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Journal of Animal Science logoLink to Journal of Animal Science
. 2023 May 19;101:skad160. doi: 10.1093/jas/skad160

Effects of a Lactobacillus fermentation product on the fecal characteristics, fecal microbial populations, immune function, and stress markers of adult dogs

Samantha A Koziol 1, Patricia M Oba 2, Katiria Soto-Diaz 3, Andrew J Steelman 4,5, Jan S Suchodolski 6, Erik R M Eckhardt 7, Kelly S Swanson 8,9,10,✉
PMCID: PMC10237232  PMID: 37208000

Abstract

The objective of this study was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, blood biomarkers, immune function, and serum oxidative stress markers of adult dogs. Thirty adult beagle dogs [23 M, 7 F; mean age = 8.47 ± 2.65 yr old; mean BW = 15.43 ± 4.17 kg] were used in a completely randomized design study. All dogs were fed a basal diet to maintain BW for 5 wk, followed by baseline blood and fecal sample collections. Dogs remained on the same diet, but then were randomly assigned to a placebo (dextrose) or LBFP supplement (Limosilactobacillus fermentum and Lactobacillus delbrueckii). Both treatments were dosed at 4 mg/kg BW via gelatin capsule for 5 wk (n = 15/treatment). Fecal and blood samples were collected at that time. Change from baseline data were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant and P < 0.10 being trends. Most circulating metabolites and immunoglobulins (Ig) were unaltered by treatment, but LBFP-supplemented dogs had lower changes in serum corticosteroid isoenzyme of alkaline phosphatase (P < 0.05), alanine aminotransferase (P < 0.10), and IgM (P < 0.10) than controls. The change in fecal scores tended to be lower (P = 0.068) in LBFP-supplemented dogs than controls, signifying firmer feces in LBFP-supplemented dogs. Regarding the fecal microbiota, alpha diversity indicators tended to be higher (P = 0.087) in LBFP-supplemented dogs than controls. One fecal bacterial phylum (Actinobacteriota) was altered by treatments, with its relative abundance tending to have a greater (P < 0.10) increase in controls than LBFP-supplemented dogs. Fifteen bacterial genera were altered (P < 0.05 or P < 0.10) by treatments, including relative abundances of fecal Peptoclostridium, Sarcina, and Faecalitalea that had a greater (P < 0.05) increase in controls than LBFP-supplemented dogs. In contrast, relative abundances of fecal Faecalibaculum, Bifidobacterium, and uncultured Butyricicoccaceae had a greater (P ≤ 0.05) increase in LBFP-supplemented dogs than controls. After week 5, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The change in serum superoxide dismutase after transport had a greater (P < 0.0001) increase in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when subjected to stress.

Keywords: canine nutrition, gastrointestinal health, postbiotic

In this study, a Lactobacillus fermentation product was tested in adult dogs and shown to stabilize stool quality, beneficially shift fecal microbiota populations, and modulate oxidative stress markers after transport stress.

Introduction

Functional ingredients are increasingly incorporated into pet diets to support health, with products targeting digestive health being quite popular. Several categories of digestive health products exist. Dietary fibers are effectively used in pet foods to maintain or improve stool characteristics, provide laxation, and modulate gastrointestinal microbiota populations (Gallaher, 2000; Fahey et al., 2004). A variety of biotics, including probiotics (i.e., live microbes; Hill et al., 2014), prebiotics (i.e., substrates for beneficial gut microbiota; Gibson et al., 2017), and synbiotics (i.e., a mixture of live microbes and substrates; Swanson et al., 2020), have also been highly studied and widely used to provide health benefits to pets over the past few decades. Although many potential mechanisms exist, biotics are thought to benefit gut health through the production of short-chain fatty acids (SCFA) and other metabolites that can aid in pathogen resistance and increase the growth of beneficial microbes.

In the past few years, another type of biotic substance (i.e., postbiotic) has increased in popularity. A postbiotic is defined as “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host” (Salminen et al., 2021). Many ingredients that may be classified postbiotics are already approved by the Association of American Feed Control Officials (AAFCO, 2022). Postbiotics may be derived from yeast (i.e., yeast culture), bacteria (i.e., dried fermentation product), or fungi (i.e., dried fermentation product), with their functionality depending on the strains incorporated, their growth conditions, and the processes used for inactivation. Many studies have reported that postbiotics may confer comparable health benefits as live organisms, while allowing for easier storage and incorporation into processed food products (Sugawara et al., 2016; Liu et al., 2022; Vinderola et al., 2022). Postbiotics may provide benefits by altering the gut microbiota, host intestinal barrier function and metabolism, immune regulation, and/or systemic signaling (Salminen et al., 2021).

Common microbes utilized as probiotics and targeted by prebiotics are lactic acid-producing bacteria (LAB). In general, LAB are commensal bacteria in the gastrointestinal tract and have been correlated with health when consumed as part of the diet or as a supplement. These bacteria have a broad range of benefits, including the potential to limit pathogenic bacteria growth, lower cholesterol concentrations, and modulate brain activity (Gilliland, 1990; Kapila et al., 2007; Kiso et al., 2013; Tillisch et al., 2013). Importantly, there are microbial strain differences in regard to their functionality, mechanisms of action, and effects on the host. While LAB are commonly used as probiotics, research has discovered that their benefits are also possible after the bacteria have been made nonviable. Various studies focused on heat-killed Lactobacillus have revealed that these products possess microbiome- and immune-modulating capabilities in addition to benefits pertaining to feed conversion and diarrhea incidence in livestock species and humans (Sugawara et al., 2016; Canani et al., 2017; Burdick Sanchez et al., 2019; Seong et al., 2021). Most benefits of feeding a fermentation product are attributed to these bioactive metabolites instead of the direct effects of the bacteria itself (Mathur et al., 2020).

Lactobacillus fermentation product (LBFP) consists of two proprietary Lactobacillus bacterial strains (Limosilactobacillus fermentum and Lactobacillus delbrueckii) and culture media that have been heat-killed at the end of an optimized fermentation process, creating a stable powder. LBFP falls under the AAFCO definition of a dried Lactobacillus fermentation product (AAFCO 36.11). In addition to the nonviable bacterial cells it contains, there are many constituents of the bacterial fermentate, including the various metabolites produced by the microbes during the fermentation process. Despite the advances that have occurred in recent years, there is more to understand in regard to the mechanisms by which postbiotics function as well as determining the dosages that are effective and safe, especially in companion animal species. The objective of this study was to determine the effects of LBFP supplementation on the fecal characteristics, fecal microbiota, immune function, and stress response of adult dogs. We hypothesized that dogs supplemented with LBFP would exhibit beneficial shifts in fecal microbial populations characterized by an increase in relative abundance of generally beneficial bacteria (e.g., Prevotella, Lactobacillus, and Bifidobacterium) and/or a decrease in proteolytic bacterial taxa (Warda et al., 2019, 2021). Additionally, we hypothesized that LBFP would modulate immune cell functionality and immunoglobulin (Ig) concentrations. Finally, we hypothesized that dogs supplemented with LBFP would have reduced markers of oxidative stress and increased antioxidant activity compared with controls when subjected to travel stress.

Experimental Design and Methods

All experimental procedures were approved by the Kennelwood Inc., IACUC prior to experimentation and were performed in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Animals and diets

Thirty adult beagle dogs (23 M, 7 F; 8.47 ± 2.65 yr; 15.43 ± 4.17 kg) were used in a completely randomized design study. All dogs were housed individually in an environmentally controlled animal facility. Dogs had free access to fresh water at all times and were fed once a day to maintain body weight (BW) throughout the study. BW and body condition scores were measured once a week. All dogs were fed an extruded kibble diet (Purina ONE Adult Chicken and Rice Formula; Nestlé Purina PetCare, St. Louis, MO; Supplementary Table S1) for 5 wk and then allotted to one of two treatments based on age, BW, and sex: basal diet + a placebo control (4 mg dextrose/kg BW/d) (11 M, 4 F; 8.60 ± 2.18 yr; 15.95 ± 4.61 kg) or the basal diet + 4 mg LBFP/kg BW/d (12 M, 3 F; 8.33 ± 3.05 yr; 14.90 ± 3.60 kg). The LBFP tested is a postbiotic that contains 60 billion heat-killed bacterial bodies of L. fermentum and L. delbrueckii along with spent fermentation medium (Adare Biome, Houdan, France). The treatments were provided via gelatin capsules once daily prior to feeding. Dosing was derived from data in growing pigs (unpublished data).

Experimental timeline

Prior to the study, blood samples were collected for serum chemistry and complete blood count measures and a physical exam was conducted by a veterinarian to confirm health. A 5-wk adaptation phase, whereby all dogs ate the basal diet at a rate to maintain BW, preceded the study. After the adaptation phase, fecal and blood samples were collected. Dogs were then allotted to treatments and fed for an additional 5 wk. At the end of the treatment phase, fecal and blood samples were collected. Dogs were then placed into individual dog carriers, loaded into a van, and transported for approximately 45 min to evaluate the effects of travel stress. Blood samples were collected prior to and 3 h after transport for cortisol, malondialdehyde (MDA), and superoxide dismutase (SOD) measurements. Saliva samples were collected 15 min after transport for cortisol measurement.

Diet chemical analyses

The diet was ground in a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ) through a 2-mm screen and then analyzed for dry matter and ash according to AOAC (2006; methods 934.01 and 942.05), with organic matter being calculated. Crude protein was calculated from Leco (FP2000 and TruMac) total nitrogen values according to AOAC (2006; method 992.15). Total lipid content (acid-hydrolyzed fat) was determined according to the methods of theAmerican Association of Cereal Chemists (1983) and Budde (1952). Total dietary fiber was determined according to Prosky et al. (1988). Gross energy was measured using an oxygen bomb calorimeter (model 6200, Parr Instruments, Moline, IL).

Blood collection and analysis

Overnight fasted blood samples were collected via jugular puncture. Samples were immediately transferred to appropriate vacutainer tubes, including some blood into plasma tubes containing heparin (BD Vacutainer Sodium Heparin Tubes #366480; Becton Dickinson, Franklin Lakes, NJ) for peripheral blood mononuclear cell (PBMC) collection, some blood into whole blood tubes containing K2EDTA additive (BD Microtainer Tubes #363706; Becton Dickinson) for complete blood count analyses, and some blood into serum tubes containing a clot activator and gel for serum separation (BD Vacutainer SST Tubes #367988; Becton Dickinson) for serum chemistry profile, Ig, cortisol, and oxidative stress marker analyses. Serum was isolated by centrifuging tubes at 2,000 × g at 4 °C for 15 min (Beckman CS-6R centrifuge; Beckman Coulter Inc., Brea, CA). Some aliquots of the fresh extracted serum were stored at −80 °C until analysis, while some were transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for serum chemistry analysis. Samples in K2EDTA tubes were cooled (but not frozen) and submitted to the University of Illinois Veterinary Medicine Diagnostics Laboratory for complete blood count analysis.

PBMC were separated using histopaque and centrifugation. Once PBMC were collected, the responsiveness of lymphocytes to Toll-like receptor (TLR) agonists, including zymosan (TLR2 agonist; mimics yeast challenge), polyinosinic–polycytidylic acid sodium salt (TLR3 agonist; mimics viral challenge), lipopolysaccharides (TLR4 agonist; mimics bacterial challenge), and R848 (TLR7/8 agonist; mimics viral challenge) was assessed by measuring tumor necrosis factor-alpha (TNF-α) production using commercial ELISA kits (R&D Systems CATA00; Minneapolis, MN). Circulating oxidative stress markers, namely MDA (MyBioSource MBS2605193; San Diego, CA) and SOD (MyBioSource MBS2104718), as well as IgA (MyBioSource MBS018650), IgE (MyBioSource MBS2605261), IgG (MyBioSource MBS2700205), and IgM (MyBioSource MBS2606734) concentrations were measured using commercial ELISA kits.

Saliva collection and analysis

During the transport stress phase, saliva was collected using of SalivaBio Oral swabs (Salimetrics 5001.06; Salimetrics, LLC, Carlsbad, CA). Collected swabs were stored at −20 °C until analysis. Samples were used to measure pre and post-transport salivary cortisol concentrations using a commercial ELISA kit (Salimetrics 1-3002; Salimetrics, LLC).

Fecal collection and analysis

During the fecal collection phases, fecal samples were scored according to the following scale: 1 = hard, dry pellets, small hard mass; 2 = hard, formed, dry stool; remains firm and soft; 3 = soft, formed, and moist stool, retains shape; 4 = soft, unformed stool, assumes shape of container; and 5 = watery, liquid that can be poured. During the fecal collection phases, one fresh fecal sample (within 15 min of defecation) was collected for microbiota, pH, dry matter, and IgA analysis. Fecal pH was measured immediately using an AP10 pH meter (Denver Instrument, Bohemia, NY) equipped with a Beckman Electrode (Beckman Instruments Inc., Fullerton, CA), and then aliquots were collected. Aliquots of fresh feces were immediately transferred to sterile cryogenic vials (Nalgene, Rochester, NY), snap-frozen in liquid nitrogen, and stored at −80 °C until microbiota and IgA analysis. The remainder of feces was retained in a whirlpak bag and frozen at −20 °C until fecal dry matter determination.

Fecal IgA concentrations

Fecal proteins were extracted according to Vilson et al. (2016). Fecal samples (250 mg) were vortexed with 750 µL extraction buffer containing 50 mM-EDTA (ThermoFisher, Waltham, MA) and 100 µg/L soybean trypsin inhibitor (Sigma, St. Louis, MO) in PBS/1 percent bovine serum albumin (Tocris Bioscience, Bristol, UK). Phenylmethanesulphonyl fluoride (12.5 µL, 350 mg/L; Sigma) were added into each tube, followed by centrifugation for 10 min. The supernatants were collected for measurement of IgA using a commercial ELISA kit (MyBioSource MBS018650).

Fecal DNA extraction and MiSeq Illumina sequencing of 16S amplicons

Total DNA from fecal samples was extracted using DNeasy PowerLyzer PowersSoil Kits (Qiagen, Valencia, CA). Concentrations of extracted DNA samples were quantified using a Qubit 3.0 Fluorometer (Life Technologies, Grand Island, NY). 16S rRNA gene amplicons were generated using a Fluidigm Access Array (Fluidigm Corporation, South San Francisco, CA) in combination with Roche High Fidelity Fast Start Kit (Roche, Indianapolis, IN). The primers 515F (5ʹ-GTGCCAGCMGCCGCGGTAA-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ) that target a 252-bp-fragment of the V4 region of the 16S rRNA gene were used for amplification (primers synthesized by IDT Corp., Coralville, IA) (Caporaso et al., 2012). CS1 forward tag and CS2 reverse tag were added according to the Fluidigm protocol. Quality of the amplicons was assessed using a Fragment Analyzer (Advanced Analytics, Ames, IA) to confirm amplicon regions and sizes. A DNA pool was generated by combining equimolar amounts of the amplicons from each sample. The pooled samples were then size selected on a 2% agarose E-gel (Life Technologies) and extracted using a Qiagen gel purification kit (Qiagen). Cleaned size-selected pooled products were run on an Agilent Bioanalyzer to confirm appropriate profile and average size. Illumina sequencing was performed on a MiSeq using v3 reagents (Illumina Inc., San Diego, CA) at the Roy J. Carver Biotechnology Center at the University of Illinois.

Microbial data analysis

Forward reads were trimmed using the FASTX-Toolkit (version 0.0.14) and QIIME 2.0 (Bokulich et al., 2017)was used to process the resulting sequence data. Briefly, high-quality (quality value ≥ 20) sequence data derived from the sequencing process were demultiplexed. Data were then denoised and assembled into amplicon sequence variants using DADA2 (Callahan et al., 2016). The SILVA 132 database (Quast et al., 2013) was used to assign taxonomy. An even sampling depth (10,755 sequences per sample) was used for assessing alpha- and beta-diversity measures. Beta-diversity was assessed using weighted and unweighted UniFrac (Lozupone and Knight, 2005)distance measures and presented using principal coordinates analysis (PCoA) plots.

Quantitative polymerase chain reaction (qPCR) analysis

qPCR analysis of selected bacterial groups commonly present in the gastrointestinal tract of dogs was performed with specific primers targeting Faecalibacterium, Fusobacterium, Blautia, universal bacteria, Turicibacter, Escherichia coli, Clostridium hiranonis, and Streptococcus as described by AlShawaqfeh et al. (2017). Briefly, the conditions for qPCR were as follows: initial denaturation at 98 °C for 2 min, then 40 cycles with denaturation at 98 °C for 3 s, and annealing for 3 s (see Supplementary Table S2 for specific annealing temperatures for the final qPCR panel). Melt curve analysis was performed to validate the specific generation of the qPCR product using these conditions: 95 °C for 1 min, 55 °C for 1 min, and increasing incremental steps of 0.5 °C for 80 cycles for 5 s each. Each reaction was run in duplicate. The qPCR data were expressed as the log amount of DNA (fg) for each particular bacterial group/10 ng of isolated total DNA as reported previously (Suchodolski et al. 2012; Panasevich et al. 2015).

Statistical analysis

Data were analyzed using the Mixed Models procedure of SAS (SAS Institute, Inc., Cary, NC). The fixed effect of ­treatment was tested, while dog was considered a random effect. Data were tested for normality using the UNIVARIATE procedure of SAS. Log transformation was used when normal distribution was lacking. If after the logarithmic transformation of the data, the data did not reach normality, the data were analyzed using the npar1way procedure and Wilcoxon statistic. Regardless of transformation, nontransformed least squares means were reported. Statistical significance, however, was dependent on transformations. Differences between treatments were determined using a Fisher-protected least significant difference with a Tukey adjustment to control for experiment-wise error. A probability of P < 0.05 was accepted as statistically significant, with P < 0.10 being considered trends. Reported pooled standard errors of the mean was determined according to the Mixed Models procedure of SAS.

Results

All baseline data were analyzed and differences between groups are noted in Supplementary Tables S3 to S9 and Figure S1. Most baseline measures were not different (P > 0.05) between groups. One exception included serum creatinine (Supplementary Table S3), but it was relatively minor and all dogs were within reference ranges. The other exception was fecal Slackia relative abundance (Supplementary Table S9).

There were no significant changes in BW, body condition score, or food intake from baseline to the end of the study (data not shown). Change from baseline serum metabolite concentrations are presented in Figure 1 and Supplementary Table S10. While most metabolites were unaltered by treatment, dogs supplemented with LBFP had lower (P < 0.05) change in corticosteroid isoenzyme of alkaline phosphatase (CALP). Dogs supplemented with LBFP also tended to have a lower (P < 0.10) change in alanine aminotransferase (ALT) concentrations than controls. While CALP and ALT concentrations increased from baseline of control dogs (6.07 and 21.43 U/L, respectively), those supplemented with LBFP had reductions (−1.53 and −4.33 U/L, respectively). Blood cell counts and percentage were not altered by treatment, with change from baseline values presented in Supplementary Table S11. Change from baseline serum Ig concentrations are presented in Table 1. Dogs supplemented with LBFP tended to have a lower (P = 0.068) change in circulating IgM ­concentrations than controls. The other circulating Ig concentrations were not affected by treatment.

Figure 1.

Figure 1.

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Change from baseline serum corticosteroid isoenzyme of ALP (CALP), total alkaline phosphatase (ALP), and alanine aminotransferase (ALT)concentrations of dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control.

Table 1.

Change from baseline serum immunoglobulin (Ig) concentrations of dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control

△ Baseline
Item Control LBFP1 SEM2 P-value
IgG, mg/mL 0.99 0.80 0.43 0.750
IgA, µg/mL 6.55 −5.67 7.78 0.178
IgM, µg/mL 0.35x −0.10y 0.60 0.068
IgE, ng/mL 1.83 0.41 2.13 0.640
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1 Lactobacillus fermentation product.

2Pooled standard error of the mean.

x,yMeans lacking a common superscript tend to differ (P < 0.10).

TNF-α concentrations from cell cultures were used to test responsiveness of PBMC to TLR agonists, with change from baseline data being presented in Table 2. Cells isolated from the control group tended to have a greater (P = 0.065) change from baseline in response to zymosan (TLR2 agonist) than those in the LBFP group. TNF-α concentrations from cells treated with the other agonists were not affected by treatment.

Table 2.

Change from baseline TNF-α concentrations of cell cultures treated with Toll-like receptor (TLR) agonists from adult dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control

△ Baseline
Agonist Control, pg/mL LBFP1, pg/mL SEM2 P-value
Control −871 −841 264 0.788
Poly I:C3 923 708 315 0.113
LPS4 711 694 330 0.347
R848 −139 1844 2320 0.395
Zymosan 2161x 981y 641 0.065
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1 Lactobacillus fermentation product.

2Pooled standard error of the mean.

3Polyinosinic-polycytidylic acid sodium salt.

4Lipopolysaccharides.

x,yMeans lacking a common superscript tend to differ (P < 0.10).

Change from baseline fecal characteristics are presented in Table 3. Fecal pH, IgA, and dry matter percentage were not affected by treatment. However, the increase in fecal scores tended to be lower (P = 0.068) in dogs supplemented with LBFP than controls, signifying more stable and firm fecal samples in the LBFP supplemented group.

Table 3.

Change from baseline fecal characteristics and biomarkers of dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control

△ Baseline
Item Control LBFP1 SEM2 P-value
Fecal pH −0.14 0.09 0.21 0.444
Fecal score3 0.56x −0.07y 0.23 0.068
Fecal dry matter −3.25 -0.72 0.98 0.135
Fecal IgA, mg/g 6.84 2.63 11.03 0.351
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1 Lactobacillus fermentation product.

2Pooled standard error of the mean.

3Fecal samples were scored according to the following scale: 1 = hard, dry pellets, small hard mass; 2 = hard, formed, dry stool; remains firm and soft; 3 = soft, formed, and moist stool, retains shape; 4 = soft, unformed stool, assumes shape of container; and 5 = watery, liquid that can be poured.

x,yMeans lacking a common superscript tend to differ (P < 0.10).

In regard to fecal microbiota, alpha diversity indicators, represented by observed operational taxonomic units, Faith’s phylogenetic diversity, and Shannon diversity index, suggest that species richness tended to be higher (P = 0.087) in dogs supplemented with LBFP than controls as shown in Figure 2. PCoA plots representing beta diversity are represented in Figure 3. Unweighted UniFrac distances suggest a trend of bacterial population separation between treatment groups (P = 0.057), demonstrating a shift away from baseline in control animals, but stability in LBFP supplemented dogs. Weighted UniFrac distances were not different.

Figure 2.

Figure 2.

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Alpha diversity measures of fecal samples collected from dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control. Alpha-diversity is represented by observed OTU, Faith’s phylogenetic diversity (PD), and Shannon diversity index. Faith’s PD and Shannon diversity index suggest that species richness tended to be higher in dogs supplemented with Lactobacillus fermentation product than those fed the control. The observed OTU comparison, however, did not reach statistical significance.

Figure 3.

Figure 3.

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Beta diversity measures of fecal samples collected from dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control. Principal coordinates analysis (PCoA) plots of unweighted (a) and weighted (b) UniFrac distances of fecal microbial communities were performed on the 97% OTU abundance matrix using QIIME2. Unweighted UniFrac distances suggest a trend of bacterial population separation between treatment groups.

Change from baseline dysbiosis index and bacterial abundance data measured by qPCR did not identify any differences due to treatment (Table 4). When 16S sequencing data were analyzed, several fecal microbial taxa were shown to be altered by treatment (Figures 4 and 5). The only bacterial phyla affected by treatment was Actinobacteriota, which tended to have a greater increase (P < 0.10) in dogs in the control group (1.76%) than dogs supplemented with LBFP (−0.37%) (Supplementary Table S12). At the genus level, relative abundances of fecal Faecalibaculum, uncultured Butyricicoccaceae, and Bifidobacterium had a greater increase (P ≤ 0.05) and relative abundances of fecal uncultured Erysipelotrichaceae, Anaerofilum, Fusicatenibacter, and Prevotellaceae Ga6A1 group tended to have a greater increase (P < 0.10) in dogs supplemented with LBFP than in dogs fed the control (Figure 4). In contrast, relative abundances of fecal Peptoclostridium, Sarcina, and Faecalitalea had a greater increase (P < 0.05) and relative abundances of fecal Allobaculum, Erysipeloclostridium, Terrisporobacter, Collinsella, and Slackia tended to have a greater increase (P < 0.10) in dogs in the control group than in dogs supplemented with LBFP (Figure 5). The other bacterial phyla and genera were not affected by treatment (Supplementary Table S12).

Table 4.

Change from baseline fecal dysbiosis index and bacterial abundance (log DNA/gram feces) of dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control

△ Baseline
Item Control LBFP1 SEM2 P-value
Dysbiosis index 0.51 −0.07 0.51 0.431
Total bacteria 0.06 0.04 0.07 0.833
Blautia −0.07 −0.15 0.07 0.447
Clostridium hiranonis 0.08 −0.06 0.07 0.194
Escherichia coli −0.13 −0.15 0.46 0.976
Faecalibacterium −0.36 0.00 0.20 0.216
Fusobacterium −0.27 0.02 0.28 0.471
Streptococcus 0.26 0.10 0.36 0.753
Turicibacter 0.17 0.31 0.21 0.635
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1 Lactobacillus fermentation product.

2Pooled standard error of the mean.

Figure 4.

Figure 4.

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The change from baseline relative abundances (% sequences) of fecal Faecalibaculum, uncultured Butyricicoccaceae, and Bifidobacteriumwere greater (P ≤ 0.05) and change from baseline relative abundances of fecal uncultured Erysipelotrichaceae, Anaerofilum, Prevotellaceae Ga6A1 group, and Fusicatenibactertended to be greater (P < 0.10) in dogs supplemented with Lactobacillus fermentation product than control dogs.

Figure 5.

Figure 5.

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The change from baseline relative abundances (% sequences) of fecal Peptoclostridium, Sarcina, and Faecalitaleawere lower (P < 0.05) and change from baseline relative abundances of fecal Allobaculum, Erysipelatoclostridium, Terrisporobacter, Collinsella, and Slackiatended to be greater (P < 0.10) in control dogs than dogs supplemented with Lactobacillus fermentation product.

At the end of the study, dogs underwent a transport stress challenge. Before transport, serum SOD concentrations were higher (P = 0.001) in control dogs than in dogs supplemented with LBFP, but serum cortisol, salivary cortisol, and serum MDA were not different (Supplementary Table S13). The change in oxidative stress marker concentrations from ­transport stress are presented in Table 5. While changes to serum and salivary cortisol concentrations and serum MDA concentrations post-transport were not altered by treatment, serum SOD concentrations had a greater (P < 0.0001) increase in dogs supplemented with LBFP than controls following transport stress. Even though pretransport SOD concentrations were different between groups, the large change posttransport between groups suggests that the response truly occurred.

Table 5.

Oxidative stress marker concentrations of dogs supplemented with Lactobacillus fermentation product or placebo (dextrose) control before and after transport

△ Prepost transport
Item Control LBFP1 SEM2 P-value
Serum cortisol, ng/mL −2.45 14.62 15.04 0.431
Serum SOD3, ng/mL −8.51b 68.59a 12.39 0.0001
Salivary cortisol, μg/dL 1.95 1.45 0.23 0.130
Serum MDA4, nmol/mL 2.08 3.61 1.52 0.483
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1 Lactobacillus fermentation product.

2Pooled standard error of the mean.

3Superoxide dismutase.

4Malondialdehyde.

a,bMeans lacking a common superscript differ (P < 0.05).

Discussion

Anthropomorphism of pets is an increasing trend in today’s society. Our pets are no longer strictly living outdoors and being fed for convenience. People now view companion animals as part of their family, resulting in a focus and advances towards life longevity through nutrition similar to those used in humans. Owners gravitate toward high-quality pet foods, with most including functional ingredients. Foods containing these ingredients offer health benefits beyond those of essential nutrients. Health benefits of functional ingredients are diverse and may include reduction in oxidative damage, modulation of the immune system, maintenance of the gut microbiome, and support of muscle and joint health (Hasler, 2002; Wilson et al., 2017). Heat-killed fermentation products are functional ingredients that may offer many benefits from both a production and consumer standpoint. These products are easily incorporated into animal feeds and foods due to their nonviable and heat-stable nature, while simultaneously offering benefits similar to those of a live microorganism supplement. LBFP and similar products have been shown to be effective in reducing the incidence of diarrhea, modulating gut microbiota, aiding in antioxidant protection, and supporting immunity in humans and livestock (Virtanen et al., 2007; Asama et al., 2016; Moal, 2016; Warda et al., 2019).

Many of the benefits elicited by LBFP and other postbiotics have been attributed to the metabolites within the fermentation matrix rather than a direct effect of the heat-killed organism (Mathur et al., 2020). Examples of metabolites that many Lactobacillus strains produce during fermentation are angiotensin-converting enzyme (ACE)-inhibitory peptides, exopolysaccharides (EPS), and bacteriocins, all of which have been shown to elicit benefits similar to those of the LBFP (Fuglsang et al., 2003; Salazar et al., 2009; Nout, 2014). ACE-inhibitory peptides relax veins by blocking the enzyme from producing angiotensin 2, which is responsible for narrowing blood vessels. Hypertension has been associated with the release of pro-inflammatory cytokines and a localized immune response (Singh et al., 2014). Long-term hypertension, leading to chronic high blood pressure, can be a cause of systemic inflammation. The production of ACE inhibitors by LAB may be a mechanism of action in decreasing an immune response as seen by a less severe TLR response in ex-vivo challenged white blood cells. Previous studies have demonstrated an increase in TLR2 signaling or stimulation of the immune system through the use of postbiotics (Luan et al., 2014; Morita et al., 2019). Our data show an increase in TLR activity when challenged ex-vivo; however, it tended to be less severe than that of the control group. We believe that a less drastic increase in TNF-α production by TLR2 may be a sign of tolerance and modulation. TNF-α is highly pro-inflammatory cytokine and lack of regulation can lead to unspecific tissue damage (Jang et al., 2021). In these cases, a slighter increase in TLR2 signaling would allow for an adequate immune response without overproduction of pro-inflammatory cytokines.

Another metabolite class that is thought to provide postbiotic functionality is that of EPS, which have been the focus of recent LAB reviews focused on their potential mechanisms of action (Sørensen et al., 2022). EPS have been shown to possess bifidogenic effects, reduce oxidation, and modulate immunity (Dal Bello et al., 2001; Wu et al., 2010; Patel and Prajapati, 2013). EPS are produced by most lactobacilli and although their concentrations are reduced, they remain present after the microorganisms have been heat-killed (Almalki, 2020; Nachtigall et al., 2021). The Bifidobacterium response in the current study was quite small, but other postbiotics fed to dogs and humans have resulted in similar findings and possibly partially due to EPS production (Lin et al., 2019; Warda et al., 2021). An increase in Bifidobacterium is regarded as being beneficial and bifidobacteria are often used as probiotics in dogs due to their ability to beneficially alter the gastrointestinal environment (Strompfová et al., 2014). In a previous study, for instance, Bifidobacterium probiotic supplementation resulted in a decrease in idiopathic diarrhea in dogs (Kelley et al., 2009). The LBFP-supplemented dogs in the current study did not have diarrhea, but had more firm stool samples that also agree with the results of that experiment. Another bacterial taxon that was more prevalent in the LBFP-­supplemented dogs of the current study was ­Faecalibaculum. ­Certain strains of Faecalibaculum have recently been shown to be anti-tumorigenic when incorporated in the gut microbiome of a mouse model (Zagato et al., 2020). For these reasons, we conclude that these taxa are generally favorable in higher concentrations.

Many of the bacteria that increased with LBFP supplementation are lactate and butyrate producers. Butyrate is the preferred energy source of colonocytes and serves many benefits within the body, including immune modulation, oxidative stress reduction, and regulation of cellular functions (Bedford and Gong, 2018). In addition, lactate and SCFA have been used to control the growth of pathogenic bacteria in animals for many years (Kabara et al., 1972). Bacteriocins are another mentionable metabolite produced by LAB that possess anti-microbial capabilities as well. Studies have reported that bacteriocins produced by Lactobacillus are able to decrease incidence of and/or be used to treat pathogenic infections (Corr et al., 2007). In our findings, dogs supplemented with LBFP had lower prevalence of the genera Peptoclostridium, Sarcina, Collinsella, and Erysipelatoclostridium, all of which are either potential pathogens, are proteolytic in nature, or have an association with health issues (Lam-Himlin et al., 2011; Neumann-Schaal et al., 2019). A decrease in the abundance of these taxa may be due to their metabolic preferences or the presence of antimicrobials. In conjunction with a generally positive shift in the microbiome, species richness within the LBFP-supplemented dogs tended to be higher than controls as shown by alpha-diversity indicators. Increases in alpha diversity have been positively correlated with health, whereas a decrease in diversity has been linked with disease (Pickard et al., 2017). Although the effects on the microbiota were moderate, the increased relative abundance in beneficial bacteria paired with higher alpha diversity suggests that LBFP may positively modulate the gut microbiome of dogs.

Oxidative stress refers to the pro-oxidative cellular reactions outweighing the antioxidant defense system. This can lead to damage throughout the body and assist in aging and disease (Ji and Yeo, 2021). The SOD enzyme is part of an endogenous antioxidant system that catalyzes the transformation of free radicals into less reactive molecules. In the current study, we noted an increase in serum SOD concentrations in LBFP-supplemented dogs after being exposed to transport stress. High SOD concentrations are commonly viewed as being protective from free radicals and oxidative damage, whereas low levels are associated with aging and disease (Younus, 2018). It is reasonable to conclude that this increase is a sign of greater antioxidant capabilities of dogs treated with LBFP, but analysis of more biomarkers would be helpful in assuring that claim in the future. Although SOD was increased, serum MDA concentrations were unchanged between treatment groups. MDA is a biomarker of lipid peroxidation, leading to oxidative damage (Roede et al., 2010). Although MDA concentrations were unchanged due to treatment, these results suggest that LBFP may support the body’s antioxidant system and would be a worthwhile focus of future studies. Heat-killed lactobacilli have previously been shown to lower stress hormone levels in animals (Warda et al., 2019). In the current study, however, we did not observe an effect of treatment on either serum or salivary cortisol concentrations.

In conclusion, our data suggest that LBFP may offer benefits pertaining to gut microbiota modulation, stool quality maintenance, immune tolerance, and an increase in antioxidant capabilities of adult dogs when compared with those given a placebo. LBFP beneficially shifted the fecal microbiota, including an alteration of 15 microbial genera, and increased microbial alpha diversity. These microbiota changes occurred in concert with more stable fecal scores in LBFP-supplemented dogs. In addition, immune measures exhibited a less severe response to TLR2 stimulation using an ex-vivo assay. The lower response suggests that LBFP may provide a more controlled release of pro-inflammatory cytokines if challenged. Lastly, LBFP was shown to increase circulating concentrations of SOD, which is indicative of a higher antioxidant capacity. Collectively, our data suggest that LBFP may provide benefits to adult dogs by shifting the fecal microbiota, stabilizing stool quality, and reducing oxidative damage when exposed to stress.

Supplementary Material

skad160_suppl_Supplementary_File
Click here for additional data file. (209.1KB, docx)

Acknowledgment

Funding for this study was provided by Adare Pharmaceuticals SAS, 78550 Houdan, France.

Glossary

Abbreviations:

ACE

angiotensin-converting enzyme

ALT

alanine aminotransferase

BW

body weight

CALP

corticosteroid isoenzyme of alkaline phosphatase

Ig

immunoglobulin

LAB

lactic acid-producing bacteria

LBFP

Lactobacillus fermentation product

MDA

malondialdehyde

OTU

operational taxonomic units

PBMC

peripheral blood mononuclear cell

PCoA

principal coordinates analysis

qPCR

quantitative polymerase chain reaction

SCFA

short-chain fatty acids

SOD

superoxide dismutase

TLR

toll-like receptor

TNF-α

tumor necrosis factor-alpha

Contributor Information

Samantha A Koziol, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Patricia M Oba, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Katiria Soto-Diaz, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Andrew J Steelman, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Jan S Suchodolski, Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, Texas A&M University, College Station, TX 77843, USA.

Erik R M Eckhardt, Adare Biome, 78550 Houdan, France.

Kelly S Swanson, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, Texas A&M University, College Station, TX 77843, USA.

Conflict of Interest Statement

The authors have no conflict of interest.

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