Ryan Neil Dilger

Human lactoferrin (hLF) is a glycoprotein of commercial interest as a food ingredient for gut health. We conducted an exploratory analysis comparing the effects of Helaina hLF (effera^®), produced by Komagataella phaffii, and bovine LF (bLF) on the adult gut microbiome and fecal metabolites. In a randomized, double-blind, parallel-arm, controlled trial, 66 healthy adults received either high-dose (HD) effera^® (3.4 g/day), low-dose (LD) effera^® (0.34 g/day), or bLF (3.4 g/day) supplementation for 28 days. Fecal samples collected at Days 0 (baseline), 28, 56, and 84 were analyzed for microbial diversity, taxonomic shifts, and volatile fatty acids (VFA). Alpha-diversity remained stable across all groups. Beta-diversity showed no main effect of treatment; however, bLF was associated with significant visit-related shifts, as assessed by weighted UniFrac. At the phylum level, significant changes associated with effera^® were observed, including decreases in Bacillota (LD) and Verrucomicrobiota (HD), and notable genus-level increases in Lachnospira, Paraprevotella, and Faecalibacterium (HD), while bLF was associated with an increase Bacteroidota at the phylum level and with an increase in the genus Roseburia. Both effera^® and bLF were associated with decreases in Blautia and Dorea. VFA analysis revealed that bLF increased absolute concentrations of total short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs); both effera^® treatments produced proportional changes in SCFAs as a percent of total VFA, acetate, and individual BCFAs. In healthy adults, effera^® supplementation promoted a proportional increase in acetate and supported potentially beneficial taxa while maintaining microbial diversity, without disrupting community structure. As a pre-specified exploratory outcome, microbiome and VFA findings should be interpreted as hypothesis-generating. (clinicaltrials.gov: NCT06012669).

Mushroom-derived β-glucans and bovine colostrum have potential roles in modulating gut health and immune function, but limited data are available regarding their effects on the gut microbiome, microbial metabolites, and immune responsiveness in adult dogs. Twenty healthy adult beagle dogs (8 females, 12 males; age: 5.6 ± 2.5 yr old; body weight: 9.55 ± 0.92 kg; body condition score: 5.7 ± 0.5) were used in a randomized, blinded, placebo-controlled study. After a 3-wk wash-in phase, dogs were assigned to one of two treatment groups (n = 10/group) and fed for 12 wk: placebo consisting of cellulose, rice bran, liver, and natural food coloring or a mushroom complex consisting of mushroom (turkey tail mushroom, red reishi, and lion’s mane) water extracts and bovine colostrum (MC). Each treatment (1.8 g/d) was top dressed on the diet. Fecal samples (characteristics, microbial fermentation metabolites, immunoglobulin A, and microbiota) and blood samples (serum chemistry, hematology, immunoglobulins, oxidative stress markers) were collected after the wash-in phase (wk 0) and after 6 and 12 wk. Immune function was evaluated ex vivo by stimulating whole blood cells with toll-like receptor agonists and measuring tumor necrosis factor-alpha production. Data were analyzed using mixed models, using baseline data used as a covariate. Statistical significance was set at P < 0.05 and trends were considered at P < 0.10. All dogs remained healthy throughout the study, with fecal characteristics, serum chemistry, hematology, and serum immunoglobulins and oxidative stress markers being unchanged. Dogs fed MC had greater fecal acetate, propionate, and total short-chain fatty acid concentrations than controls. Fecal bacterial alpha and beta diversity metrics remained stable over time. Significant treatment*time interactions were observed for five fecal bacterial genera (Atopobiaceae unclassified, Prevotella_9, Lactobacillus, Megamonas, and Phascolarctobacterium). Relative abundances of fecal Candidatus_Stoquefichus, Epulopiscium, and Negativibacillus tended to be increased by MC (P < 0.10). The relative abundances of several genera were affected by time. In summary, the mushroom complex was well tolerated and did not adversely affect gut health, immune status, or microbial diversity in healthy dogs. Our results suggest that it may slightly alter gut microbiota populations and microbial fermentation metabolites.

The objectives of this study were to determine how diets distinctly processed from the same raw batch to mimic common pet food formats may affect apparent total tract macronutrient digestibility (ATTD), gastrointestinal transit time (GTT), and fecal characteristics, metabolites, and the microbiota of healthy dogs. Ten spayed female adult beagles (age: 6.10 ± 1.20 yr; body weight: 8.63 ± 0.77 kg) were used in a replicated 5 × 5 Latin square design to test the following diet formats: retorted (RT), mildly cooked (sous vide [SV], steamed [ST]), and raw (high-pressure processed [HPP], freeze-dried [FD]). The study comprised five 28-d periods, including a 7-d diet transition, 14-d treatment, and 7-d collection phase. All feces excreted during the collection phase were pooled and analyzed for dry matter (DM), organic matter (OM), crude protein (CP), lipids, and gross energy. On the first day of fecal collections, one fresh fecal sample was collected to measure pH, DM, fermentative metabolite concentrations, and microbiota composition. Data were analyzed by Mixed Models using SAS 9.4, with P < 0.05 being statistically significant. Dogs consuming the RT and FD diets had a higher (P < 0.05) as-is fecal output to DM intake ratio than dogs consuming the ST and HPP diets. The HPP and ST diets had higher (P < 0.05) DM and OM ATTD than the RT, FD, and SV (ST not different) diets. All diets had greater (P < 0.05) CP ATTD than the RT diet and higher (P < 0.05) fat ATTD than the FD diet. Energy ATTD was higher (P < 0.05) in the ST, HPP, and SV diets than in the FD and RT (SV not different) diets. Fecal acetate and butyrate concentrations were higher (P < 0.05) in dogs consuming the RT diet than the ST and SV (butyrate only) diets. Fecal isobutyrate, isovalerate, total branched-chain fatty acid, indole, and ammonia concentrations were higher (P < 0.05) in dogs consuming the RT diet than all other diets (ST not different for indole and ammonia). Alpha and beta diversity measures were not impacted, but diet altered the relative abundances of three bacterial phyla and 19 genera (P < 0.05). In summary, processing method influenced nutrient digestibility as well as the fecal metabolite concentrations and microbiota populations of dogs, but did not impact GTT, fecal score, pH, DM, and alpha and beta diversity measures. More research is needed to evaluate additional factors such as packaging size, ingredient inclusion levels, macronutrient concentrations, different processing parameters, and other formats.

Mushroom complex (MC) is a blend of mushroom water extracts including β-glucans and bovine colostrum that may provide benefits to cats. The objective of this study was to test the effects of MC supplementation on gut health and immune function outcomes of adult cats. Twenty healthy adult cats (7.40 ± 1.54 yr old; 4.25 ± 0.62 kg) were used in a randomized, blinded, placebo-controlled study using a completely randomized design. After a 3-wk wash-in phase, cats were assigned to one of two treatment groups (n = 10/group) and fed for 12 wk: placebo consisting of cellulose, rice bran, liver, and natural food coloring or MC consisting of mushroom (turkey tail mushroom, red reishi, and lion’s mane) water extracts and bovine colostrum. Each treatment (900 mg/d) was top-dressed on the diet. Fecal samples [characteristics, metabolites, microbiota, immunoglobulin (Ig) A] and blood samples (serum chemistry, hematology, oxidative stress markers, immunoglobulins) were collected after the wash-in phase (wk 0) and after 6 and 12 wk. Data were analyzed using the GLIMMIX model of SAS, with P < 0.05 accepted as significant and P < 0.10 as trends. Fecal characteristics (pH, dry matter, scores, frequency) were not affected by treatment, but treatment-time interactions (P < 0.05) were observed for serum IgA and blood urea nitrogen (BUN). Serum IgA increased over time in controls but decreased over time in cats fed MC. Serum BUN tended to increase in all cats over time but to a higher level in cats fed MC. Serum triglycerides were lower (P < 0.05) in cats fed MC. Fecal ammonia, isovalerate, and total branched-chain fatty acids were greater (P < 0.05) in cats fed MC than controls. A treatment-time interaction (P < 0.05) was observed for alpha diversity measures (Fisher’s Alpha, Shannon Diversity Index, and Observed Features), with all being reduced over time but to a lower extent in cats fed MC. The weighted PCoA plot for fecal beta diversity highlighted a significant treatment-time interaction, with some shifts in the microbiome being observed. The relative abundance of fecal Ruminococcus torques remained fairly stable over time in controls but increased in cats fed MC, while fecal Fusobacterium remained stable in cats fed MC but decreased in controls. Over 30 bacterial genera were affected by time. Our results suggest that without affecting fecal characteristics, MC supplementation slightly modified fecal microbiota and metabolite concentrations and reduced serum triglycerides in cats.

Background/Objectives: Human lactoferrin (hLF) is glycoprotein of commercial interest as a food ingredient for gut health. Here, we report an exploratory analysis evaluating the effects of Helaina hLF (effera ^® ), produced by Komagataella phaffii , on the adult gut microbiome and fecal metabolites in comparison to bovine LF (bLF). Methods: In a randomized, double-blind, parallel-arm, controlled trial, 66 healthy adults received either high-dose (HD) effera ^® (3.4 g/day), low-dose (LD) effera ^® (0.34 g/day), or bLF (3.4 g/day) supplementation for 28 days. Fecal samples were collected at baseline (Day 0), Day 28, Day 56, and Day 84 and analyzed for microbial diversity, taxonomic shifts, and volatile fatty acids (VFA). Results: Alpha-diversity remained stable across all groups. Beta-diversity showed no main effect of treatment; however, bLF was associated with significant visit-related shifts, as assessed by weighted UniFrac. At the phylum level, significant changes associated with effera ^® were observed, including decreases in Bacillota (LD) and Verrucomicrobiota (HD), and notable genera increases in Lachnospira , Paraprevotella , and Faecalibacterium (HD), while bLF was associated with an increase in Roseburia . Both effera ^® and bLF were associated with decreases in Blautia and Dorea . VFA analysis revealed that bLF increased absolute total short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) concentrations, while both effera ^® groups produced proportional changes in SCFAs, individual BCFAs, and acetate. Conclusions: In healthy adults, effera ^® supplementation promoted a proportional increase in acetate and supported potentially beneficial taxa while maintaining microbial diversity, without disrupting community structure. (clinicaltrials.gov: NCT06012669 ).

The increasing demand for functional ingredients has drawn attention to yeast and yeast-derived ingredients in pet foods. This experiment aimed to evaluate the in vitro fermentation characteristics of common dietary fibers using fecal inoculum from dogs fed either a control diet (CTRL) or a diet supplemented with dried brewers yeast at 1.5% inclusion (BY). Sixteen healthy adult dogs were acclimated for 7 d and then randomly assigned to the two treatments (n = 8/group) for 21 d. Fecal samples collected at the end of the treatment period, were preserved in 20% glycerol and later used as inoculum for in vitro fermentation of beet pulp, pectin, and cellulose over 0, 6, 12, or 18 h. Fermentation patterns differed among fiber substrates. Pectin was highly fermentable, beet pulp showed moderate fermentation, and cellulose was minimally fermented, as indicated by SCFA production and pH changes. Within each fiber, BY inocula produced more SCFA and a lower pH than CTRL after 18 h, with the largest SCFA differences observed in beet pulp tubes (3549 vs. 2980 μmole/g) and the greatest pH differences in pectin tubes (−1.66 vs. –1.54). Microbiota analysis revealed increased relative abundances of SCFA-producing genera, along with reduced relative abundances of Fusobacterium, Streptococcus, and Sutterella. Overall, dried brewers yeast supplementation can influence canine gastrointestinal microbiota composition and fermentative activity in vitro.