WO2014068338A1

WO2014068338A1 - Diverticulitis treatment

Info

Publication number: WO2014068338A1
Application number: PCT/GB2013/052879
Authority: WO
Inventors: Barry Edward SMITH
Original assignee: Multigerm UK Enterprises Ltd
Current assignee: Multigerm UK Enterprises Ltd
Priority date: 2012-11-05
Filing date: 2013-11-04
Publication date: 2014-05-08
Anticipated expiration: 2015-05-05
Also published as: GB201219873D0

Abstract

The invention relates to a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non- dairy liquid substrate, for use in the treatment of diverticulitis and/or diverticular disease.

Description

Diverticulitis Treatment

Field of the invention

The invention relates to treatment of diverticulitis and/or diverticular disease using probiotic preparations containing viable, metabolically active probiotic bacteria.

Background to the invention

Diverticulitis and diverticular disease are extremely common digestive diseases particularly found in the large intestine and develop from diverticulosis . Diverticulosis is characterized by the formation of pouches (diverticula) that bulge to the outside of the colon, presumably through areas of weakness. Inflammation (diverticulitis) results if one of these diverticula becomes infected and / or obstructed. It is commonly accompanied by gross or microscopical

perforation, ranging in severity from a single, mild, acute attack of diverticulitis to more severe attacks

characterized by perforation and abscess formation,

occasionally resulting in chronic complications such as obstruction and fistula formation (Fox et al . , 2010, Sopeha et al . , 2011) . Many patients with diverticulitis then develop periodic changes in bowel openings, from diarrhoea and constipation, and many patients have abdominal pain and a symptom complex that resembles Irritable Bowel Syndrome (IBS) .

Summary of the invention

In accordance with a first aspect of the invention there is provided a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate, for use in the treatment of diverticulitis and/or diverticular disease.

The invention further provides a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate, for use in reducing the incidence and/or severity of diarrhoeal attacks in a patient previously diagnosed with diverticulitis or diverticular disease .

The invention further provides a method of treating diverticulitis and/or diverticular disease in a human patient comprising administering to a patient in need thereof an effective amount of a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate.

The invention further provides a method of reducing the incidence and/or severity of diarrhoeal attacks in a human patient previously diagnosed with diverticulitis or

diverticular disease, comprising administering to a patient in need thereof an effective amount of a probiotic

preparation comprising viable, metabolically active

probiotic bacteria in a non-dairy liquid substrate.

In certain embodiments the probiotic preparation is capable of delivering viable, metabolically active probiotic bacteria to the intestinal tract of a human subject without triggering digestion.

In certain embodiments the probiotic bacteria in the probiotic preparation are stable when maintained in culture at pH 3.0 for a period of at least 6 hours.

In accordance with a second aspect of the invention there is provided a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate comprising a mixture of complex carbohydrates and simple sugars, wherein the ratio of total carbohydrate content to reducing sugar content of the probiotic preparation is in the range of from 8:1 to 2:1, for use in the treatment of diverticulitis and/or

diverticular disease.

The invention further provides a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate comprising a mixture of complex carbohydrates and simple sugars, wherein the ratio of total carbohydrate content to reducing sugar content of the probiotic preparation is in the range of from 8:1 to 2:1, for use in reducing the incidence and/or severity of diarrhoeal attacks in a patient previously diagnosed with diverticulitis or diverticular disease.

The invention further provides a method of treating diverticulitis and/or diverticular disease in a human patient comprising administering to a patient in need thereof an effective amount of a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate comprising a mixture of complex carbohydrates and simple sugars, wherein the ratio of total carbohydrate content to reducing sugar content of the probiotic preparation is in the range of from 8:1 to 2:1.

preparation comprising viable, metabolically active

probiotic bacteria in a non-dairy liquid substrate

comprising a mixture of complex carbohydrates and simple sugars, wherein the ratio of total carbohydrate content to reducing sugar content of the probiotic preparation is in the range of from 8 : 1 to 2:1.

The probiotic preparation used in the treatment of diverticulitis and/or diverticular disease as described herein may be prepared by growing one or more strains of probiotic lactic acid bacteria in a growth substrate is derived from malted cereal grains. More specifically, the probiotic preparation may be prepared by growing one or more strains of probiotic lactic acid bacteria in an extract of germinated barley.

Therefore, in a further aspect the invention provides a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate, for use in the treatment of diverticulitis and/or

diverticular disease, wherein the probiotic preparation is prepared by growing one or more strains of probiotic

bacteria in an extract of germinated barley.

The invention further provides a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate, for use reducing the incidence and/or severity of diarrhoeal attacks in a human patient previously diagnosed with diverticulitis or

bacteria in an extract of germinated barley.

The invention further provides a method of treating diverticulitis and/or diverticular disease in a human patient comprising administering to a patient in need thereof an effective amount of a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate comprising an extract of germinated barley.

preparation comprising viable, metabolically active

probiotic bacteria in a non-dairy liquid substrate

comprising an extract of germinated barley.

The invention further provides use of a probiotic preparation comprising viable, metabolically active

probiotic bacteria in a non-dairy liquid substrate in the manufacture of a medicament for treating or preventing diverticulitis and/or diverticular disease.

Detailed description of the invention

It is estimated that between one-third and a half of the population of Western Europe and North America will develop diverticula in the colon during their lifetime with the likelihood of having the condition increasing with age: Less than one person in 20 has the condition before the age of 40, rising to a quarter by 60 years of age and two-thirds by the age of 85. Nevertheless, most people with diverticula suffer no symptoms or complications and it is known that as many as three people in four with diverticula are unaware they have the condition.

According to available guidelines, treatment of

symptomatic, but uncomplicated, diverticular disease aims to reduce the frequency and severity of diverticular-related symptoms (abdominal pain, bloating, alterations in bowel habit) and to prevent complications (Sopeha et al . , 2011, Maconi et al . , 2011) . Different agents have been proposed, such as bulking agents, antispasmodics, and non-absorbed topical antibiotics, on the basis of different potential pathophysiological mechanisms; that is, abnormal colonic motility, inadequate intake of dietary fibers, intestinal bacterial overgrowth, and mucosal inflammation. However few of these measures are of proven efficacy (Petruzziello et al . , 2006, Beckham et al . , 2009) . There is some indication that 5-amino-acid preparations can reduce the prevalence of recurrent diverticulitis after an attack (Di Mario et al . , 2005) . In addition, changes in intestinal microflora have been proposed as possible mechanisms responsible for low grade inflammation as bacterial overgrowth has been shown to occur in this disease.

Because the large majority of patients with

diverticulosis will remain entirely asymptomatic, there is no need for treatment. However, based on the pathogenesis of diverticular disease, a diet rich in vegetables, fruit and fiber is recommended in order to accelerate colonic transit and reduce intraluminal pressure. Current therapeutic approaches to prevent recurrence of symptoms of

uncomplicated diverticulosis are mainly based on

nonabsorbable antibiotics and / or probiotics (Maconi et al . , 2011) .

The use of probiotics for the purpose of health and disease prevention, first proposed in the 19th Century and Metchnikoff, suggested that a high concentration of

lactobacilli in the intestinal flora is important for the health and longevity of humans. There has recently been renewed interest in the possibility that the intestinal microflora plays a role in patients with gastrointestinal diseases, largely fuelled by makers of yogurts that are claimed to contain live and desirable bacteria. However although ingestion of these foodstuffs are certainly not harmful the effectiveness of this dietary modification remains questionable, not least for the fact that the stability of the preparations is highly variable and the microbes do not withstand storage. There have been major developments in these respects, largely confined to non- dairy based products. Studies investigating the effect of probiotic treatment of patients with diverticular disease have found that the duration of remission (time to recurrent diverticulitis) was longer after treatment with the

probiotic than without the probiotic or more patients remained asymptomatic in the probiotic treated groups than on standard treatments such as anti-inflammatories or antibiotics (Beckham et al . , 2009, Quigley et al . , 2010) . A further study using SCM-III symbiotic mixture (Lactobacillus acidophilus 145 and Bifidobacterium spp) , three times a day, was effective in preventing recurrence of symptomatic uncomplicated diverticular disease of the colon, especially in those patients with constipation-predominant features (Lamiki et al . , 2010) . Although the main shortcomings of these studies are that they were small and often without appropriate control groups, and secondly there is continuous concern on the viability of the preparations used, they do suggest probiotics may have a positive effect on the

recurrence of symptomatic diverticular disease (Beckham et al . , 2009) . The most positive aspects of these studies are that they appear to demonstrate efficacy and that the treatments are without significant side effects. The use of probiotics in diverticular disease is generally thought to work by altering the local microflora in and around the diverticula of the colon and improve immune responses, thus potentially having a beneficial effect on the microscopic colitis associated with the diverticula (Beckham et al . , 2009, Quigley et al . , 2010, Ouwehand et al . , 2003).

Probiotics come in several formats and the principle challenge is delivering the bacteria effectively and

documenting a health benefit clinically in appropriately designed trials. Calculating a specific daily amount or viable colony-forming unit (CFU) at a level proven to confer a health benefit is difficult. However, it is even harder to produce a product that can cope with changes in ambient temperatures, or the pH levels of the host environment and still hold the same number of viable count at - the point where they are needed - and not at the time of manufacture. For dairy and freeze-dried probiotic formats, this is particularly challenging as yoghurts trigger digestion in the stomach, and powders and capsules need a few hours to rehydrate before they can be activated.

Until today, format may be one of the reasons why probiotic companies have struggled to prove efficacy for IBS supported by strong evidence.

Microbiological organisms are frequently used as food supplements. Examples are probiotic bacteria which are known to have beneficial effects on the intestinal microflora increasing the resistance to infectious disease such as diarrhoea .

Probiotic bacteria can be found in dairy products such as yoghurt and species known to have health benefits include those from the genera Enterococcus and Lactobacillus .

However, probiotic dairy products have a short shelf life.

Current methods for storing bacterial cultures

typically use lyophilization, also called freeze-drying . In this process, water is removed from the organism by

sublimation and the organism and can be revived after the addition of water. However, freeze-dried bacteria are not metabolically active and it is well known that freeze-dried products typically lose much of their resilience after a few weeks of storage at room temperature (Fonseca et al and Murga et al ) .

Furthermore, many commercial probiotics do not seem to contain all of the species mentioned on the labels, and where bacteria are present the numbers of viable bacteria are often very low (J . Hamilton-Miller) .

The present invention provides effective treatment of diverticulitis and/or diverticular disease using a probiotic preparation. In particular, the invention provides an effective means of reducing the incidence and/or severity of diarrhoeal attacks in a human patient previously diagnosed with diverticular disease or diverticulitis.

The probiotic preparation used in the effective

treatment of diverticulitis and/or diverticular disease is a liquid-based product (and typically a water-based product) which is non-dairy and is not freeze-dried. The probiotic preparation contains viable, metabolically active probiotic bacteria in a liquid substrate which is capable of

delivering viable, metabolically active probiotic bacteria to the intestinal tract, where the probiotic bacteria rapidly begin to establish and multiply. The probiotic bacteria in the preparation are therefore "alive" and ready to function immediately after the preparation is swallowed.

Prior art probiotic products are predominately

available in dairy or freeze-dried formats such as powders or capsules. In order for probiotics to work effectively and to their optimum, they need to be directed to specific sites within the intestinal tract without triggering digestion as well as avoiding extremes. If digestion is triggered, the stomach acids can weaken or destroy probiotic bacteria. For some probiotics this is a challenge as they are contained in yoghurt type drinks which the stomach initially 'sees' as food. On the other hand, freeze-dried probiotics have been taken down to a temperature of around minus 80 °C and the fimbria can be broken off during the freezing process.

Freeze-dried probiotics have to rehydrate before they are able to function adequately. This can take several hours. Finding an effective "transport system" for probiotic bacteria is a key objective for manufacturers producing probiotic supplements or functional food.

The probiotic preparation described herein does not suffer from these drawbacks, as the probiotic bacteria are carried in a liquid-based substrate, typically a water-based substrate, which shields and transports the probiotic bacteria safely through the stomach (without triggering digestion) to the intestines where they rapidly begin to establish and multiply. Without wishing to be bound by any particular theory, it is believed that the viable nature of the probiotic bacteria in the preparation described herein allows them to establish rapidly in the patient's

gastrointestinal tract, perhaps within 15 to 20 minutes of ingestion.

Although the preparations and methods described herein are indicated to be for use in the treatment of diverticular disease and/or diverticulitis, the prevention of these conditions using the same preparations and methods is also contemplated. Prevention may be particularly desirable in individuals known to have developed diverticulosis . Definitions

"Diverticulosis" - as used herein the term "diverticulosis relates to a condition characterised by the formation of pouches (diverticula) that bulge to the outside of the colon.

"Diverticular disease" and "Diverticulitis" - as used herein the terms "diverticular disease" and "diverticulitis" relate to related conditions affecting the colon, involving the inflammation and/or infection of pouches (diverticula) that bulge to the outside of the colon. The most common symptom of diverticular disease is intermittent pain in the lower abdomen, usually in the lower left-hand side. Other

symptoms of diverticular disease include (but are not limited to) :

• a change in normal bowel habits, such as constipation or diarrhoea, or episodes of constipation that are followed by diarrhoea

• stomach cramps

· bloating

• rectal bleeding

The main symptom of diverticulitis is a constant and severe pain. The pain usually starts below the navel, before moving to the lower left-hand side of the abdomen. Besides severe stomach pain, other symptoms of diverticulitis include (but are not limited to) :

• a high temperature (fever) of 38°C (100.4°F) or above

• nausea

· vomiting

• constipation • rectal bleeding

"Probiotic" - as used herein the term "probiotic" is to be interpreted according to the FAO/WHO joint report and guidelines for use of probiotics, in which probiotics are defined as "live microorganisms which when administered in adequate amounts confer a health benefit to the host". The term "probiotic bacteria" refers to any bacterial strain which fulfils this definition of a "probiotic".

"Lactic acid bacteria (LAB)" - as used herein the term

"lactic acid bacteria (LAB)" refers to a group of Gram positive, catalase negative, non-motile anaerobic bacteria that ferment carbohydrates to lactic acid. This group includes the genera Lactobacillus, Lactococcus, Pediococcus, Bifidobacterium, and Enterocoecus .

"Probiotic lactic acid bacteria" - as used herein the term "probiotic lactic acid bacteria" refers to lactic acid bacteria which also satisfy the definition of a "probiotic" as used herein. Exemplary probiotic lactic acid bacteria include, but are not limited to, those in the genera

Lactobacillus and Enterococcus . "Treatment" of diverticular disease - as used herein in the specific context of diverticular disease, the term

"treatment" refers to an improvement in one or more symptoms associated with diverticular disease. In addition,

prevention of diverticular disease is also contemplated, particularly in individuals known to have diverticulosis . "Treatment" of diverticulitis - as used herein in the specific context of diverticulitis, the term "treatment" refers to an improvement in one or more symptoms associated with diverticulitis. In addition, prevention of

diverticulitis is also contemplated, particularly in

individuals known to have diverticulosis .

"Complex carbohydrates" - as used herein the term "complex carbohydrates" includes both oligosaccharides and

polysaccharides. "Oligosaccharides" are saccharide polymers containing 3 to 10 saccharide units; whereas the term

"polysaccharides" includes longer polymeric structures, such as those formed from repeating saccharide (or disaccharide ) units .

"Simple sugars" - as used herein the term "simple sugars" refers to both monosaccharides and disaccharides , unless otherwise stated. "Reducing sugars" - as used herein the term "reducing sugars" refers to sugars which either have an aldehyde group or are capable of forming an aldehyde group in solution through isomerisation . The presence of reducing sugars may be determined by means of the Nelson-Somogyi method using glucose as the reference standard (Somogyi, M. (1052)

Journal of Biological Chemistry., Vol. 195., p.19;

reproduced in many standard textbooks of carbohydrate chemistry) . Although certain complex carbohydrates (e.g. starches) may contain reducing ends, and therefore fulfil the definition of "reducing sugars", a determination of the content of "reducing sugars" in a given sample (e.g. a sample of probiotic preparation as described herein) using the Nelson-Somogyi method may be taken as an approximation of the amount of simple sugars in the sample, since the simple sugars contain a greater proportion of reducing ends per unit mass than complex carbohydrates.

"Total carbohydrate content" - as used herein the terms "total carbohydrate" or "total carbohydrate content" refer to the total amount of complex carbohydrate and simple sugars present in a given product (e.g. a probiotic

preparation as described herein) . Total carbohydrate content may be measured using the phenol-sulphuric acid assay, using glucose as a reference standard (Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956) Analytical Chemistry, vol. 28., p. 350).

Where reference is made herein to the ratio of total

carbohydrate content to reducing sugar content of a liquid- based product (e.g. in a probiotic preparation) then this is to be determined by calculating the ratio of total

carbohydrate content of the product, as measured by the phenol-sulphuric acid method described herein (result expressed in mg/ml), to reducing sugar content of the product, as measured by the Nelson-Somogyi method described herein (result expressed in mg/ml) .

Probiotic bacterial strains

In exemplary embodiments of each aspect of the

invention, the probiotic bacteria included in the probiotic preparation may be lactic acid bacteria.

In exemplary embodiments, the probiotic bacteria may be of the genus Lactobacillus or Enterocoecus . In specific embodiments, the lactic acid bacteria may be of at least one of the following species: Enterococcus faecium, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus acidophilus . In other exemplary embodiments, a combination of two or three of the species Enterococcus faecium, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus acidophilus may be used. In an exemplary embodiment, a combination of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus and

Lactobacillus casei is used. In another exemplary

embodiment, a combination of Enterococcus faecium,

Lactobacillus plantarum, Lactobacillus rhamnosus and

Lactobacillus acidophilus is used. This combination of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus acidophilus was shown to be particularly effective in reducing IBS symptom severity, and also improving quality of life scores for IBS patients, in a clinical study carried out by the inventors.

A key factor in achieving therapeutic efficacy in the treatment of diverticulitis and/or diverticular disease, is the ability to deliver a high count of viable, metabolically active probiotic bacteria to the gastrointestinal tract of the patient undergoing treatment. In order to achieve this, in exemplary embodiments, the total population of

metabolically active bacteria in the probiotic preparation may be in the range of from 1.0 x 10⁶ to 1.0 x 10⁹ viable cells per millilitre, preferably in the range of from 1.0 x 10⁷ to 1.0 x 10⁹ viable cells per millilitre. Each

individual strain of metabolically active bacteria present in the probiotic preparation may be present in the range of from 1.0 x 10⁵ to 1.0 x 10⁹ viable cells per millilitre, more preferably in the range of from 1.0 x 10⁷ to 1.0 x 10⁹ viable cells per millilitre. In the case of probiotic preparations comprising a combination of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus rhamnosus, it is preferred that at least one and preferably each of these strains is present in the range of from 1.0 x 10⁶ to 1.0 x 10⁹ viable cells per millilitre, preferably in the range of from 1.0 x 10⁷ to 1.0 x 10⁹ viable cells per millilitre

The population of L. acidophilus, if included, may be lower than 1.0 x 10⁵ viable cells per millilitre.

In an exemplary embodiment the preparation may comprise a combination of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus, wherein the bacterial count for each of these bacterial strain is in the range of from 1.0 x 10⁵ to 1.0 x 10⁹ viable cells per millilitre, more preferably in the range of from 1.0 x 10⁷ to 1.0 x 10⁹ viable cells per millilitre, and may optionally contain Lactobacillus acidophilus . The population of L.

acidophilus, if included, may be lower than 1.0 x 10⁵ viable cells per millilitre.

Although the total bacterial count in the presently described preparation may appear to be lower than that of typical freeze-dried probiotic preparations, it is important to note that the bacteria are viable. As noted elsewhere herein, it is believed that the viable nature of the

bacteria in the probiotic preparation described herein and shown to be effective in treating IBS allows them to establish more rapidly in a patient's gastrointestinal tract, perhaps within 15 to 20 minutes of ingestion. It is believed that such rapid establishment contributes to the beneficial effects of the preparation in the treatment of diverticulitis and/or diverticular disease. Another key advantage of the probiotic preparation described herein, is that the probiotic strains are

significantly more stable at pH3 than proprietary milk-based or freeze-dried probiotic products, and therefore more likely to be capable of tolerating the harsh conditions encountered in the human GI tract. In particular, it has been shown that the probiotic bacteria in the preparation described herein are stable both at pH6 and at pH3. In exemplary embodiments the probiotic bacteria in the

probiotic preparation are stable when maintained in culture at pH 3 for a period of at least 6 hours. In this context "stable" can be taken to mean that when the bacteria are cultured at pH 3, 37°C, in standard culture medium (e.g. MRS broth) then over a period of at least 6 hours the bacterial count (cfu/ml) does not fall by more than 0.5 logio units below the bacterial count (cfu/ml) at time zero. In

specific embodiments, the bacterial count should remain above 10⁶ cfu/ml for at least 6 hours when cultured at pH 3, 37°C, in standard MRS broth. In fact, the bacterial count may be observed to increase when cultured under these conditions .

Composition of the probiotic preparation

The probiotic preparation described herein comprises viable, metabolically active probiotic bacteria in a non- dairy liquid substrate. The function of the substrate is to support, and maintain viability of, the probiotic bacteria, particularly during storage of the preparation, such that the probiotic bacteria can be maintained, and then delivered to the patient, in a viable, metabolically active form. To achieve this objective, the liquid substrate typically contains a mixture of complex carbohydrates and simple sugars. In particular, the substrate may comprise a mixture of polysaccharides, oligosaccharides, disaccharides and monosaccharides .

In certain embodiments, the probiotic preparation may be characterised by the ratio of total carbohydrate content to reducing sugar content of the preparation, reflecting the complex mix of polysaccharides, oligosaccharides,

disaccharides and monosaccharides present. In exemplary embodiments, the ratio of total carbohydrate content to reducing sugar content of the preparation is in the range of from 8:1 to 2:1, more typically in the range of from 5:1 to 2.5:1, or in the range of from 4:1 to 3:1. Means of

measuring the total carbohydrate content and reducing sugar content, and calculating the ratio between them, are as defined above.

In further exemplary embodiments of the probiotic preparation, the total carbohydrate content of the

preparation may be in the range of from 20 mg/ml to 40 mg/ml, or in the range of from 20 mg/ml to 30 mg/ml and the total reducing sugar content may be in the range of from 5 mg/ml to 20 mg/ml, or in the range of from 5 mg/mg to 10 mg/ml .

The probiotic preparation preferably also comprises protein and peptide components. Typically the total amount of protein and peptides present in the probiotic preparation is in the range of from 1 mg/ml to 2 mg/ml and the total amount of high molecular weight peptides (molecular weight greater than 5000 Daltons) is in the range of from 100 g/ml to 300 g/ml. In a specific embodiment, the concentration of protein and peptides may be about 2mg/ml and the

concentration of high molecular weight peptides may be about 250μg/ml . The probiotic preparation may contain further

components such as, for example, cellulose, starch, β- glucans, pentosans, polyphenols, ribonucleic acids, lipids, phosphates, flavenoids, amino acids, vitamins (Βχ, B₂, C and E), silicates and trace elements.

The probiotic preparation may comprise an extract of germinated barley containing the desired probiotic bacterial strains .

An exemplary embodiment of the probiotic preparation comprises extract of germinated barley and a combination of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus, wherein the bacterial count for each of these bacterial strain is in the range of from 1.0 x 10⁵ to 1.0 x 10⁹ viable cells per millilitre, more preferably in the range of from 1.0 x 10⁷ to 1.0 x 10⁹ viable cells per millilitre, and optionally contains Lactobacillus

acidophilus .

Additional components may be added to the probiotic preparation, such as flavourings and/or colourings, to improve palatability for human patients.

The pH of the preparation can be conveniently

controlled by the addition of a suitable buffer or

combination of buffering agents. Preferred buffers include, for example, tri-sodium citrate or phosphate buffers. The pH of the liquid-based preparation described herein is

typically maintained in the range of from 3.8 to 4.5, and in particular at about pH 4.0, during long-term storage. The probiotic preparation may be stored at any temperature from 4°C up to ambient temperature (about 25 °C) . The Symprove™ product described herein has been shown to remain stable (in terms of bacterial count) for a period of at least 6 months when stored at about 4°C, and for at least 4 months when stored at 25°C.

The preparation may additionally comprises an antifungal agent, such as, for example, sterilised potassium sorbate and/or and anti oxidant, such as vitamin C.

In a preferred embodiment the growth substrate may contain particulate matter, for example particles not exceeding 1mm in diameter.

The most preferred embodiment of the probiotic

preparation, shown to be effective in the treatment of IBS in a clinical study carried out by the inventors, is the product denoted Symprove™, containing viable, metabolically active cells of Enterococcus faecium, Lactobacillus

plantarum, Lactobacillus rhamnosus and Lactobacillus

acidophilus, which may be prepared according to the examples provided herein.

The Symprove™ product is water-based, with a typical water content of about 95% (v/v) . This high water content is advantageous since it may result in the product being perceived by the human GI system as a drink rather than "food", thus avoiding triggering digestion.

The probiotic preparation may also be lactose-free, since it is not milk-based, and gluten-free. In particular the Symprove™ product is demonstrated to fulfil the accepted criteria for classification as "gluten-free" based on

Gliadin ELISA. Batches of the product typically contain less than lOppm gluten when measured by Gliadin ELISA.

Treatment of diverticulitis and/or diverticular disease

Preferably, the treatment of diverticulitis and/or diverticular disease comprises amelioration of the symptoms associated with the condition. Thus, treatment of diverticulitis and/or diverticular disease may include amelioration of one or more of stomach pain, stomach cramps, diarrhoea, constipation, bloating and alterations in bowel habit .

The precise dosage will depend on many factors, not least the severity of an individual patient's condition. The most appropriate dose can be determined by a medical practitioner and generally should be a dose which results in a beneficial effect, e.g. improvement in INS symptom

severity. Typically dosing of the Symprove™ product

prepared according to the examples could be lml/kg body weight per day or 50ml per day for an adult patient. The frequency and duration of treatment will vary from patient to patient. A typical treatment period is 12 weeks, but the period may be longer or shorter. Typically the dosage frequency would de daily, although other dosage frequencies may also be used.

For treatment of human patients with diverticulitis and/or diverticular disease, the liquid probiotic

preparation is typically administered orally, e.g. as a drink. If convenient, the probiotic preparation may be incorporated into foodstuffs or beverages for human

consumption, provided that this does not affect the

viability of the bacteria in the preparation.

For human use, in the treatment of diverticulitis and/or diverticular disease, it may be convenient to

formulate the probiotic preparation into unit dosage form, e.g. a single dose aliquot of the liquid preparation which is ready-to-drink.

The probiotic preparation to be used in the treatment of diverticulitis and/or diverticular disease as described herein is typically a liquid-based preparation which is advantageously not freeze dried.

Preparations of freeze-dried bacteria which are

currently used typically contain few, if any, metabolically active bacteria. Furthermore, preparations of freeze-dried bacteria lose some of their characteristics and activity upon rehydration. Also, the reactivated bacteria have a short shelf life and can be kept in storage for a short time only. Freeze-dried bacteria are often not rehydrated before use in a host animal. If they are rehydrated, then they generally have to be used on the same day, otherwise there may be rapid loss in viability and spoilage from

contaminating organisms.

A further problem with prior art freeze-dried

preparations is that they are hygroscopic (attract moisture from the air) and therefore a packet has to be used within a few days of opening, otherwise the partial hydration will cause rapid loss of viability.

The liquid-based probiotic preparation provided herein for use in treating diverticulitis and/or diverticular disease avoids the above-listed problems associated with the use of freeze-dried bacterial cultures. In addition, it has also been shown that the liquid-based bacterial cultures prepared as described herein are significantly more robust than bacteria prepared by methods known in the prior art, which enables the bacteria to establish more rapidly in host animals and to tolerate the harsh environment of the

mammalian digestive tract. Manufacture of the probiotic preparation

The probiotic preparation for use in treatment of diverticulitis and/or diverticular disease as described herein may be prepared by growing one or more probiotic bacterial strains in a liquid growth substrate, such as for example an extract of germinated barley. The growth

substrate may be itself prepared starting from seed or malting sample barley using the manufacturing process described hereinbelow. This method is substantially as described in WO 2006/035218, the contents of which are incorporated herein by reference.

The growth substrate may be prepared according to a method comprising:

subjecting malted cereal to a mashing step in which the malted cereal is mixed with aqueous liquid and subjected to conditions of time and temperature which limit the extent of conversion of complex carbohydrates to simple sugars such that a mixture of complex carbohydrates, simple sugars, proteins and peptides is obtained; and

separating the mixture of complex carbohydrates and simple sugars, proteins and peptides from the spent malted cereal to obtain a growth substrate.

The terms "malted cereal" or "malt" as used herein refers to the product of a malting process applied to cereal grains. Malting is a process well known in the art of brewing .

In a typical malting process cereal grains (e.g. seed or malting sample barley) are germinated to induce the mobilisation of storage nutrients. Germinating seeds produce a number of enzymes to mobilise storage proteins and

carbohydrates, including oc-amylases which hydrolyse starch into maltose. An exemplary cereal is barley, but other cereals, such as rice, wheat, corn and oats or even mixtures thereof, may also be used. The process of germination is generally well known. It will be appreciated that the method may be carried out by providing malted grains or a synthetic substrate comprising a mixture of carbohydrates, proteins and enzymes .

Typically the malting grains are rolled before further processing. Cracking of the grains by rolling facilitates access of water and extraction of nutrients during the mashing step, whilst avoiding shattering of the grains assists in the subsequent separation of the growth substrate from spent malted grains.

The prepared malt is subjected to a mashing step which resembles the mashing-in step used in methods for brewing (see, for example, Kunze, W. Technology Brewing and Malting (1996)) . The term "mashing-in" is well known in the field of brewing and relates to a process wherein malted grains are agitated in the presence of water heated to defined

temperatures in order to prepare a wort. During the mashing in step complex carbohydrates in the malted grains are broken down into maltose.

The method described herein also involves a mash step in which malted cereal grains are mixed with an aqueous liquid (typically water) and the mixture heated to various defined temperatures. This mash step does not, however, conform to a typical brewer's mashing-in process. Brewers aim to convert as much carbohydrate to simple sugars as possible, for subsequent fermentation to alcohol. In contrast, the conditions of the mash step described herein are specifically chosen to limit the extent of conversion to simple sugars, leaving significant amounts of carbohydrates in more complex oligomeric and polymeric forms.

The extent of conversion of carbohydrate may be limited such that the amount of reducing sugars present in the resulting growth substrate, expressed as a percentage (w/w) of total carbohydrate content, is in the range of from 10% (w/w) to 50% (w/w) . As noted above, the reducing sugar content measured by the Nelson-Somogyi method is a good approximation of the total amount of simple sugars present.

The desired limited conversion of complex to simple sugars may be achieved by increasing the temperature in the mash step over a short period of time, typically 30 minutes, without allowing the mixture of malted grains/water to rest at intermediate temperatures. Traditional brewing processes include rests at 60-65°C and 70-74°C, as this allows enzymes to produce high concentrations of simple sugars (mainly maltose) . Accordingly, the mashing-in step described herein does not comprise rests at temperatures in the range of from 60°C to 65°C and/or at temperatures in the range of from 70°C to 74°C.

The mash step may comprise mixing the malted cereal with water at a temperature in the range of from 30°C to 45°C, resting the mixture for 1 to 2 hours, increasing the temperature to a temperature in the range of from 75°C to 85°C over a time period in the range of from 20 to 60 minutes, preferably 30 minutes, and then resting the mixture at a temperature in the range of from 75°C to 85°C for a period of time in the range of from 30 to 90 minutes. At the higher temperature, any enzymes present in the mixture are inactivated and nutrients can be extracted. Higher

temperatures in the range of from 76°C to 80°C and

specifically 78°C are generally preferred. The temperature in this step needs to be sufficiently high for sufficiently long to inactivate all enzymes present in the preparation. It will be appreciated that the precise temperature and times used can vary somewhat according to the type of cereal used . The process described here specifically limits the amount of conversion of complex sugars to simple sugars. Furthermore, the initial rest at a temperature in the range of from 30°C to 45°C provides an additional advantage in that it maximises the release of amino acids and peptides.

Temperatures towards the higher end of this range, i.e. from 40°C to 45°C or specifically about 45°C, are generally preferred. The purpose of this step is to hydrolyse storage proteins present in the cereal grains into available amino acids and peptides. The optimum combination of time and temperature to achieve the desired hydrolysis may vary somewhat depending on the type of grains used.

The presence of high concentrations of proteins, peptides and amino acids is desirable in a growth substrate to be used to support the growth of microorganisms as it provides a useful a source of nitrogen. Typical mashing-in processes used in traditional brewing would generally not include a rest at a temperature in this range, since brewers do not normally seek to achieve high protein or amino acid content in a wort intended for fermentation to produce alcohol .

When the mash step is complete, e.g. all the desired nutrients have been extracted from the now "spent" malted grains, the resulting mixture comprising complex

carbohydrates and simple sugars, proteins and peptides may be separated from the spent grains to obtain a growth substrate using any suitable means. Typically this will involve coarse filtration, for example using a 1mm filter such as a wedge-wire basket, yielding a solution containing coarse particles. It is a feature of the process described herein that the growth substrate is not clarified, as would generally be the case with a wort prepared during standard brewing. In traditional brewing the wort is generally clarified to remove all coarse particles, thereby producing a clear liquid.

The presence of some particulate matter in a growth substrate prepared according to the process described herein is advantageous as regards subsequent use in supporting the growth of bacteria since it provides both slow release nutrients and particulate surfaces for the adhesion of bacterial cultures.

If required, the growth substrate prepared according to the invention can be sterilised prior to further usage. As will be appreciated, this can be carried out by boiling for about one hour or by autoclaving at 120°C for about 20 minutes .

A buffer, or several buffering components may be added to the growth substrate if required. Preferred buffers are tri-sodium citrate or phosphate buffers. If the growth substrate is to be sterilised, the buffer (s) may be added prior to, during or after sterilisation as convenient.

The prepared growth substrate is then inoculated with the desired probiotic bacterial strains and grown until they reach the desired bacterial count required in the probiotic preparation. In a specific embodiment, the probiotic bacteria are grown in the growth substrate until they reach a concentration of between 1 x 10⁶ and 1 x 10⁹ colony forming units per millilitre. At this point the culture (or fermentation) can be cooled down to about 4°C and the combination of temperature, pH, phase of growth and residual substrate composition provides equilibrium conditions which allows for maintenance of a near equilibrium growth state during long term storage. In this near equilibrium state, which can be maintained for a period of at least 5-6 months, the population of metabolically active probiotic bacteria is maintained, typically in the range of from 10⁶ to 10⁹ viable cells per millilitre. It will be appreciated that the precise number of viable cells at the near equilibrium state can vary somewhat depending on the species of probiotic bacteria used. Typically the bacterial count (cfu/ml) is "stable" meaning that it does not vary by more than 1 logio unit, or more preferably by more than 0.5 logio unit, when the product is stored at 4°C for 5 months and/or when stored at 25°C for 4 months.

This manufacturing method involves a growing step in which the probiotic bacteria are grown in a growth substrate until they reach a concentration which enables the near- equilibrium condition to be achieved during subsequent storage. It is an important feature of the method that the growth substrate used provides a balanced nutrient supply containing a mixture of complex carbohydrates and simple sugars wherein a high proportion of carbohydrate content is in the form of complex carbohydrates that may be used as an energy source by the probiotic bacteria. This mix helps to limit immediately available energy during the growing step. The composition and preferred features of the growth

substrate is/are preferably as described above. The growth substrate is preferably prepared from malted cereal grains using the manufacturing method described herein, and is typically an extract of germinated barley. It will be appreciated, however, that it is not strictly necessary to use growth substrate prepared according to this method.

Similar results can be achieved using growth substrate prepared synthetically by mixing the required proportions of complex carbohydrates and simple sugars, proteins and peptides . The growing step must be carried out in such a way that care is taken not to grow the probiotic bacteria for too long, to avoid producing acid conditions which would

otherwise inhibit further growth as well as limiting the period of storage of the resulting preparation. On the other hand, if the growing step is terminated prematurely, this will limit biomass production. The pH of the preparation during growth of the probiotic bacteria can be used as an indicator of the near equilibrium state. In a typical embodiment, cultures of lactic acid bacteria may be grown until a pH of 4.5 +/- 0.3 units is reached.

It will be appreciated that the pH of the growth substrate may vary somewhat depends on the optimal pH range for the probiotic bacteria used. In a typical embodiment (suitable for lactic acid bacteria) pH should be maintained in the range of from 3.8 to 4.5 during storage. Preferably buffers, such as tri-sodium citrate or phosphates are added to the growth substrate to control the pH during growth of the probiotic bacteria and subsequent storage.

It will be appreciated that the exact timing of the step of growing the probiotic bacteria to a near equilibrium state depends on the species used. Growth of the probiotic bacteria can be carried out in any suitable culture

apparatus. In specific embodiments the growing step can be carried out by way of fermentation in a fermentation vessel.

In exemplary embodiments, the probiotic bacteria used are lactic acid bacteria. Preferred species are of the genera Enterococcus and Lactobacillus, and mixtures thereof, as described above. If such species were to be grown using "conventional" growth media consisting largely of

fermentable simple sugars the excess energy supply would lead to excess acid production by lactic acid bacteria, which would limit biomass production and shelf life of the resulting culture. In contrast, the mix of complex

carbohydrates and simple sugars present in the growth substrate used in the manufacturing method described herein supplies energy for growth in the initial growing step and also for maintenance during storage of the product.

The growing step can be carried out using a single species of probiotic bacteria, and two or more different bacterial species may be grown independently in separate growth media and then blended together before or after the chilling step. The growth substrates used for independent culture of two or more different bacterial species may be the same or of different composition. Thus, it is possible to optimise the growing conditions for cultures of

individual bacterial species and then combine the cultures together for storage under conditions which permit

maintenance of equilibrium growth for each of the individual species in the culture.

In other manufacturing methods, two or more different bacterial species can be grown together in a single culture in the same growth substrate, provided that their growth rates are comparable so that one species does not dominate the population.

Following the growing step, the probiotic preparation can be analysed by standard methods to determine the

microbiological purity and enumeration of probiotic

bacteria. It is also possible to grow two or more different combinations of probiotic bacteria together in separate growth substrates and then combine the cultures together in a final probiotic preparation. A combination culture could similarly be blended with a culture of a single bacterial species . Starter cultures for the growing step of the method include, for example, freeze-dried bacteria or liquid cultures .

It will be appreciated that the probiotic preparation may be used immediately but more typically the preparation will be stored before use. The optimum temperature for storage in order to maintain the near equilibrium growth state in the culture is about 4°C but it will be appreciated by the skilled reader that the storage temperature could vary somewhat. As noted above, the probiotic preparation is stable fro at least 4 months even when stored at ambient temperature (about 25°C) . For convenience, aliquots of the probiotic preparation produced by the method may be

dispensed into suitable sterile packaging prior to long term storage.

It is a key advantage of the probiotic preparation described herein that the product maintains viability and integrity [of the probiotic bacteria] when stored for extended periods, typically for at least 5 or 6 months.

However, it will be appreciated that it is not essential to the method that the probiotic preparation must actually be stored for 5-6 months prior to use.

To avoid the growth of fungi or yeast, an anti fungal agent, such as sterile potassium sorbate, may be added prior to storage of the probiotic preparation. The anti fungal agents are primarily to prevent spoilage by yeast or fungi during use by end-users, once a container of the product has been opened. Furthermore, an anti oxidant, for example, vitamin C can be added to help to prevent spoilage of the product during storage. It will be appreciated that other agents well known in the art can also be used as anti fungal agents or anti oxidants. An alternative method of producing the probiotic preparation which does not require an active growing step simply involves inoculating the growth substrate (e.g. the extract of germinated barley prepared as describe herein) with starter culture (s) of probiotic bacteria. Starter cultures for the method include, for example, freeze-dried bacteria or liquid cultures. The growth substrate may be inoculated with more than one bacterial species. Preferably, the concentration of the viable cells in the starter culture is in excess of 10⁶ viable cells per millilitre.

The invention will be further understood with reference to the following non-limiting experimental examples:

Example 1: Production of a growth substrate, using a barley- based medium.

(A) Germination (malting) Day 1

Barley (seed or malting sample grains) was steeped in water for 2 to 24 hours, depending on the batch of grain. 0.1% (w/v) sodium hypochlorite (bleach) can be included in the water to inhibit growth of contaminants during the

germination phase. After 4 hours, the water was drained from the grains and the grains left to stand at room temperature from 10 to 30°C for approximately 1 day.

Day 2

The grains were steeped in clean water for another 4-hour period. Hydrogen peroxide (0.1% w/v) may be added to the water for this and subsequent soaks. Hydrogen peroxide provides oxygen for the germinating grains, and act as a disinfectant. Alternatively, sodium hypochlorite may be used. After 4 hours, water was drained from the grains and the grains left to stand for approximately 1 day. Occasional (e.g. every 4 hours) agitation of the grains may improve germination by increasing gaseous exchange, providing oxygen and removing carbon dioxide.

Day 3 onwards

The cycle of steeping, draining and standing was continued until the emerging rootlets on the germinating grains were 2 to 4 mm long. This state of growth indicated that the grains had produced enzymes for the mobilisation of stored

nutrients. These enzymes are central to the subsequent production of the growth medium, during ' mashing-in . ' More extensive modification of the grains could be allowed, leaving the germination phase until the rootlets are several millimetres long. However, too much growth will merely convert nutrients into plant roots and shoots, which cannot be used in the fermentation.

(B) Rolling

When the grains had germinated sufficiently, they were then milled with a roller mill. The mill was adjusted such that the grains were cracked open but not shattered or completely flattened. Cracking of the grains allows access of water and extraction of nutrients during mashing in, whilst avoiding shattering of the grains assists in filtration steps.

(C) Preparation of a growth substrate. The germinated, milled, grains were mixed with sufficient water to cover them, at 45°C, and the mixture held at 45°C for 1 hour. After 1 hour at 45°C, the temperature was increased to 78° C over a period of 30 minutes.

The mixture was allowed to stand at 78°C for 1 hour. After the 78 °C stand, the spent grains were separated out by filtration. A relatively coarse filter (e.g. 1mm gap wedge- wire basket) was used yielding a solution containing significant amounts of suspended solids. The spent grains were discarded and the liquid then heat-treated to

pasteurise or sterilise it. In this particular experiment the liquid was boiled for 45 minutes. A buffer (0.5% (w/v) tri-sodium citrate) was then added and the mixture boiled for a further 15 minutes to yield the final growth

substrate . Example 2: Analysis of the growth substrate

(a) Carbohydrate Analyses:

Total carbohydrate content was determined using the phenol- sulphuric acid assay, with glucose as reference standard.

(Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956) Analytical Chemistry, vol. 28., p.

350) . Reducing sugars were determined using the Nelson-Somogyi method, again with glucose as the reference standard.

(Somogyi, M. (1952) Journal of Biological Chemistry., vol. 195., p. 19) . Both complex and simple sugars have reducing ends, but simple sugars have a greater number of reducing ends per unit mass. Thus, whilst the Nelson-Somogyi method does detect complex sugars, the signal is so low so as to be negligible. The Nelson-Somogyi method therefore gives a good approximation of the level of simple sugars in a sample .

Results :

Total sugars in substrate are in the range 20 to 40 mg/ml (milligrams per millilitre)

Reducing sugars are in the range 5 to 20 mg/ml.

(b) Protein and Peptide Analyses:

Total protein was determined using two assays, with bovine serum albumin as reference standard:

(i) The Biuret Reagent (Itzhaki, R. F & Gill, D. M. (1964) Analytical Biochemistry, Vol. 9., p. 401-410.

(ii) The Lowry Method, as modified by Ohnishi and Barr .

(Ohnishi, S. T. & Barr, J. K. (1978) Journal of Biological Chemistry, Vol. 193, p. 265).

Peptides of molecular weight above 5000 daltons were

determined using the Bradford Reagent (Bradford, M. M.

(1976) Analytical Biochemistry, Vol. 72, p. 248-254).

Results : Total protein and peptides are in the range 1 to 2

milligrams per millilitre.

High molecular weight peptides (greater than 5000 daltons) are in the range 100 to 300 micrograms per millilitre.

Example 3 : Production of a metabolically active bacterial culture (A) Fermentation

The growth substrate prepared according to example 1 was cooled to 37°C and the bacterial cultures added. Examples of a suitable inoculum are freeze-dried bacteria or liquid starter cultures (typically 1% (v/v) of an overnight culture in nutrient broth) .

In this example the following bacteria were grown in two vessels :

(i) Enterococcus faecium, Lactobacillus plantarum;

(ii) Lactobacillus rhamnosus, Lactobacillus acidophilus

Fermentation was carried out for 16-20 hours, until the pH reached 4.5 +/- 0.3 units. The fermentation mixture was then cooled to 4°C and subjected standard techniques to assess quality (to determine microbiological purity and enumeration of bacteria) .

At this point, sterile potassium sorbate (0.005% w/v final concentration) may be added to the fermented broth. This acts to inhibit the growth of any fungi or yeast that may arise due to contamination during handling by the end-user. Vitamin C may also be added (0.01% w/v final concentration) as an anti-oxidant .

Following quality assurance tests, different batches can be blended to give products with complex mixtures of bacterial species, if required.

In the example presented here, blending of the two batches of bacteria yielded a final product containing the bacteria Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus acidophilus .

Batches of this final product typically contain 2.3g

carbohydrate per 100ml, of which 0.7g is sugars and 1.6g is starch. The bacterial count in the final product is

typically less than 1.0 x 10⁵ for L. acidophilus and

typically at least 1.0 x 10⁷ for each of L. rhamnosus, L. plantarum and E. faecium . Batches of the final product were tested for the presence of gluten using the Gliadin ELISA test and were determined to contain less than 15ppm gluten, and more typically less than lOppm gluten, satisfying the Codex Alimentarius definition of gluten-free.

Example 4 : Assessment as a dietary supplement in symptomatic diverticular disease

The primary objective of this study is to study the

proportion of patients with clinically proven episodes of diverticular disease achieving an improvement in intestinal symptoms, as assessed clinically by a validated symptom severity score questionnaire following 3 months treatment with the final product of example 3 (identified herein as Symprove™, a multiple strain probiotic containing viable, metabolically active bacterial cells) (lml/kg/day) as

compared with placebo.

Inclusion/Exclusion Criteria

All patients with diverticular disease attending the

Gastroenterology-Colorectal surgery outpatients at King's will be considered for participation in this double blind placebo controlled trial provided that they fulfil the inclusion criteria which are: · Have a documented episode of diverticulitis as assessed clinically, on CT scans and raised serological markers of inflammation

• Have problematic symptoms associated with established diverticular disease

· Aged between 18 and 80 years;

• Willing and able to provide a written informed consent.

Specific exclusion criteria include those:

• Aged less than 16 years and greater than 80 years;

· Severe disease (ongoing severe active diverticulitis) as defined by hemoglobin < 8.0 g/dl, white blood cell count >20,000 cells/mm3, temperature >38.5°C, albumin < 25 g/dl,

• Diverticular complications such as recto-vaginal or bladder fistula, abscess, etc.

· Severe respiratory, cardiovascular, neurological, psychiatric, rheumatological diseases;

• Undergone major intestinal resections; Patients with malignancy;

On NSAIDs;

Pregnancy or actively seeking pregnancy;

History of intolerance or allergy to probiotics

Current drug or alcohol dependence syndrome.

Pregnancy

Patients with severe learning difficulties

Concomitant Therapy

Continued treatment with oral or rectal mesalasine will be allowed if the dose has been stable for 3 months or more. Oral or rectal antibiotics are not allowed. Treatment with antidiarrheal agents (loperamide, diphenoxylate, and

opiates) are allowed, provided that the doses have remained stable during the previous 4 weeks. Patient receiving investigational therapies within 30 days are not eligible for the study, such as rifaximine. Patients consenting to the study will undergo a faecal calprotectin test and routine haematology and biochemistry, to be repeated at the end of the study. Each will fill out symptom severity score sheets, quality of life and sleep quality assessment. They will then be randomised to receive Symprove (lml/kg/day for 3 months) or placebo (lml/kg/day) that contains all the ingredients of Symprove apart from the bacteria. Other medication will be taken as usual. Return visit to the outpatient clinic will be a 1, 2 and 3 months and the patients will repeat the laboratory investigations (3 months) and fill out the clinical disease activity scores at these times. Patients who were on placebo will be offered the active preparation for 5 months and those on the active treatment will be offered a further 2 month supply free of charge .

Outcome measures

A standardised outcome questionnaire i.e. a symptom severity assessment measure used as a means for clinicians to monitor and assess IBS patients will be used as the primary outcome measures (as there are no widely available symptom severity scores for patients with diverticular disease (who have the same symptoms as those with IBS) ) (8) . The maximum

achievable score is 500, moderate severity is defined as between 175-300 points and severe as > 300 points.

Reductions in 50 points are considered clinically

significant as is a symptom severity score of < 150. A quality of life questionnaire and sleep quality assessments will also be made as secondary endpoints.

Safety

Laboratory parameters will be assessed. Each patient will be alerted to possible side effects of the probiotic including abdominal bloating, diarrhoea and constipation. In case of new symptoms developing the patients will have 24 hour access to a specialist diverticular nurse, specialist registrar or consultant. All patients will undergo routine bloods (haematology and biochemistry) before and after treatment. All patients will be assessed clinically 1 month after their completion of the trial. Randomisation and sample size

The randomization to active treatment and placebo will be done by a computer program. Equal number of patients will receive the active treatment and placebo. The number of patients is estimated as 96 (48 on active treatment and 48 on placebo) . The current study has assuming a 50% reduction in symptomatic score in both groups with a 10% reduction in the placebo group and a 35% reduction in the active group, both groups having started at 300 points. This has a 90% power of showing a significant (p < 0.05) difference between the two treatments. Patient testimonial 1

Patient A (female, post-retirement age) was diagnosed with diverticular disease after suffering violent diarrhoea over an extended period.

Diverticula is the medical term used to describe the small pouches that stick out of the side of the large intestine (colon) . Diverticula are very common and associated with ageing. It is estimated that 50% of people have diverticula by the time they are 50 years old, and 70% of people have them by the time they are 80 years old.

The majority of people with diverticula will not have any symptoms. However, 1 in 4 people with diverticula experience symptoms such as abdominal pain and diarrhoea. People who experience symptoms are said to have diverticular disease. With no sign of the diarrhoea stopping, patient A went straight to her GP . "In fact I saw a couple of GPs in the practice over the coming weeks as they weren't sure what was causing the diarrhoea." Eventually patient A was prescribed Loperamide, an opioid drug which is used against diarrhoea. This increases the amount of time substances stay in the intestine, allowing for more water to be absorbed out of the fecal matter. Loperamide also decreases colonic mass movements and suppresses the gastrocolic reflex. The

diarrhoea stopped, however, within weeks it began again. As the diarrhoea was now progressively worse, patient A went back to her GP, who prescribed an antibiotic. This didn't make any difference. Patient A went back to her GP and was referred to King's College Hospital where she had a

colonoscopy and was told she had diverticular disease.

Patient A began taking Symprove™ daily at the recommended dose of lml/kg/day. Within 3 days her diarrhoea stopped and she has remained symptom-free, with no a diarrhoea attacks, for over 6 months.

Patient testimonial 2 After experiencing symptoms associated with diverticulitis, specifically abdominal pain and diarrhoea, patient B

(female) visited her Consultant. She was prescribed a course of antibiotics and told to take codeine phosphate to relieve pain and reduce muscle contractions of the intestine to stop diarrhoea. However, these had no effect. A second course of antibiotics was prescribed and the results remained

negative .

Patient B began taking Symprove™ daily at the recommended dose of lml/kg/day and experienced a positive result

extremely quickly.

"I am absolutely convinced that Symprove™ stopped my

Diverticulitis as I was on no other medication at the time. It was amazing that Symprove worked within 3 days, having lived with pain and diarrhoea for many months." References

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Claims

1. A probiotic preparation comprising viable,

metabolically active probiotic bacteria in a non-dairy liquid substrate, for use in the treatment of diverticulitis and/or diverticular disease.

2. A probiotic preparation comprising viable,

metabolically active probiotic bacteria in a non-dairy liquid substrate, for use in reducing the incidence and/or severity of diarrhoeal attacks in a patient previously diagnosed with diverticulitis or diverticular disease.

3. The probiotic preparation for use according to claim 1 or claim 2, wherein the probiotic preparation is capable of delivering viable, metabolically active probiotic bacteria to the intestinal tract of a human subject without

triggering digestion.

4. The probiotic preparation for use according to any one of claims 1 to 3, wherein the probiotic bacteria in the preparation are stable when maintained in culture at pH 3 and 37°C for a period of at least 6 hours.

5. The probiotic preparation for use according to any one of claims 1 to 4, wherein the probiotic preparation

comprises a mixture of complex carbohydrates and simple sugars, wherein the ratio of total carbohydrate content to reducing sugar content of the probiotic preparation is in the range of from 8:1 to 2:1.

6. The probiotic preparation for use according to any one of the preceding claims wherein the probiotic preparation comprises at least one strain of probiotic lactic acid bacteria .

7. The probiotic preparation for use according to any one of the preceding claims wherein the probiotic preparation comprises two or more different strains of probiotic lactic acid bacteria.

8. The probiotic preparation for use according to claim 6 or claim 7 wherein the probiotic preparation comprises at least one probiotic bacterial strain of the genus

Lactobacillus .

9. The probiotic preparation for use according to claim 6 or claim 7 wherein the probiotic preparation comprises at least one probiotic bacterial strain of the genus

Enterococcus .

10. The probiotic preparation for use according to claim 6 or claim 7 wherein the probiotic preparation comprises at least one of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus or Lactobacillus casei.

11. The probiotic preparation for use according to claim 7 wherein the probiotic preparation comprises a mixture of Enterococcus faecium, Lactobacillus plantarum and

Lactobacillus rhamnosus.

12. The probiotic preparation for use according to claim 11 wherein the probiotic preparation comprises a mixture of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus acidophilus .

13. The probiotic preparation for use according to any one of the preceding claims, wherein the total count of

metabolically active bacteria in the preparation is in the range of from 1.0 x 10⁶ to 1.0 x 10⁹ viable cells per millilitre .

14. The probiotic preparation for use according to claim 11 or claim 12 wherein the total count of at least one of, and more preferably each of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus rhamnosus in the preparation is at least 1.0 x 10⁶ viable cells per millilitre.

15. The probiotic preparation for use according to claim 11 or claim 12 wherein the total count of at least one of, and more preferably each of Enterococcus faecium,

Lactobacillus plantarum and Lactobacillus rhamnosus in the preparation is at least 1.0 x 10⁷ viable cells per

millilitre .

16. The probiotic preparation for use according to any one of the preceding claims, wherein the probiotic preparation has a total carbohydrate content in the range of from 20 mg/ml to 40 mg/ml and a reducing sugar content in the range of from 5 mg/ml to 20 mg/ml.

17. The probiotic preparation for use according to any one of the preceding claims which additionally comprises

proteins and peptides, wherein the total amount of protein and peptides is in the range of from 1 mg/ml to 2 mg/ml and wherein the total amount of high molecular weight peptides is in the range of from 100 g/ml to 300 g/ml.

18. The probiotic preparation for use according to any one of the preceding claims wherein the pH of the preparation is maintained in the range of from 3.8 to 4.5.

19. The probiotic preparation for use according to any of the preceding claims wherein the probiotic preparation is prepared by growing the probiotic bacterial strain (s) in an extract of germinated barley.

20. The probiotic preparation for use according to any one of the preceding claims wherein the preparation further comprises an anti-fungal agent.

21. The probiotic preparation for use according to claim 20 wherein the anti-fungal agent is sterilised potassium sorbate .

22. The probiotic preparation for use according to any one of the preceding claims wherein the preparation further comprises an anti oxidant.

23. The probiotic preparation for use according claim 22 wherein the anti-oxidant is vitamin C.

24. A method of treating diverticulitis and/or diverticular disease in a human patient, comprising administering to a patient in need thereof an effective amount of a probiotic preparation comprising viable, metabolically active

probiotic bacteria in a non-dairy liquid substrate.

25. A method of reducing the incidence and/or severity of diarrhoeal attacks in a human patient previously diagnosed with diverticulitis or diverticular disease, comprising administering to a patient in need thereof an effective amount of a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate.

26. The method according to claim 24 or claim 25 wherein the probiotic preparation is capable of delivering viable, metabolically active probiotic bacteria to the intestinal tract of a human subject without triggering digestion.

27. The method according to any one of claims 24 to 26 wherein the probiotic bacteria in the preparation are stable when maintained in culture at pH 3 and 37°C for a period of at least 6 hours.

28. The method according to any one of claims 24 to 27, wherein the probiotic preparation comprises a mixture of complex carbohydrates and simple sugars, wherein the ratio of total carbohydrate content to reducing sugar content of the probiotic preparation is in the range of from 8:1 to 2:1.

29. The method according to any one of claims 24 to 28 wherein the probiotic preparation comprises at least one strain of probiotic lactic acid bacteria.

30. The method according to any one of claims 24 to 29 wherein the probiotic preparation comprises two or more different strains of probiotic lactic acid bacteria.

31. The method according to claim 29 or claim 30 wherein the probiotic preparation comprises at least one probiotic bacterial strain of the genus Lactobacillus .

32. The method according to claim 29 or claim 30 wherein the probiotic preparation comprises at least one probiotic bacterial strain of the genus Enterocoecus .

33. The method according to claim 29 or claim 30 wherein the probiotic preparation comprises at least one of

Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus or Lactobacillus casei.

34. The method according to claim 30 wherein the probiotic preparation comprises a mixture of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus rhamnosus.

35. The method according to claim 34 wherein the probiotic preparation comprises a mixture of Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus and

Lactobacillus acidophilus .

36. The method according to any one of claims 24 to 35, wherein the total count of metabolically active bacteria in the preparation is in the range of from 1.0 x 10⁶ to 1.0 x 10⁹ viable cells per millilitre.

37. The method according to claim 34 or claim 35 wherein the total count of at least one of, and more preferably each of Enterococcus faecium, Lactobacillus plantarum and

Lactobacillus rhamnosus in the preparation is at least 1.0 x 10⁶ viable cells per millilitre.

38. The method according to claim 34 or claim 35 wherein the total count of at least one of, and more preferably each of Enterococcus faecium, Lactobacillus plantarum and

Lactobacillus rhamnosus in the preparation is at least 1.0 x 10⁷ viable cells per millilitre.

39. The method according to any one of claims 24 to 38, wherein the probiotic preparation has a total carbohydrate content in the range of from 20 mg/ml to 40 mg/ml and a reducing sugar content in the range of from 5 mg/ml to 20 mg/ml .

40. The method according to any one of claims 24 to 39 which additionally comprises proteins and peptides, wherein the total amount of protein and peptides is in the range of from 1 mg/ml to 2 mg/ml and wherein the total amount of high molecular weight peptides is in the range of from 100 g/ml to 300 μg/ml.

41. The method according to any one of claims 24 to 40 wherein the probiotic preparation is prepared by growing the probiotic bacterial strain (s) in an extract of germinated barley .

42. Use of a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate in the manufacture of a medicament for treating or preventing diverticulitis and/or diverticular disease .

43. Use of a probiotic preparation comprising viable, metabolically active probiotic bacteria in a non-dairy liquid substrate in the manufacture of a medicament for reducing the incidence and/or severity of diarrhoeal attacks in a patient previously diagnosed with diverticulitis or diverticular disease.