Effects of commercial and traditional kefir supplementation on apparent total tract macronutrient digestibility and the fecal characteristics, metabolites, and microbiota of healthy adult dogs

Abstract

Kefir is a fermented dairy beverage that has been consumed by humans for centuries, but poorly studied in pets. The objective of this study was to determine the effects of commercial or traditional kefir supplementation on apparent total tract macronutrient digestibility (ATTD) and fecal characteristics, microbiota populations, and metabolite and immunoglobulin (Ig) A concentrations of healthy adult dogs. Twelve healthy adult dogs (5.67 ± 1.72 yr, 7.27 ± 1.15 kg) were used in a replicated 3 × 3 Latin square design (n = 12/group). All dogs were fed a commercial diet and allotted to 1 of 3 treatments (60 mL/d): 2% reduced-fat milk treated with lactase [CNTL; 4.57E + 03 lactic acid bacteria (LAB) colony-forming units (CFU)/mL], commercial kefir (C-Kefir; 6.95E + 04 LAB CFU/mL), or traditional kefir brewed daily from 2% reduced-fat milk and kefir grains (T-Kefir; 1.79E + 09 LAB CFU/mL). The experiment was composed of three 28-d periods, with each consisting of a 22-d transition phase, a 5-d fecal collection phase, and 1 d for blood collection. Fecal samples were collected for determination of ATTD and fecal pH, dry matter, microbiota, and metabolite, and IgA concentrations. Data were analyzed using the Mixed Models procedure of SAS 9.4. The main effects of treatment were tested, with significance set at P ≤ 0.05 and trends set at P ≤ 0.10. Kefir products differed in microbial density and profile, but fecal microbiota populations were weakly impacted. Bacterial alpha diversity tended to be greater (P = 0.10) in dogs fed T-Kefir than those fed CNTL. Bacterial beta diversity analysis identified a difference (P < 0.0004) between dogs-fed CNTL and those fed C-Kefir. Dogs-fed C-Kefir tended to have a greater (P = 0.06) relative abundance of Fusobacteriota than those fed CNTL or T-Kefir. Dogs-fed T-Kefir had a greater (P < 0.0001) relative abundance of Lactococcus than those fed CNTL or C-Kefir. Dogs-fed T-Kefir also tended to have a lower (P = 0.09) relative abundance of Escherichia Shigella and greater (P = 0.09) relative abundance of Candidatus stoquefichus than dogs-fed CNTL or C-Kefir. Dogs-fed C-Kefir tended to have lower (P = 0.08) fecal valerate concentrations than those fed CNTL or T-Kefir. All other measures were unaffected by kefir treatments. Our results suggest that kefir supplementation had minor effects on the fecal microbiota populations and fecal metabolite concentrations of healthy adult dogs without impacting ATTD, fecal characteristics, or fecal IgA concentrations.

This study was conducted to determine the effects of commercial or traditional kefir supplementation on apparent total tract macronutrient digestibility and the fecal characteristics, microbiota populations, and metabolite and immunoglobulin A concentrations of healthy adult dogs. Our results suggest that kefir supplementation had minor effects on the fecal microbiota populations and fecal metabolite concentrations without impacting stool quality or apparent total tract macronutrient digestibility.

Introduction

Kefir is a fermented milk beverage traditionally produced from the fermentation of kefir grains with milk. Kefir has been consumed for centuries, originating from the Caucasus Mountains and Tibet (Rosa et al., 2017). Kefir products fall into the category of fermented foods, which have been defined by the International Scientific Association for ­Probiotics and Prebiotics to be “foods made through desired microbial growth and enzymatic conversions of food components” (Marco et al., 2021). DNA sequencing has shown that traditionally produced kefirs contain bacteria such as Lactobacillus kefiranofaciens, Lentilactobacillus kefiri, and Lentilactobacillus parakefiri and yeasts such as Saccharomyces, Kluyveromyces, Naumovozyma, and Candida (Guzel-Seydim et al., 2011; Nejati et al., 2022). In contrast to traditional kefirs, commercial kefirs sold in stores are based on bacterial cultures (typically no yeast) that ferment milk. Because the commercial kefir products are not brewed from kefir grains, but from defined cultures, they do not possess the traditional kefir microbial profiles (Nejati et al., 2022). Human kefir products sold in the United States have microbial profiles containing Lactobacillus, Lactococcus, Streptococcus, and/or Leuconostoc (Metras et al., 2021). Similarly, commercial companion animal kefir products primarily consist of Bacillus, Streptococcus, Lactobacillus, and Lactococcus (Metras et al., 2020). Although fermented foods and beverages are believed to provide health benefits, further work is needed to better understand the potential physiological benefits they provide to companion animals.

In addition to microbiota, kefirs contain other fermentation byproducts, such as exopolysaccharides, water-soluble polysaccharides, kefir-fermented peptides, and kefir cell-free supernatants that may provide host benefits (Simova et al., 2002; Medrano et al., 2008). Traditional grain-based kefirs have been shown to modulate the gut microbiome and improve cholesterol metabolism in mice (Bourrie et al., 2016). A handful of randomized clinical trials assessing traditional kefir intervention have been conducted in healthy adult humans (Pražnikar et al., 2020; Wang et al., 2020) as well as in patients who are overweight (Fathi et al., 2017), diabetic (Judiono et al., 2014; Ostadrahimi et al., 2015; Bellikci-Koyu et al., 2019; El-Bashiti et al., 2019), or hypercholesteremic (St-Onge et al., 2002). A recent meta-analysis determined that kefir has a significant impact on reducing insulin (P < 0.05) and fasting blood sugar concentrations (P < 0.05) when at least 200 mL were consumed for at least 4 wk consecutively (Salari et al., 2021). In one study, Praznikar et al. (2020) reported that kefir consuming people had lower serum C-reactive protein (P < 0.018), high density lipoprotein (P < 0.013), and fasting blood sugar (P < 0.001) concentrations than those consuming milk. Other human clinical trials conducted, however, have not observed consistent physiological outcomes, which could be attributed to the large microbial variations among traditional and commercial kefir products used.

Research on the health effects of various kefir products has been lacking, especially in companion animals. This is especially important, as there are commercial kefir products that are advertised for dogs. A couple studies have investigated kefir in dogs, but neither specified if traditional or commercial kefir was used, providing uncertainty to kefir’s overall attributes. In one study, healthy adult dogs receiving 200 mL/day of traditional grain kefir [9.32 ± 0.23 log colony-forming units (CFU)/mL of lactic acid bacteria (LAB); 7.12 ± 0.36 log CFU/mL of yeast] for 2 weeks had some shifts in the fecal microbiota (Kim et al., 2019). While alpha diversity indices were unchanged, kefir consumption led to shifts in beta diversity and changes to several individual bacterial taxa. Based on sequencing data, the relative abundances of several bacterial families (Prevotellaceae, Selenomonadaceae, and Sutterellaceae) were increased (P < 0.05), while the relative abundances of other bacterial families (Clostridiaceae, Fusobacteriaceae, and Ruminococcaceae) were decreased (P < 0.05) after kefir consumption. Based on qPCR data, kefir consumption was shown to increase (P = 0.012) LAB abundance, increase (P = 0.04) the LAB:Enterobacteriaceae ratio, and decrease (P = 0.03) the Firmicutes:Bacteroidetes ratio. In another study, healthy adult dogs were fed Lentilactobacillus kefiri (from Kefibios; 1 × 10⁷ CFU/day) for 30 d, but no significant differences in fecal IgA or microbiota were observed (Gaspardo et al., 2020).

Given the lack of research on kefir products in dogs, especially in comparison of grain-based and commercial kefirs, further in vivo research in this area is justified. The objective of this study was to determine the effects of commercial or traditional kefir supplementation on apparent total tract digestibility (ATTD) and the fecal characteristics, microbiota, and metabolite and immunoglobulin A (IgA) concentrations of healthy adult dogs. Without negatively affecting ATTD or fecal characteristics, we hypothesized that kefir supplementation would beneficially shift the fecal microbiota and metabolites and increase fecal IgA concentrations of adult dogs compared with controls. We also hypothesized that the traditional kefir would have greater effects than the commercial kefir product.

Materials and Methods

All procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation. All methods were performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Animals, treatments, and experimental design

Twelve healthy adult spayed female dogs (age: 5.67 ± 1.72 yr; body weight: 7.27 ± 1.15 kg) were used in a replicated 3 × 3 Latin square design (n = 12/group). The experiment consisted of three 28-d periods, each consisting of a 22-d transition phase, a 5-d fecal collection phase, and 1 d for blood collection. All dogs were housed individually in pens (1.0 m wide by 1.8 m long) in an environmentally controlled facility at the University of Illinois Urbana-Champaign. Dogs had free access to fresh water at all times. All dogs were fed a commercial diet without probiotics or prebiotics, having low levels of fermentable fiber, and formulated to meet all essential nutrients recommended by the Association of American Feed Control Officials (AAFCO, 2022) for adult dogs at maintenance (Best Dog 21/12, Coker Feed Mill, Inc., Goldsboro, NC). On the basis of the maintenance energy requirement for adult dogs and information from previous feeding records, an amount of food to maintain body weight was offered, and intake was measured twice daily (8:00 a.m.; 4:00 p.m.). Dogs were weighed, and body condition scores were assessed (9-point scale) once a week prior to feeding.

The following treatments were tested: diet + 2% milk treated with lactase (CNTL; 2% Reduced Fat Milk, Great Value Farm, Fort Wayne, IN; 60 mL/d); diet + commercial kefir (C-Kefir; Champions Choice Kefir; Champions Choice, Millersburg, IN, 60 mL/d); and diet + traditional kefir (T-Kefir; Traditional Kefir; Kefir Garden Grains, Hamilton Ontario, Canada; 60 mL/d). Whole milk and kefir products were stored at 4°C. Before feeding, milk and kefir product containers were inverted 10 times to homogenize the products. In addition, 1 gallon of 2% reduced fat milk was treated with 14 lactase drops 24 h before being fed to dogs (Lactase Drops Seeking Health, Bellingham, WA). Servings of 30 mL were aliquoted and given to dogs at each meal (60 mL/dog/d).

The commercial kefir product was ordered from a local vendor, shipped in liquid form, and refrigerated immediately upon arrival. The product was refrigerated at 4 °C between feedings and was thoroughly homogenized before feedings. The kefir brewing process to prepare the traditional kefir began with the addition of 5 % w/v kefir grains to 2% reduced-fat dairy milk (Great Value Farm). Kefir grains and kefir mixtures were incubated at 25 °C in a sterilized lightly sealed glass jar for 24 h. The pH of the kefir was measured after 24 h of fermentation using a pH meter equipped with a pH and temperature probe (Orion Triode 3-in-1 pH/Automatic Temperature Compensation Probe, ThermoFisher Scientific, Waltham, MA), with an ideal pH range maintained between pH 4–5. After 24 h, grains were removed using a stainless-steel strainer, with the kefir refrigerated at 4 °C until it was fed. Kefir grains were rinsed with fresh 2% reduced-fat milk and stored in 2% reduced-fat milk at 25 °C for 24 h, when the process was repeated. Each batch of grain kefir was viable for use up to 72 h after the initial product was refrigerated. Fresh grain kefir batches were prepared daily to maintain freshness.

Milk and kefir bacterial quantification

To begin, all milk and kefir products were inverted 10 times for homogenization, with 1 mL of product added to 9 mL of phosphate-buffered saline (PBS), and the product-PBS mixture inverted 10 times again. Serial 10-fold dilutions were prepared with PBS. A 50-μL volume of 10⁵ and 10⁷ dilutions were enumerated in duplicate via spiral plater (Eddy Jet Spiral Plater, Neutec Group Inc., Farmingdale, NY) onto deMan Rogosa Sharpe media (BD Difco, Franklin Lakes, NJ). deMan Rogosa Sharpe plates were incubated anaerobically at 37 °C for 48 h and aerobically at 30 °C for 48 h to culture LAB. Colony counts were measured via spiral plating software (Colony counter, IUL Flash and Go, Neutec Group Inc., Farmingdale, NY) once each experimental period, using composite treatment samples in triplicate. Average anaerobic and average aerobic counts were combined to calculate a total LAB count (CFU/mL). No attempt to distinguish organisms were made, but only enumeration.

Fecal collection, scoring, and handling

Total feces excreted during the collection phase were collected from each dog, weighed, and frozen at −20 °C until analyses. During the collection phase, all fecal samples were scored using 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 collection phase, one fresh fecal sample (within 15 min of defecation) was collected for the measurement of pH and dry matter (DM) content, microbiota populations, and metabolite and IgA concentrations. Fecal metabolites of interest included short-chain fatty acids (SCFA), which serve as an important energy source for colonocytes, and protein fermentative products [ammonia; branched-chain fatty acids (BCFA); phenols; indoles] that are responsible for fecal odor and are associated with gastrointestinal disease. Fecal aliquots for analysis of phenols and indoles were frozen at −20 °C immediately after collection. One aliquot was collected and placed in 2 N hydrochloric acid for ammonia, SCFA, and BCFA analyses. An aliquot of fresh feces was immediately transferred to sterile cryogenic vials (Nalgene, Rochester, NY), snap-frozen in liquid nitrogen, and stored at −80 °C for microbiota and IgA analysis. An additional aliquot was used for fecal DM determination.

Blood collection and analysis

Blood samples were immediately transferred to appropriate vacutainer tubes for hematology (367841 BD Vacutainer Plus plastic whole blood tube—with K₂EDTA additive) and serum collection (367974 BD Vacutainer Plus plastic serum tube—red/gray with clot activator and gel for serum separation; BD, Franklin Lakes, NJ). The blood tube for serum isolation was centrifuged at 1,300 × g at 4 °C for 10 min (Beckman CS-6R centrifuge; Beckman Coulter Inc., Brea, CA). Once serum was collected, it was transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for serum chemistry analysis. K₂EDTA tubes were cooled (but not frozen) and then transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for hematology analyses.

Chemical analyses and ATTD calculations

Before analyses, milk and kefir samples were lyophilized (Dura-Dry MP microprocessor-controlled freeze-dryer, FTS Systems, Stone Ridge, NY), and fecal samples were dried at 55 °C in a forced-air oven. All samples were then ground in a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ) through a 2-mm screen. Milk and kefir sugar (lactose, glucose, tagatose, galactose), lactate, glycerol, acetate, and ethanol concentrations were measured using high-performance liquid chromatography. Composite samples were run on a 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, Santa Clara, CA) equipped with a refractive index detector using a Rezex ROA-Organic Acid H + (8%) column (Phenomenex, Torrance, CA). The column was eluted with 0.005 N H₂SO₄ at a flow rate of 0.6 mL/min at 5 °C, with fucose, salicylic acid, 2ʹ-fucosyllactose, 3ʹ-sialyllactose, lacto-N-neotetraose, and lacto-N-triose II (Carbosynth US, San Diego, CA) used as detection standards.

Dried and ground diet, milk, kefir, kefir grain, and fecal samples were analyzed for DM and ash according to AOAC (2006; methods 934.01 and 942.05), with organic matter 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 the American Association of Cereal Chemists (1983) and Budde (1952). Total dietary fiber of the diet was determined according to Prosky et al., (1992). Gross energy was measured using an oxygen bomb calorimeter (model 6200, Parr Instruments, Moline, IL). ATTD of nutrients and energy were calculated using the following equation:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{\%}Digestibility=\frac{NutrientIntake\left(\frac{g}{d}\right)-Fecaloutput\left(\frac{g}{d}\right)}{NutrientIntake\left(\frac{g}{d}\right)}\ast 100\mathrm{\%}}\end{array}}$$

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 ethylenediaminetetraacetic acid (ThermoFisher) and 100 µg/L soybean trypsin inhibitor (Sigma, St. Louis, MO) in PBS/1% bovine serum albumin (Tocris Bioscience, Bristol, UK). Phenylmethanesulphonyl fluoride (12.5 µL, 350 mg/L; Sigma) was added into each tube, followed by centrifugation for 10 min at 2,500 × g. Supernatants were collected for the measurement of IgA using a commercial enzyme-linked immunosorbent assay kit (MBS018650; MyBioSource, Inc., San Diego, CA).

Milk, kefir, and fecal DNA extraction and MiSeq Illumina sequencing of 16S rRNA gene amplicons

Total DNA from fecal, commercial kefir product, traditional kefir product, and whole milk samples were extracted using Mo-Bio PowerSoil kits (MO BIO Laboratories, Inc., Carlsbad, CA). The concentration of extracted DNA was 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 a 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). The CS1 forward and CS2 reverse tags were added according to the Fluidigm protocol. The 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 1%–2% agarose E-gel (Life Technologies) and extracted using a Qiagen gel purification kit (Qiagen, Valencia, CA). Cleaned size-selected pooled products were run on an Agilent Bioanalyzer to confirm the 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 0.0.13), and QIIME 2.0 (Caporaso et al., 2010) was used to process the resulting sequence data. Briefly, high-quality (quality value ≥ 20) sequence data derived from the sequencing process was 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 (sequences per sample) was used for assessing alpha- and beta-diversity measures. Beta-diversity was assessed using weighted and unweighted UniFrac distance measures (Lozupone and Knight, 2005) and presented using principal coordinates analysis (PCoA) plots.

Statistical analysis

Data were analyzed using the Mixed Models procedure of SAS (SAS Institute, Inc., Cary, NC), testing for differences due to treatment. Dog and period were considered random effects. Differences among dietary 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 being statistically significant, with P ≤ 0.10 accepted as statistical trends.

Results

Milk and kefir samples were similar in moisture, protein, calcium, and phosphorus concentrations, but C-Kefir contained higher fat concentrations than CNTL and T-Kefir (Table 1). Sugar content differed among CNTL and kefir treatments. Both kefirs contained much higher lactose and ethanol concentrations than the CNTL, while the CNTL contained higher glucose and galactose concentrations. These differences were most likely the result of the lactase treatment to the CNTL. Lactate, acetate, glycerol, and tagatose concentrations were similar among CNTL and kefir samples.

Table 1.

Analyzed nutrient concentrations (as-is values) of milk and kefir treatments tested

Item	CNTL1	C-Kefir2	T-Kefir3
Moisture, %	93.30	93.03	93.43
Crude protein, %	4.86	4.46	4.90
Acid-hydrolyzed fat, %	3.47	5.27	3.93
Calcium, %	0.14	0.09	0.12
Phosphorus, %	0.11	0.08	0.09
Lactose, g/L	2.12	29.81	31.75
Glucose, g/L	13.33	0.03	0.02
Tagatose, g/L	0.00	0.01	0.01
Galactose, g/L	22.14	0.01	0.01
Lactate, g/L	8.28	7.71	9.25
Glycerol, g/L	0.52	0.37	0.39
Acetate, g/L	0.99	0.87	1.26
Ethanol, g/L	3.43	8.46	7.02
Gross energy, kcal/g	0.83	0.89	0.86

¹2% milk treated with lactase.

²C-Kefir: commercial kefir.

³T-Kefir: traditional kefir.

As predicted, the T-Kefir contained a much higher bacterial density [1.79E + 09 CFU/mL] than the CNTL (4.57E + 03 CFU/mL) or C-Kefir (6.95E + 04 CFU/mL) treatments. In regard to bacterial profile, the CNTL was primarily composed of Lactococcus (61.80%), Leuconostoc mesenteroides (20.72%), Enterobacteriaceae (11.86%), and Yersiniaceae (4.73%) (Table 2). C-Kefir was primarily composed of two genera, namely Lactococcus (91.94%) and Lactobacillus (7.97%). T-Kefir also contained high amounts of Lactococcus (72.69%) and Lactobacillus (25.18%), including species such as L. kefiranofaciens (4.99%) and Gluconobacter (1.85%). T-Kefir grains, which were used to produce T-Kefir primarily contained Lactococcus (68.38%), Lactobacillus (31.38%), and L. kefiranofaciens (30.25%).

Table 2.

Microbial composition (% of sequences) of kefir grains and milk and kefir treatments tested

Phyla	Genus	CNTL1	C-Kefir	T-Kefir	Kefir grains
Bacteroidota		0.05	0.02	0.04	0.05
	Bacteroides	0.02	0.00	0.00	0.01
	Muribaculaceae uncultured	0.03	0.01	0.01	0.04
Firmicutes		82.91	99.96	98.01	99.81
	Total Lactobacillus	0.09	7.97	25.18	31.38
	Lactobacillus spp. (unclassified)	0.09	7.86	20.19	1.13
	Lactobacillus kefiranofaciens	0.00	0.00	4.99	30.25
	Lactococcus	61.80	91.94	72.69	68.38
	Leuconostoc mesenteroides	20.72	0.00	0.00	0.00
Fusobacteriota		0.03	0.01	0.04	0.02
	Fusobacterium	0.00	0.00	0.02	0.00
Proteobacteria		17.01	0.01	1.91	0.11
	Enterobacteriaceae	11.86	0.00	0.00	0.00
	Gluconobacter	0.00	0.00	1.85	0.11
	Pseudomonas	0.38	0.00	0.00	0.00
	Yersiniaceae	4.73	0.00	0.00	0.00
Verrucomicrobiota		0.03	0.01	0.02	0.02
	Akkermansia	0.03	0.01	0.02	0.02

¹2% milk treated with lactase.

Abbreviations: C-Kefir: commercial kefir; T-Kefir: traditional kefir.

Fecal characteristics, including pH, scores, and DM percent were normal for all dogs throughout the study and were not affected by treatment (Table 3). The ATTD of DM, organic matter, crude protein, fat, and energy were not affected by treatment. Fecal valerate concentrations tended to be higher (P = 0.08) in CNTL dogs than those supplemented with C-Kefir or T-Kefir. All other fecal metabolites and fecal IgA were unaffected by treatment.

Table 3.

Fecal characteristics and metabolite concentrations of healthy adult dogs consuming commercial or traditional kefirs

	Treatment
Characteristics	CNTL1	C-Kefir	T-Kefir	SEM	P-values
pH	6.71	6.60	6.50	0.19	0.747
Fecal score2	3.13	3.33	3.17	0.11	0.385
Fecal DM (%)	29.64	30.46	30.29	0.71	0.687
Digestibility, %
Dry matter	69.47	69.71	69.25	1.73	0.982
Organic matter	74.37	74.65	74.47	1.45	0.990
Crude protein	72.59	72.26	71.94	1.66	0.976
Acid-hydrolyzed fat	87.75	87.67	86.80	0.67	0.536
Energy	74.59	74.81	74.62	1.66	0.976
Metabolites, μmole/g (DMB)
Total SCFA3	651.50	612.85	622.38	33.44	0.699
Acetate	379.62	368.21	355.54	23.65	0.773
Propionate	190.36	166.06	176.27	13.21	0.436
Butyrate	81.53	78.59	90.57	9.51	0.654
Total BCFA3	24.96	22.73	26.15	2.19	0.542
Isobutyrate	9.79	10.34	11.40	0.92	0.467
Isovalerate	12.65	11.84	13.60	1.18	0.577
Valerate	2.53	0.56	1.15	0.57	0.081
Total phenols and indoles	2.98	2.91	3.18	0.19	0.589
4-Methylphenol	0.29	0.28	0.27	0.03	0.955
Indole	1.61	1.66	1.76	0.18	0.847
Ammonia	106.23	93.27	100.36	6.16	0.342
Fecal IgA, mg/g	29.26	29.76	30.27	0.03	0.761

¹2% milk treated with lactase.

²Fecal score: 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.

³Total SCFA: total short-chain fatty acids = acetate + propionate + butyrate; total BCFA: total branched-chain fatty acids = isobutyrate + isovalerate + valerate; valerate is not technically a SCFA but is derived from amino acid fermentation, so it was included in the BCFA calculation.

Abbreviations: C-Kefir: commercial kefir; T-Kefir: traditional kefir.

Fecal bacterial alpha diversity, as measured by Faith’s phylogenetic diversity, was higher (P = 0.03) in dogs-fed C-Kefir than those fed CNTL (Figure 1a). All other measures of alpha diversity, however, were unchanged by treatment (data not shown). Fecal bacterial beta diversity, as assessed using unweighted and weighted PCoA plots, showed bacterial shifts among treatments (Figure 1b and c). The unweighted PCoA plot showed that CNTL was different (P = 0.01) from C-Kefir, while CNTL tended to be different (P = 0.10) from T-Kefir. The weighted PCoA plot, however, did not identify treatment differences. At the phyla level, the relative abundance of fecal Fusobacteriota tended to be higher (P = 0.06) in dogs-fed C-Kefir than those fed CNTL or T-Kefir (Table 4). At the genus level, dogs fed T-Kefir had a higher (P < 0.0001) relative abundance of fecal Lactococcus than those fed CNTL or C-Kefir. The relative abundance of fecal Escherichia-Shigella tended to be higher (P = 0.10) in CNTL dogs than those fed C-Kefir or T-Kefir. Finally, the relative abundance of fecal Candidatus stoquefichus tended to be greater (P = 0.09) in dogs-fed T-Kefir than those fed CNTL or C-Kefir.

Figure 1.

Bacterial alpha diversity indices of canine treatments as assessed by Faith’s phylogenetic diversity (a). Superscripts indicate a significant difference (P < 0.05). PCoA plots of (b) unweighted and (c) weighted Unifrac distances of canine fecal microbial communities performed on the 97% OTU abundance matrix using QIIME. The unweighted PCoA plot (b) showed that CNTL was different (P = 0.01) from C-Kefir, while CNTL tended to be different (P = 0.10) from T-Kefir. The weighted PCoA plot (c) did not identify treatment differences.

Table 4.

Fecal microbiota relative abundances (% of sequences) of healthy adult dogs consuming commercial or traditional kefirs

		CNTL1	Treatment			P-values
Phyla	Genus		C-Kefir	T-Kefir	SEM
Actinobacteriota		2.98	3.04	3.23	0.83	0.853
	Bifidobacterium	1.91	1.79	1.95	0.52	0.783
	Coriobacteriaceae UCG-002	0.51	0.89	0.88	0.37	0.179
Bacteroidota		26.56	29.51	26.91	1.43	0.997
	Alloprevotella	3.71	2.87	2.64	0.51	0.322
	Bacteroides	10.15	13.26	10.49	2.03	0.501
	Muribaculaceae	3.51	3.28	4.31	1.41	0.723
	Prevotella	6.80	6.93	6.64	1.62	0.986
	Prevotellaceae Ga6A1	1.31	1.41	1.51	0.21	0.751
Firmicutes		46.83	37.65	44.86	4.78	0.214
	Allobaculum	4.53	4.48	6.68	2.27	0.784
	Blautia	2.75	2.13	2.49	0.63	0.741
	Candidatus Stoquefichus	0.08	0.04	0.24	0.08	0.086
	Faecalibacterium	1.19	1.23	1.09	0.27	0.889
	Holdemanella	1.02	0.31	0.35	0.31	0.586
	Lactobacillus	3.07	2.03	2.63	0.38	0.174
	Lactococcus	0.04a	0.03a	0.39b	0.07	<0.001
	Peptoclostridium	3.93	3.67	4.26	0.84	0.850
	Phascolarctobacterium	1.10	0.95	0.75	0.17	0.379
	Romboutsia	0.77	1.07	0.98	0.27	0.548
	Ruminoccus	3.31	2.88	3.59	0.73	0.880
	Ruminococcus torques	1.34	1.06	1.60	0.22	0.279
	Streptococcus	1.09	0.31	0.22	0.27	0.173
	Turicibacter	1.42	1.79	1.38	0.56	0.869
Fusobacteriota		14.01	18.37	15.61	1.25	0.056
	Cetobacterium	0.88	1.44	1.25	0.32	0.445
	Fusobacterium	12.84	14.45	13.04	1.44	0.692
Proteobacteria		9.55	11.26	9.30	1.43	0.576
	Anaerobiospirillum	2.99	4.63	3.36	0.83	0.352
	Escherichia-Shigella	0.24	0.02	0.00	0.07	0.095
	Parasutterella	4.10	3.70	4.37	1.02	0.747
	Sutterella	1.31	1.47	1.11	0.22	0.530

¹2% milk treated with lactase.

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

Abbreviations: C-Kefir: commercial kefir; T-Kefir: traditional kefir.

All serum metabolites were within reference ranges throughout the study with the exception of the albumin:globulin ratio exceeding the reference range (0.6–1.1) in CNTL (1.13) and C-Kefir (1.13) groups (Table 5). Serum metabolites were not affected by treatment.

Table 5.

Serum chemistry of healthy adult dogs consuming commercial or traditional kefirs

		Treatment
Item	Reference Range1	CNTL2	C-Kefir	T-Kefir	SEM	P-value
Creatinine, mg/dL	0.5–1.5	0.65	0.66	0.61	0.066	0.724
Blood urea nitrogen, mg/dL	6–30	16.67	16.92	15.50	0.317	0.744
Total protein, g/dL	5.1–7.0	5.76	5.73	5.76	0.084	0.949
Albumin, g/dL	2.5–3.8	3.03	3.01	3.00	0.044	0.858
Globulin, g/dL	2.7–4.4	2.73	2.72	2.76	0.082	0.931
Albumin:globulin ratio	0.6–1.1	1.13	1.13	1.10	0.041	0.838
Ca, mg/dL	7.6–11.4	8.64	8.42	8.30	0.587	0.723
P, mg/dL	2.7–5.2	4.10	4.13	3.95	0.213	0.821
Na, mmol/L	141–152	146.75	146.08	146.42	0.402	0.510
K, mmol/L	3.9–5.5	4.76	4.70	4.86	0.156	0.709
Na: K ratio	28–36	31.17	31.67	30.33	1.021	0.651
Cl, mmol/L	107–118	112.00	112.42	111.92	0.696	0.863
Glucose, mg/dL	68–126	82.17	84.25	82.58	2.901	0.866
Alkaline phosphatase (ALP), U/L	7–92	82.17	84.25	82.58	2.901	0.710
Corticosteroid-induced ALP, U/L	0–40	13.42	9.33	7.33	7.576	0.564
Alanine transaminase, U/L	8–65	25.25	24.33	24.75	2.735	0.857
Gamma-glutamyl transferase, U/L	0–7	2.75	2.75	3.08	0.374	0.769
Total bilirubin, mg/dL	0.1–0.3	0.25	0.26	0.24	0.028	0.986
Creatine phosphokinase, U/L	26–310	111.83	105.33	109.83	0.908	0.961
Cholesterol, mg/dL	129–297	212.67	206.42	211.33	0.511	0.898
Triglycerides, mg/dL	32–154	42.75	42.92	43.75	0.199	0.723
Bicarbonate, mmol/L	16–24	21.58	21.00	21.58	0.471	0.604
Anion gap	—	17.92	17.42	17.67	0.854	0.723
Hemolytic indicator	—	0.35	0.621	0.45	0.235	0.715

¹University of Illinois Veterinary Diagnostic Laboratory Reference Ranges.

²2% milk treated with lactase.

Abbreviations: C-Kefir: commercial kefir; T-Kefir: traditional kefir.

Discussion

Because companion animals are considered to be members of the family in many households worldwide, pet owners seek to feed them high-quality ingredients and provide them with supplements that are believed to promote health. Dogs are metabolically omnivorous and can tolerate a range of diets varying in ingredient profile, nutrient composition, and forms (Lee et al., 2022). In addition to their metabolic flexibility, the canine gastrointestinal system contains complex microbial communities that provide many benefits to the host. In their search for health promotion, many owners look for products that may target the gastrointestinal tract and/or microbiota. Key outcomes include fecal scores, fecal metabolites (SCFA; phenols and indoles), and microbiota populations. Traditionally, dietary fibers, probiotics (Bifidobacterium, Lactobacillus, Streptococcus), prebiotics (galactooligosaccharides, fructooligosaccharides, inulin), synbiotics (prebiotic + probiotic), and postbiotics (bacterial and yeast fermentation products) have been used in this fashion (Swanson et al., 2002; Lin et al., 2019; Lee et al., 2022; Palade et al., 2022; Koziol et al., 2023). Several studies have shown benefits on satiety, reduced blood lipid and glucose concentrations, improved stool quality, greater fecal SCFA concentrations, reduced fecal protein catabolites, and altered microbiota populations (de Godoy et al., 2013; Panasevich et al., 2013; Palmqvist et al., 2023). Many of the same potential benefits may be derived from probiotics and prebiotics (Lin et al., 2022).

A recent addition to this area is that of fermented foods and beverages (Vinderola et al., 2023). Fermented foods have been consumed for centuries by humans, as this process typically extends the duration by which a food can be stored and consumed. Lactic acid bacteria are often responsible for the fermentation process within foods, as is the yeast Saccharomyces. These organisms work, often in concert, to transform dairy, vegetables, meats, and cereals into new products full of live microorganisms (Marco et al., 2021). Such foods may not only be more stable and delicious but may provide health benefits as well. The regular consumption of yogurt, for example, has been shown to reduce symptoms of type 2 diabetes in humans (Ostadrahimi et al., 2015). Consistent physiological outcomes from other fermented foods have yet to be observed, but consumers have become more health-conscious so interest in fermented foods has increased. Given the success of the yogurt industry, companies have expanded to produce commercialized versions of the fermented dairy beverage kefir. Traditional kefir is made through the fermentation of kefir grains with milk to produce a fermented milk beverage. In the United States, commercial kefir production does not use kefir grains in order to avoid yeast-related ethanol issues and to provide product consistency possible with the use of defined starter cultures. Consequently, commercial kefir products will have a different microbial communities and thus might have different potential impacts on host health (Metras et al., 2021; Nejati et al., 2022).

Similar to humans, several commercial kefir products for companion animals exist (Metras et al., 2020). To our knowledge, only a couple studies on kefir have been conducted in dogs. Kim et al. (2019) reported mild changes to the fecal microbiota, showing changes in beta diversity and a few ­bacterial taxonomic groups. The study conducted by Gaspardo et al. (2020), however, did not identify treatment differences. Treatment duration, dosage, animals and housing conditions, or other factors may explain the differences between studies. In both studies, the microbial profiles and nutrient compositions of test products were not presented, making direct comparisons challenging.

Because kefir products are being sold commercially, but are not well studied, we aimed to conduct the current study in hopes of gaining a better understanding of what effects these products may have on the stool quality, fecal metabolites and microbiota, and serum metabolites of dogs. Our initial focus was on microbial and nutrient differences among the milk, commercial kefir, and traditional kefir treatments. The kefir and milk treatments were not dramatically different in regard to nutrient composition, but sugar, lactate, and ethanol concentrations were slightly different. Many commercial kefir products claim to be lactose-free, but low amounts were detected in both kefir products. The lactase treatment of the milk, which was done in an attempt to avoid a lactose effect, converted most of the lactose into glucose and galactose. Because the kefirs were not treated with lactase, they ended up containing much higher lactose concentrations than the milk. Both kefirs contained about twice the ethanol that milk contained.

Variation in the microbial profiles of treatments was also of great interest given that commercial and traditional kefirs have been reported to differ (Nejati et al., 2022). Most importantly, the traditional kefir brewed in our laboratory contained much higher CFU/mL than the commercial kefir and milk. Based on the results of our previous work in this area (Metras et al., 2020), this was expected. Lactococcus was the primary genera present in kefir grains, kefir products, and milk. Not surprisingly, however, the microbial profile of the traditional kefir was more similar to that of the kefir grains. Most notable was that the traditional kefir contained higher amounts of Lactobacillus in general and was the only treatment to contain L. kefiranofaciens that is present in the kefir grains. The kefirs tested in this study did not contain any measurable Bacillus, Streptococcus, and Leuconostoc, which were measured in a few commercial kefir products in our previous study (Metras et al., 2020). Despite the large differences in bacterial counts and different microbial profiles of kefir products, minor changes were noted in fecal microbiota. It is interesting to note that although the C-Kefir product had the highest relative abundance of Lactococcus, it was only the T-Kefir product that increased fecal Lactococcus in the dogs studied. Overall, minor changes to the fecal microbiota were observed, while serum metabolites, fecal metabolites, and fecal IgA were unchanged. Further study is required to determine whether kefirs derived from different kefir grains or containing higher microbial densities or different microbial profiles may provide benefits to dogs.

In conclusion, we observed that the traditional kefir brewed in our laboratory contained significantly more live ­microorganisms and a different microbial profile than the commercial kefir tested. Of note, the traditional kefir was the only one to contain L. kefiranofaciens that is derived from kefir grains. The supplementation of commercial and traditional kefir products had minor effects on fecal microbiota populations and metabolite concentrations of healthy adult dogs. In addition to slight changes in the bacterial beta diversity for both kefirs, the traditional kefir led to an increase in the relative abundance of fecal Lactococcus. The commercial kefir tended to reduce fecal valerate concentrations. Stool quality and all other physiological measures were unaffected by treatments. Further research is needed to test whether kefirs derived from different kefir grains or those containing higher microbial densities or different microbial profiles may provide benefits to dogs.

Glossary

Abbreviations:

ATTD

apparent total tract digestibility

BCFA

branched-chain fatty acids

CFU

colony-forming units

C-Kefir

commercial kefir treatment

CNTL

milk treatment

DM

dry matter

IgA

immunoglobulin A

LAB

lactic acid bacteria

PBS

phosphate-buffered saline

PCoA

principal coordinates analysis

SCFA

short-chain fatty acids

T-Kefir

traditional kefir treatment

Conflicts of interest statement

All authors have no conflicts of interest.