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Title:
SYNBIOTIC COMPOSITIONS FOR GUT MICROBIOTA
Document Type and Number:
WIPO Patent Application WO/2021/081362
Kind Code:
A1
Abstract:
The present disclosure relates to a synbiotic compositions, comprising a plurality of preselected strains of probiotic bacteria with a sterilized organic vegetable and fruit juice liquid medium which may be used for colon health and improving overall immune function and intestinal health. Methods for preparing the probiotic blend and juice liquid medium to form the synbiotic composition are also disclosed. The methods can achieve a desirable shelf-life while maintaining bacterial viability.

Inventors:
ROBINS HOWARD F (US)
KAMAREI A REZA (US)
KOSCHITZ MICHAEL W (US)
Application Number:
PCT/US2020/057111
Publication Date:
April 29, 2021
Filing Date:
October 23, 2020
Export Citation:
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Assignee:
NEWGEN 27 LLC (US)
International Classes:
A23L2/02; A23L33/135; A61K35/745; A61K35/747; A61P1/00
Attorney, Agent or Firm:
CAMPAGNA, Pina M. (US)
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Claims:
CLAIMS

1. A synbiotic composition comprising: a plurality of probiotic bacterial strains of the genus Bifidobacterium and genus

Lactobacillus in a sterilized liquid fruit and vegetable juice medium, wherein the plurality of probiotic bacterial strains of the genus Bifidobacterium include the species B. bifidum, B. breve, B. lactis, B. longum, B. infantis and mixtures thereof, wherein the plurality of probiotic bacterial strains of the genus Lactobacillus include the species L· acidophilus, L. brevis, L. bulgaricus, L. casei, L. gasseri, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius and mixtures thereof, and wherein the synbiotic composition provides a synergistic benefit to the user’s gut and overall wellbeing.

2. The synbiotic composition according to claim 1, wherein the sterilized liquid fruit and vegetable juice medium includes at least one of mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, lemon juice, and mixtures thereof, wherein all of the juices being organic without pesticides, chemicals or additives.

3. The synbiotic composition according to claim 1, further comprising a probiotic.

4. The synbiotic composition according to claim 1, wherein the composition forms a drinkable beverage with a desirable shelf-life while maintaining bacteria viability without refrigeration or cooling of the beverage. 5. The synbiotic composition according to claim 1, wherein the composition comprises from 1 to about 100 billion Colony Forming Units (CFUs) of pre-hydrated, actively living probiotics in a 2 fluid ounce bottle of a juice beverage at the time of manufacture.

6. The synbiotic composition according to claim 1, wherein the synbiotic composition inhibits the growth of undesirable bacteria and pathogens in a user’s gut and helps ameliorate Clostridium difficile infection (CDI).

7. The synbiotic composition according to claim 1, wherein the selected plurality of probiotic bacterial strains releases bioactive compounds into the selected sterilized liquid fruit and vegetable juice medium and is configured to inhibit growth of

C. diff.

8. The synbiotic composition according to claim 7, wherein the effect of the bioactive compounds being released reduces the pH of the user’s gut to an acidic range unfavorable for spore germination and/or growth of C. diff.

9. A synbiotic composition comprising: a plurality of probiotic bacterial strains of the genus Bifidobacterium and genus

Lactobacillus in a liquid carrier, wherein the plurality of probiotic bacterial strains of the genus Bifidobacterium include the species B. bifidum, B. breve, B. lactis, B. longum, B. infantis and mixtures thereof, wherein the plurality of probiotic bacterial strains of the genus Lactobacillus include the species L· acidophilus, L. brevis, L. bulgaricus, L. casei, L. gasseri, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius and mixtures thereof, and wherein the sterilized liquid carrier includes at least one of mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, lemon juice, and mixtures thereof, wherein the juices being organic without pesticides, chemicals or additives.

10. The synbiotic composition according to claim 9, wherein the synbiotic composition forms a drinkable beverage with a desirable shelf-life while maintaining bacteria viability without refrigeration or cooling of the beverage.

11. The synbiotic composition according to claim 9, wherein the synbiotic composition comprises from 1 to about 100 billion Colony Forming Units (CFUs) of prehydrated, actively living probiotics in a 2-ounce bottle of a juice beverage at the time of manufacture.

12. The synbiotic composition according to claim 9, wherein the synbiotic composition inhibits the growth of undesirable bacteria and pathogens in a user’s gut and helps ameliorate Clostridium difficile infection (CDI).

13. The synbiotic composition according to claim 9, wherein the selected plurality of probiotic bacterial strains releases bioactive compounds into the selected sterilized liquid fruit and vegetable juice medium and is configured to inhibit growth of

C. diff.

14. The synbiotic composition according to claim 13, further comprising a prebiotic.

15. A method of manufacturing a synbiotic composition comprising: combining a plurality of probiotic bacterial strains of the genus Bifidobacterium and genus Lactobacillus to form a probiotic blend; and mixing the probiotic blend in a liquid carrier to form a juice beverage, wherein the plurality of probiotic bacterial strains of the genus Bifidobacterium include the species B. bifidum, B. breve, B. lactis, B. longum, B. infantis and mixtures thereof, wherein the plurality of probiotic bacterial strains of the genus Lactobacillus include the species L acidophilus, L. brevis, L. bulgaricus, L. casei, L. gasseri, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius and mixtures thereof, and wherein the sterilized liquid carrier includes at least one of mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, lemon juice, and mixtures thereof, wherein the juices being organic without pesticides, chemicals or additives.

16. The method according to claim 15, wherein the synbiotic composition comprises from 1 to about 100 billion Colony Forming Units (CFUs) of pre-hydrated, actively living probiotics in a 2 fluid ounce bottle of a juice beverage at the time of manufacture. 17. The method according to claim 15, wherein the synbiotic composition inhibits the growth of undesirable bacteria and pathogens in a user’s gut and helps prevent and/or ameliorate Clostridium difficile infection (CDI).

Description:
SYNBIOTIC COMPOSITIONS FOR GUT MICROBIOTA

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to U.S. Patent

Application No. 62/925,983 filed October 25, 2019, and U.S. Patent Application No.

63/048,739, filed July 7, 2020, the entire contents of each of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to synbiotic compositions comprising probiotic bacterial strains with a juice-based medium that when combined exhibit synergistic behavior. The synergistic compositions may ameliorate, repair and/or rehabilitate the gut microbiome to improve a user’s immune response before and/or after experiencing gut infections such as those caused after antibiotic associated diarrhea, Clostridium difficile

(C. diff.) infection and other gut infections.

BACKGROUND

[0003] The human gut, or gut microbiome, has become the epicenter of scientific research recently after learning that up to 90 percent of all diseases can be traced in some way back to the gut and health of the microbiome, resulting in the affiliation of the gut as

“the second brain”. The microbiome is defined as a “community of microbes.” The vast majority of the bacterial species that make up our microbiome live in our digestive systems. The human microbiota consists of the 10-100 trillion microbial cells harbored by each person, primarily bacteria in the gut. Our skin and digestive system alone host about 2,000 different types of bacteria. Accordingly, a healthy gut is so important to a person’s overall wellbeing because of the role that bacteria has in fostering a strong immune system and keeping us healthy. Not only are all bacteria not detrimental to our health, but some are actually crucial for boosting immunity, keeping our digestive systems running smoothly, our hormone levels balanced and our brains working properly.

A person’s good gut bacteria is also responsible for producing vitamin B12, butyrate and vitamin K2, crowding out bad microbes, creating enzymes that destroy harmful bacteria, and stimulating secretion of IgA and regulatory T cells, which support immune function.

[0004] A person’s diet plays a big part in establishing gut health and supporting the microbiome’s good bacteria. In turn, a person’s gut health can impact how the body extracts and uses nutrients from a person’s diet. Historically, people had plenty of probiotics in their diets from eating fresh foods from good soil and by fermenting foods to keep them from spoiling. The term "probiotics" means live microorganisms having a beneficial effect on the health of hosts such as humans, animals, or the like, or a component thereof, and it has been known that the probiotics provide a beneficial effect, for example, maintenance of a balance in intestinal microflora, to the host taking the probiotics. Specifically, the probiotics in our gut have been proven effective in supporting immune function, reducing inflammation, promoting healthy digestion, as well as maintaining beautiful skin, especially when combined with prebiotics. Today, however, because of refrigeration and agricultural practices like soaking our foods with chlorine, much of our food contains little to no probiotics as a result of sanitation.

Actually, many foods contain dangerous antibiotics, hormones, and pesticides, that kill off the good bacteria in our bodies, may contribute to an increased incidence of illnesses such as gastrointestinal tract infections, C. diff., constipation, irritable bowel syndrome

(IBS), inflammatory bowel disease (IBD), Crohn's Disease, ulcerative colitis, nosocomial infection, colonic polyps, constipation and other digestive irregularities such as bloating, and discomfort, food allergies, diarrhea, certain cancers such as colorectal cancer, as well as even effecting a person’s mood and mental wellbeing.

[0005] With this new research and education, consumers are realizing the important role dietary supplements play in achieving these health goals since we are not able to get all the nutrients the body needs from just foods especially in such a fast-paced society.

Probiotic supplements are a very important part of a person’s dietary supplement regimen to improve overall health. More importantly, the role of any medicinal or immunotherapeutic intervention must be to counter-balance the loss of optimum gut function. Many of the current medicinal approaches are imperfect for improving gastrointestinal health and are even harmful to the gut microbiome.

[0006] For example, Clostridium Difficile Infection (CDI) causes life-threatening diarrhea which is usually a side-effect of taking antibiotics. Clostridium difficile (“C. difficile ” or “C. diff’) is a Gram-positive, spore-forming, anaerobic, motile bacterium which produces two types of toxins, enterotoxin A and cytotoxin B. C. diff can easily spread from person to person and is considered a major health threat. In 2017, there were an estimated 223,900 cases in hospitalized patients and 12,800 deaths in the United

States. There has been considerable scientific research in using various probiotic strains against C. diff with inconsistent and ineffective results. [0007] Accordingly, there is a need for a synbiotic/probiotic product that inhibits growth and pathogenicity of C. diff and other diseases caused by an unhealthy microbiome as well as a product to ensure optimal gut health.

SUMMARY

[0008] Accordingly, the present disclosure provides a synbiotic composition containing a plurality of strains of probiotic bacteria and methods of making the composition. The synbiotic composition comprises a beverage medium which may include fruit and vegetable juices and optionally herbs that provides a synergistic benefit to the host or user.

[0009] In embodiments, the present disclosure is directed to the probiotic bacterial strains of the genus Bifidobacterium, in embodiments the species Bifidobacterium breve,

Bifidobacterium lactis, Bifidobacterium iongum, Bifidobacterium bifidum, B. adolescentis, B. thermophilum, and Bifidobacterium infantis, and mixtures thereof, and probiotic bacteria of the genus Lactobacillus, in embodiments the species Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus paracasei,

Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus brevis, and mixtures thereof.

[0010] In embodiments, the optimal synbiotic composition of the present disclosure includes five (5) strains of Bifidobacteria probiotic bacteria and ten (10) strains of Lactobacilli probiotic bacteria. In embodiments, the five strains of the genus

Bifidobacteria probiotic bacteria may include Bifidobacterium lactis BI-07,

Bifidobacterium breve BB-03, Bifidobacterium bifidum BB-06, Bifidobacterium Iongum BL-05, Bifidobacterium infantis BI-26, and mixtures thereof. In embodiments, the ten strains of the genus Lactobacilli may include Lactobacillus plantarum LP-115,

Lactobacillus paracasei LPC-37, Lactobacillus casei LC-11, Lactobacillus salivarius LS-

33, Lactobacillus rhamnosus LR-32, Lactobacillus acidophilus LA-14, Lactobacillus brevis LBR-35, Lactobacillus gasseri LG-36, Lactobacillus reuteri 1 El, Lactobacillus bulgaricus LB-87, and mixtures thereof.

[0011] In embodiments, the composition may also include probiotic strains of the genus Bifidobacterium such as B. adolescentis, B. thermophilum and others known in the art now and any species found in the future. In embodiments, the composition may also include probiotic strains of the genus Lactobacillus such as Lactobacilli johnsonii,

Lactobacilli lactis, and others known in the art now and any species found in the future.

[0012] In embodiments, the synbiotic composition of the present disclosure may include

15 strains in an optimum carrier such as an excipient or liquid medium forming a drinkable beverage with a desirable shelf-life and while maintaining bacteria viability without refrigeration or cooling of the beverage.

[0013] In embodiments, the liquid medium or carrier may include the sterilized juices of at least one fruit, vegetable and/or herb or spice.

[0014] In embodiments, the liquid medium or carrier may comprise at least one of the following: mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice, all of the juices being organic without pesticides, chemical or additives.

In embodiments, the optimum liquid carrier may include a blend of hundred (100) percent organic green vegetable and fruit juices comprising mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, lemon juice, and mixtures thereof. [0015] In aspects, the synbiotic composition of the present disclosure comprises from 1 to about 100 billion Colony Forming Units (CFUs) of pre-hydrated, actively living probiotics in a 2-ounce bottle of a juice beverage at the time of manufacture.

[0016] It is envisioned that the composition of the present disclosure may replenish the human gut microbiome with special bacteria referred to as “Smart bacteria” (SB). It is believed that these “Smart bacteria” may change the genetic composition of the gut to create a healthier environment by attaching to the wall of the gut where they will live and thrive helping to repair damage to it caused by unhealthy biomes that have been ingested and caused to overgrow in numbers such as Candida albicans (yeast) and harmful bacteria.

[0017] It is also envisioned that the present synbiotic composition may inhibit the growth of undesirable bacteria and pathogens and/ or help ameliorate serious medical problems caused by the use of antibiotics, i.e., Clostridium difficile infection (CDI), a worldwide medical problem. Specifically, it is envisioned that the specific choice of probiotics in the synbiotic compositions of the present disclosure release bioactive compounds into the specific liquid medium or carrier disclosed herein that may inhibit growth of C. diff.

For example, it is envisioned that a bioactive compound released in the medium may be lactic acid which may reduce the pH of the gut environment to an acidic range unfavorable for spore germination and/or growth of C. diff, playing a decisive inhibitory role against growth of C. diff and potentially other possible diseases which can arise from an unhealthy or unbalanced microbiome.

[0018] Other aspects, features, and advantages will be apparent from the description, the drawings, tables, examples, and the claims that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 illustrates an enlarged image of the Gram-positive, spore-forming, anaerobic, motile bacterium Clostridium difficile (“C. diff”) cells under a microscope

(Source: US Center for Disease Control and Prevention (CDC));

[0020] FIG. 2 illustrates the synbiotic composition pre-centrifugation during a study as described in the Examples;

[0021] FIG. 3 illustrates the synbiotic composition shown in FIG. 2 postcentrifugation/microfiltration during a study as described in the Examples;

[0022] FIG. 4 illustrates the presence of C. diff. colony forming units during a study as described in the Examples; and

[0023] FIG. 5 illustrates the absence of C. diff. colony forming units during a study as described in the Examples when synbiotic composition’s supernatant is used for serial dilution.

DETAILED DESCRIPTION

[0024] The scientific definition of the term ‘probiotic’ was proposed in a 2001 joint report of the Food and Agriculture Organization of the United Nations and the World

Health Organization (F AO/WHO), and confirmed by an expert panel convened by

International Scientific Association for Probiotics and Prebiotics (ISAPP) held in 2013.

The currently accepted definition of probiotics is “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” This means that if the microorganisms are not “live”, they cannot be considered a probiotic and if their amount is not adequate enough to confer a health benefit, they cannot be considered a probiotic either. “Next Generation Probiotics” (NGP) are bacteria that due to their development act as live biotherapeutic products and lend themselves to act more like pharmaceuticals than traditional dietary supplements.

[0025] Certain strains of NGP “Smart Bacteria” (SB) consistently achieve better results in helping to prevent and assist in GDI amelioration along with the usual treatment regimens. SB can sense they are attached to intestinal cells, and then remodel their expression of specific genes, including those involved in metabolism, to beneficially exploit cells and colonize the gut.

[0026] Bacteria have evolved to develop remarkable systems that can sense neighboring cells called “target” cells, and can initiate upon contact a series of changes that enhance their ability to survive and grow at the expense of these “target” cells. SB do this by employing a mechanism called “Horizontal Gene Transfer” (HGT). Essentially, HGT shares genetic material between organisms that are not in a parent-offspring relationship and is a widely recognized mechanism for adaptation in bacteria.

[0027] With this concern in mind, about 80 percent or more of these NGP SB’s were configured to survive the stomach acids and digestive enzyme. Also, to ensure that they are “living” and thriving at the time of ingestion, an appropriate environment and media, would be required meeting all the above-mentioned needs including the range of acidity needed for survival and growth, such as the sterilized liquid medium presently disclosed herein. The NGP SB’s that were specifically chosen in the present disclosure were required to be compatible with each other so as not to alter or interfere with each other’s growth and benefits. The SB or probiotics of the present disclosure also needed to be in a sufficient number of Colony Forming Units (CFU) to repopulate the colon and attack or prevent CDI or any other gut initiated disease effectively, or alternatively maintain optimal gut health in those without a gut-related disease.

[0028] The term “synbiotics” is defined as a mixture comprising live microorganisms and substrates selectively utilized by host microorganisms that confers a health benefit on the host. Within this definition, ‘host’ microorganisms comprise both autochthonous

(resident or colonizing the host) and allochthonous (externally applied, such as probiotics) microorganisms, either of which can be targets for a user of the substrate contained in the synbiotic. Two subsets of synbiotics may be defined as complementary and synergistic. A ‘synergistic synbiotic’ is a synbiotic in which the substrate is designed to be selectively utilized by the co-administered microorganism(s). A ‘complementary synbiotic’ is a synbiotic composed of a probiotic combined with a prebiotic, which is designed to target autochthonous microorganisms.

[0029] It is contemplated that the specifically chosen probiotics, i.e., the NGP SB, of the present disclosure together with the sterilized liquid medium or carrier of the NGP SB probiotics presently disclosed form a synbiotic relationship with a synergistic benefit on the user.

[0030] Bacteria of the genus Bifidobacterium are known to promote a healthy microbiome. Bifidobacteria may lead to increased levels of lactic and acetic acids, which decreases the pH in the digestive tract, inhibiting the growth of harmful bacteria such as

Clostridium perfringens, Clostridium difficile and certain pathogenic Escherichia coli.

Bacteria of the Lactobacillus genus are also known to promote a healthy microbiome and help prevent or alleviate many disorders and diseases including antibiotic-induced imbalances in gastrointestinal microflora, hypercholesterolemia, vaginal infections, E. coli infection and depressed inununity.

[0031] The synbiotic composition of the present disclosure may include probiotic bacterial strains of Bifidobacterium, in embodiments the species B. breve, B. animalis

(lactis), B. longum, B. bifidum, B. adolescentis, B. thermophilum, B. infantis, and mixtures thereof. In embodiments, the synbiotic composition of the present disclosure may include Bifidobacterium lactis BI-07, Bifidobacterium breve BB-03, Bifidobacterium bifidum BB-06, Bifidobacterium longum BL-05, Bifidobacterium infantis BI-26, and mixtures thereof. Probiotic bacteria of the genus Lactobacillus may also be used, in embodiments the species L. acidophilus, L. casei, L. rhamnosus, L, paracasei, L. johnsonii, L. reuteri. L. plantarum, L. lactis, L. salivarius, L. bulgaricus, L. brevis, L gasseri, and mixtures thereof. In embodiments, the synbiotic composition of the present disclosure may include Lactobacillus plantarum LP-115, Lactobacillus paracasei LPC-

37, Lactobacillus casei LC-11, Lactobacillus salivarius LS-33, Lactobacillus rhamnosus

LR-32, Lactobacillus acidophilus LA-14, Lactobacillus brevis LBR-35, Lactobacillus gasseri LG-36, Lactobacillus reuteri 1 El, Lactobacillus bulgaricus LB-87, and mixtures thereof. In embodiments, the composition may comprise bacteria from multiple species.

Suitable strains are available commercially by Dupont Nutrition and Biosciences.

[0032] The synbiotic composition of the present disclosure may include five strains of

Bifidobacterium and ten strains of Lactobacillus, in embodiments, Bifidobacterium lactis

BI-07, Bifidobacterium breve BB-03, Bifidobacterium bifidum BB-06, Bifidobacterium longum BL-05, Bifidobacterium infantis BI-26, and mixtures thereof together with

Lactobacillus plantarum LP-115, Lactobacillus paracasei LPC-37, Lactobacillus casei LC-11, Lactobacillus salivarius LS-33, Lactobacillus rhamnosus LR-32, Lactobacillus acidophilus LA-14, Lactobacillus brevis LBR-35, Lactobacillus gasseri LC-36,

Lactobacillus reuteri 1 El, Lactobacillus bulgaricus LB-87, and mixtures thereof

[0033] In embodiments, the synbiotic compositions of the present disclosure may include the 15 strains in an optimum carrier such as an excipient or sterilized liquid medium forming a drinkable beverage with a desirable shelf-life and while maintaining bacteria viability without refrigeration or cooling of the beverage.

[0034] In embodiments, the sterilized liquid medium or carrier may include at least one of or a portion of a plant selected from fruits, vegetables, herbs, spices, seeds, other suitable plant substances, or a mixture thereof More specifically, the selected portion of a plant may be in a solid or in a liquid form (e.g. a vegetable juice or a fruit juice) or a combination thereof.

[0035] Examples of the plant in fruit form are apple, orange, lemon, lime, banana, watermelon, papaya, mango, pineapple, blueberry, raspberry, strawberry, cherry, peach, cucumber, grape, coconut and any other fruits or juices or extracts produced therefrom, some which may be already known in the art. Examples of the plant in vegetable form may include kale, spinach, broccoli, asparagus, cauliflower, lettuce, mushroom, celery and any other vegetables or juices or extracts produced therefrom, some which may be already known in the art.

[0036] Examples of the plant in herb form may include basil, mint, oregano, ginger, black pepper, parsley, garlic, onion, fennel, celery seed, bay leaf, lemon grass, rosemary, sage, chives and any other herbs or juices or extracts produced therefrom, some which may be already known in the art. Examples of the plant in spice form may include cardamom, cinnamon, allspice, cloves, nutmeg, pepper, turmeric, ginger, mace, saffron, vanilla, cumin, dill seed and other like fleshy spice products. It is worth mentioning that in embodiments, seeds may include fruits, roots, barks, or other plant substances primarily used for flavoring, coloring or preserving food

[0037] In embodiments, the sterilized liquid medium or carrier may comprise at least one of the following: mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice, all of the juices being organic without pesticides, chemical or additives. In embodiments, the optimum sterilized liquid carrier may include a blend of hundred (100) percent organic green vegetable and fruit juices comprising mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice.

[0038] In embodiments, the sterilized beverage or liquid medium may alternatively be a tea-based beverage with natural sweeteners added to the beverage such as organic cane sugar, honey, stevia, monk fruit, erythritol, or any other natural sweetener known in the art.

[0039] Viable bacteria, which may be included in the beverage, generally are presented in numbers which are often reported as CFU, or colony forming units. One colony is formed by a single viable bacterium when the bacteria are plated at a suitable dilution for single colony formation. This is a standard technique known to microbiologists.

Typically, the amount is expressed as the number of CFU in a liquid measure such as milliliters (ml) or fluid ounces (fl. oz). U.S. regulation 21 CFR 101.9(b)(5)(viii) defines a fluid ounce as exactly 30 ml. Sufficient numbers of viable bacteria may be necessary to obtain the beneficial effects of the probiotic bacteria. Often bacteria are packaged at a certain level of viable bacteria; however, before consumption, the levels may decrease preventing the consumer from acquiring a beneficial dose of bacteria. Indeed, the

National Center for Complementary and Alterative Medicine (NCCAM) has identified several issues relating to the quality of probiotic products including, viability of the bacteria in the product, types and titer of bacteria in the product, and stability under storage.

[0040] In embodiments, the synbiotic composition of the present disclosure comprises from 1 to about 100 billion CFUs of pre-hydrated, actively living probiotics in a 2-ounce bottle of a sterilized beverage at the time of manufacture. In selected embodiments, the synbiotic composition comprises from about 20 to about 30 billion CFUs, in embodiments, about 27 Billion CFUs of pre-hydrated, actively living probiotics in a 2- ounce bottle of a beverage at the time of manufacture. It is contemplated that as the product ages the bacteria may continue to grow and form additional colonies providing the ambient temperature and shelf life is not exceeded.

[0041] Further, the synbiotic composition of the present disclosure may comprise prebiotics, such compositions also known as a complementary synbiotic. A

‘complementary synbiotic’ is a synbiotic composed of a probiotic combined with a prebiotic, which is designed to target autochthonous microorganisms. Minimum criteria for the existing probiotic and prebiotic must be met for both components of a complementary synbiotic.

[0042] Prebiotics are substances that may not be digestible for some humans. More specifically, some human’s gastrointestinal tract cannot process or digest prebiotics.

Generally, the human gastrointestinal tract stimulates, preferentially, the growth of certain bacteria. Known prebiotics include: fructans such as inulin and fructooligosaccharides (FOS); galacto-oligosaccharides (GOS) and lactulose.

Fructooligosaccharides are not hydrolyzed in the small intestine and instead pass through into the large intestine where they preferentially support the growth of probiotic strains of

Lactobacillus and Bifidobacterium, increasing colonization of the colon by these probiotic bacteria. By contrast, fructooligosaccharides do not support the growth of undesirable bacteria such as species of bacteroides, Clostridia, and fusobacteria .

[0043] In selected embodiments, the synbiotic composition or beverage may include selected types of probiotics (optionally used in products for human consumption) which may include inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose and arabinoxylan plant fiber extract.. These prebiotics may be naturally produced in a plant, semi-synthetic, synthetic, recombinant, etc. Typically, these prebiotics will be used in a semi-purified state, in which other components of the plant, fruit, flower, or vegetable source, or other components of the synthetic or semi-synthetic reaction are diminished in concentration and/or removed.

[0044] Inulin is a naturally occurring soluble fiber composed of a mixture of oligomers of varying degrees of polymerizations. Inulins are mainly comprised of fructose units and typically have a terminal glucose. Plant inulins generally contain between 2 to 140 fructose units. Inulin can be obtained from a variety of sources including Jerusalem artichoke, dahlia, onion, garlic and chicory tubers. Lactulose is a synthetic sugar, which does not occur naturally. The disaccharide lactulose (galacto-fructose) is synthesized from lactose (galacto-glucose) by isomerization of glucose to fructose. Galacto- oligosaccharides (GOS) can also be synthesized from lactose; for example, by using β- galactosidase enzymes purified from Lactobacillus reuteri L103 as a catalyst. [0045] Fructooligosaccharides may be prepared by any of several methods known in the art. For example, fructooligosaccharides can be extracted from natural substances.

Fructooligosaccharides occur in many kinds of plants including dahlias, chicory, onions, garlic, shallots, wheat rye, artichokes and tomatoes. Fructooligosaccharides may also be produced enzymatically through chemical techniques. Fructooligosaccharides are particularly well-known for use in promoting the growth of Bifidobacterium species.

[0046] Fructooligosaccharides are typically linear chains of fructose bound to a terminal glucose. The fructooligosaccharides can be a mixture of short chain polymers. The length of the fructose chain, also called the degree of polymerization or DP, can be from about 2 to about 5. Typically, the fructose chain length varies from 2 to 4. Such short-chain fructooligosaccharides may also be referred to as GF2 (1-kestose), GF 3 (nystose), and

GF4 (Ι-β-fructofuranosyl nystose). Suitable commercially available fructooligosaccharides may be used, for example, Nutraflora® by GTC Nutrition

(Golden, Colo. 80401).

[0047] In embodiments, the synbiotic composition may also include any vitamin or mineral known in the art. For example, vitamins A, B, C, D, and/or E may be included in specified amounts to enhance the benefits of the composition.

[0048] In embodiments, the synbiotic composition may also include an active ingredient such as polyunsaturated fatty acids, omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, conjugated fatty acids, coenzyme Q-containing active ingredients, oil soluble vitamins other than a vitamin E, alpha lipoic acid, medium chain triglycerides, carotenoid-containing active ingredients and phytochemicals and cannabinoids. [0049] The preselected bacteria which may be included in the synbiotic composition may be prepared in a variety of ways known in the art, including, for example, the bacteria may be grown without casein, providing a completely dairy-free bacterial preparation. During manufacturing, the selected bacteria may be stored via known methods of storage or methods specifically designed to maintain cultures alive such as refrigeration, freezing, or freeze-drying without diminishing viability below a selected level (the selected level may be a level selected from temperate, volume, time, or freshness). Still during manufacturing, the selected bacteria may be added to the beverage while in the same state as they were stored, such as while frozen, freeze-dried, or refrigerated. Optionally, the selected bacteria may be thawed prior to adding to the beverage. The selected bacteria may be frozen after growth and maintained in a frozen state until they are added to the beverage.

[0050] In embodiments, the resulting beverage comprising the synbiotic composition of the present disclosure may be a shelf stable beverage. The shelf stable beverage may be kept or have an expected lifetime of about three months at room temperature (about 68- 72 degrees Fahrenheit), and/or about four months at refrigerated temperature (about 35- 40 degrees Fahrenheit).

[0051] While in use, the synbiotic composition of the present disclosure may compete with and exclude all harmful bacteria and yeasts e.g., Clostridium difficile and Candida albicans. Further, the synbiotic composition may aid in bringing a user’s gut- environment to optimal balance. Moreover, the synbiotic composition may help to reestablish a healthy gut and normalize digestion and absorption of nutrients. [0052] Further aspects of the invention relate to methods of manufacturing and storage associated with the synbiotic composition while maintaining a high number of viable bacteria. In an exemplary embodiment, to manufacture the synbiotic composition, in a sanitary environment, bacteria, plant, and /or synbiotic products (such as the ones noted above) are combined to form the beverage. More specifically, at least one strain from the genus Bifidobacterium and at least one strain from the genus Lactobacillus which may be frozen, freeze-dried, or refrigerated in powder form are mixed together to uniformity and then combined with a sterilized liquid medium or carrier, i.e., the organic fruit/vegetable juice mixture described herein.

[0053] As noted above in one exemplary embodiment and more specially referring to the plurality of strains, in embodiments, at least 15 different strains, of freeze-dried probiotics are combined. In embodiments, the synbiotic composition of the present disclosure may include five strains of Bifidobacterium and ten strains of Lactobacillus, in embodiments, Bifidobacterium lactis BI-07, Bifidobacterium breve BB-03,

Bifidobacterium bifidum BB-06, Bifidobacterium longum BL-05, Bifidobacterium infantis

BI-26, and mixtures thereof, together with Lactobacillus plantarum LP-115,

Lactobacillus paracasei LPC-37, Lactobacillus casei LC-11, Lactobacillus salivarius LS-

33, Lactobacillus rhamnosus LR-32, Lactobacillus acidophilus LA-14, Lactobacillus brevis LBR-35, Lactobacillus gasseri LG-36, Lactobacillus reuteri 1 El, Lactobacillus bulgaricus LB-87, and mixtures thereof. The preselected 15 strains are combined and blended for a selected time, of from about 20 minutes to about 40 minutes, and further stored in a selected environment, i.e., an environment including temperature control means. In embodiments, the preselected 15 strains are blended for about 30 minutes and placed in a refrigerator at a temperature of about 35 degrees to about 40 degrees Celsius.

[0054] Furthermore, in embodiments, the probiotic blend may include a specific concentration or strain of bacteria. More specifically, the specific concentration of each strain of probiotic may be from about 0.5% to about 15%, in embodiments from about

1.5% to about 12.5% in the probiotic blend.

[0055] After manufacturing, in embodiments, the synbiotic composition may include less than 5 grams of the preselected probiotic blend (which can be a mixture of probiotics in powder forms) per liter of sterilized juice. In selected embodiments, the synbiotic composition may include about 2 grams to about 3 grams of a combination or mixture per liter of sterilized juice. In embodiments, the liquid medium or carrier may include any of the above noted plants. In selected embodiments, the synbiotic composition may include organic fruit and juice blend of the present disclosure. In embodiments, the liquid medium or carrier may comprise at least one of the following: mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice, all of the juices being organic without pesticides, chemicals or additives. In exemplary embodiments, the optimum liquid carrier may include a blend of hundred (100) percent sterilized organic green vegetable and fruit juices comprising mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice which may be mixed with a blend of preselected probiotics for about 20 minutes to about 30 minutes until reaching a uniform texture for the resulting beverage. In another exemplary embodiment, the synbiotic composition may include a stock of the preselected probiotic blend already mixed together and the preselected sterilized organic fruit/juice blend. The stock of mixed probiotic blend and the sterilized organic juice may be stored in a refrigerated environment, and at some later time, additional organic juice may be added in a kettle, with the stock mixture and blended for an additional 20 minutes to about 30 minutes or until the stock reaches a uniform consistency.

[0056] In embodiments, the synbiotic composition may include about 20 billion CFUs to about 30 billion CFUs. For example, the synbiotic composition may include 27 Billion CFUs of pre-hydrated, and actively living probiotics in about 2-ounces of the composition or beverage at the time of manufacture.

[0057] In embodiments, the resulting beverage of the present disclosure has a substantially pleasant taste. More specifically, the resulting beverage may be slightly sweet, the sweetness generally coming from the fruit juices masking the less desirable taste of the vegetable juices.

[0058] The methods and compositions of the present disclosure may be effective in supporting immune function, reducing inflammation, promoting healthy digestion, as well as maintaining beautiful skin, and promoting an overall healthier microbiome.

[0059] It is envisioned that the compositions of the present disclosure may replenish the human gut microbiome with special bacteria referred to as “Smart bacteria” (SB). It is believed that these “Smart bacteria” may change the genetic composition of the gut to create a healthier environment by attaching to the wall of the gut where they will live and thrive helping to repair damage to it caused by unhealthy biomes that have been ingested and caused to overgrow in numbers such as Candida albicans (yeast) and harmful bacteria. [0060] It is also envisioned that the present synbiotic composition may inhibit the growth of undesirable bacteria and pathogens and/ or help ameliorate serious medical problems caused by the use of antibiotics, i.e., Clostridium difficile infection (CDI), a worldwide medical problem. Specifically, it is envisioned that the specific choice of probiotics in the synbiotic compositions of the present disclosure release bioactive compounds into the specific liquid medium or carrier disclosed herein that inhibits growth of C. diff. For example, it is envisioned that a bioactive compound released in the medium may be lactic acid which may reduce the pH of the gut environment to an acidic range unfavorable for spore germination and/or growth of C. diff, playing a decisive inhibitory role against growth C. diff and potentially other possible diseases which can arise from an unhealthy or unbalanced microbiome.

[0061] In embodiments, it is contemplated that the presence of lactic acid in the synbiotic composition of the present disclosure, and upon addition of the preselected probiotic strains to the liquid medium of the present disclosure, i.e., the fruit and vegetable juice blend, can potentially reduce the pH of the environment to an acidic range unfavorable for spore germination and/or growth of pathogens such as C. difficile. However, other potential bioactives present in the composition could also have a contributing role.

[0062] In contrast to probiotics and other living cultures described above, it is worth mentioning that most probiotics in capsules and tablets are not actively living. Probiotics in capsules and tablets are generally noted “alive” at the time of manufacture however these probiotics are in a so-called state of “suspended animation”, hoping to come back to life when hydrated in the highly acidic environment of the stomach. [0063] The following Examples are noted to illustrate exemplary embodiments associated with the present disclosure. Some or all the examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure. In addition, quantities, ratios, percentages, and/or values are generally a weight measurement unless otherwise indicated.

EXAMPLES

Example A- Manufacture of the Exemplary Synbiotic Composition of the Present

Disclosure

[0064] The synbiotic composition of the present disclosure may include a preselected powder probiotic blend and a sterilized organic juice product which can be prepared as follows.

[0065] As a first step, prior to manufacturing, a user checks if a certified HEPA filter or an equivalent thereof is installed and functioning in the room where the composition is to be prepared.

[0066] A working surface is required to aid in the preparation of the composition that is disinfected and clean such as a bench which may have been treated with ethanolic paper.

[0067] A mixer is required such as a KitchenAid 5-Quart (4.7) liter tilt-head stand mixer or the like with at least 275 horsepower. The mixer may include additional accessories such as a stainless-steel bowl, 6-wire whip and plastic lid, opening devices, scissors and spoon which are completely cleaned and disinfected.

[0068] Next, the user preselected a probiotic blend of bacterial probiotic strains, such as the ones noted below in Table 1. Probiotics are generally stored in a closed, sealed container free of contaminants. After the selection, the user opened the probiotic container and extracted a selected amount of the pre-selected freeze-dried probiotic in powder form. Alteratively, the probiotics used may be in liquid form.

[0069] Through a pouring shield, 100g of individual strain probiotics were transferred into the mixer bowl (100g of 15 individual strain probiotics = 100 x 15 = 1,500g probiotic blend). The specified portions of each of the strains of the probiotic blend are provided in Table 1 below.

[0070] During the mixing of the freeze-dried probiotics, the stirring option of the mixer was turned on and the rate was gradually increased. Occasionally, the mixer was stopped and the blend was mixed with a long spoon. The mixture was stirred in the bowl with the mixer for about 30 min or until a desired uniform mixture was formed.

[0071] Using a clean spoon, the resultant probiotic blend was transferred into a clean moisture-proof container. The container was capped tightly, labeled and dated. The probiotic blend was kept in the refrigerator at approximately 4 degrees Celsius until ready to mix with a sterilized liquid medium or carrier.

TABLE 1 [0072] About 2.14 grams of the probiotic powder blend was mixed with about one (1) liter of sterilized organic fruit/vegetable juice in a 600-liter stainless steel mixer for about

20 minutes to 30 minutes until uniformity. The organic fruit/vegetable juice comprised the ingredients listed in Table 2:

[0073] The nutrition facts of the organic fruit/vegetable juice (commercially available by Elite Naturel Organik Gida San ye Tic. A.S.) is provided as follows in Table

3: TABLE 3

[0074] Each two (2) fluid ounce serving of the resulting beverage composition provides about 27 billion CFUs at the time of manufacturing. Every batch produced is tested by an independent, FDA-registered accredited microbiology lab for safety and to confirm the absence of pathogens. Example B- Study of the Effects of the Svnbiotic Composition on C. diff.

[0075] Culture Propagation and Strain Verification. A sterile inoculating loop of

Clostridium difficile ATCC #9689, illustrated in FIG. 1, was streaked onto a reinforced

Clostridial agar slant, broth tubes, and two sheep blood agar plates. The slant and broth tubes and one sheep blood agar plate were incubated anaerobically at 36-38°C for 48 ± 2 hours. The second sheep blood agar plate was incubated aerobically at 36-38°C for 48 ± 2 hours and served as the contamination check. Following incubation, the agar plates and tube were observed for colony growth and turbidity. The sheep blood agar plate incubated anaerobically was biochemically identified using the VITEK ANC card, i.e, a test card kit for identifying anaerobic bacteria, to verify purity.

[0076] Inoculum Preparation. A well isolated colony from the sheep blood agar plate was used to inoculate a reinforced Clostridial broth, which was then incubated anaerobically at 36-38°C for 18 to 20 hours. Following incubation, the culture was stored overnight at 3 ± 1 °C.

[0077] Sample Preparation. A blend of five strains of Bifidobacteria shown in

Table 4a and ten strains of Lactobacilli shown in Table 4b from DuPont Nutrition &

Biosciences was prepared for the synbiotic composition. Six, individually packaged, intact 2-ounce glasses with the synbiotic composition of the present disclosure were composited into a sterile 500 mL glass bottle under a laminar flow hood. This bottle was mixed by gentle swirling and stored at ambient temperature (20-25°C) until testing. At that time, the composited product was homogenized. Then 10 mL was pipetted into a test tube for a before (FIG. 2) and after comparison photo with the centrifuged product.

Following centrifugation at 4500 RPM for 15 minutes, the supernatant liquid was filtered through a 0.22 pm filter to ensure that the supernatant liquid was free of cells. A 10 mL portion was pipetted into a test tube and photographed (FIG. 3). The filtered sample was used as the test product sample and the 9 mL diluent tubes for its ten-fold serial dilution.

The inoculated control was analyzed with sterile 0.1% peptone buffer.

Table 4a Bifidobacteria of the Synbiotic Composition

(registered trademarks of DuPont-Danisco)

[0078] Test Procedure. The test procedure was performed according to the

Compendium of Methods for the Microbiological Examination of Foods, 5th Edition,

Chapter 33, Section 33.7 (Editors: Yvonne Salfinger and Mary Lou Tortorello, American

Public Health Association). [0079] 1. Control Test Procedure: The inoculum used for the sample test procedure was vortexed for 5 seconds. 1 mL of the inoculum was transferred to a 9 mL tube of 0.1% peptone buffer and vortexed for 5 seconds. This served as the 10 -1 dilution tube. 1 mL from the 10 -1 dilution tube was transferred in duplicate into sterile petri dishes. With the same pipet, 1 mL was transferred into a second 9 mL tube containing the 0.1% peptone buffer for the 10 -2 dilution tube and vortexed for 5 seconds. 1 mL from the 10 -2 dilution tube was transferred in duplicate into sterile petri dishes. With the same pipet, 1 mL was transferred into a third 9 mL tube containing the 0.1% peptone buffer for the 10 -3 dilution tube and vortexed for 5 seconds. Serial dilution was repeated until a 10 -8 dilution plate was reached. Approximately 20 mL of tempered (44-46°C) reinforced

Clostridial agar was added to the petri dishes and gently swirled to evenly disperse inoculum throughout the agar. After solidification, 15 mL of reinforced Clostridial agar was overlaid onto each plate. The plates were incubated anaerobically in an upright position at 35-37°C for 18-24 hours.

[0080] 2. Sample Test Procedure: The refrigerated anaerobic chamber containing the inoculum was equilibrated to ambient temperature prior to inoculation. The inoculum was vortexed for 5 seconds to homogenize. 1 mL of the inoculum was transferred to a 9 mL tube of the supernatant test sample and vortexed for 5 seconds. This served as the 10- 1 dilution tube. 1 mL from the 10 -1 dilution tube was transferred in duplicate into sterile petri dishes. With the same pipet, 1 mL was transferred into a second 9 mL tube containing the supernatant test sample for the 10 -2 dilution tube and vortexed for 5 seconds. 1 mL from the 10 -2 dilution tube was transferred in duplicate into sterile petri dishes. With the same pipet, 1 mL will be transferred into a third 9 mL tube containing the supernatant test sample for the 10 -3 dilution tube and vortexed for 5 seconds. Serial dilution was repeated until a 10 -8 dilution plate was reached. Approximately 20 mL of tempered (44-46 °C) reinforced Clostridial agar was added to the petri dishes and gently swirled to evenly disperse inoculum throughout the agar. After solidification, 15 mL of reinforced Clostridial agar was overlaid onto each plate. The plates were incubated anaerobically in an upright position at 35-37°C for 18-24 hours.

[0081] RESULTS: Following incubation, photographs were taken of the serially diluted agar plates of the control to visually show presence of Clostridium difficile colony forming units (FIG. 4). The plates which exhibited less than 300 colonies were enumerated and representative colonies for Clostridium difficile were biochemically identified via VITEK ANC (Table 5 below). [0082] Similarly, following incubation, photographs were taken of the serially diluted agar plates of the sample to visually show absence of Clostridium difficile colony forming units due to the inhibitory effect of the synbiotic composition (see FIG. 5 and

Table 5).

[0083] Conclusions and Findings. The obtained results of the in-vitro study are consistent with several published investigations in PubMed. We believe inhibition of C. difficile by the presently disclosed synbiotic composition should be considered from both quantitative and qualitative points of view.

[0084] From a quantitative point of view, it is contemplated that a general mechanism of microbial inhibition is through the “competitive exclusion principle”. This means that when two species compete in a limited environment (e.g., colon) and compete for the same limited amount of nutrients, the species that has advantage over the others

(e.g., larger numbers) will dominate the environment and lead to the exclusion of the weaker competitor. For example, in the present study example, it is contemplated that the probiotic dose of 27 billion live and active CFU for a daily serving of the presently disclosed synbiotic composition is considered among the higher quantities commercially available.

[0085] From a qualitative point of view, it is contemplated that the chosen blend of

Bifidobacteria and Lactobacilli have functioned in a complementary, additive and possibly synergistic way to completely inhibit growth of C. difficile. In other words, the chosen probiotics have released some bioactive compounds into the sterilized organic liquid medium or fruit and vegetable juice that has inhibited growth of C. difficile. One such compound could possibly be lactic acid which can reduce the pH of the environment to an acidic range unfavorable for spore germination and/or growth of C. difficile. Thus, it is contemplated that the bioactive compounds secreted from the chosen 15 probiotic strains into the sterilized organic vegetable and fruit juice has played a decisive inhibitory role against growth of C. difficile.

[0086] Accordingly, it is contemplated that the combined quantitative and qualitative properties of the juice-based synbiotic composition is expected to positively and impressively impact prevention and/or treatment of CDI.

[0087] It will be appreciated that variations of the above-disclosed and other features and functions, or alteratives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alteratives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.