A “berry” small inclusion: 40 types of commercial dog and cat kibble with added blueberries provide low levels of quercetin, free phenolics, and alkali-labile phenolics

Abstract

Blueberries provide dietary polyphenols and are often included in dog and cat kibble as a source of antioxidants. For this study, we investigated the polyphenol content in commercial dog and cat extruded food that lists blueberries on their ingredient deck. We sampled 40 bags of kibble (18 cat and 22 dog) from four pet food stores in Guelph, Canada. High-performance liquid chromatography was employed for the determination of quercetin, free phenolics (caffeic acid; cinnamic acid; ferulic acid; gallic acid; hesperetin; naringin; p-coumaric acid; p-hydroxybenzaldehyde; protocatechuic acid; syringaldehyde; syringic acid; vanillic acid; vanillin), and antioxidant-protected alkali-labile phenolics (caffeic acid; chlorohenic acid; cinnamic acid; ferulic acid; gallic acid; hesperetin; naringin; p-coumaric acid; p-hydroxybenzaldehyde; protocatechuic acid; sinapic acid; syringaldehyde; syringic acid; vanillic acid; vanillin). Mean concentration ± SE was calculated for all assessed polyphenol types. The ANOVA procedure was used to determine if the intended species (cat or dog) affected quercetin and antioxidant-protected alkali-labile polyphenol concentrations. Quercetin concentrations were found at 5.05 ± 4.32 µg/g across all bags. Cat kibble had lower average concentrations of quercetin and alkali-labile phenolics compared to dog kibble, which is likely linked to the quantity of added fruits and vegetables. Concentrations of free phenolics were minimal to nonexistent for all kibble types. Dog and cat kibble containing blueberries do not provide significant amounts of dietary polyphenols and, therefore, do not contribute to enhanced sources of antioxidants and anti-inflammation. Future research should assess optimal polyphenol doses in healthy cats and dogs to determine the target doses to achieve physiological benefits.

Forty bags of cat (n = 18) and dog (n = 22) kibble with added blueberries contained low levels of quercetin and free and antioxidant-protected polyphenols. The tested extruded kibbles that list blueberries on their ingredient deck likely do not provide enough polyphenols from blueberries to supply enhanced sources of antioxidants and anti-inflammatory properties.

Introduction

Blueberries are an excellent source of polyphenols that provide antioxidant and anti-inflammatory benefits to humans and animals in a dose-dependent manner. Given that pet foods follow human food trends, blueberries are often included in cat and dog kibble as a source of antioxidants; however, little scientific evidence supports this claim. Given the dose-dependency of these compounds and the metabolic idiosyncrasies of dogs and cats, the impact of blueberry supplementation and functional dose should be assessed in each species separately. Further, the amount and types of polyphenols provided by commercial cat and dog kibble with added blueberries have not been surveyed.

Polyphenols are secondary metabolites found in the cell walls of plants. Based on their structure, they can be classified into five to six subcategories (phenolic acids, stilbenes, flavonoids, lignans, tannins, and other polyphenols) (Li et al., 2014). Polyphenols can be bound to cell wall components (released through alkali hydrolysis) or unbound (free) (Beckman, 2000). Sources of dietary polyphenols have been studied primarily for their antioxidant benefits across species. For example, sled dogs fed blueberries before an exercise challenge showed an increase in total antioxidant power (TAP) when compared to a control group immediately following exercise; however, these effects were no longer observed 24 h postexercise (Dunlap et al., 2006), likely due to a short supplementation period (4 d) or a potentially low dose. This said, the impact of dietary polyphenols on oxidation status has been studied in other species—such as sheep, pigs, cattle, and poultry—and can be attributed to their ability to scavenge free radicals (Pandey and Rizvi, 2012; Alov et al., 2015), increase vitamin E regeneration (Pazos et al., 2007; Fabre et al., 2015), inactivate metal ions (Wu et al., 2024), and inhibit pro-oxidizing enzymes (Gladine et al., 2007; Hussain et al., 2016; Wu et al., 2024).

Polyphenol content found in blueberries depends on the cultivar strain (Bunea et al., 2011; Gavrilova et al., 2011; Giovanelli and Buratti, 2009; Häkkinen et al., 1999; D. Prior et al., 1998; Li et al., 2017; Wang et al., 2019), processing method (Häkkinen et al., 1999; Lee and Wrolstad, 2004; Li et al., 2013), ripeness (Gibson et al., 2013) and environmental factors such as altitude (Prior et al., 2001; Li et al., 2017), UV exposure (Gavrilova et al., 2011), and soil pH (Ochmian et al., 2020). While the total phenolic content varies, research reports high levels of flavonoids, most commonly anthocyanins and flavonols, in blueberries. Other antioxidant sources found in blueberries include ascorbic acid, tocopherols, and carotenoids. However, total phenolic content is known to have a strong linear relationship with total antioxidant capacity (TAC) in both wild and cultivated blueberries (Prior et al., 1998; Giovanelli and Buratti, 2009). Additionally, quercetin, which appears in high concentrations in blueberries, has one of the highest antioxidant capacities. In previous work, quercetin exhibited a strong potential to scavenge free radicals, as measured by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging (Heřmánková et al., 2019). Therefore, it can be estimated that the concentration of polyphenols, especially quercetin, can be used to evaluate some of the antioxidant abilities of food sources. We focused on blueberries due to their widespread growth across North America and use in the pet food market. Therefore, the objective of this study is to analyze the polyphenol profile (including quercetin and various free and alkali-protected phenolics) in cat and dog kibble with blueberries listed on their ingredient deck and to compare the profiles between the kibbles intended for each species. We hypothesize that dog and cat kibble will contain low levels of free and alkali-labile phenolics and quercetin, with dog kibble containing greater concentrations of all assessed polyphenol types when compared to cat kibble.

Materials and Methods

Pet food collection, storage, and transport conditions

Forty bags of pet food (18 cat and 22 dog; Supplementary Table S1) were purchased from four pet stores (Pet Valu, Global Pets, Rens Pet Depot, and Pet Smart) in Guelph, ON, in October 2023. An internet search of available formulas with blueberries was conducted to determine which stores carried the relevant foods. Then, the ingredient lists of all dog and cat kibbles in each pet food line were screened in-store. All selected pet foods listed blueberries in their ingredient lists. The selected kibble was formulated for healthy adults, including both dogs or cats, and only one product was selected per product line for each species. Once collected, subsamples (a minimum of 400 g) of the kibble were taken and stored in labeled plastic bags to prepare for analysis. The kibble was frozen at −20 °C to help prevent fat oxidation and leaching through the plastic bags. The bags of kibble were transported to the testing facility at the University of Illinois (Urbana, IL) on ice for phenolic analysis.

Sample preparation

Kibble was ground using a model 4 mill with a 1-mm screen (Thomas Wiley Mills, Hoboken, NJ). Each ground sample was then analyzed for organic matter (AOAC, 942.05) and dry matter (DM; AOAC, 934.01). Methods to quantify free and alkali-labile phenolics (Jung et al., 1983; Hefni et al., 2019) and quercetin (Mikkonen et al., 2001) have previously been described.

Quercetin extraction

To prepare samples for quercetin analysis, 400 to 500 mg of ground kibble was weighed, and 25 mL of 50% methanol solution containing 300 μg morin and 20 mg tert-butylhydroquinone was added. The resulting mixture was vortexed and placed in a 35 °C water bath for two hours. The supernatant was then transferred to 50 mL centrifuge tubes and spun at 950 × g for 10 min. From this mixture, 10 mL was added to a clean 25 × 150 mm Teflon-lined test tube, followed by the addition of 2 mL of 6 M hydrochloric acid solution. Tubes were placed in a 90 °C water bath for 2 h. Next, 2 mL of 6 M sodium hydroxide solution and 20 mL of ethyl acetate were added, and samples were placed on a roller mixer for 15 min. Then, the upper layer (ethyl acetate) was transferred to 20 × 150 mm tubes. Samples were evaporated to dryness at 35 °C with N₂ gas, then redissolved in 1 mL of methanol. Extracts were stored at −20 °C until high-performance liquid chromatography (HPLC) analysis.

Free phenolics extraction

To prepare samples for analysis of free phenolics, 1 g of ground kibble was weighed, and 40 mL of ethyl ether was added. The samples were purged with N₂ gas and vortexed every 2 h during the initial 8 h of the 24-h extraction period, which was conducted in the dark. After the extraction, samples were vortexed once more immediately before being filtered through Whatman No. 54 filter paper and rinsed with 5 mL of ethyl ether. They were then evaporated to dryness with N₂ gas at 40 °C and then redissolved in 2 mL of methanol. Extracts were stored at −20 °C until HPLC analysis.

Alkali-labile phenolics extraction

To prepare samples for analysis of alkali-labile phenolics, approximately 200 mg of ground kibble was weighed, and 5 mL of 2N NaOH (O₂ free) containing 40 ppm dimethylaminocinnamaldehyde (DMCA) was added. Samples were purged with N₂ gas and vortexed every 2 h during the initial 8 h of the 24-h extraction period, which was conducted in the dark. After the extraction, samples were transferred to centrifuge tubes, and 4 mL of water was added, followed by 1 mL of phosphoric acid. Samples were cooled to 4 °C and centrifuged at 20,000 × g for 20 min. Supernate was transferred to a C18 solid phase extraction cartridge, and phenolic compounds were eluted with 5 mL 50% methanol solution. Extracts were stored at −20 °C until HPLC analysis.

Analysis of quercetin, free phenolics, and alkali-labile phenolics

Each polyphenol type was assessed using HPLC (model ICS-5000; Dionex Corp., Sunnyvale, CA), and all samples were tested in duplicate. In brief, free and alkali-labile phenolic compounds were separated using a GL Sciences Inertsil ODS-1 column (4.6 × 250 mm, 5 µm) fitted with a matching guard column. The mobile phase consisted of 10 mM potassium phosphate buffer (pH 3.25) and methanol, delivered at a constant flow rate of 1.0 mL/min. The injection volume was 25 µL, and detection was carried out at a wavelength of 305 nm using a UV detector. The column was maintained at ambient temperature throughout the analysis. The gradient elution was programmed as follows: 0 to 10 min, 78% buffer and 22% methanol; 10 to 20.6 min, 74% buffer and 26% methanol; 20.6 to 35.0 min, 70% buffer and 30% methanol; 35.0 to 40.0 min, 30% buffer and 70% methanol; 40.0 to 46.0 min, 15% buffer and 85% methanol; 46.1 to 53.0 min, 100% methanol; followed by a re-equilibration to initial conditions (78% buffer and 22% methanol) from 53.1 to 70.0 min. The flow rate was briefly increased to 1.2 mL/min at 46.1 min to aid elution before returning to 1.0 mL/min for re-equilibration.

Quercetin analysis was conducted using a Waters Spherisorb ODS-1 column (4.6 × 250 mm, 5 µm) with a matching guard column. The mobile phase consisted of 10 mM potassium phosphate buffer (pH 3.0) and acetonitrile, delivered at a 1.0 mL/min flow rate. The injection volume was 25 µL, and detection was carried out at a wavelength of 360 nm using a UV detector. The column temperature was maintained at ambient conditions. A gradient elution program was employed as follows: 0 to 10 min, 95% buffer and 5% acetonitrile; 10 to 20 min, 60% buffer and 40% acetonitrile; 20.0 to 20.1 min, 30% buffer and 70% acetonitrile, followed by 100% acetonitrile from 22.0 to 23.0 min. The flow rate was increased to 1.2 mL/min at 20.1 min and maintained until 25.0 min, after which the initial conditions (95% buffer and 5% acetonitrile) were restored and maintained until 32.0 min for re-equilibration. Final concentrations for quercetin, free phenolics, and alkali-labile phenolics were calculated using the following equations:

$${\displaystyle \begin{array}{l}\begin{array}{c}\mathrm{Q}\mathrm{u}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{n}(\mu \mathrm{g}/\mathrm{g}\mathrm{D}\mathrm{M})=[\mathrm{H}\mathrm{P}\mathrm{L}\mathrm{C}\mathrm{p}\mathrm{p}\mathrm{m}(\mu \mathrm{g}/\mathrm{m}\mathrm{l})\times \mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}]/[\mathrm{D}\mathrm{r}\mathrm{y}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}(\mathrm{g})]\end{array}\end{array}}$$

$${\displaystyle \begin{array}{l}\begin{array}{c}\mathrm{F}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{p}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{s}(\mu \mathrm{g}/\mathrm{g}\mathrm{D}\mathrm{M})=[\mathrm{H}\mathrm{P}\mathrm{L}\mathrm{C}\mathrm{p}\mathrm{p}\mathrm{m}(\mu \mathrm{g}/\mathrm{m}\mathrm{l})\mathrm{x}2\mathrm{m}\mathrm{L}]/[\mathrm{D}\mathrm{r}\mathrm{y}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}\right)]\end{array}\end{array}}$$

$${\displaystyle \begin{array}{l}\begin{array}{c}\mathrm{A}\mathrm{l}\mathrm{k}\mathrm{a}\mathrm{l}\mathrm{i}\text{-}\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{e}\mathrm{p}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{c}\mathrm{s}(\mu \mathrm{g}/\mathrm{g}\mathrm{D}\mathrm{M})=\mathrm{H}\mathrm{P}\mathrm{L}\mathrm{C}\mathrm{p}\mathrm{p}\mathrm{m}(\mu \mathrm{g}/\mathrm{m}\mathrm{l})\mathrm{x}5\mathrm{m}\mathrm{l}/[\mathrm{D}\mathrm{r}\mathrm{y}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}(\mathrm{g})]\end{array}\end{array}}$$

Statistical analysis

Descriptive statistics were conducted to determine the mean concentrations and SE of each polyphenol class, as well as to assess the highest and lowest concentrations within each class. A post hoc analysis was performed using the proc ANOVA procedure in SAS Studio (Version 3.81; Enterprise Edition) to determine if the intended species (cat or dog) affected quercetin and antioxidant-protected alkali-labile concentrations. Given the low inclusion and high variability of free phenolics, further statistical analyses were not conducted for these compounds.

Results

Dog kibble had higher polyphenol concentrations than cat kibble (Table 1); however, different polyphenol profiles were observed in individual foods.

Table 1.

Average concentration of quercetin, free phenolics, and alkali-labile phenolics (µg/g) on a DM basis in dog (n = 22) and cat (n = 18) kibble with added blueberries

Phenolic group	Phenolic acid	Cat kibble (µg/g)	±SE	Dog kibble (µg/g)	±SE
Quercetin	Quercetin	3.2a	0.44	6.57b	1.1
Free	Gallic acid	0.13	0.13	ND	ND
	Protocatechuic acid	0.04	0.02	ND	ND
	p-Hydroxybenzaldehyde	0.03	0.02	0.01	0.01
	Vanillic acid	0.06	0.03	ND	ND
	Caffeic acid	ND	ND	ND	ND
	Syringic acid	0.02	0.01	ND	ND
	Syringaldehyde	0.04	0.02	0.01	0.01
	p-Coumaric acid	0.07	0.07	ND	ND
	Ferulic acid	ND	ND	0.01	0.01
	Naringin	0.05	0.03	ND	ND
	Hesperetin	0.01	0.01	ND	ND
	Cinnamic acid	0.25	0.15	ND	ND
Alkali-labile	Gallic acid	11.28	1.59	14.61	2.08
	Protocatechuic acid	97.74a	24.29	244.36b	42.77
	Chlorogenic acid	23.38a	5.94	4.79b	1.9
	Vanillic acid	29.89	8.12	45.29	5.55
	p-Hydroxybenzaldehyde	15.65	2.15	11.65	0.99
	Caffeic acid	3.91a	0.83	11.23b	1.87
	Syringic acid	46.41	9.73	29.38	4.59
	Vanillin	2.10a	0.82	4.81b	0.59
	Syringaldehyde	1.87	0.56	0.69	0.22
	p-Coumaric acid	75.37	43.37	84.39	13.86
	Ferulic acid	84.46a	16.42	268.05b	41.83
	Sinapic acid	20.02	5.11	22.69	6.67
	Cinnamic acid	4.01	1.37	1.45	0.56
	Naringin	2.74	1.01	2.41	0.83
	Hesperetin	2.46a	0.91	0.10b	0.08

Abbreviation: ND, not detected.

Different letters within the same row for quercetin and alkali-labile phenolics indicate a significant difference at P < 0.05. Due to reported low levels of free phenolics, no statistical analyses were conducted.

Quercetin

Average quercetin concentrations for the assessed cat and dog kibbles were reported as 3.20 µg/g and 6.57 µg/g on a DM basis, respectively. The highest and lowest concentrations were reported as 25.13 µg/g and 0.32 µg/g in a high-protein dog and grain-free cat kibble, respectively (Supplementary Table S2). Cat kibble showed lower average concentrations of quercetin when compared to dog kibble (P < 0.05).

Free phenolics

Low and often untraceable concentrations of free phenolics were found in the assessed cat and dog kibble. The range in concentration for each free phenolic was: cinnamic acid = 0 to 2.44 µg/g, ferulic acid = 0 to 0.11 µg/g, gallic acid = 0 to 2.29 µg/g, hesperetin = 0 to 0.17 µg/g, naringin = 0 to 0.39 µg/g, p-coumaric acid = 0 to 1.17 µg/g, protocatechuic acid = 0 to 0.29 µg/g, p-hydroxybenzaldehyde = 0 to 0.12 µg/g, syringaldehyde = 0 to 0.38 µg/g, syringic acid = 0 to 0.17 µg/g, vanillic acid = 0 to 0.47 µg/g. Free caffeic acid and ferulic acid were not detected in cat kibble. Free caffeic acid was also not detected in dog kibble (Supplementary Table S2). Free syringaldehyde was detected the most often across all assessed pet foods (17.5%; 7/40). Most of the detected free phenolics appeared as outliers and produced high variability in measurements, especially for protocatechuic acid, p-coumaric acid, and hesperetin, detected in cat kibble.

Alkali-labile phenolics

Greater concentrations of protocatechuic acid, caffeic acid, vanillin, and ferulic acid and lower concentrations of chlorogenic acid and hesperetin were found in dog kibble compared to cat (P < 0.05 for all). No other significant differences between cat and dog kibble were found for other assessed alkali-labile phenolics. The range in concentration for each alkali-labile phenolic was: caffeic acid = 0 to 38.4 µg/g, chlorogenic acid = 0 to 94.4 µg/g, cinnamic acid = 0 to 20.2 µg/g, ferulic acid = 20.1 to 831.4 µg/g, gallic acid = 0 to 49.9 µg/g, naringin = 0 to 12.7 µg/g, hesperetin = 0 to 13.4 µg/g, p-coumaric acid = 1.6 to 782.2 µg/g, p-hydroxybenzaldehyde = 1.2 to 31.5 µg/g, protocatechuic acid = 3.1 to 595.4 µg/g, sinapic acid = 0 to 102.7 µg/g, syringaldehyde = 0 to 8.8 µg/g, syringic acid = 0 to 179.5 µg/g, vanillic acid = 0 to 118.0 µg/g, and vanillin = 0 to 13.2 µg/g (Supplementary Table S3). Alkali-labile ferulic acid, p-hydroxybenzaldehyde, and p-coumaric acid were detected in all 40 pet foods, whereas hesperetin was detected the least often (22.5%; 9/40).

Discussion

Many current commercial dog and cat kibbles containing blueberries do not provide substantial amounts of dietary polyphenols; therefore, adding blueberries likely does not enhance the food’s potential antioxidants and anti-inflammatory properties. Additionally, dog kibble contained higher levels of quercetin and antioxidant-protected alkali-labile phenols than cat kibble, likely due to varying levels of fruits, vegetables, and grains included. Specifically, while ingredients like blueberries are often marketed for their polyphenol content, their inclusion in feline diets is limited due to their high-soluble fiber content. High-soluble fiber diets can reduce the digestibility of protein, fat, and energy in cats (Fekete et al., 2004), which leads pet food companies to include less fruit in cat diets to limit the soluble fiber content in particular. However, some studies suggest that adding ingredients higher in insoluble fiber can improve gastrointestinal function. For example, obese and overweight cats fed beet pulp showed increased fecal production of acetate, propionate, and lactate (Fischer et al., 2012). Moreover, adding a high-fiber bundle with pecan shells, flaxseed, beet pulp, citrus pulp, and cranberries to the diet of healthy adult cats resulted in a shift in the microbiome, improving their ability to break down carbohydrates (Jewell et al., 2022a). The same fiber bundle added to the diet of healthy dogs showed a similar shift towards saccharolysis as well as increased fecal butyric, valeric, and hexanoic acid (Jewell et al., 2022b). Therefore, to provide cats and dogs with functional levels of polyphenols as a source of antioxidants, alternative ingredients that can be included in greater quantities should be explored.

Effects of processing and cooking

Previous studies that report phenolic content in blueberries report an average of 76.7, 144.2, and 46.4 mg/kg (converted from mg/100 g; DM) quercetin in cultivated highbush, wild rabbiteye, and frozen unsweetened blueberries, respectively (Haytowitz et al., 2018). In addition, ~150 µg/g of quercetin was found in a blueberry powder tested using the same analytical methods (unpublished results). These levels are considerably higher than the polyphenol content detected in the dog and cat kibble assessed in the present study. This observation is especially relevant given that most pet food manufacturers include whole fresh blueberries in their formulations. The final polyphenol content from blueberries in cat and dog kibble likely depends on multiple factors, including quantity, format, and potential losses. Although assessing the polyphenol content of blueberries before and after inclusion in extruded kibble is beyond the scope of this study, our results may suggest a reduction in polyphenol content due to cooking (extrusion) or ingredient interactions (e.g., phenol oxidase activity), as well as the quantity and format (dried, extract, powder, or fresh) of blueberries added to the pet food blend.

The effects of processing and cooking on polyphenol profiles vary depending on the class and source. Previous research has yielded similar results, with a decreased bound-free ratio of polyphenols and total phenolic content in gluten-free pasta after boiling (Rocchetti et al., 2017). However, this is not consistent across all sources of dietary polyphenols. For instance, while cooking generally reduces polyphenol concentration and TAC, results from some studies have shown the opposite. An increase (of 100% to 200%) of total polyphenols and TAC was found in leafy green vegetables (Gunathilake et al., 2018a). While these studies have included total polyphenols in their analyses, our research aimed to determine the specific polyphenol profile of cat and dog kibble, as cooking does not appear to affect all classes of polyphenols uniformly. For instance, the concentrations of chlorogenic acid, caffeic acid, total polyphenol content, and TAC in sweet potatoes decreased after boiling; however, the concentration of dicaffeoylquinic acid remained unchanged compared to raw sweet potatoes (Padda and Picha, 2008). Further, concentrations of 4-O-caffeoylquinic acid and 1,5-di-O-caffeoylquinic acid were not significantly different when comparing raw and steamed artichokes. In contrast, many other caffeoylquinic acids increased, and apigenin derivatives decreased after boiling, steaming, or frying (Ferracane et al., 2008). Different forms of vegetables and fruits in pet kibble are expected to respond differently to processing and cooking. In addition, the cooking method can impact the bioavailability of dietary polyphenols (Arfaoui, 2021), further affecting their function once consumed. Future research should assess the polyphenol content of kibble both before and after extrusion to help determine the processing controls when high-polyphenol ingredients are considered. Notably, a decreased polyphenol content resulting from cooking and processing has also been associated with a reduced TAC. For example, soluble polyphenols and TAC decreased after boiling, microwaving, and steaming conventional and organic vegetables (Faller and Fialho, 2009), as well as baking or boiling sweet potatoes (Padda and Picha, 2008). In contrast, some research has shown improved anti-inflammatory activity after steaming leafy vegetables due to the lower protein denaturation activity compared to the raw form (Gunathilake et al., 2018b).

Impact on antioxidation and anti-inflammation properties

The current study’s findings suggest that pet foods containing blueberries may provide minimal anti-inflammatory or antioxidant benefits, given the generally low concentrations of polyphenols observed, especially when compared to whole, unprocessed blueberries. Although a decrease in polyphenol content has not been directly linked to a reduction in anti-inflammatory properties, polyphenols are widely recognized for their ability to modulate inflammatory pathways in humans and animals. For example, supplementation with 0.96% g/kg or 1.92% g/kg of green tea polyphenols decreased pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, in obese dogs (Li et al., 2020). In addition, supplementation with 0.02%, 0.04%, or 0.08% gallic acid decreased IFN-y and IL-1β in healthy dogs (Yang et al., 2022). Similar results have also been reported in humans who have been supplemented with blueberries (Kay and Holub, 2002; Park et al., 2018). However, there is a dearth of knowledge on effective polyphenol doses in companion animals. For example, dogs supplemented with blueberries at an unspecified dose for four days showed an increase in TAP immediately following exercise; however, this effect was no longer evident 24 h postexercise (Dunlap et al., 2006). Moreover, although quercetin exhibits strong antioxidant potential in vitro, the extract alone has limited bioavailability in dogs (Reinboth et al., 2010). The bioavailability of dietary polyphenols from blueberries, such as quercetin, is unknown in cats. As this study focuses on the quantification of polyphenols in dog and cat kibble, future research is necessary to determine the effective levels and most bioavailable forms of dietary polyphenols in animals.

Other considerations

While consuming blueberries alone provides a source of polyphenols, ingredient interactions should be considered in a complete diet. For example, previous research has reported a reduction in flavanol metabolites in a blueberry smoothie with added banana compared to a control blueberry-only smoothie (Ottaviani et al., 2023) due to phenol oxidase, a compound involved in the enzymatic browning of fruits and vegetables. Other known food sources with high levels of phenol oxidase include apples, peaches, pears, apricots, berries, mangos, avocados, grapes, potatoes, lettuces, burdock, guava, melon, eggplant, and mushrooms (Mayer and Harel, 1979), some of which are included in extruded kibble. Consuming blueberries alone can provide high levels of dietary polyphenols; however, including additional fruit and vegetable sources may affect the level of polyphenols in pet foods. In the current study, the only pet food with blueberries listed among the first five ingredients had the lowest concentration of quercetin, which may suggest an interaction between ingredients. Future studies should comparatively assess the optimal doses of specific polyphenols (such as quercetin) in cats and dogs to contribute to our broader understanding of animal nutrition and to facilitate evidence-based recommendations for future pet food formulations. Given the differences in gastrointestinal physiology and metabolism among animals, the bioavailability of heat-treated polyphenol-containing ingredients should be investigated separately in dogs and cats.

Glossary

Abbreviations

ABTS

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

DM

dry matter

DMCA

dimethylaminocinnamaldehyde

DPPH

1,1-diphenyl-2-picrylhydrazyl

GSH-Px

glutathione peroxidase

HPLC

high performance liquid chromatography

OM

organic matter

TAC

total antioxidant capacity

TAP

total antioxidant power

Author Contributions

Pauline Kosmal (Conceptualization, Investigation, Methodology, Visualization, Writing—original draft, Writing—review & editing), Ryan Dilger (Data curation, Formal analysis, Methodology, Writing—review & editing), Laura Bauer (Data curation, Formal analysis, Methodology, Writing—review & editing), Jennifer Saunders-Blades (Conceptualization, Resources, Writing—review & editing), and Anna Shoveller (Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing—review & editing)

Conflict of interest statement. J.S.B. works for Champion Petfoods Holdings Inc. A.K.S. has received honoraria and research funding from various pet food manufacturers, ingredient suppliers, and provincial and federal granting agencies. They declare that they serve on Champion Petfoods’ Scientific Board. All other authors report no conflicts of interest.