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. 2020 Sep 11;98(9):skaa301. doi: 10.1093/jas/skaa301

Assessment of commercial companion animal kefir products for label accuracy of microbial composition and quantity

Breanna N Metras 1, Maxwell J Holle 2, Valerie J Parker 3, Michael J Miller 1,2, Kelly S Swanson 1,4,5,✉
PMCID: PMC7523595  PMID: 32914845

Abstract

Kefir is a fermented beverage containing yeast and bacteria produced by the fermentation of water or milk with kefir grains. Lack of regulation for probiotic-containing fermented food sold for companion dogs and cats creates the potential for misreporting on viable microbial counts, taxonomy, and label claims. In this study, the microbiota of six companion animal kefir products were measured quantitatively using standard plating techniques. Microbial composition of these products was also characterized by using high-resolution, long-read amplicon sequencing of the 16S rRNA gene. Five products (83%) listed specific microorganisms, and four products (66%) guaranteed colony forming units (CFU)/g on their label. To enumerate viable lactic acid bacteria (LAB), two lots of each homogenized product were plated upon opening and following 14 d on deMan Rogosa and Sharpe (MRS) agar and incubated under anaerobic and aerobic conditions. Results from point of opening revealed that all commercial kefir products with a guaranteed CFU/g overstated the number of microorganisms present by at least 1 log, with only one product exceeding 1 × 109 CFU/g. Sequencing results demonstrated that none of the labels claiming specific bacterial genera and species on their labels were correct, and all products contained at least three additional bacterial species above the minimum detectable threshold (0.001% relative abundance) that were not disclosed by the manufacturer. In addition to the incorrect viable CFU and bacterial taxonomies, several of the product labels and websites contained a wide range of health claims, none of which are supported by the companion animal literature. Our results demonstrate a low level of accuracy in the labeling of commercial kefir products intended for use in dogs and cats. Regulatory agencies, veterinarians, pet food professionals, and pet owners must scrutinize these products and demand a higher level of accuracy and quality in the future.

Keywords: bacterial enumeration, Lactobacillus, LoopSeq, pet nutrition, fermented food

Introduction

Probiotics are living microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al., 2014). Probiotics are becoming increasingly popular, as more information is learned of their potential health benefits, with annual worldwide revenue predicted to reach 64 billion dollars by 2023 (Freedman et al., 2020). Probiotics may come in many forms, including capsules, fermented foods, and dairy supplements. In addition to their potential benefits, their ease of intake, low cost, and low incidence of associated adverse events make them appealing to a broad range of consumers (Reid et al., 2019). For companion animals, veterinarians often prescribe probiotics for those with gastrointestinal (GI) disorders, under stress, or during antibiotic treatment. There is mixed support in regard to research demonstrating the impact of introduced bacterial species in animals with GI disease (Suchodolski and Jergens, 2016) or after a round of antibiotics (Reid et al., 2019), but one study did show reduced antibiotic-associated GI signs following probiotic and yeast treatment after antibiotics in cats (Stokes et al., 2017).

Due to the differences that exist between companion animal and human gut microbiomes (Deng and Swanson, 2015), niche markets for probiotic supplements, probiotic foods (e.g., yogurt), and kefirs made specifically for companion animals have emerged. Kefir contains both yeast and bacteria produced by the fermentation of water or milk with kefir grains (Rosa et al., 2017); it may serve as an alternative to probiotic supplements in companion animals.

The US Food and Drug Administration (FDA) considers probiotic products to be dietary supplements, and according to the FDA Code of Federal Regulations Title 21, kefir is categorized as a cultured milk that contains aroma- and flavor-producing microbial cultures. Where there are nomenclature and manufacturing regulations in place mandating the fermented beverage to be identified as “Kefir cultured milk,” there is little existing regulation regarding the added probiotic quality, quantity, or viability. This lack of regulation has resulted in widespread use of structure-function claims for included microbial cultures that come without or with little evidence of clinical efficacy. Several companion animal kefir product labels contain claims of supporting healthy skin and coat or cognitive function, providing benefit for candidiasis and heart problems, providing anti-fungal properties, preventing allergies, alleviating gas, bloating, and heartburn, and numerous other claims. The popularity of these fermented products in the human consumer market has incentivized misleading label claims resulting in higher sales (Freedman et al., 2020). Randomized clinical trials testing the effects of kefir consumption on humans have been published (Bellikci-Koyu et al., 2019; Wang et al., 2019). Results have suggested that the beverages improve elements of metabolic syndrome, but more clinical trials are needed to understand their effects.

Currently, there are only two published clinical trials that report on testing the effects of kefir in dogs. One study demonstrated that kefir modulated the gut microbiota, including increases in LAB (Kim et al., 2019). The other study reported that dogs supplemented with Lactobacillus kefiri did not have changes to fecal immunoglobulin A concentrations or microbiota populations (Gaspardo et al., 2020). Because neither study reported health benefits, it is clear that more clinical research is needed to test what benefits may be provided by kefir products in companion animals. Recent studies have demonstrated the discrepancies and inaccuracies that exist in regard to bacterial counts and identity listed on probiotic labels compared to those measured in a laboratory (Weese and Arroyo, 2003; Weese et al., 2005; Weese and Martin, 2011). Genomic sequencing of probiotic supplements and foods has been limited in the past due to assay and database limitations that made it difficult to identify bacteria at the species level. Recent advances, however, have resulted in the development of assays able to sequence the majority of the bacterial 16S rRNA gene, which is commonly used for identification purposes.

The objective of this study was to investigate the accuracy of label claims made by commercial kefir products designed for companion animals. Given the lack of regulation and results from previous studies testing companion animal probiotic supplements, we hypothesized that the products tested would not match label claims of viable colony counts or bacterial taxonomy.

Materials and Methods

Sample preparation and culture conditions

Companion animal kefir products were purchased from online vendors and local stores in Urbana, IL. This is a niche market and at the time of this study, six products were available for purchase. For each product, two separate lots were ordered to address batch variability, and experiments on each lot were performed in duplicate. Products included the following: Product A: The Honest Kitchen Daily Boosters Instant Goat’s Milk w/ Probiotics for Dogs & Cats (Chicago, IL); Product B: Open Farm Raw Organic Grass-Fed Kefir Topper for Dogs & Cats (Toronto, Canada); Product C: Answers Pet Food Raw Cow’s Milk Kefir (Fleetwood, PA); Product D: Coco Love Coconut Kefir for Dogs Nuggets (Chenango County, NY); Product E: Champions Choice Natural Kefir (Millersburg, IN); and Product F: Only Natural Raw Goat Milk Kefir (Boulder, CO). A Chobani Plain Yogurt 0% Milk Fat (YOG; South Edmeston, NY) was included as a standard for assessing technique validity. Claims for CFU/g, bacterial taxonomy, lot numbers, and expiration dates were recorded as stated on the labels. Labels and product websites were evaluated for a clear description of microbial taxa (genus and species) and health claims. Products B, C, D, and F were shipped frozen and were kept frozen until 24 h before analysis. These products were thawed, then refrigerated at 4 °C before analysis as per individual manufacturer instructions. Product E and YOG arrived in refrigerated liquid form and were stored at 4 °C until analysis. Product A was refrigerated at 4 °C and rehydrated prior to analysis as recommended on the label. This was done with DNA-free water to avoid foreign bacterial contamination.

To begin, all products were vortexed and thoroughly homogenized, 1-g product was added to 9 mL of phosphate-buffered saline (PBS), and the product-PBS mixture was vortexed 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 (MRS) media (BD Difco, Franklin Lakes, NJ). MRS plates were incubated anaerobically at 37 °C for 48 h and aerobically at 30 °C for 48 h to culture lactic acid bacteria (LAB). Colony counts were measured via spiral plating software (Colony counter, IUL Flash and Go, Neutec Group Inc., Farmingdale, NY). No attempt to distinguish organisms was made, only enumeration. Fourteen days after product opening, enumeration was repeated for all product lots in identical conditions. Results at time of opening and results at 14 d are reported as the average of each duplicate from both product lots.

DNA extraction and sequencing

Samples were vortexed until homogenized, then 250 mg of each kefir product sample on the day of opening was used for DNA extraction (DNeasy PowerLyzer PowerSoil Kit, Qiagen, Germantown, MD) following manufacturer’s instructions. DNA quantity was determined using a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA) and DNA quality was assessed via gel electrophoresis (E-Gel Power Snap and E-Gel EX 1% Agarose, Invitrogen by Thermo Fisher Scientific, Carlsbad, CA). DNA extracts were sequenced at the W. M. Keck Center for Biotechnology at the University of Illinois at Urbana-Champaign using the LoopSeq 16S Long Read Kit (Loop Genomics, San Jose, CA) at a 1 ng/μL concentration. All samples were processed following the standard Loop Genomics protocol for full length 16S sequencing. Sequence results were processed through the SILVA132 Large Subunit rRNA Database. All taxa with a relative abundance greater than 0.001% were retained for analysis.

Statistical analysis

Statistical analyses were conducted using R x64 version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). Data obtained from enumeration samples on 1 and 14 d, and relative abundance of Lot 1 and relative abundance of Lot 2 were tested for significance via paired Student’s t-test. A P value less than 0.05 was considered statistically significant.

Results

Bacterial enumeration

Bacterial plating on MRS media results is presented in Table 1. Anaerobic conditions at 37 °C were maintained to culture LAB, and aerobic conditions were held at 30 °C to culture aerobes such as Streptococcus thermophilus and Lactobacillus lactis. The label for Product A claimed that each sachet contained 5 × 109 CFU/g at point of rehydration. Using our culture media and conditions, Lot 2 failed to reach the 2.0 × 106 threshold of the assay on 1 and 14 d, yet Lot 1 reached 2.04 × 108 ± 1 × 103 and 1.53 × 109 ± 1.87 × 109 in anaerobic conditions. Product B is sold as a kefir topper for dogs and cats, claiming 1.5 × 1010 CFU/g. Using our culture media and conditions, Lot 1 and Lot 2 contained colonies much lower than the counts claimed on the label. Although Product C claimed 1 × 1010 CFU/g, our culture methods showed that neither lot surpassed 1.4 × 108 CFU/g at opening. Colony counts decreased when tested 14 d later. Product D’s label had no CFU/g claims. Upon enumeration, both Lot 1 and Lot 2 contained less than our detection limit (2.0 × 106 CFU/g). Product E did not make claims for amount of viable CFU on their label, but had the highest cell counts of the kefir products tested. Our enumeration results at time of opening showed that Lot 1 contained 9.68 × 108 ± 3.69 × 107 and Lot 2 contained 9.14 × 108 ± 7.11 × 107 when tested under aerobic conditions. Retesting 14 d after opening showed that viable colony counts significantly decreased. Product F claimed to contain 1.00 × 106 CFU/g at opening, yet neither lot contained enough bacteria to exceed the detectable colony threshold. After 14 d, viable colonies were still below the threshold. The standard product used to validate techniques (YOG) claimed to contain 1 × 1010 CFU/g. Our enumeration tests estimated that Lot 1 and Lot 2 met these claims. When products were tested again 14 d after opening, the values were largely unchanged.

Table 1.

Comparison of bacterial cell counts (colony forming units/gram) label claims and enumeration results on deMan Rogosa and Sharpe (MRS) media in aerobic (AER) and anaerobic (ANA) conditions

Day 1 Day 14
Product1 Claimed Lot MRS AER MRS ANA MRS AER MRS ANA
A 5.00 × 109 1 2.04 × 108 ± 1 × 103 < 2.0 × 106 5.10 × 108 ± 1.44 × 108 1.53 × 109 ± 1.87 × 109
2 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106
B 1.50 × 1010 1 3.87 × 107 ± 8.65 × 106 4.79 × 107 ± 4.32 × 106 3.21 × 106 ± 1.01 × 105 3.29 × 106 ± 3.03 × 105
2 4.08 × 107 ± 2.88 × 106 < 2.0 × 106 7.22 × 106 ± 7.81 × 105 6.96 × 106 ± 5.68 × 105
C 1.00 × 1010 1 9.88 × 107 ± 2.16 × 107* 1.4 × 108 ± 5.33 × 107 < 2.0 × 106* 6.93 × 107 ± 1.44 × 107
2 1.06 × 108 ± 1.15 × 107* 1.06 × 108 ± 2.02 × 107 < 2.0 × 106* 1.22 × 108 ± 1.15 × 107
D N/A 1 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106
2 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106
E N/A 1 9.68 × 108 ± 3.69 × 107* 8.82 × 108 ± 5.41 × 107 1.43 × 107 ± 1 × 103 1.12 × 107 ± 1.44 × 106
2 9.14 × 108 ± 7.11 × 107* 6.68 × 108 ± 8.29 × 107 4.48 × 107± 1 × 103* 3.87 × 107 ± 2.88 × 106
F 1.00 × 106 1 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106
2 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106 < 2.0 × 106
YOG 1.00 × 1010 1 5.12 × 109 ± 1 × 103 5.71 × 109 ± 2.51 × 108 3.14 × 109 ± 2.12 × 108 4.08 × 109 ± 7.94 × 107
2 9.00 × 109 ± 1.08 × 109 1.19 × 1010 ± 6.96 × 109 4.64 × 109 ± 1.01 × 109 1.09 × 1010 ± 1.30 × 109
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1Product A: The Honest Kitchen Daily Boosters Instant Goat’s Milk w/ Probiotics for Dogs & Cats (Chicago, IL); Product B: Open Farm Raw Organic Grass-Fed Kefir Topper for Dogs & Cats (Toronto, Canada); Product C: Answers Pet Food Raw Cow’s Milk Kefir (Fleetwood, PA); Product D: Coco Love Coconut Kefir for Dogs Nuggets (Chenango County, NY); Product E: Champions Choice Natural Kefir (Millersburg, IN); Product F: Only Natural Raw Goat Milk Kefir (Boulder, CO); YOG: Chobani Plain Yogurt 0% Milk Fat (South Edmeston, NY).

*A significant difference from 1 to 14 d (P < 0.05 by paired Student’s t-test).

Sequencing bacterial DNA

Loop Genomics sequencing was used to assess the 16S (bacteria) rRNA components of the test products (Table 2). The most common and abundant genera were Lactococcus, Streptococcus, and Bacillus. For some products, the bacterial genus and species listed on the labels appeared to differ significantly from our results. Product A claimed to contain four bacterial species (B. coagulans, L. casei, L. acidophilus, and L. rhamnosus), which were all detected in the sequencing results. In addition, nearly 10 unclaimed species were detected. For Product B, sequencing results only detected the Lactobacillus and Propionibacterium species claimed on its label. However, the Bifidobacterium, Enterococcus and Leuconostoc spp. listed were not detected. Product C claimed nine bacterial species on their label, yet sequencing detected 15 bacterial species among seven bacterial genera. For Product D, sequencing results confirmed seven bacterial species, all LAB, yet none were claimed on the label. Product E claimed to contain four bacterial genera and one bacterial species, yet only DNA from the Lactococcus, Streptococcus, and Lactobacillus genera were detected. No Acetobacter were detected (less than 0.1% abundance). Product F only claimed one bacterial species, yet our analyses detected nine bacterial genera, including 10 bacterial species but not the species on the label. The yogurt sample claimed to contain five bacterial species. Our sequence data were successful for Lot 1 but failed to generate sufficient data for Lot 2 due to extremely low DNA concentrations. Four of the five species claimed were clearly identified via sequencing. Sequencing also identified a significant abundance of unidentified lactobacilli, which may explain the lack of L. bifidus in the sequencing results. An additional three bacterial species were detected via sequencing but each is closely related to another identified species, suggesting a limitation for using Loop for species discrimination of very similar species.

Table 2.

Comparison of label claims and 16S rRNA sequencing results (relative abundance) of commercial kefir products

Product1 Claimed Species Lot 1 Lot 2
A Bacillus coagulans Bacillus coagulans 0.793 0.759
Lactobacillus casei Lactobacillus sp. 0.098 0.144
Lactobacillus acidophilus Lactobacillus casei 0.044 0.060
Lactobacillus rhamnosus Lactobacillus acidophilus 0.023 0.008
Lactobacillus paracasei 0.012 0.005
Lactobacillus plantarum 0.011 0.006
Lactobacillus rhamnosus 0.010 0.004
Pseudomonas stutzeri 0.003 0.003
Bacillus sp. 0.002 0.002
Lactobacillus pentosus 0.001 0.000
Staphylococcus sp. 0.000 0.001
Lactococcus sp. 0.000 0.001
Streptococcus salivarius 0.000 0.001
Streptococcus thermophilus 0.000 0.001
B Lactobacillus delbrueckii Streptococcus thermophilus 0.420 0.352
Lactobacillus casei Lactobacillus paracasei 0.285 0.174
Lactobacillus acidophilus Lactococcus sp. 0.137 0.125
Propionibacterium freudenreichii Lactobacillus delbrueckii 0.055 0.041
Propionibacterium freudenreichiilacti Streptococcus salivarius 0.037 0.130
Leuconostoc mesenteroides Lactobacillus casei 0.025 0.068
Bifidobacterium animalis Lactococcus lactis 0.014 0.046
Enterococcus cremoris Lactobacillus sp. 0.010 0.014
Enterococcus diacetyenterococcus thermophilus Lactobacillus rhamnosus 0.007 0.015
Enterococcus diacetylacti Streptococcus sp. 0.007 0.031
Enterococcus thermophilus Lactobacillus acidophilus 0.003 0.002
Propionibacterium sp. 0.001 0.000
C Lactococcus lactis Lactococcus sp. 0.753 0.724
Lactobacillus kefir Lactococcus lactis 0.079 0.079
Leuconostoc mesenteroides Streptococcus thermophilus 0.070 0.065
Lactobacillus acidophilus Lactobacillus kefiranofaciens 0.053 0.089
Lactobacillus bulgaricus Pseudomonas sp. 0.012 0.010
Lactobacillus caucasicus Leuconostoc mesenteroides 0.009 0.010
Lactococcus cremoris Streptococcus salivarius 0.005 0.006
Leuconostoc cremoris Streptococcus uberis 0.004 0.003
Saccharomyces kefir Citrobacter freundii 0.004 0.003
Lactobacillus kefiri 0.003 0.004
Streptococcus sp. 0.003 0.002
Lactobacillus sp. 0.001 0.001
Lactococcus raffinolactis 0.001 0.002
Raoultella ornithinolytica 0.001 0.001
Pseudomonas fluorescens 0.001 0.001
D N/A Streptococcus thermophilus 0.567 0.669
Lactobacillus sp. 0.122 0.097
Lactobacillus acidophilus 0.113 0.094
Streptococcus salivarius 0.091 0.067
Lactobacillus paracasei 0.035 0.027
Lactobacillus rhamnosus 0.026 0.014
Lactobacillus casei 0.016 0.009
Streptococcus sp. 0.015 0.010
Lactobacillus delbrueckii 0.012 0.012
Lactococcus sp. 0.001 0.000
E Lactobacillus Lactococcus sp. 0.823 0.881
Streptococcus Lactococcus lactis 0.169 0.112
Acetobacter Lactobacillus sp. 0.004 0.002
Lactobacillus caucasicus Streptococcus uberis 0.002 0.002
Leuconostoc Lactobacillus casei 0.001 0.000
Saccharomyces kefir Lactobacillus plantarum 0.001 0.000
Streptococcus sp. 0.000 0.001
Streptococcus thermophilus 0.000 0.001
F Lactobacillus acidophilus Lactococcus sp. 0.891 0.878
Lactococcus lactis 0.075 0.090
Lactobacillus kefiranofaciens 0.003 0.004
Streptococcus sp. 0.002 0.001
Enterococcus faecalis 0.001 0.001
Lactobacillus paracasei 0.001 0.000
Streptococcus thermophilus 0.001 0.001
YOG Streptococcus thermophilus Streptococcus thermophilus 0.775
Lactobacillus acidophilus Streptococcus salivarius 0.087
Lactobacillus casei Lactobacillus paracasei 0.046
Lactobacillus bulgaricus Lactobacillus sp. 0.044
Lactobacillus bifidus Lactobacillus acidophilus 0.014
Streptococcus sp. 0.014
Lactobacillus casei 0.009
Lactobacillus rhamnosus 0.008
Lactobacillus delbrueckii 0.002
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1Product A: The Honest Kitchen Daily Boosters Instant Goat’s Milk w/ Probiotics for Dogs & Cats (Chicago, IL); Product B: Open Farm Raw Organic Grass-Fed Kefir Topper for Dogs & Cats (Toronto, Canada); Product C: Answers Pet Food Raw Cow’s Milk Kefir (Fleetwood, PA); Product D: Coco Love Coconut Kefir for Dogs Nuggets (Chenango County, NY); Product E: Champions Choice Natural Kefir (Millersburg, IN); Product F: Only Natural Raw Goat Milk Kefir (Boulder, CO); YOG: Chobani Plain Yogurt 0% Milk Fat (South Edmeston, NY).

*A significant difference from relative abundance of Lot 1 to relative abundance of Lot 2 (P < 0.05 by paired Student’s t-test).

Discussion

The market for companion animal probiotic products has increased in demand in recent years but has little regulatory oversight. Previous studies have demonstrated deficiencies in companion animal probiotic quality, quantity, and label accuracy (Weese and Martin, 2011). After analyzing six pet kefir products, all three brands that made CFU/g claims failed to provide the guarantees stated on their labels. These results suggest a lack of quality control, accurate testing, and/or deficiencies in bacteria stability during storage. Even if label claims were met at the point of manufacture, viability should be maintained until the expiration date is reached. Literature supports that bacteria do not need to be alive to provide an anti-inflammatory response, but must be viable to modulate the microbiota (Adams, 2010). Certain products had counts up to 107–108 CFU/g, so it is reasonable to assume that these products with higher counts would be more likely to be effective than those with lower counts. It should be noted that cultivation of LAB was attempted in both anaerobic and aerobic conditions to enumerate as many species as possible in each product. Previous in vivo probiotic studies in pets have tested doses of 107–109 CFU/g and observed effective responses as measured by microbial culture (Strompfová et al., 2006; Huys et al., 2013). Of the three brands that did not make claims, two of them did not have enough viable colonies to meet our minimum detection threshold of 2.0 × 106 CFU/g.

Despite there being only two published clinical trials in dogs whereby kefir response was minimal (Kim et al., 2019; Gaspardo et al., 2020), the six kefir products tested in this study all made health claims on their websites or on their product labels. Product A claimed to support digestion and the immune system. Several claims were made by product B, including support for strong teeth, soft skin and coat, improved gut health and digestion, as well as a strong immune system. Product C claimed to assist in the ease of digestion, and that consuming live enzymes will maximize nutrient absorption. A range of health claims were made by product D, including support for healthy skin and coat, cognitive function, delayed osteoarthritis, and improved blood circulation. Product E made the most health claims for their product, stating anti-fungal properties, restoration of the digestive tract after a course of antibiotics, prevention of allergies, promotion of healthy skin, alleviation of gas, bloating, and heartburn, and several more diseases. Product F claimed to settle a sensitive stomach, support a healthy immune system, and entice picky eaters. Clinical trials have been performed to understand the effects of kefir on humans, but these conclusions cannot be directly translated to dogs. Like claims for probiotics (Hill et al., 2014) and prebiotics (Gibson et al., 2017), kefir products should be tested in the target host animal. Until these studies are conducted, these broad claims are not supported by the published literature.

Product A contained high amounts of Bacillus coagulans. Bacillus coagulans are spore-forming bacteria, which when sporulated can withstand processing and acidic environments such as the stomach thus able to reach the small intestine for germination (Ara et al., 2002). If present as spores in the products, it is possible that the spores did not germinate and form colonies on our plates and thus underrepresent the counts in product A. This species has been demonstrated to assist companion animals in carbohydrate utilization and to create an environment inhospitable to pathogens (Jäger et al., 2018). However, further studies are needed to fully understand the safety and dose of B. coagulans probiotics for pets. In Products B and D, the main bacteria detected were of the Streptococcus genera (Table 2). Streptococci are Gram-positive bacteria and are normal inhabitants of animal and human GI microbiota. Streptococcus thermophilus is often essential in industrial dairy production as yogurt starters, so it is unsurprising that these bacteria appear in high amounts in kefir products. Other pathogenic members of streptococci such as Streptococcus mutans and Streptococcus pneumoniae are pathogenic to mammals, but Streptococcus thermophilus has adapted to the dairy environment and lost functionality of genes not involved in basic cellular functions (Price et al., 2012).

The genera Lactococcus made up the majority of sequences measured in Products C, E, and F, and is a LAB common in dairy products. The species L. lactis is commonly used as a starter culture for dairy products, but no companion animal studies have researched effective dose or observed its impacts on the animal’s GI tract and health indices. Many of the tested kefir products contained numerous members of the Lactobacillus genera, including L. acidophilus, L. casei, L. delbrueckii, L. paracasei, and L. rhamnosus, several of which have been investigated individually and as “cocktails” for use in cats and dogs (Baillon et al., 2004; Garcia-Mazcorro et al., 2011; Ferandez et al., 2019; Fusi et al., 2019). It should be noted that in Product D and YOG, their labels claim to contain Lactobacillus bulgaricus whose proper name is Lactobacillus delbreuckii subsp. bulgaricus. Because the names of many Lactobacillus spp. have now been changed (Zheng et al., 2020), labels for many commercial probiotic products will require revision in the near future.

Considering our results, it would be very difficult to detect and enumerate all claimed bacteria present in equal amounts. Identification of Enterococci and Leuconostoc genera by LoopSeq was extremely low; this suggests poor survival in the final fermented product, potential bias in the DNA extraction procedure, or limitations in the primers used for sequencing. Products were thoroughly homogenized before samples were taken, but sonification might have broken up cell clusters better to provide a more accurate enumeration of bacteria. Further tests using real-time polymerase chain reaction for individual genera could confirm these hypotheses. Many bacterial species not claimed by the kefir labels were detected in sequencing, suggesting those species might originate from the manufacturing process and were not unintentionally listed, or were misidentified by SILVA132 data base due to high sequence similarity with other species. For example, Lactobacillus casei and L. paracasei have very similar 16S rRNA sequences and may lead to misidentification when only one of these is present in the sample such as with products A, B, and YOG. Some labels had the issue of inconsistency in labeling bacterial genera and species properly, while others had issues with correct spellings and proper capitalization.

In conclusion, our analysis of companion animal kefir products via culture enumeration and sequencing revealed many deficiencies in label accuracy. Better quality control of these products and clinical trials to demonstrate and understand their potential health benefits for animals are required. Given the various health claims, inconsistent product quality, and absence of regulatory scrutiny, consumers should be wary of this segment of the market and demand better quality commercial probiotics for their pets.

Glossary

Abbreviations

CFU/g

colony forming units/gram

GI

gastrointestinal

FDA

Food and Drug Administration

LAB

lactic acid bacteria

MRS

deMan Rogosa and Sharpe

PBS

phosphate-buffered saline

Funding

This study was funded by USDA Hatch Grant ILLU-538-937.

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

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