Microbiome intervention

Abstract

The present disclosure relates to compositions comprising an array of ingredients useful for regulating and / or treating companion animals, such as canines (e.g., dogs) or felines (e.g., cats), and their microbiomes, monitoring tools, and diagnostic methods for determining the health of companion animals and their microbiomes.

Description

Technical Field

[0001] The present disclosure relates to compositions for regulating and / or treating companion animals and their microbiomes, monitoring tools, and diagnostic methods for determining the health of companion animals and their microbiomes. Background Art

[0002] The microbiome is described as all the microorganisms in any given environment, and more specifically, the combined genetic material of the microorganisms in that environment. In mammals, microorganisms are found on the skin, in the gut, and in the mouth, in a symbiotic relationship with their hosts; in fact, these microorganisms play an important role in the health of the host, forming a barrier to prevent colonization by foreign microorganisms, thus protecting the animal from pathogens, and in the gut helping to break down nutrients, releasing energy and producing vitamins necessary for life.

[0003] Although the impact of pathogenic and probiotic microorganisms on their mammalian hosts has long been recognized, the influence of the overall community composition, specific microbial balance, and genetic potential of the resident microbiota is now increasingly recognized. Non-invasive studies of the human gut microbiome have been primarily performed using stool surrogate samples that specifically represent the distal colon, the primary site of bacterial growth within the intestine. Bacterial growth in the colon occurs via fermentation, a process that uses dietary components that are not digested and absorbed by the host and can be used to influence the gut microbial community and microbiome. Thus, a state of equilibrium is achieved where the predominant microbial community types remain relatively stable in individual animals over time in the absence of significant changes in diet or medications or incoming infectious agents.

[0004] Although a combination of maternal or paternal inoculation and environmental inoculation and bacterial interactions with the immune system are thought to play a role in immune priming for future recognition of pathogenic microorganisms and intestinal physiology, the development of the microbiome occurs soon after mammalian birth. Based on studies of human infants, it is now believed that these early developments in the gut microbiome influence health throughout later life. In the weeks and months after birth, diversity increases rapidly, representing the early establishment of intestinal microbiome development, and the microbiome and microbial communities present during this stage are thought to be plastic or changeable. In part due to this pre-diversity development stage in early life, puppies have an incompletely developed intestinal barrier, which includes the gastrointestinal microbiota as well as the histology and immune function associated with the gut. As a result, puppies and young dogs are more susceptible to gastrointestinal diseases such as soft, malformed stools, diarrhea, flatulence, and disease. The increase in diversity during maturation leads to the development of an adult intestinal microbiome that is resistant to colonization by even beneficial microorganisms such as probiotics, and therefore the adult microbiome is relatively resilient in resisting large changes in community structure and intestinal dysbiosis. The microbiome of adult animals contained similar bacterial communities, but was more diverse and well-developed with an adapted community structure that represented a more robust and resistant microbiota compared to other life stages, with the intestinal microbiota showing increased resistance.

[0005] Older and geriatric dogs, as well as older and geriatric humans, are also more likely to experience digestive issues, such as diarrhea and gastrointestinal infections, which can have a higher severity and greater impact. These gastrointestinal issues may, in part, be triggered by a deterioration in the gut microbiome.

[0006] In adult dogs, changes in the gut community can also be triggered by larger-scale changes in composition over time or due to environmental and dietary factors. Infectious agents entering the digestive system or altering the nutrients available to the microbial community due to dietary changes can disrupt the balance of the microbiome, leading to dysbiosis. Dysbiosis is described as an imbalance in the microbial community and can lead to clinical symptoms including gastrointestinal discomfort, diarrhea, vomiting, nutrient deficiencies and weight loss in the intestine. Although the microbiome and microorganisms in the intestine exist in a continuous abundance from birth or shortly after weaning to late life stages in old age, challenges to the microbiome caused by, for example, infection, antibiotic clearance, drug treatment or extreme dietary changes can alter community composition throughout life. In some cases, there can be lasting effects on the health of the host, such as chronic diarrhea, lactose intolerance. In cases of dogs with recurrent or chronic diarrhea, characteristic patterns, signatures or fingerprints can be detected in the microbiome, which can indicate the possibility of effective treatment through changes in dietary habits and the subsequent changes in the microbiome.

[0007] Cats and dogs are particularly well suited to studying the effects of nutrition on the microbiome, as complete and balanced diets can serve as the sole food source for extended periods, thereby reducing the confounding effects of dietary preferences between individuals. That said, despite decades of efforts to improve gastrointestinal (GI) health and optimize stool quality, knowledge of bacterial taxa and functional genomes associated with health parameters such as stool form, overt GI health (lack of clinical signs, resistance to challenge, and recovery from infection), and GI dysfunction remains very limited. This is particularly evident in hosts other than humans, such as dogs and cats.

[0008] A variety of ingredients have been reported to alter host health, and functional foods such as prebiotics and fibers are particularly useful for gastrointestinal health and stool quality or consistency. In particular, complex mixtures of soluble and insoluble fibers, prebiotic oligosaccharides, polysaccharides, or β-glucans may represent long-established functional dietary ingredients that help correct intestinal health conditions such as chronic and acute diarrhea or bloating of the digestive system and improve health status and vitality of the host.

[0009] Since the microbiota in the large intestine selectively ferments resistant fibers, there are particularly many studies evaluating the effects of dietary fiber. Therefore, a large amount of data can be obtained from in vitro and in vivo tests on specific food ingredients, including soluble and insoluble fibers and prebiotics. It is also known that such ingredients affect the microbiota in the intestine, especially the microbiota in the colon, and they have different effects on the microbiota that enhances health through fermentation of resistant fibers that are not digested by the host. There are many studies evaluating the effects of dietary fibers that are considered important for bulking properties and intestinal transit, including soluble fibers, prebiotics, and insoluble composite resistant dietary fibers, on the microbiota, and it has been demonstrated that the dietary fibers have an effect on stool consistency and the microbial content or microbial-derived content in the stool.

[0010] In part because of the variability and complexity of multiple ingredients and fiber sources, study results can be difficult to compare, and therefore revealing consistent trends remains problematic. Most naturally occurring dietary fiber sources represent a mixture of soluble and insoluble fiber, with a significant amount of research effort focused on prebiotic oligosaccharides, polysaccharides, and β-glucans. Specific products, inclusion levels, and host species are also confounding factors in trend assessments, but these ingredients have been shown to have an effect on both stool consistency and the microbial or microbial-derived content of stool (Ferrario et al., 2017; Wakshlag et al., 2011; Simpson et al., 2001; Sunvold et al., 1995; Vickers et al., 2001). Although source-specific characteristics such as dose and amount of intake are key to validity, a meta-analysis of studies on fiber and gastrointestinal health in humans revealed an association between fiber intake and colorectal cancer incidence. Although products / ingredients and specific inclusion levels have been described as reducing the risk of colorectal cancer, high fiber intake, particularly consumption of whole grains and grain-derived fibers, has been reported to have a potential protective effect.

[0011] To date, the research literature describing fiber and prebiotic dietary supplementation in pet animals has largely defined bacterial changes associated with specific ingredients, measured by selective bacterial culture or now outdated molecular techniques such as denaturing gradient gel electrophoresis, which typically measure taxa known to be beneficial to human health. Whether these insights apply to different hosts and are therefore relevant to canine health remains unclear. However, a small number of studies have been published that address changes in the microbiota measured by deep sequencing after dogs were fed specific fiber sources and prebiotics. Middlebos et al., (2010) fed dogs a diet containing 7.5% beet pulp and found that this amount significantly altered the structure of the gut microbiota compared to a reference diet. Although the data only supported changes at the phylum level, a significant increase in Firmicutes and a significant decrease in Fusobacteria were observed when dogs received a diet containing beet pulp.

[0012] Understanding the long-term effects of diet on the microbiome and the impact on host health parameters such as clinical biomarkers of health and gastrointestinal resistance and resilience represents a major advance in this area of ​​research as we manipulate the fecal microbiota to enhance health.

[0013] Stool consistency, particularly the extremes of loose or dry stool, is a key indicator of pet health for owners, and objectionable stool quality, diarrheal episodes, and poor gastrointestinal health leading to hygiene and lifestyle inconveniences are reported to be major barriers to pet ownership, which can have a significant impact on human-animal interactions and, consequently, quality of life issues for people and their pets. Therefore, the impact of food and ingredients on dog and cat stool quality is of great significance for optimizing pet health, well-being, lifestyle, vitality, and nutrition. Dietary intake, including dry matter volume, water content, and nutrient content, can influence stool consistency.

[0014] However, research on the intestinal microbiome and its connection with animal health is limited. Therefore, it is necessary to understand the relationship between the intestinal microbiome and animal health and vitality. It would be more advantageous to measure, monitor and track intestinal bacteria in feces over time or through nutritional intake or intervention to enhance or support the healthy intestine of pets, because this enables pet owners to observe the effects of compositions, ingredients or nutritional factors and provide pet owners with the motivation to continue to provide health-promoting factors. In addition, new methods and compositions are needed to enhance the gastrointestinal resistance, intestinal health, and treatment of chronic and acute intestinal diseases of healthy pets, such as acute self-limiting diarrhea infections and chronic enteropathy associated with microbiome. In addition, it is necessary to support gastrointestinal resistance after veterinary treatment and drugs (such as compositions, ingredients and dietary supplements or topper) that affect the microbiome to reduce the occurrence of dysbiosis and the resulting diarrhea or soft stools. Compositions or ingredients with this utility provide additional benefits to help veterinarians manage gastrointestinal symptoms treated by veterinarians.

[0015] Given the importance of the microbiome to health and well-being, it is important to find ways to influence and monitor the state of the animal's microbiome and enable owners to observe benefits by tracking health status over time, as intrinsic changes in the gut barrier, resistance to diarrhea, and gastrointestinal health can occur as a function of the nutritional intake provided by the owner, altering the microbiome content and influencing the health and vitality of the pet. Summary of the invention

[0016] The present disclosure relates to compositions for altering the microbiome of a companion animal, such as a canine, and methods involving the administration and use of the compositions.

[0017] The compositions and methods affect, optimize and enhance the intestinal microbiome of companion animals, and affect gastrointestinal health and resilience, thereby improving the health, wellness and vitality of the animals. In addition, the present disclosure relates to methods for monitoring microbiome by single-point and multi-point testing methods, so that the effects of the compositions on the microbiome can be tested, thereby generating microbiome-based intestinal health and resistance care pathways that enhance the health of pets. As shown in the examples, the methods of the present disclosure can achieve this with high precision.

[0018] In some embodiments, the present disclosure provides a composition, e.g., a pet food, e.g., a complete nutritional pet food, e.g., a dry (e.g., kibble), semi-moist or moist pet food; an incomplete nutritional pet food, e.g., a supplement, a functional topper, a functional food booster, or a nutritional composition or pharmaceutical composition for a companion animal, comprising at least one combination selected from the following ingredients: about 0.05 g / day to about 5.0 g / day of galactooligosaccharides (GOS); a total amount of about 0.1 g / day to about 10 g / day of beet pulp and / or chicory pulp; and about 0.2 g / day to about 30.8 g / day of cellulose.

[0019] In some embodiments, the product comprises at least two of the ingredients, preferably at least three of the ingredients, combined at their minimum levels. In some embodiments, the concentrations of the combined ingredients are up to the maximum levels as described above.

[0020] In some embodiments, the composition further comprises additional prebiotics. In some embodiments, the composition further comprises additional fiber or other functional food ingredients.

[0021] In some embodiments, the composition further comprises a probiotic species of lactic acid bacteria, such as Bifidobacterium, Lactobacillus, or Enterococcus. In some embodiments, the composition further comprises a probiotic species of yeast, such as a species from the genus Saccharomyces. In some embodiments, the composition further comprises a probiotic species that forms spores, such as a species from the genus Bacillus.

[0022] In some embodiments, the composition improves the intestinal health of the companion animal within about 3-21 days after the composition is administered to the companion animal. In some embodiments, the composition is a dietary supplement, also known as a reinforcing agent. In some embodiments, the dietary supplement is added to the outermost part of a pet food as an ingredient. In some embodiments, the dietary supplement is then mixed into the entire product. In some embodiments, the composition is a dog's food.

[0023] In some embodiments, the subject matter of the present disclosure provides a method of improving intestinal health and resistance in a healthy companion animal or an animal in need of improved gastrointestinal robustness (e.g., an animal suffering from acute or recurrent diarrhea), thereby improving resistance, health, and well-being. In some embodiments, the method comprises administering to a companion animal an effective amount of any composition disclosed herein.

[0024] Also provided is a method for determining the health of a companion animal microbiome, comprising quantifying one, two, three, four or more bacterial taxa (e.g., certain families or genera or species) to determine their abundance; and comparing the abundance with the abundance of the same bacterial taxa in a control data set; wherein an increase or decrease in the abundance of four or more bacterial taxa relative to the control data set indicates the health of the microbiome. A healthy microbiome can reduce pathogen load and the production of short-chain fatty acids. Reduced pH in the intestinal lumen is associated with reduced intestinal barrier permeability and gastrointestinal resistance. An unhealthy microbiome with a higher bacterial load of pathogenic microorganisms is associated with a variety of health conditions. Therefore, it is necessary to monitor the health of the intestinal microbiome or diagnose an unhealthy microbiome.

[0025] Further provided is a method of determining the health of a companion animal's microbiome, comprising detecting at least one, at least two, preferably at least three, and preferably at least four bacterial taxa in a sample obtained from the companion animal; wherein the presence of at least four bacterial taxa indicates an unhealthy microbiome.

[0026] The health of a companion animal microbiome can also be determined by a method comprising the steps of calculating a diversity index for species within the companion animal microbiome, and comparing the diversity index to a diversity index of a control data set.

[0027] The present invention also provides a method for monitoring a companion animal such as a canine, comprising the step of determining the health of the companion animal's microbiome at at least two time points by the method of the present invention. In one embodiment, health can be determined by comparison with physical characteristics such as stool morphology, stool consistency and / or gastrointestinal resistance, or by the stability of the microbiome. This is particularly useful in cases where the companion animal is undergoing treatment to alter the microbiome, as it can monitor the progress of the treatment. It is also useful for monitoring the health of companion animals.

[0028] Also provided is a method for monitoring the health of the microbiome in a companion animal that has received a composition of the present invention that can change the composition of the microbiome, comprising measuring the health of the microbiome according to the methods of the present disclosure. The methods allow the technician to determine whether the treatment is successful or not. Preferably, these methods include measuring the health of the microbiome before and after treatment, because this helps to assess the success of the treatment. These methods also help to predict the possible success or outcome of other pets with similar symptoms (i.e., inform prognostic methods). The subject matter of the present disclosure therefore provides a method by which a composition of dietary ingredients can be received through a main meal or supplemental pet care or pet food and combined with a method for measuring the gastrointestinal health of the animal to enhance health, so that the owner or attending veterinarian can observe the effects on gastrointestinal health and resistance, thereby being able to observe the effects of feeding the composition on the animal, and determining whether the animal will benefit or has benefited from intervention measures that restore the microbiome to its healthy state compared to feeding the composition or pet food, based on the timing of the test.

[0029] The disclosed subject matter also provides a method for assessing the intestinal health status of a healthy companion animal with no signs of gastrointestinal distress and determining whether the canine would benefit from an intervention to restore the microbiome to its healthy state. In some embodiments, the disclosed subject matter provides a method for determining the intestinal health status of a companion animal in need thereof (e.g., an animal with clinical symptoms such as diarrhea or poor stool quality or with intestinal dysbiosis such as chronic enteropathy or IBD).

[0030] In some embodiments, the disclosed subject matter provides a method for determining the intestinal health status in a companion animal before and / or after receiving a composition of the present invention, thereby assessing the need for receiving a pet care product. For example, intestinal health status can be measured by using a stool scoring system, such as the Waltham stool scoring system. https: / / www.waltham.com / sites / g / files / jydpyr1046 / files / 2020-05 / waltham-scoring.pdf

[0031] In some embodiments, the presently disclosed subject matter provides a method of assessing intestinal health in a companion animal before, during, and after receiving a pet care product (e.g., a composition, such as a supplement, a pet food functional ingredient or enhancer, or a complete nutritional dry kibble), thereby determining and monitoring the gastrointestinal health of the animal before and after receiving the product, thereby allowing the success of the pet care product to be assessed.

[0032] In some embodiments, the amount of bacterial taxa is measured from a fecal sample of a companion animal.

[0033] In some embodiments, bacterial taxa are measured at time points separated by about 1 week, about 2 weeks, about 21 days, about 28 days, about 1 month, about 56 days, about 2 months, about 3 months, about 4 months, about 84 days, about 5 months, or about 6 months. In some embodiments, the treatment regimen includes a dietary regimen. In some embodiments, the dietary regimen includes administering an effective amount of any composition disclosed herein.

[0034] In some embodiments, the amount of bacterial taxa is determined using DNA sequencing technology.

[0035] In some embodiments, RNA sequencing technology is used to determine the amount of bacterial taxa.

[0036] In some embodiments, the amounts of bacterial taxa are determined using microarrays.

[0037] In some embodiments, the amount of a bacterial taxa is determined using polymerase chain reaction technology (eg, quantitative PCR). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Figure 1 is a graphic representation of the study design used to compare the effects of individual dietary ingredients on the fecal microbiome and blood markers of intestinal permeability.

[0039] Figure 2 : Figure 2 is a graphical representation of daily feces wet weight and feces number measured as described in Example 1.

[0040] Figure 3 : Figure 3 is a graphical representation of daily stool wet weight and stool frequency measured as described in Example 1.

[0041] Figure 4 : Figure 4 is a graphical representation of the Shannon diversity of samples collected from a cohort of dogs while they were fed a control diet and a microbiome supplement as described in Example 2.

[0042] Figure 5 : Figure 5 is a graphical representation of the relative abundance (Logit transformed) of bacterial families significantly affected by the SBP / GOS / cellulose furnish treatment as described in Example 2 (p-values ​​are indicated in the corner of each graph).

[0043] Figure 6 : Figure 6 is a graphical representation of the relative abundance (Logit transformed) of bacterial genera significantly affected by the SBP / GOS / cellulose furnish treatment as described in Example 2 (p-values ​​are indicated in the corner of each graph). DETAILED DESCRIPTION

[0044] In some aspects, the present disclosure relates to compositions for altering the gut microbiota of a companion animal to confer a healthier gut microbiome. The present disclosure also relates to methods for monitoring the effects of the compositions on the health status of the host.

[0045] In some aspects, the present disclosure relates to a composition suitable for administration to a companion animal. In some embodiments, the composition comprises at least one ingredient selected from the following: about 0.05 grams (g) / day to about 5.0 g / day of galacto-oligosaccharides (GOS); a total amount of about 0.1 g / day to about 10 g / day of beet pulp and / or chicory pulp; and about 0.2 g / day to about 30.8 g / day of cellulose. In one embodiment, the composition includes at least one ingredient selected from the following: about 0.05g to about 3.6g / day of GOS; a total amount of about 1.65g / day to about 9.52g / day of beet pulp and / or chicory pulp; and about 5.32g to about 25.98g / day of cellulose.

[0046] In other aspects, the present disclosure relates to methods of altering the gut microbiota and microbiome of a companion animal by a composition for use as a dietary intervention, such as a pet food, a supplement, a pet food functional ingredient or enhancer; or a nutritionally complete dog dry kibble containing the following combination of GOS, cellulose, beet pulp and / or chicory pulp.

[0047] GOS is also referred to as galacto-oligosaccharide, galacto-oligosaccharide, lacto-oligosaccharide or transgalactooligosaccharide (TOS). It is produced by enzymatic conversion of lactose, and the chain length and the type of connection between the monomer units are different. They can also be naturally present in plants and dairy products or fractions at a lower level. Cellulose is an insoluble substance and is the main component of plant cell walls and plant fibers (such as cotton). It is a polysaccharide composed of glucose monomer chains. Examples of cellulose suitable for the present invention include, but are not limited to, plant fibers, such as those naturally present in leaves, seeds, straw, grass and wood. Specifically, ingredients can include fibrous plant hydrocolloids that combine fat and water to produce swelling activity without anti-nutritional factors, such as lettuce fiber or leaf fiber or other plant fibers (including pea fiber), shell fiber (including psyllium shell fiber, peanut shell fiber and citrus peel fiber) or pure natural grade fiber.

[0048] In some embodiments, the chicory pulp may be in the form of chicory pulp fibers. In some embodiments, the beet pulp may be beet pulp and / or beet pulp fibers.

[0049] Specific ingredients are described that can produce changes in the composition of the gut microbiome of companion animals that reflect an increase in gut microbiome health and immunity. Accordingly, combinations of ingredients for administration to companion animals via pet foods, treats, supplements, enhancers, or ingredients are described, as well as methods for demonstrating, monitoring, and tracking the effects of the compositions on individual companion animals.

[0050] The experiments described below show that feeding the compositions described herein to companion animals results in one or more of the following: a reduction in unacceptable feces, a reduction in fecal pH, an increase in fecal quantity, an increase in fecal wet weight that is capable of removing metabolic toxins, a reduction in lymphocyte counts, and an alteration in the intestinal microbiome as evidenced by the diversity of fecal bacterial taxa in the fecal microbiome, particularly a reduction in bacterial diversity, an increase in total bacterial taxa from the families Bacteroidaceae and Fusobacteriaceae, a reduction in Erysipelotrichaceae, a reduction in taxa of the genera Peptostreptococcaceae and Peptostreptococcus, and an increase in the bacterial genus Anaerobiospirillum.

[0051] monitor

[0052] In some aspects, the compositions described herein can be used as part of a companion animal monitoring program. In other aspects, the present disclosure relates to a method of altering the microbiome of a companion animal by administering a composition described herein to a companion animal. In one embodiment, the first step is to determine the health of the companion animal's microbiome, and when it is determined that a healthy or unhealthy microbiome, preferably an unhealthy microbiome, is detected in the first step, the composition is administered to the companion animal.

[0053] The method of the present disclosure is performed more than once, for example, twice, three times, four times, five times, six times, seven times or more than seven times. This allows the health of the microbiome to be monitored over time. For example, when a companion animal is receiving treatment to change the microbiome, or when the owner believes that the gastrointestinal health and resistance of the animal needs to be improved, this can be useful to reduce or even prevent or resolve the occurrence of diarrhea when clinical symptoms are detected. When the method is performed for the first time, the health of the microbiome is determined, and after the dietary intervention or administration of the composition of the present invention, the method is repeated to evaluate the effect of the composition on the microbiome. The health of the microbiome can also be determined for the first time after the companion animal receives treatment, and then the method is repeated to evaluate whether the health of the microbiome has changed. In this case, the effect of the treatment is indicated by a change in the microbial taxa, where the level of one or more of the bacterial taxa described herein changes significantly compared to the previous measurement, or moves outside the normal range compared to the average range of the control data set outside the 95% range.

[0054] The method can be repeated a few days, a week, two weeks, three weeks, a month, two months, three months, four months, five months, six months, 12 months, 18 months, 24 months, 30 months, 36 months, or more than 36 months apart.

[0055] Companion Animals

[0056] The compositions disclosed herein can be used to alter the microbiome of a companion animal. Companion animals include pets and / or livestock, such as canines and felines.

[0057] In some embodiments, the companion animal is a Canis lupusfamiliaris. This genus includes Canis lupusfamiliaris, wolves, coyotes, foxes, jackals, dingos, and the present invention can be used with all of these animals. Most preferably, the subject is a Canis lupusfamiliaris, referred to herein simply as a dog.

[0058] The companion animal may be healthy. "Healthy" may refer to a companion animal that has not been diagnosed with a disease known to be associated with the microbiome. Examples of such diseases include chronic and acute diarrhea, irritable bowel syndrome, ulcerative colitis, Crohn's disease, and inflammatory bowel disease. Preferably, the companion animal does not suffer from dysbiosis.

[0059] There are many different breeds of domestic dogs, which exhibit different behaviors. Different breeds also have different life expectancies, with smaller dogs generally living longer than larger dogs. Therefore, different breeds are considered to be junior, adult, senior, or geriatric at different points in their lives. The following table provides a summary of the different life stages.

[0060]

[0061] The distinction between toy, small, medium and large breeds is known in the art. In particular, toy breeds include different breeds such as Affenpinscher, Australian Silky Terrier, Bichon Frise, Bolognese, Cavalier King Charles Spaniel, Chihuahua, Chinese Crested, Coton De Tulear, English Toy Terrier, Griffon Bruxellois, Havanese, Italian Greyhound, Japanese Chin, King Charles Spaniel, Lowchen (Little Lion Dog), Maltese, Miniature Pinscher, and many more. Pinscher, Papillon, Pekingese, Pomeranian, Pug, Russian Toy Terrier and Yorkshire Terrier.

[0062] Miniature breeds are larger on average than toy breeds, with average weights up to about 10 kg. Exemplary breeds include the French Bulldog, Beagle, Dachshund, Pembroke Welsh Corgi, Miniature Schnautzer, Cavalier King Charles Spaniel, Shih Tzu, and Boston Terrier.

[0063] Medium-sized dog breeds have an average weight of about 10 to about 26 kg, preferably 10 to 25 kg. These dog breeds include Bulldog, Cocker Spaniel, Shetland Sheepdog, Border Collie, Basset Hound, Siberian Husky and Dalmatian.

[0064] Large breeds are breeds with an average weight equal to or greater than about 25 kg, preferably at least 27 kg. Examples include Great Dane, Neapolitan mastiff, Scottish Deerhound, Dogue de Bordeaux, Newfoundland, English mastiff, Saint Bernard, Leonberger and Irish Wolfhound.

[0065] Hybrids can usually be classified based on weight as toy, small, medium, and large dogs.

[0066] sample

[0067] The methods disclosed herein generally use fecal samples or samples from the gastrointestinal cavity of a companion animal. Fecal samples are convenient because their collection is non-invasive and can also be easily repeated over a period of time. Other samples may also be used in the present invention, such as ileal, jejunal, duodenal samples and colonic samples.

[0068] The sample may be a fresh sample. Prior to use in the methods of the invention, the sample may also be frozen, or stabilized by other means, such as by addition to a storage buffer, or by dehydration using methods such as freeze drying.

[0069] Before being used in the methods of the present disclosure, the sample is usually processed to extract DNA. Methods for isolating DNA are well known in the art, such as those described in reference (Hart et al. (2015) PLoS One. Nov 24; 10(11): e0143334). Suitable methods include, for example, QIAamp Power Faecal DNA kit (Qiagen). To date, there is still a need for new methods and compositions for treating intestinal flora imbalance and other intestinal diseases, which target the intestinal microbiome to improve health and gastrointestinal resistance.

[0070] For purposes of clarity and not limitation, the detailed description of the subject matter of the present disclosure is divided into the following subsections:

[0071] 1. A healthy microbiome and good stool quality;

[0072] 2. Composition;

[0073] 3. Treatment methods; and

[0074] 4. Testing, tracking and monitoring methods

[0075] 1. Microbiome Health and Good Stool Quality

[0076] In some embodiments, changes in a companion animal's bacterial taxa can be used to indicate its intestinal health and possible stool quality. In some embodiments, the bacterial taxa are associated with a subject's health status or intestinal flora imbalance, or good or bad stool quality.

[0077] In some embodiments, after feeding the composition of the present invention, the change or stability or reduced variability of the fecal microbial community diversity or microbial community Shannon diversity level can indicate that the health of the companion animal is improved. In some embodiments, after feeding the composition of the present disclosure, the changes in the levels of Bacteroidaceae, Erysipelotrichaceae, Fusobacteriaceae, and Peptostreptococcaceae indicate that the health of the companion animal is improved. In one embodiment, after feeding the composition of the present disclosure, the levels of Bacteroidacea and Fusobacereicaea are significantly increased. In another embodiment, after feeding the composition of the present disclosure, the levels of Erysipelotrichaeceae and Peptostreptococcaecea are significantly reduced.

[0078] In some embodiments, a change in Peptostreptococcus species or taxa from the Escherichia / Shigella group or Anaerobiospirillium sp. after feeding the compositions of the present invention may indicate an improvement in the health of the companion animal.

[0079] 2. Composition

[0080] In some aspects, the present disclosure relates to a composition suitable for administration to a companion animal, the composition comprising at least one ingredient selected from the group consisting of: about 0.05 grams (g) / day to about 5.0 g / day of galacto-oligosaccharides (GOS); a total amount of about 0.1 g / day to about 10 g / day of beet pulp and / or chicory pulp; and about 0.2 g / day to about 30.8 g / day of cellulose.

[0081] In one embodiment, the composition is a food, a nutraceutical, a medicament, a supplement, a enhancer, or a topper. In one embodiment, the composition is suitable for administration to a companion animal.

[0082] As used herein, "companion animal" includes any animal that may be found in a domestic environment, including mammals, such as canines and felines.

[0083] In one embodiment, the composition is suitable for use in medicine.

[0084] In one embodiment, the composition is suitable for treating gastrointestinal disorders in companion animals. In one embodiment, the composition is suitable for changing the microbiome of companion animals. In one embodiment, the composition is suitable for increasing the number of bacteria present in the gastrointestinal tract or feces of companion animals compared to the number of Bacteroidaceae, Fusobacteriaceae and Anaerobiospirillum present in the companion animal before administering the composition. In one embodiment, the composition is suitable for reducing the number of Erysipelotrichaeceae and Peptostreptococcaceae. In one embodiment, the composition is used to reduce the fecal pH of companion animals.

[0085] In some embodiments, the composition comprises an effective amount of a pulp. In some embodiments, the pulp is beet pulp, such as beet pulp, preferably unprocessed beet pulp. In another embodiment, the pulp can be chicory pulp, preferably chicory pulp fiber. In some embodiments, the pulp is cooked or sterilized, or included in an extruded or processed product. In one embodiment, the pulp of the composition can be derived from at least one plant, such as chicory and beet.

[0086] In some embodiments, the concentration of the slurry is between about 0.5% w / w and about 10% w / w, between about 0.5% w / w and about 5% w / w, between about 0.5% w / w and about 4% w / w, between about 0.5% w / w and about 3% w / w, between about 0.5% w / w and about 2% w / w, between about 0.5% w / w and about 1.5% w / w, between about 0.5% w / w and about 1.2% w / w, between about 0.5% w / w and about 1% w / w, between about 0.5% w / w and about 0.9% w / w, or between about 0.5% w / w and about 0.8% w / w. In some embodiments, the concentration of the slurry is between about 0.8% w / w and about 10% w / w, about 0.8% w / w and about 5% w / w, about 0.8% w / w and about 4% w / w, about 0.8% w / w and about 3% w / w, about 0.8% w / w and about 2% w / w, about 0.8% w / w and about 1.5% w / w, about 0.8% w / w and about 1% w / w, about 1% w / w and about 10% w / w, about 1% w / w and about 5% w / w, about 2% w / w and about 5% w / w, or about 1% w / w and about 2% w / w. In some embodiments, the concentration of the slurry is about 0.8% w / w.

[0087] In some embodiments, the composition comprises any of the bacteria disclosed herein in an amount effective to correlate with a healthy intestinal state in a subject.

[0088] In some embodiments, the bacteria contained in the composition is between about 1000 CFU and about 10 trillion CFU. In some embodiments, the bacteria is between about 1000 CFU and about 1 trillion CFU, between about 1 million CFU and about 1 trillion CFU, between about 100 million CFU and about 100 billion CFU, between about 1 billion CFU and about 1 trillion CFU, between about 1 billion CFU and about 100 billion CFU, between about 100 million CFU and about 100 billion CFU, between about 1 billion CFU and about 50 billion CFU, between about 100 million CFU and about 50 billion CFU, or between about 1 billion CFU and about 10 billion CFU. In some embodiments, the composition comprises at least about 1000 CFU, at least about 1 million CFU, at least about 10 million CFU, at least about 100 million CFU, at least about 100 million CFU, at least about 1 billion CFU, at least about 10 billion CFU, at least about 100 billion CFU, or more.

[0089] In some embodiments, the composition further comprises an effective amount of a pulp, such as chicory pulp or beet pulp (eg, beet pulp).

[0090] In some embodiments, the composition is a dietary supplement, e.g., applied to the composition, as a pet food ingredient or subsequently mixed in the entire product. In some embodiments, the composition is a treat product or a chew or a kibble treat or a supplement product. In some embodiments, the composition is a cat food or a dog food. In some embodiments, the food is a dog food. In some embodiments, the composition is a dry pet food. In some embodiments, the composition is a wet pet food.

[0091] In some embodiments, the formulations of the presently disclosed subject matter may also include additional active agents. Non-limiting examples of additional active agents that may be present in the formulations of the presently disclosed subject matter include nutrients (e.g., amino acids, peptides, proteins, fatty acids, carbohydrates, sugars, nucleic acids, nucleotides, vitamins, minerals, etc.), prebiotics, probiotics, antioxidants, and / or agents that enhance the microbiome, improve gastrointestinal health, and improve animal health.

[0092] In some embodiments, the composition comprises one or more probiotics. In some embodiments, the probiotics are animal probiotics. In some embodiments, the animal probiotics are cat probiotics. In some embodiments, the animal probiotics are canine probiotics. In some embodiments, the probiotics are from Bacteroidaceae and / or Fusobacteriaceae or Anaerobiosprillum. In some embodiments, the animal probiotics are further selected from Bifidobacterium, Lactobacillus, lactic acid bacteria and / or Enterococcus. In some embodiments, the probiotic is selected from any organism in the lactic acid bacteria, and more specifically from the following bacterial genera: Lactococcus spp., Pediococcus spp., Bifidobacterium spp. (e.g., Bifidobacterium longum, B. bifidum, B. pseudolongum, B. animalis, B. infantis), Lactobacillus spp. spp.) (e.g., Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, Enterococcus spp. (e.g., Enterococcus faecium), Prevotella spp., Fusobacterium spp., Alloprevotella spp.) and any combination thereof. In some embodiments, the probiotics are administered to companion animals in an amount of about 1 colony forming unit (CFU) to about 100 billion CFU per day to maintain GI microbiota or microbiome or gastrointestinal health. In some embodiments, the probiotics are administered to companion animals in an amount of about 1 colony forming unit (CFU) to about 20 billion CFU per day to maintain GI microbiota or microbiome or gastrointestinal health. In some embodiments, the probiotics are administered to companion animals in an amount of about 1 billion to about 20 billion CFU per day to maintain GI microbiota or microbiome or gastrointestinal health.In some embodiments, the probiotics are administered to a companion animal in an amount of about 100 million to about 100 billion live bacteria per day. In some embodiments, the probiotics are administered to a companion animal in an amount of about 100 million to about 10 billion live bacteria per day. In some embodiments, the probiotics are administered to a companion animal in an amount of about 100 million to about 10 billion live bacteria per day. 4 CFU to 1×10 14 CFU amount is given to companion animals. In some embodiments, additional prebiotics, such as oligofructose (FOS), oligoxylose (XOS), soy oligosaccharides (SO), glucan, galactan, arabinogalactan, inulin and / or oligomannosaccharide, may be included. In some embodiments, additional prebiotics are given in an amount sufficient to significantly stimulate the microbiome or GI microbiome and / or cause proliferation of one or more probiotics.

[0093] In some embodiments, the composition may also include additives known in the art. In some embodiments, such additives exist in an amount that does not damage the purpose and effect provided by the subject matter of the present disclosure. The example of the additive considered includes but is not limited to functionally beneficial substances that improve health, substances with stabilizing effects, organoleptic substances, processing aids, substances that enhance palatability, coloring substances, and substances that provide nutritional benefits. In some embodiments, stabilizing substances include but are not limited to substances that can increase the shelf life of the product. In some embodiments, such substances include but are not limited to preservatives, synergists and chelating agents, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants. In some embodiments, emulsifiers and / or thickeners include, for example, gelatin, cellulose ethers, starch, starch esters, starch ethers, and modified starches.

[0094] In some embodiments, additives for coloring, palatability and nutritional purposes include, for example, colorants; iron oxide, sodium chloride, potassium citrate, potassium chloride and other edible salts; vitamins; minerals; and flavoring agents. The amount of such additives in the product is generally up to about 5% (based on dry product).

[0095] In some embodiments, the composition is a dietary supplement. In some embodiments, dietary supplements include, for example, feeds used together with another feed to improve nutritional balance or overall performance. In some embodiments, supplements also include such compositions, which are fed undiluted as supplements to other feeds, freely selected with other parts of animal rations that can be obtained separately, or diluted and mixed with the conventional feed of the animal to produce complete feed. For example, AAFCO provides a discussion of supplements in the 220th page (2003) of the official publication of the American Feed Control Association (American Feed Control Officials, Incorp.). Supplements can be in various forms, including, for example, powders, liquids, syrups, pills, tablets, encapsulated compositions, etc.

[0096] In some embodiments, the composition is a treat. In some embodiments, treats include, for example, compositions given to animals to induce the animal to eat at non-meal times. In some embodiments, the composition is a treat for canines, including, for example, dog bones. Treats can be nutritional, wherein the product contains one or more nutrients, and can, for example, have a composition for food as described above. Non-nutritional treats include any other non-toxic treats.

[0097] In some embodiments, the bacteria and / or sugar beet pulp of the presently disclosed subject matter can be incorporated into the composition during the formulation process (eg, during and / or after mixing of the other components of the product). These ingredients can be dispensed into the product by conventional means.

[0098] In some embodiments, the compositions of the disclosed subject matter can be prepared in canned or wet form using conventional companion animal food processes. In some embodiments, ground animal (e.g., mammal, poultry and / or fish) protein tissue is mixed with other ingredients, such as milk fish oil, grains, other nutritional balance ingredients, special purpose additives (e.g., vitamin and mineral mixtures, inorganic salts, cellulose and beet pulp, fillers, etc.); and water sufficient for processing is added. These ingredients are mixed in a container suitable for heating while mixing these ingredients. The mixture can be heated in any suitable manner, such as by direct steam injection or by using a container equipped with a heat exchanger. After the last ingredient is added, the mixture is heated to a temperature range of about 50°F to about 212°F. Temperatures beyond this range are acceptable, but may not be commercially achievable without the use of other processing aids. When heated to a suitable temperature, the material is generally in the form of a thick liquid. The thick liquid is placed in a can. Cover the lid and seal the container airtight. The sealed can is then placed in a conventional device designed to sterilize the contents. This is typically accomplished by heating to a temperature greater than about 230°F for an appropriate time, depending upon, for example, the temperature and composition used.

[0099] In some embodiments, the composition of the present disclosure theme can be prepared into a dry form using conventional methods. In some embodiments, dry ingredients including, for example, animal protein sources, plant protein sources, cereals, etc. are ground and mixed together. In some embodiments, then, wet or liquid ingredients including fat, oil, animal protein sources, water, etc. can be added to the dry mixture and mixed with it. In some embodiments, the mixture is then processed into coarse grinding or similar dry pieces. In some embodiments, the composition is coarse grinding. In some embodiments, coarse grinding is formed using an extrusion process, in which a mixture of dry and wet ingredients is mechanically processed under high pressure and high temperature, and is pushed through a small opening and cut into coarse grinding by a rotating knife. In some embodiments, the wet coarse grinding is then dried and optionally coated with one or more local coatings (coating), which may include, for example, flavorings, fats, oils, powders, etc. In some embodiments, coarse grinding can also be made from dough using a baking process rather than extrusion, in which the dough is placed in a mold before dry heat processing.

[0100] In some embodiments, the snack foods of the presently disclosed subject matter can be prepared by, for example, an extrusion or baking process similar to the processes described above for dry foods.

[0101] In some embodiments, the compositions of the present invention further comprise Royal Canin TM Indoor Life Pet Food.

[0102] 3. Treatment Methods

[0103] In some non-limiting embodiments, the subject matter of the present disclosure provides a method for enhancing or improving a microbiome, improving intestinal health, and / or treating intestinal flora imbalance in a subject in need thereof. In some embodiments, the subject is a companion animal, such as a dog or cat. In some embodiments, the method can improve the immunity, digestive function, stool quality and quantity of a companion animal, and / or reduce dysbiosis.

[0104] In some embodiments, the method includes administering to a subject an effective amount of any composition of the present disclosure. In some embodiments, the method further includes monitoring any bacterial taxa of the present disclosure in a subject. In some embodiments, the bacterial taxa are measured in a fecal sample of the subject. In some embodiments, the bacterial taxa are measured in a sample from the intestinal tract of the subject.

[0105] In some embodiments, the composition can be given to the subject from 20 times a day to 1 time a day, from 10 times a day to 1 time a day, or from 5 times a day to 1 time a day. In some embodiments, the composition can be given to the subject from 1 time a day, from 2 times a day, from 3 times a day, from 4 times a day, from 5 times a day, from 6 times a day, from 7 times a day, from 8 times a day, from 9 times a day, from 10 times a day or more. In some embodiments, the composition can be given to the subject once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, or once a month. In some embodiments, the composition can be given to animals in a continuous manner, for example, wherein animals are fed with the continuous available supply of this theme composition.

[0106] In some embodiments, the dosage of the composition is between about 1mg / kg body weight and about 5000mg / kg body weight every day. In some embodiments, the dosage of pet food is between about 5mg / kg body weight to about 1000mg / kg body weight every day, between about 10mg / kg body weight to about 500mg / kg body weight every day, between about 10mg / kg body weight to about 250mg / kg body weight every day, between about 10mg / kg body weight to about 200mg / kg body weight every day, between about 20mg / kg body weight to about 100mg / kg body weight every day, between about 20mg / kg body weight to about 50mg / kg body weight every day or any intermediate range thereof. In some embodiments, the dosage of pet food is at least about 1mg / kg body weight every day, at least about 5mg / kg body weight every day, at least about 10mg / kg body weight every day, at least about 20mg / kg body weight every day, at least about 50mg / kg body weight every day, at least about 100mg / kg body weight every day, at least about 200mg / kg body weight every day or more. In some embodiments, the dosage of the pet food is no more than about 5 mg / kg body weight per day, no more than about 10 mg / kg body weight per day, no more than about 20 mg / kg body weight per day, no more than about 50 mg / kg body weight per day, no more than about 100 mg / kg body weight per day, no more than about 200 mg / kg body weight per day, no more than about 500 mg / kg body weight per day, or more.

[0107] In some embodiments, the amount of the composition decreases during feeding of the companion animal. In some embodiments, the concentration of the composition increases during feeding of the companion animal. In some embodiments, the concentration of the composition changes based on the age of the companion animal.

[0108] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for determining the intestinal health of a companion animal in need thereof.

[0109] In some embodiments, the amount of bacterial taxa can be determined by any method known in the art. In some embodiments, the method includes, but is not limited to, antibody-based detection methods for detecting proteins / antigens associated with microorganisms, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, western blot; and methods for detecting 16s rRNA associated with microorganisms, such as real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), DNA sequencing, and microarray analysis. In some embodiments, the microarray includes probes for detecting any intestinal microorganisms disclosed herein.

[0110] In some embodiments, the treatment regimen can be any treatment regimen for dysbiosis known in the art.In some embodiments, the treatment regimen comprises a treatment method disclosed herein.

[0111] In some embodiments, the amount of intestinal bacteria is measured from a stool sample from the subject.

[0112] 4. Testing, tracking and monitoring methods

[0113] Additionally or alternatively, the health of the companion animal's microbiome can be assessed by determining the diversity of the species of bacteria in the companion animal's microbiome. To this end, a diversity index of the species of bacteria in the companion animal's microbiome is determined and compared to a diversity index of a control data set. The stability of the microbiome is assessed by repeated sampling, wherein the stability of the diversity is a sign of the health of the microbiome.

[0114] Comparison data set

[0115] The abundance of the bacterial species is compared to a control data set from a companion animal, such as a canine, of similar chronological age (e.g., a young canine, an adult canine, an older canine, or a geriatric canine). PCT / US2020 / 014292 or WO2020 / 150712 are incorporated herein by reference in their entirety, and they provide suitable control data sets that can be compared to the microbiome composition from a canine.

[0116] Alternatively or additionally, a control data set can be prepared. To this end, the microbiome of two or more (e.g., 3, 4, 5, 10, 15, 20 or more) healthy canines can be analyzed to determine the abundance of species contained in the microbiome. In this case, a healthy canine is a canine that has not been diagnosed with a disease known to affect the microbiome. Examples of such diseases include irritable bowel syndrome, ulcerative colitis, Crohn's disease, and inflammatory bowel disease. Preferably, the canine does not suffer from dysbiosis. Dysbiosis refers to an imbalance in the microbiome in the body, which is due to insufficient levels of keystone bacteria (e.g., bifidobacteria, such as longum subspecies infantis (B. longum subsp. infanttis)) in the intestine or excessive levels of harmful bacteria. Methods for detecting dysbiosis are well known in the art. Two or more canines are typically from a specific life stage. For example, they can be young canines, adult canines, older canines, or elderly canines. This is useful because the microbiome changes throughout the life of a canine, so the microbiome needs to be compared to a canine at the same life stage. In the case where the canine is a dog, the control dataset can also be from a dog of the same breed, or in the case where the dog is a mutt, from the same breed of one of the dog's direct ancestors (parent or grandparent).

[0117] Specific steps of preparing a control data set may include analyzing the microbiome composition of at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more) young canines, and / or at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more) adult canines, and / or at least two (e.g., 3, 4, 5, 6, 7, 8 9, 10, 15, 20 or more) older canines and / or at least two (e.g., 3, 4, 5, 6, 7, 8 9, 10, 15, 20 or more) elderly canines; determining the abundance of bacterial species (particularly those discussed above); and compiling these data into a control data set.

[0118] For embodiments in which the diversity index of a microbiome is determined, a control data set can be prepared in a similar manner. In particular, the diversity index can be determined in two or more healthy canines at a particular life stage (puppy, adult, older, or geriatric). The results can then be used to determine the average range of the diversity index for canines at that life stage.

[0119] It should be understood that it is not necessary to prepare a control data set each time the method of the present invention is performed. Instead, the skilled person can rely on an established control set.

[0120] A control data set can also include data from the same companion animal at different time points.

[0121] Techniques that allow a skilled person to detect and quantify bacterial taxa are well known in the art. These techniques include, for example, 16S rDNA amplicon sequencing, shotgun sequencing, metagenomic sequencing, Illumina sequencing, and nanopore sequencing. Preferably, bacterial taxa are determined by sequencing 16s rDNA sequences.

[0122] In some embodiments, the bacterial taxonomic group is determined by sequencing the V4-V6 region, for example using Illumina sequencing.

[0123] The species of bacteria can also be detected by other means known in the art, such as RNA sequencing, protein sequence homology, or other biomarkers that indicate the species of bacteria.

[0124] Then, sequencing data can be used to determine the presence or absence of different bacterial taxa in the sample. For example, sequences can be clustered at 98%, 99% or 100% identity, and then the relative proportions of the taxa (e.g., those representing more than 0.001 of the total sequence) of abundance can be assessed. Suitable techniques are known in the art and include, for example, logistic regression, partial least squares discriminant analysis (PLS-DA) or random forest analysis and other multivariate methods.

[0125] Overview

[0126] The term "comprising" encompasses "including" as well as "consisting", for example a composition "comprising" X may consist of X only or may include additional things, such as X+Y.

[0127] The term "about" in connection with a numerical value x is optional and means, for example, x ± 10%.

[0128] The term "substantially" does not exclude "completely", for example, a composition "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definitions of the methods and compositions of the present disclosure.

[0129] Reference to the percent sequence identity between two nucleotide sequences refers to the percentage of nucleotides that are identical when the two sequences are compared when aligned. Software programs known in the art can be used to determine the alignment and homology percentage or sequence identity. Preferred alignments are determined using the BLAST (Basic Local Alignment Search Tool) algorithm or the Smith-Waterman homology search algorithm, using an affine gap search, a gap opening penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. Alignment can be performed over the entire reference sequence, i.e., it can be performed over 100% of the length of the sequence disclosed herein.

[0130] Unless otherwise specified, a process or method including multiple steps may include additional steps at the beginning or end of the method, or may include additional intermediate steps. In addition, if appropriate, steps may be combined, omitted or performed in an alternative order.

[0131] Various embodiments of the disclosed compositions and methods are described herein. It should be understood that the features specified in each embodiment can be combined with other specified features to provide further embodiments. In particular, embodiments that are emphasized as suitable, typical or preferred herein can be combined with each other (unless they are mutually exclusive).

[0132] Embodiments of the present invention

[0133] Example 1: Fiber-based compositions for influencing stool pH and gastrointestinal health and immunity via intestinal microbiota

[0134] introduce

[0135] The colonic microbiome is critical for the integrity of the intestinal barrier and for the provision of essential metabolites to the host. Although the bacterial ratios of the gut microbiota vary, studies of the gut microbiome in pets have been primarily conducted using fecal surrogate samples. This represents a similar approach to non-invasive human gut microbiome methods, where fecal samples are analyzed as a standard surrogate for luminal samples to gain insight into the gut microbiome.

[0136] A feeding study was conducted to determine the presence of a prebiotic / soluble and insoluble fiber combination in a group of animals. The composition was formulated with feed grade cellulose, sugar beet pulp, and galacto-oligosaccharides (GOS) and the study was designed to evaluate whether an altered gut microbial composition or microbiome was associated with feeding these ingredients in the form of unprocessed dietary ingredients and whether effects on systemic health were observed. Systemic / physiological measurements included stool consistency and stool characteristics, complete blood count (CBC), and blood endotoxin (lipopolysaccharide) levels.

[0137] method

[0138] A group of 32 dogs were assigned to the study, which utilized a crossover design comparing test and control treatments to evaluate whether the ingredients fed as dietary ingredients altered the composition or microbiota characteristics of the dogs compared to a control reference diet. In addition to assessing fecal microbiota, whether systemic treatment effects were observed was assessed by hematological measurements of blood leukocyte populations, which can indicate immune effects induced by feeding the composition.

[0139] Consistent high-quality commercial pet dry pet food (RC Indoor AdultDog) was fed throughout the study. At the beginning of each study period, the dogs were fed a control diet or a control diet with one of the test pet food dietary ingredient compositions (cellulose, beet pulp, and GOS) applied to the diet for each meal each day. The animals were fed twice daily, and each treatment (control basal diet or basal diet containing fiber dietary ingredients; Table 2.1) was fed for 21 days to allow for the collection of fecal quality data over a long period of time and to stabilize the gastrointestinal microbiota after the diet change.

[0140] Fecal mass was recorded daily, and fecal samples for microbiome assessment were collected on days 2-4 and 18-20 (or +1 day if no sample was produced for collection on the target day). Fresh fecal samples were collected no more than 15 minutes after each dog defecated and stored at -80 degrees Celsius before processing for analysis of microbial composition in feces using a deep sequencing approach to determine bacterial community composition by amplicon sequencing of the 16S rDNA region V3-6.

[0141] A 6 ml fasting blood sample was collected once at the end of each period from a consistent subset of the cohort (10 animals) to assess complete blood count (CBC) and blood endotoxin (lipopolysaccharide) levels.

[0142] Table 2.1 Composition of cellulose, beet pulp and GOS

[0143]

[0144] Statistical Methods for Experimental Methods

[0145] The trial followed a two-way crossover design with a 21-day period. Thirty-two older dogs were divided into two experimental groups; A and B. Group A received the test diet (basal + cellulose, beet pulp and GOS) in the first period and then the control diet (basal only), while Group B received the control diet in the first period and then the test diet (basal + cellulose, beet pulp and GOS).

[0146] Statistical models: For daily intake (g) and fecal pH, linear mixed-effects models were fitted, modeling response to diet, phase date, and their interaction as fixed effects and animal as a random effect. To determine the significance of the proportion of unacceptable feces (% diarrheal feces; all data) and the proportion of unacceptable feces (last 14 days of each phase), binomial generalized linear mixed-effects models were fitted, modeling the binary response to diet (acceptable = 0, unacceptable = 1) as a fixed effect and pen pairs with phase date nested in pen pairs as a random structure. For all other measures, linear mixed-effects models were fitted, modeling response to diet as a fixed effect, animal as a random effect for blood measurements, and pen pairs with phase date nested in pen pairs as a random structure for fecal wet weight measurements. Visual inspection of (linear mixed) model residuals was used to assess consistency with model assumptions. Log statistic was applied when necessary. 10 Transformed to satisfy normality assumption.

[0147] Output: From all models, report the mean with 95% confidence intervals for each diet or for each diet at each time (if appropriate). 10 In the case of transformation, the reported means and CIs were transformed back to the original scale. For binomial GLMMs, the reported means and CIs represent proportions. Contrasts were made between diets or, if appropriate, between each diet, between the times of each diet, and between the times of the comparisons between diets. These contrasts were reported as fold changes with 95% confidence intervals and when log was applied. 10 The p-values ​​and 95% confidence intervals for the difference from 1 are provided for the transformed case, and the odds ratios are provided for the difference from 1 and 95% confidence intervals for the binomial GLMM, and the p-values ​​and 95% confidence intervals for the difference from 0 for all other models.

[0148] result

[0149] A total of 32 dogs were assigned to and completed the study. The table below shows the balance of breed, sex, age, and location between the two experimental groups. All animals participating in the study were sterilized. The dogs were all purebred and were from three breeds, namely Beagle, Brittany Spaniel, and Labrador Retriever. The animals assigned to the study ranged in age from 8.9 to 12.9 years, 14 were female, and 18 were male. The groups were well balanced in terms of sex and breed size.

[0150] For each measure, means and contrasts are reported in tables with accompanying graphs showing means and confidence intervals. Significant contrasts are highlighted in the graphs with stars and bars.

[0151] Table 2.2 Group details: Gender

[0152]

[0153] Cohort details:

[0154]

[0155] Group Details:

[0156]

[0157] Dogs fed the formula had significantly lower fecal scores and percentage of unacceptable feces (p < 0.001; Figure 2 Dogs fed the formula had significantly lower fecal pH at both the start and end phases (p<0.001).

[0158] The combination of cellulose, beet pulp, and GOS significantly reduced stool mass, percentage of unacceptable stool, and stool pH, suggesting changes in the dogs' microbial metabolites and microbiome. When the daily feeding amounts were the same between treatment groups, the dogs also experienced a significant increase in daily stool wet weight and stool volume when receiving the combination of ingredients cellulose, beet pulp, and GOS (p<0.001; P<0.001). Figure 2 ).

[0159] Fecal pH

[0160] diet Stage time Mean (95% CI) Royal Canin Indoor Life Baseline 6.32(6.25,6.4) Royal Canin Indoor Life Endpoints 6.34(6.27,6.42) Royal Canin Indoor Life+SBP / GOS / Cell Baseline 6.04(5.97,6.11) Royal Canin Indoor Life+SBP / GOS / Cell Endpoints 6.12(6.05,6.2)

[0161] Fecal pH comparison

[0162]

[0163] Table 2.3: Complete blood cell counts of 10 dogs collected at the end of each period *p<0.05

[0164]

[0165] When the dogs received the dietary supplement of cellulose, beet pulp and GOS at the end of the period, the blood lymphocyte counts of the dogs were significantly reduced (p=0.042). The treatments had no significant effect on the other blood parameters measured.

[0166] in conclusion

[0167] The cellulose, beet pulp, and GOS combination significantly reduced stool mass, percentage of unacceptable stool, and stool pH, indicating changes in the dogs' microbial metabolites and microbiome. Dogs also experienced significant increases in daily stool wet weight and stool count when they received the combined ingredients cellulose, beet pulp, and GOS. Ingredients and enhanced functional supplemental products that can be provided in addition to the main meal can provide a platform that can enhance the flexibility of interventions outside of the main meal product.

[0168] Example 2: Fiber-based compositions for influencing the gut microbiome and gastrointestinal health

[0169] This example describes a combination of ingredients that alter the microbiome and impart physiological effects to dogs via altered gut microbiota measured in feces. This altered gut microbiota correlates with the beneficial effects observed in Example 1, i.e., enhancement of the microbiome was associated with a reduced incidence of diarrhea, altered stool consistency, and changes in lymphocyte levels, which are key white blood cells or leukocytes involved in antigen clearance and inflammation.

[0170] Materials and methods

[0171] The study design and analysis methods were as in Example 1.

[0172] method

[0173] The method involves extracting DNA from freshly produced stool samples by methods such as the QIAamp Power Faecal DNA kit (Qiagen), followed by the use of molecular biology techniques to detect the presence of 16S rDNA or rRNA or other genetic signatures to determine the bacterial abundance and taxonomic or species richness of the microbial community in stool or other gastrointestinal samples. After DNA is extracted from freshly produced stool and sequenced by techniques such as 16S rDNA amplicon, shotgun, metagenomic, Illumina, nanopore or other DNA sequencing technologies, the resulting DNA sequences are clustered to the species (>98% ID) level and the relative abundance of taxa of individual taxonomic units (OTUs) is determined as a proportion of the total sequences. The total number and relative abundance data of OTUs are used to calculate Shannon diversity, which describes the abundance and evenness of the species detected. Shannon diversity can be calculated by the following method:

[0174]

[0175] After determining the diversity of the microbiota using functions such as alpha diversity, including the Shannon diversity index and the total number of OTUs in the sample, the diversity can be compared between samples collected from the group at the beginning and end of the control diet and samples collected from the group at the beginning and end of the SBP / GOS / Cell (beet pulp / galacto-oligosaccharides / cellulose) diet.

[0176] Microbiota Analysis

[0177] After bioinformatics processing, a total of 5160 OTUs were identified, which was reduced to 404 OTUs after removing rare sequences. After removing rare sequences, the table density (proportion of non-zero values) was 0.752, and the average number of reads per sample was 82,798 (minimum: 26,610, maximum: 167,912). Taxonomic assignment using the Silva database (v132, 2020) showed that the classification in the dataset was divided into 8 phyla, 33 families, and 84 genera. While 158 OTUs (39%) were assigned to the species level. Bacterial alpha diversity was estimated using the Shannon index to illustrate the abundance and evenness of species present in the sample. Shannon diversity (Shannon, 1997) was estimated at the species level and presented as + / - 95% confidence intervals. To explore the OTU composition when dogs were subjected to altered microbiota composition, non-metric multidimensional scaling (nMDS) was performed on logit alt. transformed data using Bray-Curtis ratios and Phate (Moon et al., 2019).

[0178] Results and discussion

[0179] Microbial diversity

[0180] The estimated mean bacterial diversity in the fecal microbiota was lower when animals received the microbiome supplement components compared to when they received the control diet alone (p=0.023), as measured by the Shannon index. Statistical analysis showed that diversity decreased significantly over time within both feeding phases, but there was no significant interaction between diet and time on diet, suggesting that changes in microbial diversity occurred early in the feeding phase. When receiving the control diet at the end of the 21-day feeding phase (-), several outlying data points showed a decrease in diversity relative to most groups (SI ≤ 2.1), but the changes within groups were less significant ( Figure 4, control—end). This suggests that the variation in fecal diversity within the group was reduced after receiving the dietary supplement. No increase in diversity was observed across the group, suggesting a general stabilizing effect on the microbiota of some animals during the 21-day supplementation period.

[0181] These data are consistent with the observed reduction in diarrheal stools, increased stool consistency, decreased stool pH, and increased transit and bowel movement frequency (see Example 1; "Fiber-based compositions for influencing stool pH, and influencing gastrointestinal health and resistance via intestinal microbiota").

[0182] Bacterial abundance in fecal microbiota

[0183] Due to the number of phylogenies within the microbiota and individual differences in the resident microbiota, consistent differences across groups may be difficult to identify. When animals receive supplements, the average abundance of several microbial families is significantly different. Similarly, these families do not change over time in every case during the feeding phase, and even after a short 2-4 day feeding period, the effect on the estimated group mean is observed. When animals receive supplements, the OTU abundance of Bacterioidaceae associated with insoluble carbohydrate / fiber consumption, leanessness, and solid feces consistency is observed to increase. At the same time, the immunogenic Erysipelotrichaceae is significantly reduced throughout the period when dogs receive microbial composition component supplements. The increase in the immunoactive population in the fecal microbiota of the older group and the significant changes in the blood leukocyte population (lymphocyte data see Example 1) indicate that dietary supplements can also be effective for young animals during immune priming. The Peptostreptococcaeae family, which is associated with human gastrointestinal health due to its role in mucin turnover, was reduced in the animals' feces when the animals received the supplement combination. These data warrant further investigation, as mucin metabolism by Peptostreptococcaeae, as well as other members of the Phylum Firmicutes, can be a rich source of energy for the colonic epithelium and contribute to a positive energy balance in the host. Therefore, these data may suggest an effect on satiety and obesity.

[0184] When considering microorganisms assigned to the genus Peptostreptococcus, the same supplement combination was not associated with a decrease in the abundance of Peptostreptococci relative to the total fecal microbiome ( Figure 6), however, a significant interaction over time was detected in animals that also received a control diet, where abundance decreased over time. This suggests that changes in related organisms with highly similar 16S rDNA sequences may be responsible for the uniformity of the microbiota across stages. Of note is the closely related family Mogibacteriacaeae, which has only minor differences in its 16S rDNA biomarker sequence compared to Peptostreptococcus and has been associated with enhanced energy balance and consequent fatty plaques in red meat from production animals.

[0185] Other contributors to energy balance and immune function were detected in the genus Anaerobiospirillium, where the abundance of these organisms in the fecal communities of animals was consistently increased when receiving a control diet compared to feeding the supplement. Species of bacteria in this genus, such as Anaerobiospirillium succiniproducens, are prolific producers of succinate, a key metabolite in the prevention and treatment of inflammation, bacterial metabolism, and an intermediate in the tricarboxylic acid (TCA) cycle. Finally, Escherichia / Shigella microbial biomarkers from the phylum Proteobacteria are considered to be commensal bacteria with opportunistic pathogenic potential and are genetically identical to many serious gastrointestinal pathogens (by analyzing 16S rDNA biomarker sequences). Similarly, the relative abundance of the Escherichia / Shigella (including E. coli, E. fetus, and all Shigella subtypes) microbiome showed an interaction over time compared to Peptostreptococcus. During the 21-day period when the animals received the control diet, the abundance of Escherichia / Shigella increased relative to the total population, whereas, conversely, levels decreased during the period when the dogs received the supplement ( Figure 6 ). These data again suggest that the role of supplementation in increasing the microbiota in feeding is not limited to older animals but can actively increase and stabilize the colonic microbiota in dogs across the age range, particularly where diarrhea, stool consistency, immune priming, or energy balance are desired physiological effects.

Claims

1. A composition suitable for treating soft stools, loose stools, gastrointestinal disorders or enhancing the resistance of the intestinal microbiome in dogs, the composition comprising the following ingredients: 0.05 g / day to 3.6 g / day of galacto-oligosaccharides, abbreviated as GOS; a total amount of 1.65 g / day to 9.52 g / day of sugar beet pulp; and 5.32 g / day to 25.98 g / day of cellulose, wherein the concentration of the pulp present in the composition is 0.5% w / w to 10% w / w, and wherein the ratio of the ingredients is 1:3.3:0.3 for total sugar beet pulp:cellulose:GOS.

2. The composition of claim 1, suitable for dogs weighing 5 to 10 kg, wherein the ingredients are: a total amount of 1.65 g / day to 5.38 g / day of beet pulp; 5.32 g / day to 12.68 g / day of cellulose; and 0.05 to 3.6 g / day of GOS.

3. The composition of claim 1, suitable for dogs weighing 10 to 25 kg, wherein the ingredients are: a total amount of 2.14 g / day to 6.36 g / day of beet pulp; 6.89 g / day to 15.86 g / day of cellulose; and 0.1 to 3.52 g / day of GOS.

4. The composition of claim 1, suitable for dogs weighing 25 kg or more, wherein the ingredients are: a total amount of 3.72 g / day to 9.52 g / day of beet pulp; 11.95 g / day to 25.98 g / day of cellulose; and 1.44 to 3.52 g / day of GOS.

5. The composition according to any one of the preceding claims 1-4, wherein the composition is a food, a supplement, a nutraceutical or a medicine.

6. The composition according to claim 5, wherein the composition is a nutritionally complete food and at least a portion of the food comprises dry matter.

7. The composition according to claim 6, wherein The nutritional profile of the food comprises, as a percentage of dry matter in the food, 7.5% to 19.8% dietary fiber, 10.0% to 19.8% crude fat, 2.5% to 44.0% protein and 0.08% to 4.4% indigestible protein.

8. The composition of claim 5, wherein the composition is a supplement or a nutraceutical and at least a portion of the supplement or nutraceutical comprises dry matter.

9. The composition according to claim 6, wherein The nutritional profile of the food is 0.9% to 99.9% dietary fiber, 1.0% to 19.8% crude fat, 2.5% to 44.0% protein and 0.08% to 4.4% indigestible protein, based on the percentage of dry matter in the composition, and the sum of the dry matter components is 100%.

10. Use of the composition of any one of claims 1 to 9 in the preparation of a medicament for treating soft stools, loose stools, gastrointestinal disorders or enhancing the resistance of intestinal microbiota in dogs.

11. Use of the composition according to any one of claims 1 to 9 in the preparation of a medicament for lowering the pH of dog feces.

12. A composition as claimed in any one of claims 1 to 9 when prepared for use in increasing the number of Bacteroidetes (Bacteroides spp.) present in the gastrointestinal tract or feces of a dog compared to the number of bacteria present in the dog before administration of the diet. Bacteriodaceae ) and / or Fusobacteriaceae ( Fusobacteriaceae ) and / or for the reduction of Erysipelothrixaceae ( Erysipelotrichaceae ) and / or Peptostreptococcus family ( Peptostreptococcaceae ) of bacterial counts in medicines.

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