Anti-aging food that improves markers of health in senior dogs by modulating gut microbiota and metabolite profiles

Dysbiosis is one of the major changes in aging that leads to an accumulation of toxic microbial metabolites. The aim of this study was to evaluate the effect of a test food containing components of citrus, carrot, spinach and tomato on gut microbiota and age-related metabolites in senior dogs. The study was conducted on 36 dogs between 8 and 13 years of age. All dogs were maintained on a control food (control 1), which used corn as major source of fiber. After 30 days, the dogs were divided into two groups of 18 dogs. One of the groups received the test food for 30 days while the other group received the control 2 food, containing multiple whole grains as the test food but without the above added sources of fiber present in the test food. After a washout period on the control 1 food for 30 days, a cross-over was performed so that the test or the control 2 food was fed for 30 days to those dogs which had not yet been fed that food. Samples from feces and blood were collected after each 30 days period to analyze changes in gut microbial composition and metabolites. The consumption of the test food led to increased proportions of Adlercreutzia, Oscillospira, Phascolarcobacteria, Faecalibacterium and Ruminococcus, Christensenellaceae, Ruminococcaceae, Cyanobacteria and Acidobacteria and decreased proportions of Megamonas, Salmonella, Enterobacteriaceae and Fusobacterium. Pets had higher levels of glycerol and fatty acids and lower levels of pyrraline and mucin amino acids in feces. The test food also reduced circulating levels of pyrraline, symmetric dimethylarginine and phenolic uremic toxins, including the microbial brain toxin, 4-ethylphenyl sulfate. Christensenellaceae abundance was strongly associated with the observed health benefits. Fermentable fibers from fruits and vegetables enhance health in senior dogs by modulating the gut bacteria and metabolites involved in aging, kidney, brain and gut health.


Introduction
Aging is associated with shifts in the composition of gut microbiota. An example of this is the 42 increase in the number of facultative anaerobes and a decline in the proportion of beneficial bacteria 43 associated with aging (1,2). This shift in the microbial composition leads to the accumulation of toxic 44 microbial metabolites in the body causing inflammation, oxidative stress and contributing to various 45 diseases prominent in the aging condition (3). The reduction in the proportion of beneficial bacteria 46 may lead to constipation, mal-absorption and longer colonic transit time. Decreased absorption of 47 dietary protein in the upper intestine and longer colonic transit times encourage increased abundance 48 of proteolytic bacteria, whose fermentation products deteriorate intestinal barrier integrity (4). 49 50 Foods containing fermentable fibers are known to benefit dogs by increasing nutrient 51 absorption and reducing enteric infection (5). In an in vitro study, Swanson et al. (6) confirmed the 52 fermentability of fruits and vegetables by canine fecal microflora with the resulting production of short 53 chain fatty acids. This study evaluates the effect of a test food containing components of citrus, carrot, 54 spinach and tomato on the microbial composition as well as metabolites associated with aging, kidney, 55 brain and gut health in senior dogs. A recent study by Hall et al (7) showed that the consumption of a 56 food with similar composition as the test food employed in the current study led to improvement of 57 markers of kidney health in geriatric dogs with early stage kidney disease. This study was designed to 58 evaluate changes in fecal microbial composition and age-related markers of health attributed to the 59 consumption of the test food by healthy senior dogs.  All dogs were spayed or neutered. A summary of the description of the dogs included in this study is 70 shown in Table 1.   The reads were de-multiplexed using Miseq built-in workflow to obtain FASTQ files 129 processed using Mothur, version 1.32 (10). Sequences were retained based on criteria such as having   Phascolarctobacterium increased by 77% and 68%, respectively. and Cyanobacteria and led to a significant reduction in Fusobacteria.

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Although they did not meet the statistical significance, Oscillospira and Ruminococcus 190 increased by 73% and 39%, respectively, after the consumption of the test food. This was 191 accompanied by reductions in the proportions of the genera Salmonella, Megamonas,192 Peptostreptococcus and an unknown genus (OTU_10001 in the family Enterobacteriaceae) by 58%, 193 81%, 32%, 80%, respectively. S1 Table provides    Changes that were statistically significant (P<0.05) are marked grey.  The relative fecal levels of amino acids that make up the mucin layer, such as aspartate, 214 proline, serine and threonine were significantly affected by the type of food consumed by the senior 215 dogs. Compared to the control 2 food, the consumption of the test food led to a 28 -61% reduction in 14 216 levels of these amino acids in the feces (Table 5).  Changes that were statistically significant are marked grey.

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Changes in circulating uremic toxins 235 Uremic toxins are among the major toxic metabolites that lead to renal and associated diseases 236 in aging. Some uremic toxins originate from protein fermentation in the colon by proteolytic bacteria.

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Products of the putrefaction process are absorbed and converted to toxic derivatives causing an 238 increased burden on kidney function. We detected a total of 14 phenolic and indolic uremic toxins in 239 plasma (Fig 2)  and methylation of arginine (15,16). The test food resulted in a significant reduction in blood 256 concentrations of SDMA (P=0.035, SE=0.2) in the senior dogs compared to the control 2 food (Fig 3).   Table 7). The genera Oscillospira and Adlercreutzia also had a negative  (Table 7).  properties and prevent various age-related diseases including diabetes and obesity (18)(19)(20). Equol is 282 also associated with a decreased risk of certain types of cancer; therefore increasing the abundance of 283 equol-producing gut microbiota has been recommended to reduce this risk (21). In this study, although Although not statistically significant, the relative proportions of the genera Ruminococcus and 315 Oscillospira increased by 43% and 73%, respectively, after the consumption of the test food. Both 316 genera belong to the family Ruminococcaceae, which increased significantly after the consumption of 317 the test food. These bacteria are known to produce short-chain fatty acids (SCF) that are beneficial to 318 the host mainly due to their anti-inflammatory effects (28,29). They also serve as an energy source for 319 enterocytes, regulate intestinal motility and ameliorating leaky gut syndrome (30). Bacteria in the 320 genus Ruminococcus are fiber degraders and major producers of butyrate, which serves as an energy 21 321 source for intestinal epithelial cells and has anti-inflammatory effects (31). Members of the genus also 322 produce bacteriocins, which have anti-microbial effects against a wide variety of pathogenic bacteria 323 (32).

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Oscillospira are known to produce butyrate by relying on fermentation products secreted by 325 other bacterial species (33). In humans, Oscillospira have been associated with leanness or lower 326 body mass index in both infants and adults (34,35).

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The consumption of the test food reduced levels of the advanced glycation end product, 22 344 pyrraline. AGE are a complex group of compounds derived from the non-enzymatic glycation of 345 proteins, lipids, and nucleic acids. They can also be acquired from food; thus restriction of foods with 346 high levels of AGE has been recommended to decrease circulating AGE in the body (40). AGE are 347 known to accelerate the process of aging and they are linked to a number of age-related diseases such 348 as diabetes, vascular and renal diseases mainly by inducing inflammation and oxidative stress (41,42). 349 Fecal microorganisms have been shown to be capable of degrading various AGE including pyrraline the level of CML in the blood did not change. This may suggest that fecal levels of CML may not be 360 biologically significant.

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The level of glycerol in the feces of the senior dogs was higher when they were fed the test 362 food. Glycerol is known to increase water retention in the colon and thus it is used to treat constipation 363 (45). Prolonged transit times are risks to develop various diseases due to the exposure to toxic 364 products accumulating due to putrefaction (46). A shorter transit time leads to a limited accumulation 365 of such products that may cause various diseases (46). People with functional constipation have been 366 shown to contain bacteria with more abundant genes to degrade glycerol (47). The strong negative  (Table   381 2), the levels of these fatty acids in feces increased after the consumption of the test food. This 382 supports the possible increased microbial lipase activity attributed to the change in the microbial 383 composition.

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Uremic toxins are among the major metabolites that cause age-related complications. There 385 was a marked improvement in markers of kidney health after the consumption of the test food.

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Circulating symmetric dimethylarginine (SDMA) has been shown to be a good biomarker of kidney 387 function in dogs as it detects reduction in glomerular filtration rate (GFR) much earlier than serum 388 creatinine (16). The reduction in circulating concentration of SDMA in the senior dogs after the 389 consumption of the test food may thus indicate an increased GFR and improved kidney function.
Furthermore, the test food reduced several phenolic uremic toxins originating from microbial 391 fermentation of protein (49). One of these metabolites was 4-ethylphenyl sulfate (4-EPS), which is 392 also known to have a negative impact on brain health by causing anxiety-like symptoms (50,51). The 393 role of foods containing fruits and vegetables in improving kidney health has been reported (7,52).

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The significant reduction in such metabolites in the senior dogs after the consumption of the test food 395 may be due to the changes in the microbial composition. The two indolic uremic toxins, 5-396 hydroxyindole sulfate and 7-hydroxyindole sulfate, increased after the consumption of the test food.

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Indolic uremic toxins originate from colonic fermentation of the amino acid tryptophan (53). The test 398 food was formulated to contain 53% higher tryptophan compared to the control 2 food. The presence 399 of more substrate may have led to increased levels of the two indolic metabolites. However, the typical 400 indolic uremic toxin, 3-indoxyl sulfate, was not affected by the consumption of the different diets.

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In conclusion, old dogs fed fiber sources from vegetables and fruits containing high soluble 402 fiber benefit by having a gut microbial composition promoting healthier metabolic profiles.