MULTI STRAIN PROBIOTIC BLENDS FOR TREATMENT OF GASTROINTESTINAL CONDITIONS AND IMPROVING OR MAINTAINING GASTROINTESTINAL HEALTH

FIELD OF THE INVENTION

[0001] One or more probiotic and prebiotic blends are provided that allow survival through the GI tract and provide desired physiological results. The probiotic blends may be used to treat one or more conditions in humans and animals.

BACKGROUND

[0002] Technological progress in the food industry (heat treatment, cooling, freezing, etc.) has resulted in a dramatic decrease of consumption of food-borne microbes and has coincided with an increased number of disorders including inflammatory bowel disease and atopic disorders such as asthma and food allergies. Foods and food additives containing probiotics (living beneficial microbes) may support the restoration of the healthy balance of the gut microflora. Further, compared to single strain probiotics, multistrain preparations may express an improved functionality by combining benefits of each probiotic strain and support the mixed culture that is normal for humans.

[0003] According to the guidelines for the evaluation of probiotics in food reported by a Joint FAO/WHO working group (Joint report: "Probiotics in Food," FAO/WHO report. Chesson et al., 2002), two of the currently most widely used in vitro tests are resistance to gastric acidity and bile salts, as based on both survival and growth studies. These studies are also useful for nutraceutical probiotic products. The predictability of these in vitro tests is limited but functional. The use of sophisticated and dynamic, computer-controlled models of the gastrointestinal tract, like the one developed by Marteau et al. J. Dairy Science (1997) 80(6): 1031-1037, is beyond the scope of most all laboratories, including ours.

[0004] Several studies have revealed that some probiotic products in the market have deficiencies in the viability of probiotic strain(s), especially in products containing bifidobacteria. This may be due to storage conditions, manufacturing or food technologies setbacks such as inappropriate packaging materials that could affect probiotic stability through variations in oxygen permeability. In the past two decades, there has been renewed interest in the study of the nutritional and therapeutic aspects of probiotic products.

[0005] It is widely accepted that probiotics may exert positive influence on the host through modulation of the endogenous ecosystem and stimulation of immune system as well as maintaining a healthy intestinal microflora. However, research suggests that health benefits are strain specific and vary by amount ingested and duration administered. Further, probiotic applications are limited because these bacteria are likely to be in a stressed state when they reach the colon due to exposure to diverse barriers in the host such as gastric acid and bile acids. Commercial probiotics should be able to recover and compete with established microflora in the colon to provide colonization and benefits for the host, but such products can often fail to achieve the desired benefits.

[0006] In view of the above, it would be desirable to provide unique blends of prebiotic or probiotic products for children, adults, or domesticated pet animals, respectively, having specific probiotic strain blends that can survive the GI tract and deliver the physiological effects desired per each blend which are geared toward each subject group.

[0007] Furthermore, if a way could be found to test such probiotic products in an in vitro system that includes oral cavity simulation, this would represent a valuable contribution to the art.

SUMMARY OF THE INVENTION

[0008] One or more probiotic and prebiotic blends are provided that allow survival through the human gastrointestinal (GI) tract and provide desired physiological results and/or improvements in the host individual. One or more embodiments in accordance with the present invention demonstrate that a unique blend of prebiotic with specific probiotic strain blends can survive the GI tract and deliver the physiological effects desired per each blend, for example to treat conditions selected from irritable bowel syndrome (IBS), diarrhea, and constipation.

[0009] In another embodiment, a process is provided to determine in vitro functionality of probiotics and prebiotics for modeling survival in the human GI tract. In a particular improvement, an in vitro simulation of a human gastrointestinal tract includes an oral cavity simulation. The processes may be adapted for use based on the age of the human subject. Certain probiotic formulations may be selected for use based in part on the age of the human subject.

[0010] In another embodiment, a process is provided to determine in vitro functionality of probiotics and prebiotics for modeling survival in the GI tract of a domesticated pet animal. In a particular improvement, an in vitro simulation of an animal gastrointestinal tract includes an oral cavity simulation.

[0011] In another embodiment a method of treating an individual for a condition selected from the group consisting of irritable bowel syndrome (IBS), diarrhea, constipation, bowel irregularity, allergic reactions, dermatitis, and reactions to antibiotic treatment is provided comprising the step of administering to the individual in need of such treatment a probiotic composition, and optionally a prebiotic. The probiotic composition may include a blend of at least two bacterial strains selected from the group consisting of Lactobacillus plantarum, Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bacillus coagulans.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 depicts Trial #1 of one embodiment of an in vitro method. The Flow chart provided shows the study design and transport through the various stages of digestion in the human gastrointestinal (GI) tract and the conditions and materials used to simulate in vitro the in vivo conditions.

[0013] FIG. 2 in Table 1 depicts initial results in Trial #1 demonstrating survival of probiotic blends through a simulated human gastrointestinal tract (viability expressed in CFU/g). The top portion of the table lists for each product: for each component strain the potency/dose (CFU) and the total bacteria (CFU) per product dose.

[0014] FIG. 3 depicts Trial #2 of another embodiment of an in vitro method. The Flow chart provided shows the study design and transport through the various stages of digestion in the human gastrointestinal (GI) tract and the conditions and materials used to simulate in vitro the in vivo conditions.

[0015] FIG. 4A in Table 2 depicts final results in Trial #2 demonstrating survival of probiotic blends through a simulated human gastrointestinal tract (viability expressed in CFU/g). The top portion of the table lists for each product: for each component strain the potency/dose (CFU) and the total bacteria (CFU) per product dose. NT = not tested.

[0016] FIG. 4B in Table 2 (cont'd.) depicts final results in Trial #2 demonstrating survival of probiotic blends through a simulated human gastrointestinal tract (viability expressed in CFU/g). The top portion of the table lists for each product: for each component strain the potency/dose (CFU) and the total bacteria (CFU) per product dose.

[0017] FIG. 5 depicts total probiotic blend strain survival (total CFU) as a function of time during the stomach and intestinal simulation, respectively in an adolescent/pre-teen model. This generally demonstrates survival of probiotic blends through a simulated preteen GI tract.

[0018] FIG. 6 in Table 3 depicts final results in Trial #4 demonstrating survival of probiotic blends through a simulated canine (dog) gastrointestinal tract (viability expressed in CFU/g). The top portion of the table lists for each product: for each component strain the potency/dose (CFU) and the total bacteria (CFU) per product dose.

[0019] FIG. 7 depicts total probiotic blend strain survival (total CFU) as a function of time during the stomach and intestinal simulation, respectively in an older adult / senior model. This generally demonstrates survival of probiotic blends through a simulated aged adult / senior GI tract.

[0020] FIG. 8 in Table 4 depicts final results in Trial #5 demonstrating survival of probiotic blends through a simulated aged adult/ senior gastrointestinal tract (viability expressed in CFU/g). The top portion of the table lists for each product: for each component strain the potency/dose (CFU) and the total bacteria (CFU) per product dose.

DETAILED DESCRIPTION

[0021] In one aspect, a unique blend of prebiotic with specific probiotic strain blends is described herein which can survive the GI tract and deliver the physiological effects desired per each blend. The present disclosure provides unique blends of prebiotic or probiotic products for children, adults, or domesticated pet animals, respectively, having specific probiotic strain blends that can survive the GI tract and deliver the physiological effects desired per each blend which are geared toward each subject group.

[0022] As used herein, the term "GI tract" refers to the gastrointestinal tract or pathway in an individual including human, mammal, or domesticated animal. The GI tract incudes at least the stomach and small intestine, and for test purposes can include the alimentary canal. Passage or transit through or residence in the GI tract is understood to proceed starting from the mouth (via chewing, mastication, liquid delivery, or swallowing, for example) and followed by ingestion to the stomach, then proceeding to the intestines. Colonization, growth, and maintenance of probiotic bacteria can occur in the GI tract, particularly in the intestines.

[0023] Currently there is a rapid growth of interest in probiotics to promote better health and well-being which shows a substantial promise to expand the food industry into new fields. Strains from genera of Lactobacillus and Bifidobacterium species, both of which are indigenous to the human intestine, are predominantly selected for use although some other species have also been used as well. Probiotics, also termed as functional foods, are commonly found in dairy products such as yogurt and cultured milk drinks or even in the form of health supplements.

[0024] Useful bacterial strains for probiotic compositions can include, but are not limited to: Lactobacillus plantarum, Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bacillus coagulans, and the like. The term "multistrain" refers to a blend of two or more strains of viable bacterial species.

[0025] The term "probiotic" as used herein refers to viable microorganisms that promote or support a beneficial balance of the autochthonous microbial population of the gut. Probiotics are defined as "live microorganisms that may confer a health benefit on the host." These bacterial strains (e.g. multistrains) are becoming extremely popular, not only in alternative circles, but also within the scientific community. Scientists have discovered that the microbes that live within animal intestines are important to their health. Animal and human species host at least 1000 different species of bacteria and fungi, and maintaining the right populations of each species is essential. Therefore,, maintaining intestinal flora with probiotics is a logical step.

[0026] The notion of probiotics evolved from a theory first proposed by, Elie

Metchnikoff (Nobel laureate) who associated longevity with the consumption of fermented milk products. He postulated that the bacillus present could positively modify the bacterial community structure of the colon, thus contributing to human health status. While not intending to be bound by any theory, the present disclosure is in general agreement with the current understanding of probiotics as used in foods and nutritional/dietary supplements for animals and humans.

[0027] The microbial population of the intestine is a highly dynamic and complex ecosystem having an estimated 10 ¹⁴ microorganisms representing more than 400 bacterial species. It has many functions in humans providing enzymes necessary for assimilation and/or synthesis of some nutrients, as well as in detoxifying certain harmful dietary compounds. In addition, the gastrointestinal flora provides a natural barrier against pathogens and can stimulate bowel motility and the immune system.

[0028] Probiotic formulations and blends should be able to recover and compete with established microflora in the colon to provide colonization and benefits for the host. For this purpose, they can use help from prebiotics such as inulin.

[0029] According to the guidelines for the evaluation of probiotics in food reported by a Joint F AO/WHO working group (Chesson et al., 2002), two of the currently most widely used in vitro tests are resistance to gastric acidity and bile salts, as based on both survival and growth studies. These studies are also useful for nutraceutical probiotic products. The predictability of these in vitro tests is limited but functional. The use of sophisticated and dynamic, computer-controlled models of the gastrointestinal tract, like the one developed by Marteau et al. (1997), is beyond the scope of most all laboratories. Also, methods developed by suppliers of probiotics are very sophisticated, such as the three-stage continuous culture system, SHIME®, EnteroMix, Lacroix model and TIM-2. {See, Venema, et al., "Experimental models of the gut microbiome," Best Pr act. Res. Clin. Gastroenterol. (2013) 27(1): 115-126.) Such systems are for basic research and not practical for development into a day to day screen of probiotics for survival in the GI tract and require use of a specific supplier's probiotic, or the tests are not practical to run on a cost basis.

[0030] The gut microbiome influences myriad host functions, including nutrient acquisition, immune modulation, brain development, and behavior. Although human gut microbiota are recognized to change as we age, information regarding the structure and function of the gut microbiome during childhood is limited. A . study using 16S rRNA gene and shotgun metagenomic sequencing, characterized the structure, function, and variation of the healthy pediatric gut microbiome in a cohort of school-aged, pre- adolescent children (ages 7-12 years). The results showed a difference in the microbiome of the children vs. adults on many strains of bacteria. Children were enriched in Bifidobacterium spp., Faecalibacterium spp., and members of the Lachnospiraceae, while adults harbored greater abundances of Bacteroides spp. From a functional perspective, significant differences were detected with respect to the relative abundances of genes involved in vitamin synthesis, amino acid degradation, oxidative phosphorylation, and triggering mucosal inflammation. Children's gut communities were enriched in functions which may support ongoing development, while adult communities were enriched in functions associated with inflammation, obesity, and increased risk of adiposity. (See, Hollister, et al., "Structure and function of the healthy pre-adolescent pediatric gut microbiome," Microbiome (2015) 3: 36.) [0031] Recently probiotics therapy, evidenced by numerous randomized clinical trials (RCTs) followed by meta analyses and Cochran reviews, has generated a great deal of renewed interest, due to its significant therapeutic effect on rotavirus- associated diarrhea in children in developed countries (Saavedra et al. 1994; Szajewska et al. 2000; Huang et al. 2002; Van Niel et al. 2002; Isolauri 2003; Reid et al. 2003; Allen et al. 2004; Szajewska et al. 2006). The most commonly used strains of probiotics belong to the genera Lactobacillus and Bifidobacterium, L. rhamnosus GG, Saccharomyces boulardii, Bacillus clausii, mix of L. delbrueckii var bulgaricus, Streptococcus thermophilus, L. acidophilus, and Bifidobacterium bifidum, or Enterococcus faecium SF 68. Median duration of diarrhoea was significantly shorter and frequency was lower only in those children who received mixes of four bacterial strains. (See, Dutta, et al., "Randomised controlled clinical trial of Lactobacillus sporogenes (Bacillus coagulans), used as a probiotic in clinical practice, on acute watery diarrhoea in children," Tropical Med. Internatl. Health (2011) 16(5): 555-561.)

[0032] The interest and expansion of the products for children in the probiotic area has led to extensive research in the field and the conclusion as above shows that multistrain products are superior to single strain products and the inclusion of a prebiotic or multiple prebiotics is preferable for improved health. New and effective multistrain probiotic blends for use in children are described hereinbelow.

[0033] Furthermore, the issue of the safe application of probiotics is not new or specific to older populations; however, there are aspects that are particular to this age group and that need to be addressed. As has been reviewed of late, the safety of application/consumption of a probiotic is linked to the potential vulnerability of the consumer to specific disease states. {See, Rijkers, et al., "Guidance for substantiating the evidence for beneficial effects of probiotics: current status and recommendations for future research," J. Nutr. 140(3), 671S-676S (2010).)

[0034] Older people are by definition more likely to present 'at-risk' factors, which include immune compromise, central venous catheter, impaired intestinal barrier function or consumption of broad-spectrum antibiotics to which the probiotic is resistant (Boyle, et al., "Probiotic use in clinical practice: what are the risks?" Am. J. Clin. Nutr. 83(6), 1256-1264 (2006)). Probiotics have been consumed safely for a long time by the general population, exemplified by only one case of lactobacillus septicemia in 10 million consumers in France over the course of a century (Bernardeau, et al., "Beneficial lactobacilli in food and feed: long-term use, biodiversity and proposals for specific and realistic safety assessments," FEMS Microbiol. Rev. 30(4), 487-513 (2006)).

[0035] Nevertheless, the suitability of therapeutic application of probiotics in older subjects, as distinct from consumption of foods containing probiotic bacteria, should be considered individually and focus on specific needs. Compared with younger adults, populations of older populations consume a complex array of medications, ranging from antibiotics through to pharmaceutical compounds with potential but unknown effects upon the complex bacterial community in the intestine. For example, in the first 360 subjects enrolled in the ELDERMET project, 95 subjects had consumed antibiotics in the 4 weeks prior to their baseline microbiota determination and 98% had consumed a recognized medicinal compound (Rijkers, et al, 2010). Probiotics have recognized utility to mitigate the diarrheal side effects of antibiotics and to reduce the incidence of Clostridum difficile- associated colitis (Hickson, et al., "Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind placebo controlled trial," BMJ 335(7610), 80 (2007); and Surawicz CM., "Role of probiotics in antibiotic-associated diarrhea, Clostridium difficile-associated diarrhea, and recurrent Clostridium difficile- associated diarrhea," J. Clin. Gastroenterol. 42(Suppl. 2), S64-S70 (2008)). Lifting the burden of infectious disease would be particularly beneficial in older populations. [0036] Several recent comprehensive reviews have summarized the major benefits associated with probiotic consumption in older adults, such benefits include increased levels of bifidobacteria, a reduction in the degree of constipation, enhanced innate immunity, and reduced inflammation (Pitkala, et al., "Fermented cereal with specific bifidobacteria normalizes bowel movements in elderly nursing home residents. A randomized, controlled trial," J. Nutr. Health Aging 11(4), 305-311 (2007); Gill, et al., "Enhancement of immunity in the elderly by dietary supplementation with the probiotic Bifidobacterium lactis HN019," Am. J. Clin. Nutr. 74(6), 833-839 (2001); and Ouwehand, et al.., "Bifidobacterium microbiota and parameters of immune function in elderly subjects," FEMS Immunol. Med. Microbiol. 53(1), 18-25 (2008)). Use of yoghurt fermented by L. bulgaricus to older people (n = 142; a median age of 74.5 years) significantly reduced the incidence and severity of winter colds and general upper respiratory symptoms. This improvement was accompanied by an increase in natural killer cell activity in the subjects receiving the yoghurt (Makino, et al., "Reducing the risk of infection in the elderly by dietary intake of yoghurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1," Br. J. Nutr. 104(7), 998-1006 (2010)).

[0037] Preclinical validation of beneficial effects in in vitro systems or animal models may thus be beneficial for strain selection, but obviously cannot replace human trials. Older adults as a group in society will typically span a greater range in health-status (from healthy and independent living to frail and dependent on assistance). They are known to have a microbiota in flux that varies significantly more between individuals than in a younger adult population. These factors should be borne in mind when designing clinical trials.

[0038] Recent analyses of the microbiota of older adults in Ireland confirmed that the prevalence of the genus Faecalibacterium varied significantly between individuals, supporting the notion that levels of this organism might be suitable for therapeutic intervention in older people with intestinal inflammation. Administration of prebiotics, or by administering probiotics that target competing elements in the microbiota, is conceptual at this time. {See, Cusack, S., et al., "How Beneficial is the Use of Probiotic Supplements for the Aging Gut?" Aging Health (2011) 7(2): 179-186.)

[0039] Furthermore, domesticated animals and/or pet animals can benefit from probiotics. Pet animals may include small or large domestic mammals, for example, but not limited to, dogs, cats, horses, sheep, cows or cattle (or other bovine species), horses, pigs, goats, rabbits, and the like. Also contemplated are small rodent species, including rats, mice, hamsters, gerbils, guinea pigs, and the like. [0040] All dogs can benefit from probiotics, which aid digestion and modulate the immune system. Probiotics produce short-chain fatty acids (SCFAs), which inhibit the growth and activity of harmful bacteria, such as E. coli, Salmonella, and Clostridium perfringens, as well as providing other benefits to the intestines. Human studies have documented the effectiveness of certain strains in treating diarrhea, irritable bowel, and intestinal inflammation. Probiotics used in dogs may help prevent urinary tract infections, and can even reduce allergic reactions by decreasing intestinal permeability and controlling inflammation.

[0041] Research looking at the effectiveness of probiotics in dogs is not nearly as extensive as what has been done in humans. Still, there are studies that suggest that they can help the health of our "best friends." The diseases that have been investigated so far have been acute diarrhea and contact dermatitis (skin allergy).

[0042] Acute diarrhea in dogs is diarrhea that starts suddenly and usually resolves on its own. Probiotics have been tested on several types of acute diarrhea, specifically diarrhea caused by dietary sensitivity and through the ingestion of an intestinal pathogen. In dogs with a dietary sensitivity, treatment with Lactobacillus acidophilus in combination with the diarrhea-provoking food led to some improvement in bowel movements. Better results, however, were seen when probiotics were applied as treatments for acute diarrhea caused by a stomach virus. Dogs treated with a probiotic cocktail containing multiple species healed more quickly than dogs given a placebo.

[0043] Probiotic species known to benefit dogs include Bacillus coagulans.

Bifidobacterium animalis (has been shown to reduce the time for acute diarrhea to resolve in dogs. Lactobacillus acidophilus improved frequency and quality of stools in sensitive dogs. Lactobacillus rhamnosus strain GG (LGG) is effective in preventing and treating diarrhea in humans, and may benefit dogs as well.

[0044] Bifidobacterium animalis has been studied more in detail. It was chosen for further research because initial studies showed that it had an above-average ability to bind to the gut, a characteristic often associated with beneficial bacteria. Initial studies in dogs showed that it could reduce the pathogenicity of Salmonella typhiurium and Clostridia difficile; bacteria known to induce acute diarrhea. And later during a treatment study, it was found that it could help acute diarrhea resolve faster.

[0045] Dermatitis usually caused by a skin allergy. To treat the dermatitis, one needs to address the underlying immune problems. In allergy, the immune system considers a normally harmless substance as a threat. In dogs with a skin allergy, contact of the allergen on the skin causes an immune reaction leading to the classic symptoms of inflammation: itching, redness and heat. Unfortunately, dogs that develop allergy are usually genetically predisposed to the condition. This means that prevention has to happen at a young age or even when the pups are still in the womb.

[0046] Scientists looked at the ability of L. rhamnosus to change the course of allergy in dogs with a genetic pre-disposition towards allergy. The probiotic was given during pregnancy to the mother and to the puppies during weaning. Unfortunately, while they saw some significant changes in immunological parameters, the puppies had no real improvements, but a follow up study performed three years later in the grown-up pups showed that there were differences in the long-term. The immune system was geared towards anti-inflammatory reactions, and the dogs had less dermatitis.

[0047] Additionally, many products on the market are of dubious quality. A study testing 19 commercial pet foods, all claiming to contain probiotics, determined that none of the feeds contained what was written on the package. Only 53% actually contained at least one of the probiotics species listed, and 26% of the diets had no live bacteria. This would suggest that using pet food fortified with probiotics is not the wisest route for providing one's pet dog with beneficial bacteria. The best bet would be to seek out a quality probiotic with the help of a veterinarian.

[0048] Probiotics are measured by colony forming units (CFUs). Few studies have been done to determine effective dosages, but these numbers are usually in the hundreds of millions or higher. If probiotics are being used to help with digestion, they should be taken with meals, but otherwise they may survive better if given between meals, particularly if taken with liquid that helps to dilute stomach acid and move them more quickly into the digestive tract (for example, given after the dog takes a big drink). Probiotics may be given short-term or long-term.

[0049] Several studies have revealed that some probiotic products in the market have deficiencies in the viability of probiotic strain(s), especially in products containing Bifidobacteria. This may be due to storage conditions, manufacturing or food technologies setbacks such as inappropriate packaging materials that could affect probiotic stability through variations in oxygen permeability. In the past two decades, there has been renewed interest in the study of the nutritional and therapeutic aspects of the mentioned products. It is widely accepted that probiotics may exert positive influence on the host through modulation of the endogenous ecosystem and stimulation of immune system as well as maintaining a healthy intestinal microflora. However, research suggests that health benefits can be strain specific and vary by amount ingested and duration administered, even in pets.

[0050] Many studies have shown that probiotics can survive in vitro techniques, however, all studies do not include food as part of the system, or oral cavity saliva as part of the testing to show survival. As shown herein a unique combination of probiotic and prebiotic products can enhance survival to the lower intestine, first through in vitro testing, and then also by human clinical testing.

[0051] In Vitro Protocols

[0052] The previous techniques described in the literature to test probiotic survival in the GI tract did not include oral mastication of the probiotic with food. Since this is the basic system used by humans (even animals), and in probiotics in yogurt, smoothies, sprinkled on food, etc., the in vitro method to test this should include this phase of the digestive process. The following method includes this critical step as well as including a dose of prebiotic to show support of the bacterial in the GI tract until reaching the lower intestinal phase of digestion.

[0053] Materials for In- Vitro Methods (Trial #1 and Trial #2):

[0054] PROBIOTIC BLEND #5, i.e., LACTOBACILLUS PLANTARUM and

BIFIDOBACTERIUM BIFIDUM (Nebraska Cultures) 50,000,000,000 cfu/gm

[0055] PROBIOTIC BLEND #7 i.e., LACTOBACILLUS RHAMNOSUS, BIFIDOBACTERIUM BIFIDUM, and LACTOBACILLUS ACIDOPHILUS (Nebraska Cultures) 10,000,000,000 cfu/gm

[0056] LACTOBACILLUS ACIDOPHILUS DDS-1 (Nebraska Cultures)

50,000,000,000 cfu/gm

[0057] PRODURA, i.e., BACILLUS COAGULANS (Nebraska Cultures) 15,000,000,000 cfu/gm

[0058] SIMULATED SALIVA PICKERING CHEMICALS 1700-0304

[0059] SIMULATED GASTRIC FLUID SIGMA CHEMICALS

[0060] SIMULATED INTESTINAL FLUID SIGMA CHEMICALS

[0061] PANCREATIN SIGMA CHEMICAL

[0062] AMYLASE SIGMA CHEMICAL

[0063] PEPSIN SIGMA CHEMICAL

[0064] BILE SALTS (OXGALL) MERCK CHEMICAL

[0065] INCUBATOR 37°C

[0066] pH METER [0067] THERMOMETER

[0068] HCL SIGMA CHEMICALS

[0069] SODIUM HYDROXIDE SIGMA CHEMICALS

[0070] STARCH USP

[0071] GLUCOSE USP

[0072] WHEY PROTEIN

[0073] ANALYTICAL BALANCE

[0074] HOT PLATE/ STIRRRER

[0075] IN VITRO METHOD. Tiial #1 :

[0076] Oral Cavity Simulation

[0077] To simulate the dilution and possible hydrolysis of bacteria in the human oral cavity, the probiotics and inulin were added to 15cc of the simulated saliva, one dose of each probiotic per three samples. To this was added 2gm of amylase based on literature of content in saliva and incubated for 2 min at 37°C. Pull 1 ml sample of each and plate for total plate count concentration, calculate and document.

[0078] Stomach Simulation

[0079] To simulate the stomach processing of food and the probiotics, the samples are subsequently added to three by 300cc of simulated gastric fluid and pepsin at 3.99gm and mixed. 1 ml Samples are to be pulled at 0, 60, 90 and 120 minutes as per gastric emptying timing in the literature and these were plated to determine CFU/gm surviving this artificial stomach environment. The solution is to be tested every 10 minutes for pH and maintained at 2.5pH using NaOH or HC1, depending on the need.

[0080] Concentration Step

[0081] This is to simulate the concentration of the solid matter in the gastric to intestinal environment, removing most of the aqueous phase. Test the solution withdrawn for total plate count using 1ml samples from each, and also test the solids for total plate count using the same 1 ml sample technique.

[0082] Intestinal Simulation

[0083] To simulate the dilution in the small intestine, the stomach simulation step solution is to be brought to intestinal pH using NaOH (pH 7.8). Also added were 0.5% bile salts (Oxgall) (estimated at 1 gm) and 0.1% pancreatin (estimated at 3.99gm). One milliliter samples were again removed after the time frame above (0, 8,12,24,36 hrs).

[0084] Based on the findings of 20% survival through the in vitro technique, which is more rigorous than others attempted before, the prebiotic and probiotic blends provided in this study showed far superior survival than other previously tested pre and probiotic blends. See Figure 2, which lists the following probiotic blends:

[0085] Probiotic blends for Trial #1 (treatment or effect in parenthesis).

[0086] FLORA RENEW (post antibiotic) :

[0087] Produra (18.667 mg);

[0088] Lactobacillus Acidophilus DDS-1 (420.000 mg);

[0089] Inulin (4561.333 mg).

[0090] DAILY BALANCE ORIGINAL (daily flora balance):

[0091] Probiotic Blend #7 (1500.000 mg, i.e., 300 mg each strain);

[0092] Inulin (3500 mg).

[0093] TRAVELER'S RELIEF / TRAVELER'S SUPPORT (diarrhea):

[0094] Produra (18.667 mg);

[0095] Lactobacillus Acidophilus DDS-1 (140.000 mg);

[0096] Inulin (4841.333 mg).

[0097] COLON SUPPORT (IBS treatment):

[0098] Probiotic Blend #5 (495.000 mg, i.e., 450 mg Bifidobacterium bifidum, 45 mg

Lactobacillus plantarum);

[0099] Inulin (4505.000 mg).

[00100] IN VITRO METHOD. Trial #2:

[00101] Oral Cavity Simulation

[00102] To simulate the dilution and possible hydrolysis of bacteria in the human oral cavity, the probiotics and inulin were added to 15cc of the simulated saliva, one dose of each probiotic per three samples. To this was added 2gm of amylase based on literature of content in saliva and incubated for 2 min at 37°C. Pull 1 ml sample of each and plate for total plate count concentration, calculate and document.

[00103 ] Stomach Simulation

[00104] To simulate the stomach processing of food and the probiotics, the samples are subsequently added to three by 300cc of simulated gastric fluid and pepsin at 3.99gm and mixed. 1 ml Samples are to be pulled at 0, 60, 90 and 120 minutes as per gastric emptying timing in the literature and these were plated to determine CFU/gm surviving this artificial stomach environment. The solution is to be tested every 10 minutes for pH and maintained at 2.5pH using NaOH or HC1, depending on the need.

[00105] Test the solution withdrawn for total plate count using 1ml samples from each, and also test the solids for total plate count using the same 1 ml sample technique. [00106] Intestinal Simulation

[00107] To simulate the dilution in the small intestine, the stomach simulation step solution is to be pH adjusted to pH 7.8 using NaOH. Also added were 0.5% bile salts (Oxgall) (estimated at 1 gm) and 0.1% pancreatin (estimated at 3.99gm), as above. One milliliter samples were again removed after the time frame above (0, 8,12,24,36 hrs).

[00108] It is further contemplated that the probiotic blends described herein may further contain Saccharomyses boulardii.

[00109] A safe and effective pharmaceutical or nutraceutical composition has been provided containing one or more probiotic bacterial strains as described. In one embodiment, a method of treating an individual for a condition selected from the group consisting of irritable bowel syndrome (IBS), diarrhea, constipation, bowel irregularity, and reactions to antibiotic treatment comprises the step of administering to the individual in need of such treatment a probiotic nutritional composition, and optionally a pharmaceutically or nutraceutically acceptable carrier, such as, for example a prebiotic.

[00110] The dosages of the probiotics are expected to be particularly effective when used in certain combinations, or as a blend. It is expected that certain probiotic (or prebiotic) blends containing bacterial strains will provide synergistic effects with regard to treating or preventing irritable bowel syndrome (IBS), diarrhea, constipation, bowel irregularity, reactions to antibiotic treatment, and the like. The dosage range for certain probiotic compositions can range from about 200 Million CFU per dose to about 100 Billion CFU per dose. In one preferred embodiment, the probiotic products are to range from no less about than 200 Million CFU per dose to no more than about 100 Billion CFU per dose.

[00111] Materials for In- Vitro Methods (Trial #3):

[00112] LACTOBACILLUS ACIDOPHILUS DDS-1 (ARS-B3208); dose: 4,000,000,000 CFU

[00113] BIFIDOBACTERIUM BIFIDUM (ATCC 29521); dose: 2,500,000,000 CFU [00114] LACTOBACILLUS RHAMNOSUS (ATCC 393); dose: 3,000,000,000 CFU [00115] PRODURA, i.e., BACILLUS COAGULANS (B-3208); dose: 500,000,000 CFU [00116] SIMULATED SALIVA PICKERING CHEMICALS 1700-0302 Lot 502036 [00117] SIMULATED GASTRIC FLUID SIGMA CHEMICALS Fluka Lot BCBN3288V

[001 18] PANCREATIN SIGMA CHEMICAL Lot SLBK9948V

[00119] AMYLASE SIGMA CHEMICAL Lot SLBM9658V

[00120] PEPSIN SIGMA CHEMICAL Lot SLBM3033V [00121] BILE SALTS (OXGALL) BCBN9789V

[00122] pH METER Dr. Meter PH-100

[00123] HCL SIGMA CHEMICALS Lot MKP5647

[00124] SODIUM HYDROXIDE SPECTRUM CHEMICALS Lot lea0031

[00125] STARCH USP Corn Starch, Staley

[00126] GLUCOSE USP Sigma Chemical Lot 27865

[00127] WHEY PROTEIN Maroon 80% Lot 06145

[00128] ANALYTICAL BALANCE (lOOg/O.OOlg) Ohaus

[00129] HOT PLATE, STIRRER Sintan Pecies Machinery Co. model 85-2

[00130] Sterile 3cc syringe sample tubes Covidien 8881513934 (lot 430220X)

[00131] INULIN Barrington Chemicals, 650 mg

[00132] FRUCTOOLIGOSACCHARIDE (FOS); NutraFlora, Ingredion, 1,550 mg

[00133] Fibersol 2™ Matsutani, 3,000 mg

[00134] Scope of Study:

[00135] The aim of this study is to provide an in vitro test using Probiotics from the

America's Naturals LLC line to support the hypothesis that exogenously administered probiotics (Specific blends from America's Naturals as described herein) may survive their passage through the adolescent stomach and the small intestine and affect bacterial ecology and metabolism in the large intestine and colon. An important aspect of this issue is to examine the various determinants and factors that allow for probiotic passage through the adolescent gut and enhancement of their metabolic activity. In this context, the effect of Inulin derived from Agave, Fructooligosaccharide (FOS) and soluble Maltodextrin (Fibersol 2®) which have been shown to enhance the metabolic activity of probiotics, will also be used. Diet and specific nutritional factors have been shown to shape intestinal microflora and influence health thus standard diet conditions for and materials for children were used in the experimentation.

[00136] In a similar manner as in Figure 3, the parameters determined as important for bacterial survival in the design were adopted in a model stomach/intestinal passage experiment around preteen/adolescent GI conditions in order to compare the survival of potential formulated probiotic products, based on the following parameters defined for the test (Trial #3).

[00137] 1. Stomach Capacity 900cc as per FDA/USP Parameters, 250cc used for study as per literature pH 3.6. [00138] 2. Diet of basic food at 200gm consumed with probiotic (milk, or carbs/protein) per product due to diet differences, liquid versus solid, a) Sugar intake for children: 150 calories per day (37.5 grams or 9 teaspoons). Alternatively, the sugar intake for children: 100 calories per day (25 grams or 6 teaspoons). According to the American Heart Association (AHA), these values reflect the maximum amount of added sugars you should eat in a day.

[00139] 3. Standard probiotic powder dose recommended by manufacturer used.

[00140] 4. Oral Cavity time 2 minutes' maximum with artificial saliva, Temperature 37 °C, pH 7.

[00141] 5. Gastric Emptying time of 90 minutes, samples taken at 30 min, 60 min, 90 min.

[00142] 6. Small Intestine Transit time 2 hours.

[00143] 7. Large Intestine 24 hours.

[00144] 8. Enzyme activity of each phase mimics as close as possible real world conditions.

[00145] 9. Bile dilutions were used to try and mimic normal bile addition to the intestine.

[00146] 10. 25 bites per meal at 1.2ml saliva generation per bite.

[00147] This data was used to create the experimental design for the testing in vitro, of the probiotic blends survival generally in accordance with Figure 3, through various stages of the pre adolescent GI tract and growth rate potential in the simulated intestinal fluid for the large intestine where the probiotics need to colonize to provide activity.

[00148] IN VITRO METHOD. Trial #3 :

[00149] Basic experimental design

[00150] Step 1. measure out 15cc simulated saliva add 2 gm amylase mix for 5 minutes at 34 °C;

[00151] Step 2. weigh out inulin listed per each formulation blend and add to saliva and mix for 5 minutes;

[00152] Step 3. weigh out probiotic blends required per formulations, keep at 4 °C;

[00153] Step 4. add probiotic blends to saliva mixes and label by blend and mix for 2 minutes;

[00154] Step 5. pull 1 ml sample from mix plate on msg plate incubate at 34 °C for 2 hours, calculate cfu/gm anerobic; [00155] Step 6. measure out 250 cc simulated gastric fluid for each saliva mix, add pepsin (10.3 mg/ liter) to each mix slowly at 37 °C for 2 hours, pulling 1 ml samples of each at 30, 60. 90 minutes, test for cfu/gm as before, adjust pH with HC1 or NaOH, and take to/keep at pH 3.6;

[00156] Step 7. create simulated intestinal juice, add 0.1% pancreatin and 0.5% bile salts the gastric mix, maintain pH at 6.8 with HC1 or NaOHand keep at 37 °C; and

[00157] Step 8. pull samples of mixes at 8 hr., 12 hr., 24 hr. times and test cfu/gm of each at each time point.

[00158] Oral Cavity Simulation

[00159] To simulate the dilution and possible hydrolysis of bacteria in the human oral cavity, the probiotics and inulin were added to 15 cc of the simulated saliva, one dose of each probiotic per three samples. To this was added 2 gm of amylase based on literature of content in saliva and incubated for 2 min at 37 °C. 1 ml samples of each were pulled and tested for total plate count concentration, calculate and document.

[00160] Stomach Simulation

[00161] To simulate the stomach processing of food and the probiotics, the samples are subsequently added to three by 300 cc of simulated gastric fluid and pepsin at 2.99 gm and mixed. 1 ml Samples were pulled at 30, 60 and 90 minutes as per gastric emptying timing in the literature and these were plated to determine CFU/gm surviving this artificial stomach environment. The solution was tested eveiy 10 minutes for pH and maintained at 3.6 pH using NaOH or HC1, depending on the need.

[00162] Intestinal Simulation

[00163] To simulate the dilution in the small intestine, the stomach simulation step solution was pH adjusted to pH 6.8 using NaOH. Also added were 0.5% bile salts (Oxgall) (estimated at 1 gm) and 0.1% pancreatin (estimated at 2.99gm). One ml samples were again removed after the time frame (8, 12, 24 hrs).

[00164] Various samples were tested Micrim Labs LLC (Davie, Florida). Data was reported in CFU/gm and converted based on volume to see recovery from initial inoculation. See Figure 4A (Table 2).

[00165] Discussion of Preteen/Adolescent results

[00166] The initial concept was to filter the stomach solution through a 0.45-micron filter to collect the solid mater and move to simulated Intestinal fluid, however this was not practical and did not function. Thus, the stomach solution was modified to intestinal fluid by addition of the enzymes and base to move the pH to 7.8. This is actually more realistic as what happens in the GI tract as the food moves down the system.

[00167] The idea to take into account the entire alimentary canal was used in this study. This study took into account the following key concepts for determining probiotic survivability from the mouth to the colon:

[00168] 1. Amylase and oral conditions were considered relevant and the data shows it is relevant on the amount of probiotic that reaches the stomach.

[00169] 2. Amylase as left in the stomach fluid section as it is in normal humans.

[00170] 3. The simulated stomach section was exposed to oxygen as is the normal human environment, and further experiments will reduce the level to 16% normal stomach levels of oxygen, as compared to the current uncontrolled air which is 20% oxygen.

[00171] 4. Testing at various time points shows that 8 hours into the intestinal phase is where most all probiotics show the lowest CFU counts, and if the dose is low (below 10 billion CFU) they do not survive the experiment. Further experiments will use anaerobic conditions for the intestinal section of the system to closer mimic what is real in the GI tract.

[00172] 5. Taking into account enzyme levels normally seen in humans at specific stages of the GI process.

[00173] 6. The results show a trend of this mix of probiotics to have significant CFU reduction, especially in the gastric fluid phase, which was to be expected. The transfer to the intestinal phase of Bile salt and pH adjustment also showed a drop off in CFU count of the bacteria. Again this was to be expected as previous experimentation has shown.

[00174] Figure 5 depicts results of the data. The total probiotics present in the final volume should be compared to the initial input content that was tested and it should be confirmed how many total CFU survived from input to the end time point. These results demonstrated the survival rate of probiotics in this test system. Compare also the results shown in Fig. 4 A (Table 2).

[00175] Namely, Figure 5 depicts the probiotic formulation Kids Probiotic to survive the testing with a final total CFU in the solution of 9.25 x 10 ⁹ CFU present from an initial input of 1.0 x 10 ¹⁰ CFU, which is more than sufficient for colonization.

[00176] The concept of testing probiotics for survival from the mouth to the intestine and colon is not a new concept. This experiment shows that the America's Naturals Kids Probiotic tested (Branded as Family Flora Daily Balance Junior) survived this in vitro test for probiotic survival to the colon and colonization. It appears the Inulin did support recovery over time in the system, this needs to be further tested to confirm the hypothesis based on this data since two commercial products tested did not demonstrate late stage growth during testing.

[00177] This test procedure seems viable for testing the survivability of probiotics in vitro as a simulation through the human GI tract. This needs further confirmation with (planned) human clinical studies to show that the results are reasonably consistent between the completed in vitro tests and future human testing. With certain modifications the tests can be more comparable to human conditions; however, our conclusion is that the in vitro testing is a reasonable representation of what can be expected in future human trials.

[00178] We conclude that the in vitro testing shows that the America's Naturals LLC Kids Probiotic/Prebiotic (Branded as Family Flora Daily Balance Junior) had sufficient survival to have a high probability for colonization of the GI tract as desired and expected.

[00179] Human tests are being scheduled to confirm in vivo these results as well as function.

[00180] Materials for In- Vitro Methods in Dogs (Trial #4):

[00181] PRODURA, i.e., BACILLUS COAGULANS (ARS #B3208); dose: 1,000,000,000 CFU

[00182] LACTOBACILLUS ACIDOPHILUS DDS-1 (ARS-B3208); dose: 4,000,000,000 CFU

[00183] BIFIDOBACTERIUM LACTIS (ATCC 29521); dose: 2,000,000,000 CFU

[00184] LACTOBACILLUS RHAMNOSUS (ATCC 393); dose: 2,000,000,000 CFU [00185] SIMULATED SALIVA PICKERING CHEMICALS 1700-0302 Lot 502036

[00186] SIMULATED GASTRIC FLUID SIGMA CHEMICALS Fluka Lot BCBN3288V

[00187] P ANCREATIN SIGMA CHEMICAL Lot SLBK9948 V

[00188] AMYLASE SIGMA CHEMICAL Lot SLBM9658V

[00189] PEPSIN SIGMA CHEMICAL Lot SLBM3033 V

[00190] BILE SALTS (OXGALL) BCBN9789V

[00191] pH METER Dr. Meter PH-100

[00192] HCL SIGMA CHEMICALS Lot MKP5647

[00193] SODIUM HYDROXIDE SPECTRUM CHEMICALS Lot lea0031

[00194] Dog Food Natural Balance Original Ultra Chicken Duck formula LOT

NBUD20183U JZN

[00195] ANALYTICAL BALANCE (lOOg/O.OOlg) Ohaus

[00196] HOT PLATE, STIRRER Sintan Pedes Machinery Co. model 85-2 [00197] Sterile 3cc syringe sample tubes Covidien 8881513934 (lot 430)

[00198] Fibersol 2™ Matsutani, 5,325 mg

[00199] Scope of Dog Study:

[00200] The aim of this study is to provide a test in support that Probiotics from America's Naturals line to support the hypothesis that exogenously administered probiotics (Specific blends from America's Naturals) may survive their passage through the stomach and the small intestine of a dog and affect bacterial ecology and metabolism in the large intestine and colon. An important aspect of this issue is to examine the various determinants and factors that allow for probiotic passage through the gut and enhancement of their metabolic activity. In this context, the effect of Maltodextrin (Fibersol2®), which has been shown to enhance the metabolic activity of probiotics, will also be used. Diet and specific nutritional factors have been shown to shape intestinal microflora and influence health thus standard diet conditions and materials were used in the experimentation.

[00201] The parameters determined as important for bacterial survival in the design were adopted in a model stomach intestinal passage experiment for dogs in order to compare the survival of potential formulated probiotic products, based on the following parameters defined for the test (Trial #4) .

[00202] 1. Stomach Capacity 500cc to 2,000cc as per FDA/USP Parameters, 250cc used for study as per literature.

[00203] 2. Diet of basic dog food at 300gm consumed with probiotic per product due to diet differences, liquid vs solid.

[00204] 3. Standard probiotic powder dose recommended by manufacturer used.

[00205] 4. Oral Cavity time 2 minutes' maximum with artificial saliva, Temperature 37 °C, pH 7.

[00206] 5. Gastric Emptying time of 2 hours, samples taken at 30 min, 60 min, 90 min, 120 min.

[00207] 6. Small Intestine Transit time 3 hours.

[00208] 7. Large Intestine 12 hours.

[00209] 8. Enzyme activity of each phase mimics as close as possible real world conditions.

[00210] 9. Bile dilutions were used to try and mimic normal bile addition to the intestine.

[00211] This data was used to create the experimental design for the testing in vitro of the probiotic blends survival through various stages of the canine GI tract and growth rate potential in the simulated intestinal fluid for the large intestine where the probiotics need to colonize to provide activity.

[00212] IN VITRO METHOD. Trial #4 for Pets, for example in Dog:

[00213 ] Oral Cavity Simulation

[00214] This test for dogs and cats was deleted from the protocol since very little amylase is generated in the saliva of dogs and none in cats.

[00215] Stomach Simulation

[00216] To simulate the stomach processing of food and the probiotics, the samples are subsequently added to three by 300 cc of simulated gastric fluid and pepsin at 3.99 gm and mixed. 1 ml Samples were pulled at 30, 60 and 90 minutes as per gastric emptying timing in the literature and these were plated to determine CFU/gm surviving this artificial stomach environment. The solution is to be tested every 10 minutes for pH and maintained at 1.8 pH using NaOH or HC1, depending on the need.

[00217] Test the solution withdrawn for total plate count using 1ml samples from each also test the solids for total plate count using the same 1 ml sample technique.

[00218] Intestinal Simulation

[00219] To simulate the dilution in the small intestine, the stomach simulation step solution was pH adjusted to pH 6.7 using NaOH. Also added were 0.2% bile salts (Oxgall) (estimated at .3 gm) and 0.1% pancreatin (estimated at 2.5gm). One milliliter samples were again removed after the time frame above (0, 8, 12, 24hrs). 12 hours is maximum normally for dogs, but if fiber diet is used it can be up to 27 hours.

[00220] Various samples were tested Micrim Labs LLC (Davie, Florida). Data was reported in CFU/gm and converted based on volume to see recovery from initial inoculation. See Table 6.

[00221 ] Discussion of Dog results

[00222] The idea to take into account the entire alimentary canal was used in this study. This study took into account the following key concepts for determining probiotic survivability from the mouth to the colon:

[00223] 1. Amylase and oral conditions were considered relevant and the data shows it is relevant on the amount of probiotic that reaches the stomach; however, dogs and cats have no amylase, and only recently have domestic dogs shown the amylase gene, but at low levels.

[00224] 2. Masticated food but less than humans, since dogs gulp food. [00225] 3. The stomach section was exposed to oxygen as is the normal environment, further experiments will reduce the level to 16% normal stomach levels of oxygen, as compared to the current uncontrolled air which is 20% oxygen.

[00226] 4. Testing at various time points shows that 8 hours into the intestinal phase is where most all probiotics show the lowest CFU counts, and if the dose is low (below 10 billion CFU) they do not survive the experiment. Further experiments will use anaerobic conditions for the intestinal section of the system to closer mimic what is real in the GI tract.

[00227] 5. Taking into account enzyme levels normally seen in dogs at specific stages of the GI process.

[00228] 6. The results show a trend of this mix of probiotics to have significant CFU reduction, especially in the gastric fluid phase, which was to be expected. The transfer to the intestinal phase of Bile salt and pH adjustment also showed a drop off in CFU count of the bacteria. Again this was to be expected as previous experimentation has shown.

[00229] The Pet Probiotic present in the final volume when compared to the initial input content that was tested confirmed how many total CFU survived from input to the end time point, thus demonstrating the survival rate of probiotics in this test system. See Figure 6 (Table 3).

[00230] The results show that the Pet Probiotic formulation survived to the last point, comparable to the large intestine with 10 ⁷ bacteria surviving the test protocol mimicking the GI tract of dogs. This is more than sufficient to surmise that this formulation will have sufficient probiotic survive the GI tract of dogs given at the right dose.

[00231] The concept of testing probiotics for survival from the mouth to the intestine and colon is not a new concept. This experiment shows that all the America's Naturals LLC Blends of Pet probiotic (Branded as Family Flora Pet Balance) survived this in vitro test for probiotic survival to the colon and colonization. It appears the Fibersol 2® did support recovery over time in the system as the literature suggested. This test procedure seems viable for testing the survivability of probiotics in vitro through the Dog GI tract. This needs confirmation with Dog studies to show the counts are within reason between this in vitro test and Dog clinical testing. With certain modifications the tests can be more comparable to dog GI conditions; however, the basic testing expectations held in these tests.

[00232] Thus the testing herein shows that America's Naturals LLC products (Branded as Family Flora Pet Balance) recovered and colonized the system at the end of the experiment as expected. This is what needs to happen for probiotics to survive to the lower GI tract in dogs. [00233] Materials for In- Vitro Methods in Older Adults/ Seniors (Trial #5):

[00234] LACTOBACILLUS ACIDOPHILUS POWDER (ARS-B3208); dose:

4,500,000,000 CFU

[00235] BIFIDOBACTERIUM BIFIDUM (ATCC 29521); dose: 4,500,000,000 CFU [00236] LACTOBACILLUS RHAMNOSUS (ATCC 393); dose: 2,700,000,000 CFU [00237] PRODURA, i.e., BACILLUS COAGULANS (B-3208); dose: 900,000,000 CFU [00238] L. PLANTARUM (LP-115 (400B)); dose: 900,000,000 CFU

[00239] BIFIDOBACTERIUM LACTIS; dose: 4,500,000,000 CFU

[00240] SIMULATED SALIVA PICKERING CHEMICALS 1700-0302 Lot 502036

[00241] SIMULATED GASTRIC FLUID SIGMA CHEMICALS Fluka Lot BCBN3288V

[00242] PANCREATIN SIGMA CHEMICAL Lot SLBK9948V

[00243] AMYLASE SIGMA CHEMICAL Lot SLBM9658V

[00244] PEPSIN SIGMA CHEMICAL Lot SLBM3033V

[00245] BILE SALTS (OXGALL) BCBN9789V

[00246] pH METER Dr. Meter PH-100

[00247] HCL SIGMA CHEMICALS Lot MKP5647

[00248] SODIUM HYDROXIDE SPECTRUM CHEMICALS Lot 1 ea0031

[00249] STARCH USP Corn Starch, Staley

[00250] GLUCOSE USP Sigma Chemical Lot 27865

[00251] WHEY PROTEIN Maroon 80% Lot 06145

[00252] ANALYTICAL BALANCE (lOOg/O.OOlg) Ohaus

[00253] HOT PLATE, STIRRER Sintan Pecies Machinery Co. model 85-2

[00254] Sterile 3cc syringe sample tubes Covidien 8881513934 (lot 430220X)

[00255] INULIN Barrington Chemicals, 650 mg

[00256] Scope of Study:

[00257] The aim of this study is to provide an in vitro test using Probiotics from the America's Naturals LLC line to support the hypothesis that exogenously administered probiotics (Specific blends from America's Naturals as described herein) may survive their passage through the older adult/senior stomach and the small intestine and affect bacterial ecology and metabolism in the large intestine and colon. An important aspect of this issue is to examine the various determinants and factors that allow for probiotic passage through the senior gut and enhancement of their metabolic activity. In this context, the effect of Inulin derived from Agave, which has been shown to enhance the metabolic activity of probiotics, will also be used. Diet and specific nutritional factors have been shown to shape intestinal microflora and influence health thus standard diet conditions for and materials for seniors were used in the experimentation.

[00258] In a similar manner as in Figure 3, the parameters determined as important for bacterial survival in the design were adopted in a model stomach/intestinal passage experiment around 60 to 70-year-old (senior) GI conditions in order to compare the survival of potential formulated probiotic products, based on the following parameters defined for the test (Trial #5).

[00259] 1. Stomach Capacity 900cc as per FDA/USP Parameters, 300 cc used for study as per literature pH 3.6.

[00260] 2. Diet of basic food at 200 gm consumed with probiotic (milk, or carbs/protein) per product due to diet differences, liquid versus solid, a) Sugar intake for seniors: 150 calories per day (37.5 grams or 9 teaspoons). According to the American Heart Association (AHA), these values reflect the maximum amount of added sugars seniors should eat in a day.

[00261 ] 3. Standard probiotic powder dose recommended by manufacturer used.

[00262] 4. Oral Cavity time 2 minutes' maximum with artificial saliva, Temperature 37 °C, pH 7.

[00263] 5. Gastric Emptying time of 90 minutes, samples taken at 30 min, 60 min, 90 min.

[00264] 6. Small Intestine Transit time 2 hours.

[00265] 7. Large Intestine 24 hours.

[00266] 8. Enzyme activity of each phase mimics as close as possible real world conditions.

[00267] 9. Bile dilutions were used to try and mimic normal bile addition to the intestine.

[00268] 10. 25 bites per meal at 1.2 ml saliva generation per bite.

[00269] This data was used to create the experimental design for the testing in vitro, of the probiotic blends survival generally in accordance with Figure 3, through various stages of the senior GI tract and growth rate potential in the simulated intestinal fluid for the large intestine where the probiotics need to colonize to provide activity.

[00270] · IN VITRO METHOD. Trial #5:

[00271] Basic experimental design [00272] Step 1. measure out 15cc simulated saliva add 2 gm amylase mix for 5 minutes at 34 °C;

[00273] Step 2. weigh out inulin listed per each formulation blend and add to saliva and mix for 5 minutes;

[00274] Step 3. weigh out probiotic blends required per formulations, keep at 4 °C;

[00275] Step 4. add probiotic blends to saliva mixes and label by blend and mix for 2 minutes;

[00276] Step 5. pull 1 ml sample from mix plate on msg plate incubate at 34 °C for 2 hours, calculate cfu/gm anerobic;

[00277] Step 6. measure out 250 cc simulated gastric fluid for each saliva mix, add pepsin (10.3 mg/ liter) to each mix slowly at 37 °C for 2 hours, pulling 1 ml samples of each at 30, 60. 90 minutes, test for cfu/gm as before, adjust pH with HC1 or NaOH, and take to/keep at H 3.6;

[00278] Step 7. create simulated intestinal juice, add 0.1% pancreatin and 0.5% bile salts the gastric mix, maintain pH at 6.8 with HC1 or NaOH and keep at 37 °C; and

[00279] Step 8. pull samples of mixes at 8 hi-., 12 hr., 24 hr. times and test cfu gm of each at each time point.

[00280] Oral Cavity Simulation

[00281] To simulate the dilution and possible hydrolysis of bacteria in the human oral cavity, the probiotics and inulin were added to 15 cc of the simulated saliva, one dose of each probiotic per three samples. To this was added 2 gm of amylase based on literature of content in saliva and incubated for 2 min at 37 °C. 1 ml samples of each were pulled and tested for total plate count concentration, calculate and document.

[00282] Stomach Simulation

[00283] To simulate the stomach processing of food and the probiotics, the samples are subsequently added to three by 300 cc of simulated gastric fluid and pepsin at 3.99 gm and mixed. 1 ml Samples were pulled at 30, 60 and 90 minutes as per gastric emptying timing in the literature and these were plated to determine CFU/gm surviving this artificial stomach environment. The solution was tested every 10 minutes for pH and maintained at 2.5 pH using NaOH or HC1, depending on the need.

[00284] Intestinal Simulation

[00285] To simulate the dilution in the small intestine, the stomach simulation step solution was pH adjusted to pH 6.8 using NaOH. Also added were 0.5% bile salts (Oxgall) (estimated at 1 gm) and 0.1% pancreatin (estimated at 3.99 gm). One ml samples were again removed after the time frame (8, 12, 24, 36 hrs).

[00286] Various samples were tested Micrim Labs LLC (Davie, Florida). Data was reported in CFU/gm and converted based on volume to see recovery from initial inoculation. See Figure 8 (Table 4).

[00287] Discussion of Senior/Aged adult results

[00288] The idea to take into account the entire alimentary canal was used in this study. This study took into account the following key concepts for determining probiotic survivability from the mouth to the colon:

[00289] 1. Amylase and oral conditions were considered relevant and the data shows it is relevant on the amount of probiotic that reaches the stomach.

[00290] 2. Amylase is left in the stomach fluid section as it is in normal humans.

[00291] 3. The simulated stomach section was exposed to oxygen as is the normal human environment, and further experiments will reduce the level to 16% normal stomach levels of oxygen, as compared to the current uncontrolled air which is 20% oxygen.

[00292] 4. Testing at various time points shows that 8 hours into the intestinal phase is where most all probiotics show the lowest CFU counts, and if the dose is low (below 10 billion CFU) they do not survive the experiment. Further experiments will use anaerobic conditions for the intestinal section of the system to closer mimic what is real in the GI tract.

[00293] 5. Taking into account enzyme levels normally seen in humans at specific stages of the GI process.

[00294] 6. The results show a trend of this mix of probiotics to have significant CFU reduction, especially in the gastric fluid phase, which was to be expected. The transfer to the intestinal phase of Bile salt and pH adjustment also showed a drop off in CFU count of the bacteria. Again this was to be expected as previous experimentation has shown.

[00295] Figure 7 depicts results of the data. The total probiotics present in the final volume should be compared to the initial input content that was tested and it should be confirmed how many total CFU survived from input to the end time point. These results demonstrated the survival rate of probiotics in this test system. Compare also the results shown in Fig. 8 (Table 4).

[00296] Namely, Figure 7 depicts the probiotic formulation Family Flora Daily Balance Senior Probiotic to survive the testing with a final total CFU in the solution of 9 x 10 ⁶ CFU present from an initial input of 1.0 x 10 ¹⁰ CFU, which is more than sufficient for colonization. [00297] The concept of testing probiotics for survival from the mouth to the intestine and colon is not a new concept. This experiment shows that the America's Naturals Senior Probiotic tested (Branded as Family Flora Senior Daily Balance) survived this in vitro test for probiotic survival to the colon and colonization. It appears the prebiotics (Inulin) did support recovery over time in the system, this needs to be further tested to confirm the hypothesis based on this data since two commercial products tested did not demonstrate late stage growth during testing.

[00298] This test procedure seems viable for testing the survivability of probiotics in vitro as a simulation through the human GI tract. This needs further confirmation with (planned) human clinical studies to show that the results are reasonably consistent between the completed in vitro tests and future human testing. With certain modifications the tests can be more comparable to human conditions; however, our conclusion is that the in vitro testing is a reasonable representation of what can be expected in future human trials.

[00299] We conclude that the in vitro testing shows that the America's Naturals LLC Senior Probiotic/Prebiotic (Branded as Family Flora Senior Daily Balance) had sufficient survival to have a high probability for colonization of the GI tract as desired and expected.

[00300] Human tests are being scheduled to confirm in vivo these results as well as function.

[00301 ] In another embodiment, it is expected that certain probiotic (or prebiotic) blends will provide synergistic effects with regard to maintaining or improving gut or GI health.

[00302] The composition may also include an excipient, most preferably a nutraceutical or pharmaceutical excipient. Compositions containing an excipient and incorporating one or more pribiotics can be prepared by procedures known in the art. Optionally, the composition can include one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries. For example, probiotics can be formulated into tablets, capsules, powders, suspensions, solutions for oral administration and solutions for parenteral administration including intravenous, intradermal, intramuscular, and subcutaneous administration, and into solutions for application onto patches for transdermal application with common and conventional carriers, binders, diluents, and excipients.

[00303] The nutraceutical compositions of the present invention may be administered in combination with a pharmaceutically acceptable carrier. The active ingredients in such formulations may comprise from 1 % by weight to 99% by weight, or alternatively, 0.1% by weight to 99.9% by weight. "Pharmaceutically acceptable carrier" means any carrier, diluent or excipient that is compatible with <fhe other ingredients of the formulation and not deleterious to the user. Useful excipients include, but are not limited to, microcrystalline cellulose, magnesium stearate, calcium stearate, any acceptable sugar (e.g., mannitol, xylitol), and the like, and for cosmetic use an oil-base is preferred.

[00304] Delivery System

[00305] Suitable dosage forms include tablets, capsules, solutions, suspensions, powders, gums, and confectionaries. Sublingual delivery systems include, but are not limited to, dissolvable tabs under and on the tongue, liquid drops, and beverages. Edible films, hydrophilic polymers, oral dissolvable films or oral dissolvable strips can be used. Other useful delivery systems comprise oral or nasal sprays or inhalers, and the like.

[00306] For oral administration, probiotics in combination with a prebiotic may be further combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable dosage forms. For example, the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents, absorbents, or lubricating agents. Other useful excipients include magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethylcellulose, microcrystalline cellulose, silica, gelatin, silicon dioxide, and the like.

[00307] The components of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof. Such forms include solids, and in particular tablets, filled capsules, powder and pellet forms, and liquids, in particular aqueous or non-aqueous solutions, suspensions, emulsions, elixirs, and capsules filled with the same, all for oral use, suppositories for rectal administration, and sterile injectable solutions for parenteral use. Such pharmaceutical compositions and unit dosage forms thereof many comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.

[00308] The components of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a chemical compound of the invention or a pharmaceutically acceptable salt of a chemical compound of the invention. [00309] For preparing pharmaceutical compositions from a chemical compound of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

[00310] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

[00311] The powders and tablets preferably may contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium state, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethlycellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without additional carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[00312] Liquid preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. The chemical compound according to the present invention may thus be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose for administration in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen- free water, before use.

[00313] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.

[00314] Compositions suitable for topical administration in the mouth includes lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in suitable liquid carrier.

[00315] Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be provided in single or multi-dose form. In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound will generally have a small particle size, for example, of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example, by micronization.

[00316] The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenges itself, or it can be the appropriate number of any of these in packaged form.

[00317] Tablets,- capsules and lozenges for oral administration and liquids for oral use are preferred compositions. Solutions or suspensions for application to the nasal cavity or to the respiratory tract are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred.

[00318] Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).

[00319] Routes of Administration

[00320] The compounds may be administered by any route, including but not limited to oral, sublingual, buccal, ocular, pulmonary, rectal, and parenteral administration, or as an oral or nasal spray (e.g. inhalation of nebulized vapors, droplets, or solid particles). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical, or subcutaneous administration. Also contemplated within the scope of the invention is the instillation of a pharmaceutical composition in the body of the patient in a controlled formulation, with systemic or local release of the drug to occur at a later time. For example, the drug may be localized in a depot for controlled release to the circulation, or for release to a local site.

[00321] Pharmaceutical compositions of the invention may be those suitable for oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebal, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or insufflations, including powders and liquid aerosol administration, or by sustained release systems. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices may be in form of shaped articles, e.g. films or microcapsules.

[00322] The following are general clinical studies for human trials to assess efficacy of one or more exemplary probiotic blends in treating irritable bowel conditions (e.g., IBS), diarrhea, constipation, bowel regularity, reactions to antibiotic treatment, and the like. Such trials may also be used to assess maintenance and/or improvement of gastrointestinal (GI) health by the administration of multistrain probiotic blends.

[00323] Product for testing (general condition): COLON SUPPORT (IBS). See also Figure 4B, Table 2.

[00324] The following protocol used to test DSM (L. plantarum 299v) (DSM 9843) in IBS conditions will be used as the model protocol for comparison: Clinical trial: Lactobacillus plantarum 299v (DSM 9843) improves symptoms of irritable bowel syndrome, Philippe Ducrotte, Prabha Sawant, Venkataraman Jayanthi, World J Gastroenterol :2012 August 14; 18(30) :4012-4018.

[00325] Irritable bowel syndrome (IBS) is one of the most frequent digestive tract disorders encountered by general practitioners and gastroenterologists. IBS is a functional bowel disorder characterized by chronic and relapsing abdominal pain or discomfort associated with altered bowel habits. The primary aim of any treatment is the relief of abdominal pain which can significantly impair the patient's quality of life. According to published guidelines, the main treatment options for abdominal pain include anti-spasmodics or anti-depressants at low dose while anti-diarrheal or laxative drugs are given to improve transit disturbances. However, in many cases, all these options remain disappointing for the relief of abdominal pain. Thetherapeutic efficacy in IBS is probably impacted by the heterogeneous pathogenesis of the disease which includes altered intestinal motility, visceral hypersensitivity, abnormal brain-gut interactions, food intolerance, altered intestinal permeability and postinfectious and/or inflammatory changes.

[00326] Recently, the deleterious role of qualitative or quantitative alterations of gut microbiota at the onset of symptoms has been emphasized. Therefore, a rationale exists to discuss the therapeutic use of probiotics, which are live microorganisms conferring health benefits to the host when ingested in adequate amounts. Clinical evidence regarding the efficacy of some probiotics trains to improve IBS symptoms has recently emerged, although the mechanism of action of probiotics on IBS symptoms is not completely understood. Some probiotics bind to small and large bowel epithelium and may produce substances with antibiotic properties, while others compete for attachment and thereby reduce invasion by pathogenic organisms. Probiotics also modulate gastrointestinal luminal immunity by changing the cytokine and cellular milieu from a pro-inflammatory to anti-inflammatory state. They may also convert undigested carbohydrates into short chain fatty acids, which act as nutrients for colonocytes and affect gut motility.

[00327] Multi single-centre studies will be used to test the clinical efficacy of Bifidobacterium Bifidum ATCC 29521 and Lactobacillus plantarum ATCC 8014 along with 3.35gm of Inulin from Agave in IBS patients. Two trials have demonstrated significant benefits in comparison with placebo on improvement of flatulence scores and a reduction of abdominal pain while the results of the third trial, based on only 12 patients, were not conclusive previously with DSM (L. plantarum 299v) (DSM 9843). The aim of the present randomized, double-blind, placebo-controlled clinical trial was to assess the symptomatic efficacy of Lactobacillus plantarum in combination with Bifidobacterium Bifidum and inulin in a larger subset of IBS patients fulfilling the Rome III criteria and will compare to the listed trial. .

[00328] Aim

[00329] To assess the symptomatic efficacy of Bifidobacterium Bifidum ATCC 29521 and Lactobacillus plantarum ATCC 8014 along with 3.35gm of Inulin from Agave for the relief of abdominal symptoms in a large subset of irritable bowel syndrome (IBS) patients fulfilling the Rome III criteria.

[00330] Study Design

[00331] In this double blind, placebo-controlled, parallel-designed study, subjects were randomized to daily receive either one sachet of Lactobacillus plantarum in combination with Bifidobacterium Bifidum and inulin or placebo for 4 wk. Frequency and intensity of abdominal pain, bloating and feeling of incomplete rectal emptying were assessed weekly on a visual analogue scale while stool frequency will be calculated.

[00332] This study will be designed as a multicentre double blind, placebo-controlled study with parallel groups to assess the beneficial effects of a daily consumption of Colon Support on IBS symptoms. Treatment duration will be 4 wk with 3 follow-up visits at weekly intervals. The study protocol will be conducted in accordance with the Declaration of Helsinki and approved by the local Ethics Committee. All volunteers will be given written informed consent prior to participation in the study.

[00333] Materials and methods.

[00334] Patients

[00335] Participants (n = over 200) will be recruited by general practitioners in multiple clinical centers. Subjects between 18-70 years of age with IBS according to the Rome M criteria will be eligible for inclusion. All subjects will receive a colonic examination at baseline to exclude any organic disease while an intestinal infection will be excluded by stool cultures in any patient in whom this diagnosis was suspected. Subjects with severe ■ chronic medical disease including colorectal and other gastrointestinal diseases will be excluded. Pregnant and breast-feeding women and patients with dietary habits which might interfere with the assessment of the study product or patients with known allergy to the study product components will also be excluded. Throughout the study, the subjects will not be allowed to consume any other probiotic and will be encouraged not to change their usual dietary and physical exercise habits.

[00336] Study products

[00337] The test product will be Colon Support with 15 Billion CFU Bifidobacterium Bifidum ATCC 29521 and 1.5 Billion CFU Lactobacillus plantarum ATCC 8014 along with 3.35gm of Inulin from Agave. The test product contains 16.5 billion colony-forming units (cfu) in sachet form with no other ingredients except the prebiotic Inulin. The control product contains 4gm of maltodextrin. Both the test and control products will have a similar appearance, texture and taste. Both products will be specifically prepared for the study and provided by America's Naturals (Fort Lauderdale, Florida).

[00338] Assessments and study endpoints

[00339] The primary endpoint will be the improvement of the frequency of abdominal pain episodes. Secondary endpoints are expected to be changes in severity of abdominal pain, changes in frequency and severity of abdominal bloating and in feeling of incomplete rectal emptying. Both frequency of abdominal pain and feeling of incomplete rectal emptying should be assessed weekly using a four-point scale ranging from 1 (only occasional symptom) to 4 (daily symptom). Symptom severity (abdominal pain, abdominal bloating and feeling of incomplete rectal emptying) should be rated on a visual analogue scale (VAS 1-10) and converted to a 4-point scale ranging from 0 (No pain, VAS = 0) to 3 (Severe, VAS = 8 to 10).

[00340] The daily number of stools and bloating episodes will be calculated.

[00341] Product for testing (general condition): TRAVELER'S RELIEF #2 (diarrhea). See also Figure 4B, Table 2.

[00342] The following protocol used to test in traveler's related diarrhea will be used as the model protocol: Clinical trial: A Randomized, Double Blind, Placebo-Controlled Trial of an Oral Synbiotic (AKSB) for Prevention of Travelers' Diarrhea; Abinash Virk, MD, Jayawant Mandrekar, PhD, Elie F. Berbari, MD, Thomas G. Boyce, MD, Philip R. Fischer, MD,t Mary J. Kasten, MD, Robert Orenstein, DO, Jon E. Rosenblatt, MD, Priya Sampathkumar, MD, Irene Sia, MD, Donna Springer, RN, CNS, and Thomas E. Witzig, MD; Division of Infectious Diseases; Division of Biomedical Statistics and Informatics; Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA; §Division of Infectious Diseases, Mayo Clinic, Scottsdale, AZ, USA; Department of Laboratory Medicine and Pathology; Division of Hematology and Oncology, Mayo Clinic, Rochester, MN, USA DOI: 10.1111/jtm.12008.

[00343] Acute diarrhea affects millions of persons who travel to developing countries each year. Food and water contaminated with fecal matter are the main sources of infection. Bacteria such as enterotoxigenic Escherichia coli, enteroaggregative E. coli, Campylobacter, Salmonella, and Shigella are common causes of traveler's diarrhea (TD). Parasites and viruses are less common etiologies. Travel destination is the most significant risk factor for traveler's diarrhea. The efficacy of pretravel counseling and dietary precautions in reducing the incidence of diarrhea is unproven. Empiric treatment of traveler's diarrhea with antibiotics and loperamide is effective and often limits symptoms to one day. Rifaximin, a recently approved antibiotic, can be used for the treatment of traveler's diarrhea in regions where noninvasive E. coli is the predominant pathogen. In areas where invasive organisms such as Campylobacter and Shigella are common, fluoroquinolones remain the drug of choice. Azithromycin is recommended in areas with quinolone- resistant Campylobacter and for the treatment of children and pregnant women. (Am Fam Physician 2005; 71 :2095-100, 2107-8. Copyright© 2005 American Academy of Family Physicians.) 2096 American Family Physician www.aafp.org/afp Volume 71, Number 11, June 1, 2005.

[00344] E. coli such as enteroaggregative E. coli have been recognized as common causes of traveler's diarrhea. Invasive pathogens such as Campylobacter, Shigella, and nontyphoid Salmonella are relatively common depending on the region, while Aeromonas and non-cholera Vibrio species are encountered less frequently. Protozoal parasites such as Giardia lamblia, Entamoeba histolytica, and Cyclospora cayetanensis are uncommon causes of traveler's diarrhea, but increase in importance when diarrhea lasts for more than two weeks. Parasites are diagnosed more frequently in returning travelers because of longer incubation periods (often one to two weeks) and because bacterial pathogens may have been treated with antibiotics. Rotavirus and noroviruses are infrequent causes of traveler's diarrhea, although noroviruses have been responsible for outbreaks on cruise ships.

[00345] Although travelers often are advised to "Boil it, cook it, peel it, or forget it," data on the effectiveness of dietary precautions in preventing traveler's diarrhea are inconclusive. Many travelers find it difficult to adhere to dietary recommendations. In a study3 of American travelers, nearly one half developed diarrhea despite pre-travel advice on avoidance measures; even persons who strictly followed dietary recommendations developed diarrhea. Avoiding high-risk foods and adventuresome eating behaviors may reduce the inoculum of ingested pathogens or prevent the development of other enteric diseases such as typhoid and hepatitis A and E. Boiling is the best way to purify water. Iodination or chlorination is acceptable but does not kill Cryptosporidium or Cyclospora, and increased contact time is required to kill Giardia in cold or turbid water. Filters with iodine resins generally are effective in purifying water, although it is uncertain whether the contact time with the resin is sufficient to kill viruses. Bottled water generally is safe if the cap and seal are intact.

[00346] Probiotics are a more natural approach to prophylaxis of traveler's diarrhea. Probiotics colonize the gastrointestinal tract and theoretically prevent pathogenic organisms from infecting the gut. Studies of Lactobacillus GG (Culturelle) have suggested protection rates of up to 47 percent. More studies are needed to confirm the efficacy of probiotic prophylaxis. This study of a combination of two well known probiotics and a well studied prebiotic will follow a well designed protocol to determine functionality.

[00347] Aim

[00348] To assess the symptomatic efficacy of Lactobacillus acidophilus ARS-B3208 and bacillus coagulans ARS #B3208 along with 4.85gm of Inulin from Agave for the relief of abdominal symptoms in a large subset of Traveler's diarrhea patients based on inclusion criteria.

[00349] Study Design

[00350] Healthy subjects traveling to an area of the world with an increased risk of TD were eligible. All subjects received pre-travel counseling and were provided antibiotics and antidiarrheals (loperamide) for use only if TD developed. The subjects were blinded and randomized to take two capsules of placebo or oral synbiotic (a combination of two probiotics and a prebiotic) called TRAVELER'S RELIEF #2 beginning 5 days prior to departure, daily while traveling, and for 10 days after return. All subjects kept symptom and medication diaries and submitted a stool sample for pathogen carriage within 7 days of return. The study was powered to detect a 50% reduction in the incidence of TD.

[00351] This study will be designed as a multicentre double blind, placebo-controlled study with parallel groups to assess the beneficial effects of a daily consumption of TRAVELER'S RELIEF #2 on diarrhea symptoms. Treatment duration will be length of trips of patients by patient. The study protocol will be conducted in accordance with the Declaration of Helsinki and approved by the local Ethics Committee. All volunteers will be given written informed consent prior to participation in the study.

[00352] Dose Selection, Treatment Assignment, Randomization, and Blinding Procedures

[00353] TRAVELER'S RELIEF #2 containing acidophilus ARS-B3208 and bacillus coagulans ARS #B3208 along with 4.85gm of Inulin from Agave in one Sachet of TRAVELER'S RELIEF #2 or placebo will be ingested daily with food, beginning 5 days prior to travel, throughout the trip (trip duration to be documented), and for 10 days after return. The Sachet can be taken any time of day. The TRAVELER'S RELIEF #2 and placebo sachets will be identical in color, packaging, and smell.

[00354] Subjects will be randomly chosen to receive TRAVELER'S RELIEF #2 or placebo. Randomization will be performed in a block of size 4 using a random number generator.

[00355] Materials and methods.

[00356] Patients

[00357] Subjects aged 18 years or above and traveling for 5 to 30 days to a location considered at high risk for TD will be eligible for the trial. The high-risk areas were defined as countries in the continents of Africa, South and Central America, and Asia. Additional exclusion criteria included: current use of antibiotic or antidiarrheal medication (ie, Pepto- Bismol, loperamide, etc.) or their use within 2weeks prior to departure for the trip, a history of inflammatory bowel disease (Crohn's disease or chronic ulcerative colitis), known bowel cancer, congenital or acquired immunocompromised states such as human immunodeficiency virus infection (HIV/ AIDS), current or recent chemotherapy or immunomodulating agents (corticosteroids and TNF-cc inhibitors), short-gut syndrome, use of oral typhoid vaccine within 48 hours of starting therapy, pregnancy, ongoing probiotic use, and previous participation in this study. Women of childbearing age are required to have a negative pregnancy test within 2 weeks of starting the study drug and are counseled not to get pregnant during the study period.

[00358] All enrolled subjects receive standard counseling and education about food and water precautions and self-management of TD.

[00359] Subjects are also offered antimicrobials (ciprofloxacin, levofloxacin, or azithromycin) to carry with them to treat TD if needed. They were instructed not to use antibiotics prophylactically. The subjects are instructed to continue taking the study drug even if TD develops and are initiating antibiotics and/or loperamide. A letter is provided to the patient to allow carriage of the study drug across international borders. The letter also contains telephone numbers for on-call personnel in case subjects experience side effects or have questions during their trip, and all subjects enrolled in this study will provide written informed consent.

[00360] Study products

[00361] The test product will be TRAVELER'S RELIEF #2 with 20 Billion CFU acidophilus ARS-B3208 and 0.4 Billion CFU bacillus coagulans ARS #B3208 along with 4.85gm of Inulin from Agave. The test product contains 20.5 billion colony-forming units (cfu) in sachet form with no other ingredients except the prebiotic Inulin. The control product will contain 5gm of maltodextin. Both the test and control products will have a similar appearance, texture and taste. Both products will be specifically prepared for the study and provided by America's Naturals (Fort Lauderdale, Florida).

[00362] Assessments and study endpoints

[00363] The primary endpoint will be the improvement of the frequency of diarrhea and abdominal pain episodes. Secondary endpoints are expected to be changes in severity of abdominal pain, changes in frequency and severity of abdominal bloating and in feeling of incomplete rectal emptying as well as time of diarrhea sesation. Both frequency of abdominal pain and feeling of incomplete rectal emptying should be assessed weekly using a four-point scale ranging from 1 (only occasional symptom) to 4 (daily symptom). Symptom severity (abdominal pain, abdominal bloating and feeling of incomplete rectal emptying) should be rated on a visual analogue scale (VAS 1-10) and converted to a 4-point scale ranging from 0 (No pain, VAS = 0) to 3 (Severe, VAS = 8 to 10).

[00364] The daily number of stools and bloating episodes will be calculated.

[00365] Product for testing (general condition): FLORA RENEW (post antibiotic). This study of a combination of two well known probiotics and a well studied prebiotic will follow a well designed protocol to determine functionality. See also Figure 4A, Table 2.

[00366] The following protocol used to test diarrhea treatment associated with antibiotic use, will be used as the model protocol: Clinical trial: Hickson M, D'Souza AL, Muthu N, et al. Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind placebo controlled trial. BMJ. 2007;335(7610):80.

[00367] There is increasing evidence that probiotics are beneficial in a range of gastrointestinal conditions, including infectious diarrhea and that related to antibiotic use. Probiotics are defined as "live micro-organisms which when administered in adequate amounts confer a health benefit on the host" and include Lactobacillus and Bacillus species. Diarrhea associated with antibiotic use and caused by Clostridium difficile is a complication of treatment with antimicrobial agents and occurs in about 5-25% of patients. C difficile is responsible for around 15-25%) of all cases of diarrhea associated with antibiotic use, most occurring in older patients, usually in the two to three weeks after cessation of antibiotic treatment. Lactobacilli and Bacillus species have all been evaluated for the prevention or treatment.

[00368] Aim

[00369] To assess the symptomatic efficacy of Lactobacillus acidophilus ARS-B3208 and bacillus coagulans ARS #B3208 along with 4.95gm of Inulin from Agave for the relief of abdominal symptoms in antibiotic treatment induced diarrhea patients based on inclusion criteria.

[00370] Study Design

[00371] The hypothesis is that Flora Renew will reduce the incidence of antibiotic associated diarrhea and C. difficile associated diarrhea. The primary outcome will be the occurrence of diarrhea, which will be recorded by attending staff and authenticated by the researchers. Diarrhea is defined as more than two liquid stools a day for three or more days in quantities in excess of normal for each patient. The secondary outcome will be the occurrence of C. difficile infection, defined as an episode of diarrhea combined with the detection of toxins A or B, or both, from a stool sample. [00372] The admitting medical team will identify potential patients who had been prescribed antibiotics and the researchers approached them within 48 hours of the first antibiotic dose. Once they obtained informed consent, they collected baseline data and prescribed the randomized study dose. The hospital pharmacy dispenses the dose. A baseline stool sample will be collected to screen for asymptomatic C difficile carriage. Bowel movements were monitored with stool charts, which were checked every weekday for accuracy. When there is evidence of diarrhea a stool sample was analyzed for C difficile toxin. Once the antibiotic course is finished a final week of study dose was dispensed and a final follow-up date fixed for four weeks later. Patients who will be discharged taking antibiotics were provided with enough doses on discharge to cover the period they had to take antibiotics plus one week. Researchers will follow up participants for four weeks from discharge with weekly phone calls to ask about diarrhea and compliance. If participants had diarrhea, the researchers will collect a further stool sample to check for C difficile toxin.

[00373] Randomization

[00374] An independent statistician will generate the random allocation sequence, which will stratify for hospital, sex, and two age groups (50-69 and >70). The sequence will be given to the pharmacy on each site.

[00375] Materials and methods.

[00376] Patients

[00377] Patients (ages from 18 to 75) who are taking antibiotics. Exclusions include diarrhea on admission, bowel pathology that could result in diarrhea, antibiotic use in the previous four weeks, severe illness, immunosuppression, bowel surgery, artificial heart valves, and history of rheumatic heart disease or infective endocarditis.

[00378] Intervention Consumption of a 5 g sachet containing 15 Billion CFU Lactobacillus acidophilus ARS-B3208 and 200 Million CFU bacillus coagulans ARS #B3208 along with 4.89 gm of inulin from Agave twice a day during a course of antibiotics and for one week after the course finished. The placebo group receive a 5gm sachet of maltodextrin.

[00379] Patients will be recruited mainly from orthopaedic, medical, and care of the elderly wards, and included inpatients aged over 18 who are prescribed antibiotics (single or multiple antibiotics, oral or intravenous) and will be able to take food and drink orally.

[00380] Our exclusion criteria will be diarrhea on admission or within the preceding week, reported recurrent diarrhea, or bowel pathology that could result in diarrhea; intake of high risk antibiotics (clindamycin, cephalosporins, aminopenicillins) or more than two courses of other antibiotics in the past four weeks to exclude pre-existing diarrhea associated with antibiotic use; severe life threatening illness, immune-suppression, bowel surgery, artificial heart valve, history of rheumatic heart disease, or history of infective endocarditis5; regular probiotic treatment before admission; and lactose intolerance or intolerance to dairy products.

[00381] Interventions

[00382] The treatment group receive 15 Billion CFU Lactobacillus acidophilus ARS- B3208 and 200 Million CFU bacillus coagulans ARS #B3208 along with 4.95gm of inulin from Agave. The placebo group received a sachet of 5 gm of Maltodextrin.

[00383] Participants will begin using the sachets within 48 hours of starting antibiotic therapy and continued doing so for one week after they stopped taking antibiotics. They will take one 5gm sachet twice daily half an hour before or one to two hours after meals. Researchers will verify participants' consumption and recorded missed or refused doses to assess compliance.

[00384] Study products

[00385] The test product will be Flora Renew with 15 Billion CFU acidophilus ARS- B3208 and 0.2 Billion CFU bacillus coagulans ARS #B3208 along with 4.95gm of Inulin from Agave. The test product contained 15.2 billion colony- forming units (cfu) in sachet form with no other ingredients except the prebiotic Inulin. The control product contained 5gm of maltodextin. Both the test and control products will have a similar appearance, texture and taste. Both products will be specifically prepared for the study and provided by America's Naturals (Fort Lauderdale, Florida).

[00386] Assessments and study endpoints

[00387] The primary endpoint will be the improvement of the frequency of diarrhea and abdominal pain episodes. Secondary endpoints are expected to be changes in severity of abdominal pain, changes in frequency and severity of abdominal bloating and in feeling of incomplete rectal emptying as well as time of diarrhea sesation. Both frequency of abdominal pain and feeling of incomplete rectal emptying should be assessed weekly using a four-point scale ranging from 1 (only occasional symptom) to 4 (daily symptom). Symptom severity (abdominal pain, abdominal bloating and feeling of incomplete rectal emptying) should be rated on a visual analogue scale (VAS 1-10) and converted to a 4-point scale ranging from 0 (No pain, VAS = 0) to 3 (Severe, VAS = 8 to 10). .

[00388] The daily number of stools and bloating episodes will be calculated.

[00389] Product for testing (general condition): DAILY BALANCE #2 (bowel regularity). See also Figure 4A, Table 2. [00390] The following protocol is being used to test in bowel regularity study and this study will be used as the model protocol: Clinical trial: A Randomized, Double Blind, Placebo-Controlled Trial of an Oral Synbiotic (AKSB) for Prevention of Travelers' Diarrhea; Abinash Virk, MD, Jayawant Mandrekar, PhD, Elie F. Berbari, MD, Thomas G. Boyce, MD, Philip R. Fischer, MD.J Mary J. Kasten, MD, Robert Orenstein, DO, Jon E. Rosenblatt, MD, Priya Sampathkumar, MD, Irene Sia, MD, Donna Springer, RN, CNS, and Thomas E. Witzig, MD; Division of Infectious Diseases; Division of Biomedical Statistics and Informatics; Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA; §Division of Infectious Diseases, Mayo Clinic, Scottsdale, AZ, USA; Department of Laboratory Medicine and Pathology; Division of Hematology and Oncology, Mayo Clinic, Rochester, MN, USA DOI: 10.1111/jtm.12008.

[00391] Constipation is one of the most common— not to mention annoying— health problems we can face. In the United States, constipation accounts for seven million physician visits per year. Chronic constipation affects almost one in six adults. The costs to your quality of life are equally steep. A review of the medical literature published in 2010 showed that reduced quality of life from constipation was comparable to that caused by serious chronic conditions such as osteoarthritis and diabetes.

[00392] Do probiotics work against constipation? Researchers at King's College in London scoured the medical literature and found 14 studies that met their criteria for a well- done study. All were clinical trials that randomly assigned people with constipation to take either probiotics or a placebo (or other control treatment).

[00393] By pooling the findings of the trials, the researchers found that on average, probiotics slowed "gut transit time" by 12.4 hours, increased the number of weekly bowel movements by 1.3, and helped soften stools, making them easier to pass.

[00394] But there's still not enough evidence to recommend a specific probiotic to people with constipation. Each of the small studies in the new report had different designs and widely variable results, making it difficult to glean a specific recommendation. What's needed is a large, multicenter trial, with standardized outcomes to determine which probiotic species and strain is most effective, how much to take, and for how long.

[00395] Less than two to three bowel movements per day indicates constipation according to many physicians. This is because the bowel helps the body eliminate toxins. After all, bile is eliminated through the feces, and the liver excretes bile as it's undertaking its detoxification role. For the body to heal and wellness to prevail, toxic waste absorption must be minimized. [00396] The large intestine absorbs about 90 percent of the water content it receives from the small intestine. If this slurry of food material and moisture remains in the colon too long, excess moisture is absorbed and toxins and waste burden are reabsorbed disproportionately, irritating the colonic lining. This is why, when individuals fail to have two to three bowel movements per day, they feel ill and tired. Furthermore, their stools are harder and drier because the body has reabsorbed the toxic wastewater.

[00397] Most over-the-counter remedies for constipation, such as laxatives and stool softeners, aren't all that helpful. Nearly half of users aren't satisfied with the results of such products, citing ineffectiveness or other issues. Laxatives are often the first choice to resolve constipation, but there are plenty of other lifestyle and dietary solutions that will ensure you not only resolve any existing constipation, but also that you prevent constipation from developing in the future— presuming your constipation isn't caused by a drug, After all, constipation is a sign that your colon isn't functioning properly, and laxatives only serve as a temporary patch rather than fixing the problem that's causing the constipation to begin with.

[00398] Additionally, laxatives are not ideal because of their potentially adverse side effects, such as causing tolerance so that the intestine isn't as capable at releasing the stool on its own, melanosis coli (a disorder of pigmentation of the wall of the colon caused by prolonged use of laxatives) or cathartic colon (structural damage to the colon associated with stimulant laxatives). Probiotics also help in achieving intestinal regularity. In one study, Lactobacillus plantarum improved abdominal pain and normalized stool frequency in constipated patients with irritable bowel syndrome (IBS). The effect appeared to be strain specific, as Lactobacillus rhamnosus GG had more of a beneficial effect in IBS patients who predominantly had diarrhea.

[00399] Aim

[00400] To assess the symptomatic efficacy of Lactobacillus acidophilus ARS-B3208, Lactobacillus Rhamnosus ATCC 393 and Bifidobacterium Bifidum ATCC 29521 along with 4gm of Inulin from Agave for the daily use for regularity in a large subset of patients based on inclusion criteria.

[00401] Study Design

[00402] All subjects received counseling and were provided antidiarrheals (loperamide) for use only if diarrhea developed. The subjects are blinded and randomized to take one sachet placebo or oral synbiotic (a combination of three probiotics and a prebiotic) called DAILY BALANCE #2 beginning 7 days after initial testing, daily. All subjects will keep symptom, stool time and medication diaries and submitted a stool sample for pathogen carriage within 7 days of study end. The study is set up to determine a regularity profile improvement over history.

[00403] This study will be designed as a multicentre double blind, placebo-controlled study with parallel groups of minimum 75 patients to assess the beneficial effects of a daily consumption of DAILY BALANCE #2 on regularity of bowel movements. Treatment duration will be 24 weeks with 4 follow-up visits at 6 week intervals. The study protocol will be conducted in accordance with the Declaration, of Helsinki and approved by the local Ethics Committee. All volunteers will be given written informed consent prior to participation in the study.

[00404] Dose Selection, Treatment Assignment, Randomization, and Blinding Procedures

[00405] DAILY BALANCE #2 containing Lactobacillus acidophilus ARS-B3208, Lactobacillus Rhamnosus ATCC 393 and Bifidobacterium Bifidum ATCC 29521 along with 4gm of Inulin from Agave in one Sachet of Traveler's support or placebo will be ingested daily with food, beginning 5 days prior to travel, throughout the trip(trip duration to be documented), and for 10 days after return. The Sachet can be taken any time of day. The DAILY BALANCE #2 and placebo sachets will be identical in color, packaging, and smell.

[00406] Subjects will be randomly selected to receive Traveler's Support or placebo. Randomization was performed in a block of size 4 using a random number generator.

[00407] Materials and methods.

[00408] Patients

[00409] Subjects aged 18 years or above and having mild to moderate irregularity issues are eligible for the trial. Additional exclusion criteria include: current use of antibiotic or antidiarrheal medication (ie, Pepto-Bismol, loperamide, etc.) or their use within 2 weeks prior, a history of inflammatory bowel disease (Crohn's disease or chronic ulcerative colitis), known bowel cancer, congenital or acquired immunocompromised states such as human immunodeficiency virus infection (HIV/AIDS), current or recent chemotherapy or immunomodulating agents (corticosteroids and TNF-a inhibitors), short-gut syndrome, use of oral typhoid vaccine within 48 hours of starting study, pregnancy, ongoing probiotic use, and previous participation in this study. Women of childbearing age are required to have a negative pregnancy test within 2 weeks of starting the study drug and are counseled not to get pregnant during the study period.

[00410] All enrolled subjects receive standard counseling and education about food and water precautions and self-management of diet. Subjects are also offered loperamide in case diarrhea initiated and was not controlled. A letter is provided to the patient to allow carriage of the study drug across international borders. The letter also contains telephone numbers for on-call personnel in case subjects experience side effects or have questions during any trip, and all subjects enrolled in this study provide written informed consent.

[00411] Study products

[00412] The test product will be DAILY BALANCE #2 with 6.8 Billion CFU

Lactobacillus acidophilus ARS-B3208, 6.6 Billion CFU Lactobacillus Rhamnosus ATCC 393 and 6.6 Billion CFU Bifidobacterium Bifidum ATCC 29521 along with 4gm of Inulin from Agave. The test product will contain 20 billion colony-forming units (cfu) in sachet form with no other ingredients except the prebiotic Inulin. The control product will contain 5gm of maltodextin. Both the test and control products will have a similar appearance, texture and taste. Both products will be specifically prepared for the study and provided by America's Naturals (Fort Lauderdale, Florida).

[00413] Assessments and study endpoints

[00414] The primary endpoint will be the improvement of the frequency of bowel movements, alleviation of constipation and abdominal pain episodes. Secondary endpoints are expected to be changes in severity of abdominal pain, changes in frequency and severity of abdominal bloating and in feeling of incomplete rectal emptying as well as time of diarrhea episodes and less forced evacuation. Both frequency of abdominal pain and feeling of incomplete rectal emptying should be assessed weekly using a four-point scale ranging from 1 (only occasional symptom) to 4 (daily symptom). Symptom severity (abdominal pain, abdominal bloating and feeling of incomplete rectal emptying) should be rated on a visual analogue scale (VAS 1-10) and converted to a 4-point scale ranging from 0 (No pain, VAS = 0) to 3 (Severe, VAS = 8 to 10).

[00415] The daily number of stools and bloating episodes will be calculated.

[00416] While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

[00417] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.