus casei CBS 116412 and/or Bifidobacterium lactis CBS 118529 are formulated in a food product or in a medicament, and said food product or medicament is administered to a given human being in an amount effective for preventing and/or treating diseases associated with undesirable bacterial infections. Therefore, the invention further relates to a method for preventing and/or treating infections associated with undesirable bacteria, preferably Gram positive bacteria and associated diseases in a given human being. This method comprises the step of administering the medicament or food product of the invention to said human being. It will be appreciated that the exact dosage of both essential active ingredients which are the probiotic and the polyphenol, constituting an amount effective for preventing and/or treating infections associated with undesirable bacteria and associated diseases in a human being in a need thereof may vary depending on the specific nature of the clinical conditions being treated, severity of the condition, age of human being treated, weight and condition of the human being treated, mode of administration of the dosage form, and the specific formulation being administered.

Preferably, administrating the composition or the product of the invention preferably means ingesting said product to give a daily dose of at least 10⁸ cfu probiotic and 10 mg polyphenol or EGCG per day. More preferably, an amount of the product that corresponds to 10¹⁰ cfu probiotic and 800 mg polyphenol or EGCG per day.

preservative and may be used as such. The preservative composition of the invention may additionally comprise an anti-fungal agent selected from the group consisting of a weak acid preservative, a polyene anti-fungal compound, sulphur dioxide, sulphites, nitrate, nitrite, dimethyl dicarbonate, biphenyl, diphenyl, orthophenylphenol, thiobendazole, an inorganic acid, an imidazole and mixture thereof. The different anti-fungal agents that may be present in the composition of the invention are below further defined:

1. a weak acid preservative such as sorbic acid, propionic acid, benzoic acid, a p- hydroxybenzoic acids, lactic acid, citric acid, acetic acid or an alkali metal or alkali earth metal salt thereof;

2. a polyene anti-fungal compound, preferably natamycin; 3. sulphur dioxide or sulphites;

4. nitrate and nitrite;

5. dimethyl dicarbonate;

6. biphenyl, diphenyl, orthophenylphenol or thiobendazole ;

7. an inorganic acid, such as hydrochloric acid;

8. an imidazole such as imazalil; and/or

9. any anti-fungal compound known in the art for use as a preservative for food products, crop protection or after-harvest treatment of fruits, vegetables or cereals, pharmaceutical or cosmetic products. The present invention further relates to the use of the composition of the invention comprising a probiotic and a polyphenol for killing among other undesirable bacateria such as Gram-positive bacteria or for inhibiting their growth or for preventing the germination of their spores. The composition of the invention can be used for the preparation of any types of products such as a food product or a medicament for preventing and/or treating infections caused by undesirable bacteria and associated diseases in a human being in a need thereof. The Gram-positive bacteria, the human being in need of the composition and the type of food products are as defined above including the preferences as described. The composition of the invention can be used as a preservative for the preparation of a product.

The invention will now be illustrated by the following example, which should not be construed as limiting the scope of the invention.

Examples General experimental conditions

In vitro experiments were performed to assess the effect of TEAVIGO™ on microbial growth of the probiotic strains L. acidophilus LAFTI^® L10 (CBS 116411), β. lactis LAFTp B94 (CBS 118529) and L. casei LAFTI^® L26 (CBS 116412), as well as the pathogenic bacteria, S. aureus ATCC 25923 and S. pyogenes ATCC 10389. The objectives of this study were to (i) assess the antimicrobial activity of TEAVIGO™ against pathogenic bacteria, Staphylococcus aureus and Streptococcus pyogenes in liquid medium, (ii) assess the effect of TEAVIGO™ on growth of probiotic strains Lactobacillus acidophilus LAFTI^® L10, Bifidobacteria lactis LAFϊP B94 and Lactobacillus casei LAFϊP L26 in liquid medium and (iii) explore the possibility of combined therapy for infectious diseases using TEAVIGO™ and probiotics together.

Cultures and media

The strains S. aureus ATCC 25923 and S. Pyogenes ATCC 10389 were obtained from the UNSW Microbiology Culture Collection. Three probiotic strains, L. acidophilus LAFTp L10 (CBS 116411 ), β. lactis YAFTf B94 (CBS 118529) and L. casei YAFTf L26 (CBS 116412) were supplied by DSM Food Specialties, Australia.

S. aureus was grown on nutrient agar medium under aerobic conditions. L. acidophilus, B. lactis and L. casei were grown in anaerobic jars under CO₂ + N₂ atmosphere (AnaroGen™, Oxoid) at 37 ⁰C on reinforced clostridial medium (RCM) agar media. S. pyogenes was grown on blood agar media under microaerophilic conditions. The susceptibility tests were carried out in RCM broth. All media were purchased from Oxoid, England.

[Example 1: In vitro growth responses of probiotic strains and pathogenic bacteria to TEAVIGO™

Antimicrobial effect and MIC of TEAVIGO™ in liquid medium TEAVIGO™ was obtained from DSM Nutritional Products Ltd. Kaiseraugst Switzerland. TEAVIGO™ is a green tea extract and contains minimum 90% of EGCG, a tea polyphenol. TEAVIGO™ was dissolved at a concentration of 10 mg /ml in milli-Q water, and was sterilised by passing through a 0.45 μM filter. The minimum inhibitory concentrations (MICs) of TEAVIGO™ were determined by the broth microdilution method, at an inoculum of about 1 x 10⁷ cfu per well. After incubation at 37 ⁰C for 24 h, all of the wells were examined macroscopically for bacterial growth, and the lowest concentration of two fold serially diluted TEAVIGO™ at which no visible growth occurred was defined as its MIC Anti-microbial activity of TEAVIGO™ in liquid medium

The anti-microbial effect of TEAVIGO™ was assessed using seven bacterial strains in RCM broth containing 0, 25, 50, 100, 200, 400 and 800 μg/ml TEAVIGO™. Visible growth of bacteria is illustrated in table 1.

Table 1. Visible growth (+) of bacteria in liquid medium containing various concentration of TEAVIGO™

Test strain Concentration of TEAVIGO ™ (μg /ml)

800 400 200 100 50 25 0

S. aureus ATCC 25923 - - - + + + +

S. pyogenes ATCC 10389 - - - + + + +

L. acidophilus L10 + + + + + + +

S. lactis B94 + + + + + + +

L casei L26 + + + + + + +

Legends: +: visible growth of bacteria

-: no visible growth of bacteria

Medium: reinforced clostridial medium (RCM) broth. All experiments were performed in triplicate in two separate experiments.

Minimum inhibitory concentrations of TEAVIGO™ on seven bacteria MICs were calculated from Table 1. As shown in table 2, the seven tested strains showed quite different susceptibility to TEAVIGO™. Compared to the MICs of TEAVIGO™ against S. aureus and S. pyogenes (100-200 μg/ml), the MICs of TEAVIGO™ against probiotic LAFTI^® strains were higher than 800 μg/ml. Table 2. Comparison of TEAVIGO™ MICs against four strains of pathogenic bacteria and three probiotic LAFϊP strains

Pathogenic bacteria Probiotic strains

Test strain MIC (μg/ml) Test strain MIC (μg/ml)

Gram-positive bacteria Gram-positive bacteria

S. aureus ATCC 25923 100-200 L acidophilus L10 >800

S. pyogenes ATCC 10389 100-200 B. lactis B94 >800

L. casei L26 >800

MIC, minimum inhibitory concentration. All experiments were performed in triplicate in two separate experiments.

It was recently reported that gram -positive bacteria generally are more sensitive to EGCG than gram-negative bacteria (12). However our results indicated that LAFTl^® probiotic strains, which are also gram-positive, demonstrated lower susceptibility to TEAVIGO™. Comparable result reported that EGCG shows bactericidal activities against gram -negative bacteria Vibrio (13). Although EGCG reportedly have selective bactericidal activity against pathogenic bacteria, no significant difference was observed in the MICs against bacterial phenotypes (13).

Conclusion

The minimum inhibitory concentrations (MICs) of TEAVIGO™ against S. aureus and S. pyogenes (100-200 μg/ml) were much lower than the values against the probiotic strains tested (>800 μg/ml). These results demonstrate the potential for combined therapy using TEAVIGO™ and probiotics on microbial infection caused by S. aureus and S. pyogenes etc., since probiotics and chemicals from natural products are two successful strategies as adjuncts or alternative to conventional antibiotic therapy. Example 2: Synergistic effect of Teavigo™ and probiotics on pathogenic bacteria

The probiotic strains and pathogenic strains defined in general experimental conditions were used in this example and cultivated the same way as defined in general experimental conditions and in example 1 unless otherwise indicated.

In the following tables, the synergistic effect of Teavigo™ associated with either Lactobacillus acidophilus L 10, Lactobacillus casei L26 or Bifidobacterium lactis B94 was demonstrated.

Table 3. Synergistic effect of Teavigo™ and Lactobacillus acidophilus L10 on the growth of Staph, aureus at 4h and 24 h.

Legends:

* RCM broth (fermentation broth) without Teavigo _T¹ M ^M (0) or with different concentrations of Teavigo™ (100, 200, 400 μg/ml)

** Staph, aureus was inoculated at (0 h) at a 5% concentration (inoculation ratio to L10:

1:1), or at 0.5% (inoculation ratio to L10: 0.1:1)

*** Fermentation was carried out anaerobically at 37⁰C for 4 h or 24 h

+: with L10, L10 was inoculated at 0 h at 5% (v/v) - : without L10

ND: not detected

The results (viable count of Staph, aureus) indicate that at 4 h the growth of Staph, aureus was significantly reduced by the synergistic effect of Teavigo™ and L10. At 24 h, the growth of Staph. Aureus was completely inhibited by the synergistic effect. Table 4: The effect of Teavigo™ (100-400 μg/ml) and Staph, aureus (0.1 :1 , 1 :1 inoculation ratio) on the growth of Lactobacillus acidophilus L10.

Lactobacillus acidophilus L10 was inoculated at 0 h at 5% (v/v). For other conditions see legends of Table 3.

The results (viable count of L10) demonstrate that neither Teavigo™ nor Staph, aureus had a significant effect on the growth of Lactobacillus acidophilus L10

Table 5. Synergistic effect of Teavigo™ and Bifidobacterium lactis B94 on the growth of Staph, aureus at 4h and 24 h.

Legends:

* RCM broth (fermentation broth) without Teavigo™ (0) or with different concentrations of

Teavigo™ (100, 200, 400 μg/ml)

** Staph, aureus was inoculated at (0 h) at a 5% concentration (inoculation ratio to B94:

1 :1 ), or at 0.5% (inoculation ratio to B94: 0.1:1)

*** Fermentation was carried out anaerobically at 37⁰C for 4 h or 24 h

+: with B94, B94 was inoculated at 0 h at 5% (v/v)

- : without B94

ND: not detected

The results (viable count of Staph, aureus) indicate that at 4 h the growth of Staph, aureus was significantly reduced by the synergistic effect of Teavigo™ and Bifidobacterium lactis B94. At 24 h, the growth was completely inhibited by the synergistic effect. Table 6. The effect of Teavigo™ (100-400 μg/ml) and Staph, aureus (0.1:1, 1:1 inoculation ratio) on the growth of Bifidobacterium lactis B94.

Bifidobacterium lactis B94 was inoculated at 0 h at 5% (v/v). For other conditions see legends of Table 3.

Concentration Staph, aureus was inoculated at O h at: of Teavigo 0 0.5% 5%

(μg/ml) 4 h 24 h 4 h 24 h 4 h 24 h

0 2.5 x10⁸ 3.8 x10⁸ 2.9 x10⁸ 3.9 x10⁸ 2.6 ^χ10⁸ 3.8 x10⁸

100 2.6 ^χ10⁸ 3.8 x10⁸ 2.2 ^χ10⁸ 3.8 x10⁸ 2.6 ^χ10⁸ 3.7 ^χ10⁸

200 2.2 x10⁸ 3.7^χ10⁸ 1.7 ^χ10⁸ 3.9x10⁸ 2.2 ^χ10⁸ 3.8x10⁸

400 1.6 ^χ10⁸ 1.8 x10⁸ 2.1 ^χ10⁸ 1.7 ^χ10⁸ 1.6 ^χ10⁸ 1.6 x10⁸

The results (viable count of Bifidobacterium lactis B94) demonstrate that neither Teavigo™ nor Staph, aureus had a significant effect on the growth of Bifidobacterium lactis B94.

Table 7. Synergistic effect of Teavigo™ and Lactobacillus casei L26 on the growth of Staph, aureus at 4h and 24 h.

Concentration Staph. aureus^** was inoculated at: of Teavigo^* 5% 0.5%

(μg/ml) 4 h _*** 24 h 4 h 24 h

- + L26 - + - + - +

0 7 χ 10⁵ 4 x 10⁴ 5 x 10⁷ ND 2.1 x 10⁴ 8 x 10³ 1 x 10⁷ ND

100 4.5 x 10⁴ 1.8 x 10⁴ 2 x 10⁵ ND 5 x 10² ND 1 x 10³ ND

200 2.8 x 10⁴ 1.1 x 10⁴ 1 x 10⁴ ND ND ND ND ND

400 2.5* 10⁴ 5 x 10³ ND ND ND ND ND ND

Legends

* RCM broth (fermentation broth) without Teavigo _TM (0) or with different concentrations of

Teavigo™ (100, 200, 400 μg/ml)

** Staph, aureus was inoculated at (0 h) at a 5% concentration (inoculation ratio to L26:

1 :1 ), or at 0.5% (inoculation ratio to L26: 0.1:1)

*** Fermentation was carried out anaerobically at 37⁰C for 4 h or 24 h

+: with L26, L26 was inoculated at 0 h at 5% (v/v)

- : without L26

ND: not detected

The results (viable count of Staph, aureus) indicate that at 4 h the growth of Staph, aureus was significantly reduced by the synergistic effect of Teavigo™ and Lactobacillus casei L26. At 24 h, the growth was completely inhibited by the synergistic effect. Table 8. The effect of Teavigo™ (100-400 μg/ml) and Staph, aureus (0.1:1, 1:1 inoculation ratio) on the growth of Lactobacillus casei L26.

Lactobacillus casei L26 was inoculated at 0 h at 5% (v/v). For other conditions see legends of Table 3.

The results (viable count of L26) demonstrate that neither Teavigo™ nor Staph, aureus had a significant effect on the growth of Lactobacillus casei L26.

Table 9. Synergistic effect of Teavigo™ and Lactobacillus acidophilus L10 on the growth of Strep, pyogenes at 4h and 24 h.

Legends

..TM

* RCM broth (fermentation broth) without Teavigo (0) or with different concentrations of

Teavigo™ (100, 200, 400 μg/ml)

** Strep, pyogenes was inoculated at (0 h) at a 5% concentration (inoculation ratio to L10:

1 :1), or at 0.5% (inoculation ratio to L10: 0.1 :1)

*** Fermentation was carried out anaerobically at 37⁰C for 4 h or 24 h

+: with L10, L10 was inoculated at 0 h at 5% (v/v)

- : without L10

ND: not detected

The results (viable count of Strep, pyogenes) indicate that at 4 h the growth of Strep, pyogenes was reduced by the synergistic effect of Teavigo™ and Lactobacillus acidophilus L10. At 24 h, the growth was completely inhibited by the synergistic effect. Table 10. The effect of Teavigo™ (100-400 μg/ml) and Strep, pyogenes (0.1 :1, 1 :1 inoculation ratio) on the growth of Lactobacillus acidophilus L10

The results (viable count of Lactobacillus acidophilus L10) demonstrate that neither Teavigo™ nor Strep, pyogenes had a significant effect on the growth of Lactobacillus acidophilus L10.

Table 11. Synergistic effect of Teavigo™ and Bifidobacterium lactis B94 on the growth of Strep, pyogenes at 4h and 24 h.

Legends

Teavigo™ (100, 200, 400 μg/ml)

** Strep, pyogeneswas inoculated at (0 h) at a 5% concentration (inoculation ratio to B94:

1 :1), or at 0.5% (inoculation ratio to B94: 0.1 :1)

*** Fermentation was carried out anaerobically at 37⁰C for 4 h or 24 h

+: with B94, B94 was inoculated at 0 h at 5% (v/v)

- : without B94

ND: not detected

The results (viable count of Strep, pyogenes) indicate that at 4 h the growth of Strep, pyogenes was reduced by the synergistic effect of Teavigo™ and Bifidobacterium lactis B94. At 24 h, the growth was completely inhibited by the synergistic effect. Table 12. The effect of Teavigo™ (100-400 μg/ml) and Strep, pyogenes (0.1 :1, 1 :1 inoculation ratio) on the growth of Bifidobacterium lactis B94.

B94 was inoculated at 0 h at 5% (v/v). For other conditions see Table 1a.

TM

The results (viable count of Bifidobacterium lactis B94) demonstrate that neither Teavigo nor Strep, pyogenes had a significant effect on the growth of Bifidobacterium lactis B94.

Table 13. Synergistic effect of Teavigo™ and Lactobacillus casei L26 on the growth of Strep, pyogenes at 4h and 24 h.

Legends

Teavigo™ (100, 200, 400 μg/ml)

** Strep, pyogeneswas inoculated at (0 h) at a 5% concentration (inoculation ratio to L26:

1 :1 ), or at 0.5% (inoculation ratio to L26: 0.1 :1)

*** Fermentation was carried out anaerobically at 37⁰C for 4 h or 24 h

+: with L26, L26 was inoculated at 0 h at 5% (v/v)

- : without L26

ND: not detected

The results (viable count of Strep, pyogenes) indicate that at 4 h the growth of Strep, pyogenes was significantly reduced by the synergistic effect of Teavigo™ and Lactobacillus casei L26. At 24 h, the growth was completely inhibited by the synergistic effect. Table 14. The effect of Teavigo™ (100-400 μg/ml) and Strep, pyogenes (0.1:1, 1:1 inoculation ratio) on the growth of Lactobacillus casei L26.

The results (viable count of Lactobacillus casei L26) demonstrate that neither Teavigo™ nor Strep, pyogenes had a significant effect on the growth of Lactobacillus casei L26.

References

I. Gδtz F. Staphylococci in colonization and disease: prospective targets and vaccines. Current opinion in Microbiology 2004;7:477-87. 2. Mitchell TJ. The pathogenesis of streptococcal infections: from tooth decay to meningitis. Nature Review 2003;1:219-30.

3. Gold HS, Moellering RC. Antimicrobial drug resistance. The New England Journal of medicine. 1996; 335: 1445-1435.

4. Saavedra JM, Bauman NA, Oung I, Perman JA, Holken RH. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet 1994;344: 1046-9.

5. Henriksson A, Conway PL. Isolation of the human faecal bifidobacteria which reduce signs of Salmonella infection when orogastrically dosed to mice. J Appl Microbiol 2001 ;90:223-8. 6. Kabir AM, Aiba Y, Takagi A, Kamiya S, Miwa T, Koga Y. Prevention of Helicobacter pylori infection by lactobacilli in a gnotobiotic murine model. Gut 1997;41(1):49-55.

7. Roos K, Holm SE, Grahn-Hakansson E, Lagergren L. Recolonization with selected alpha-streptococci for prophylaxis of recurrent streptococcal pharyngotonsilitis - a randomized placebo-controlled multicentre study. Scand J Infect Dis 1996;28:459-62.

8. Zhurylo OA, Turiiun SA, Drozd T. A biological model study of the effect of Aerococcus viridans on pathogenic bacteria. Mikrobiologicheskii Zhurnal 1998;60(3):56-62.

9. Lee K-M, Yeo M, Choue J-S, et al. Protective mechanism of Epigallocatechin-3- Gallate against Helicobacter pylori-induced gastric epithelial cytotoxicity via the blockage of TLR-4 Signaling. Helicobacter 2004;9(8):632-42.

10. Maliakal PP, Coville PF, Wanwimolruk S. Tea consumption modulates hepatic drug metabolizing enzymes in Wistar rats. J Pharm Pharmacol 2001 ;53:569-77.

II. lsolauri E, Sϋtas Y, Kankaanpaa P. Probiotics: effects on immunity. Am J Clin Nutr 2001;73:S444-50.

12. Yoda Y, Hu ZQ, Zhao WH, Shimamura T (2004) Different susceptibilities of Staphylococcus and Gram-negative rods to epigallocatchin. J. Infect. Chemother. 10:55-8. 13.Taguri T, Tanaka T, Kouno I (2004) Antimicrobial activity of 10 different plant polyphenols against bacteria causing food-borne disease. Biol. Pharm. Bull. 27: 1965-1969.

14. Green, LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tandenbaum SR (1982) Analysis of nitate and nitrite in biological fluids. Anal Biochem 126:131-144

15. Cheng AC, O'Brien M, Jacups SP, Anesty NM, Currie BJ (2004) GReactive protein in the diagnosis of Melioidodis. Am J Trop med Hyg 70:580-582

I Applicant's or agent's file reference number 24947WO International application No

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCTRuIe 136/s)

A. The indications made below relate to the microorganism referred to in the description Firstly mentioned on page 3 line 2.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional shie)(

Name of depositary institution

CENTRAAL BUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country) Uppsalalaan 8 P.O. Box 85167 NL-3508 AD Utrecht The Netherlands

Date of deposit 15 October 2004 Accession Number CBS 116411

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional

We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT, shall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent or, where applicable, for twenty years from the date of filing if the application has been refused, withd rawn or deemed to be withdrawn.

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E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed belowwill be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

For receiving Office use only — For International Bureau use only —

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Form PCT/RO/134 (July 1992) I Applicant's or agent's file reference number 24947WO International application No

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13fe)

The indications made below relate to the microorganism referred to in the description Firstly mentioned on page 3 line 2

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

CENTRAALBUREAUVOORSCHIMMELCULTURES

Address of depositary institution (including postal code and country)

P O Box 85167 NL-3508 AD Utrecht The Netherlands

Date of deposit 15 October 2004 Accession Number CBS 116412

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCT, shall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent or, where applicable, for twenty years from the date of filing if the application has been refused, withdrawn or deemed to be withdrawn

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g , "Accession Number of Deposit ")

For receiving Office use only ~~ For International Bureau use only ~~

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FormPCT/RO/134 (July 1992) I Applicant's or agent's file reference number 24947WO International application No

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13fe)

A. The indications made below relate to the microorganism referred to in the description Firstly mentioned on page 3 line 3

Name of depositary institution

CENTRAALBUREAUVOORSCHIMMELCULTURES

Address of depositary institution (including postal code and country)

P O Box 85167 NL-3508 AD Utrecht The Netherlands

Date of deposit 14 September 2005 Accession Number CBS 118529

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

For receiving Office use only ~~ For International Bureau use only ~~

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FormPCT/RO/134 (July 1992)

Claims

1. Composition comprising a probiotic and a polyphenol.

2. Composition according to claim 1 , wherein the polyphenol is a green tea extract.

3. Composition according to claim 2, wherein the green tea extract comprises at least 90% w/w, preferably at least 93% w/w, more preferably at least 95% w/w epigallocatechin gallate.

4. Composition according to any one of claims 1 to 4, wherein the probiotic is selected from the group consisting of a Lactobacillus, a Bifidobacterium species and mixture thereof.

5. Composition according to claim 4, wherein the Lactobacillus is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus fermentum and mixtures thereof and/or wherein the Bifidobacterium is selected from the group consisting of Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium lactis and mixture thereof.

6. Composition according to claim 5, wherein the probiotica is selected from the group consisting of the Lactobacillus acidophilus CBS 116411, the Lactobacillus casei, CBS 116412, Bifidobacterium lactis CBS 118529.

7. Composition according to any one of claims 1 to 6, which has the capacity to kill an undesirable bacterium or to inhibit its growth, or to prevent the germination of its spores in vitro in a given medium and/or in vivo in a given food product matrix, crop protection application, pharmaceutical or cosmetic product.

8. Composition according to claim 7, wherein the undesirable bacterium is a Gram positive bacteria, preferably selected from the group consisting of a Helicobacter, Streptococcus, Staphylococcus species and mixture thereof.

9. Composition according to any one of claims 1 to 8, which additionally comprises a prebiotic.

10. Composition according to claim 9, wherein the prebiotic is selected from the group consisting of pentosans, b-glucans, pectins, and pectic polysaccharides, mannans, arabinans, galactans, fructo-oligosaccharides, resistant starch and mixture thereof.

11. Composition according to any one of claims 1 to 10 for use as a medicament.

12. Composition according to any one of claims 1 to 11 for preventing and/or treating infections caused by undesirable bacteria and associated diseases in a human being in a need thereof.

13. Composition according to claim 12, wherein the human being in a need of the medicament experiences some form of fatigue, stress, bed sores, gingivitis, nose and throat infection and peptic ulcer and mixture thereof.

14. Composition according to any one of claims 1 to 13, which is a preservative.

15. Composition according to claim 14, wherein the composition additionally comprises an anti-fungal agent selected from the group consisting of a weak acid preservative, a polyene anti-fungal compound, sulphur dioxide, sulphites, nitrate, nitrite, dimethyl dicarbonate, biphenyl, diphenyl, orthophenylphenol, thiobendazole, an inorganic acid, an imidazole and mixture thereof.

16. Product comprising the composition of any one of claims 1 to 15, wherein the product is selected from the group consisting of a medicament, a feed product, a food product, a cosmetic, an oral hygiene product and an agricultural product.

17. Use of a composition comprising a probioticum and a polyphenol as defined in any one of claims 1 to 16 for killing an undesirable bacterium or for inhibiting its growth, or for preventing the germination of its spores in vitro in a given medium and/or in vivo in a given food product matrix, crop protection application, pharmaceutical or cosmetic product.

18. Use of a composition as defined in any one of claims 1 to 15 for the preparation of the product according to claim 16.

19. Use according to claim 18, for the preparation of a food product or a medicament for preventing and/or treating infections caused by undesirable bacteria and associated diseases in a human being in a need thereof.

20. Use according to any one of claim 17 to 19, wherein the undesirable bacterium is a Gram bacterium, which is preferably selected from the group consisting of a Helicobacter, Streptococcus, Staphylococcus species and mixture thereof.

21. Use according to any one of claim 17 to 20, wherein the human being in a need of said food product of medicament is selected from the group consisting of: human subjects that experience some form of fatigue, stress, bed sores, gingivitis, nose and throat infection and peptic ulcer and mixture thereof.

22. Use according to claim 17, as a preservative for the preparation of the product according to claim 16.