The pet industry is an important sector in the USA with total expenditures in 2016 of $66.75 billion and $28.23 billion of that spent on food (4). Dietary lipids in pet food can vary from 5% to 40% of the diet. In pet food, the fat source can be animal, vegetable, or sometimes a mixture of both. Fats associated with disorders and diseases have received more attention in recent years. In dogs, the requirements for omega-3 and 6 fatty acids have not been documented, but may exist at certain stages in their life cycle. Numerous fatty acid supplements are often used to reduce problems with the coat and skin in dogs (5). These supplements contain a mixture of omega-3 and 6 fatty acids, and sometimes include eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) from marine sources. Processing and storage of dietary lipids result in physical and chemical changes. High levels of unsaturated fatty acids are highly susceptible to oxidation damage and rancidification causing major sensory alterations that occur during storage (7).
Oxidation of polyunsaturated fatty acids adversely affects the flavour, texture, colour, odour, and nutritional value of food during storage. It was reported that oxidised dietary lipids in growing dogs stunted growth, deteriorated antioxidant status, and weakened some immune functions (34). Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole, and ethoxyquin are often added to prevent the oxidation process. However, in recent years, the possible toxicity of synthetic antioxidants has been considered (37). Thus, natural antioxidants obtained from fruits, vegetables, spices, grains, and herbs have been of increasing interest. Plant extracts rich in polyphenols such as curcumin, cranberry, pomegranate, grape seed, and açai berry (7, 10, 15) have exhibited antioxidative effects when added to food systems. No studies to our knowledge have been conducted on exploring the use of natural phenolic compounds in lipid retention of dog foods. Therefore, the main objective of this study was to evaluate the effects of curcumin, cranberry, pomegranate, GSE, and açai berry in dog food on reducing lipid oxidation.
A generic dry dog food (18.0% crude protein, 9.5% crude fat, max 12.0% moisture) was purchased from a local retailer and finely ground using a Thomas-Wiley laboratory mill with a 4 mm grinding disc. The food was then hand mixed with Virginia Prime Gold fish oil (Omega Protein Corporation, USA) and Barlean’s pure flaxseed oil purchased from the local Co-op (Carbondale, USA). The oils used were natural, non-hydrogenated and unaltered containing no preservatives. The five natural antioxidants screened in this study were purchased from www.bulksupplements.com. The natural antioxidants were grape (
Fish and flaxseed oils were added to the ground food at 3.1% and 1.8% of food (DM basis). The food was then divided into seven 3-kg batches and assigned a treatment (three replicates per treatment). The control consisted of finely ground food mixed with fish and flaxseed oils with no added antioxidants. Natural antioxidants were added to the batches at 0.2% food DM and synthetic BHA was added at the legal limit of 0.02% of food DM (7). Once hand mixed, the batches were divided into 1 kg amounts and placed in an oven (Isotemp 200 Series Model 255D incubator, Fisher Scientific, USA) at 55°C in open foil steam table pans. On days 0 and 12, a 150 g sample was collected from each pan into 1-gallon Zip-Loc bags, wrapped in aluminium foil, and immediately placed at –80°C until analysis. The remaining 5 kg of food was stored at –80°C for use in experiment two.
From the remaining 5 kg of food, six 500 g batches of the food were weighed and mixed with the same amounts of oils as outlined in experiment one. The treatments were a control (no added antioxidant), BHA at 0.02% of food DM, and either curcumin or GSE included at 0.1% and 0.2% of food DM. The food was then incubated at 55°C in an oven (Isotemp 200 Series model 255D incubator, Fisher Scientific, USA) for 12 days. On days 0 and 12, a 30 g sample from each pan was collected and placed into 1-gallon Zip-Loc bags, wrapped in aluminium foil, and then stored at –80°C until fatty acid analysis. Each treatment was run in triplicate.
TBARS was determined by preparing samples for spectrophotometry as described by Botsoglou
Food samples were methylated using the NaOCH3 and HCl two-step procedure as outlined by Kramer
The Statistical Analysis System (SAS Institute, Inc., USA) software package version 9.4 was used for statistical analysis. The data from experiment one was analysed using its proc GLM model with treatment as fixed effects and rep as a random effect. The data from experiment two was analysed using the proc GLM model with treatment and day as fixed effects and day by treatment as a random effect. Treatments means were separated by PDIFF using the
The effects of the six antioxidants on TBARS values are depicted in Fig. 1. After 12 d of storage, all supplements, except for açai berry, had significantly lower (P < 0.05) TBARS values than the control.
The effects of curcumin on fatty acid values are presented in Table 1. Compared with the control, the concentrations of EPA and DHA were greater (P < 0.05) in dog food incubated with curcumin. The concentrations of EPA and DHA for curcumin were also greater (P < 0.05) compared with the BHA.
Fatty acids concentrations (mg/g food DM) for BHA and the different levels of curcumin on days 0 and 12 a-b Means with different subscripts within a row are significantly different at P < 0.05 x-y Means with different subscripts within a column are significantly different at P < 0.05 EPA – eicosapentaenoic acid; DHA – docosahexaenoic acid; BHA – butylated hydroxyanisole; SEM – standard error of the mean
Fatty acid
Day
Control
BHA, 0.02%
Curcumin 0.1%
Curcumin, 0.2%
SEM
C18:2
0
9.67x
9.49x
9.61
9.77
0.165
12
ab8.49y
a9.27y
ab8.74
b8.02
0.174
C18:3n3
0
5.99
6.05
6.09
5.97
0.097
12
5.73
5.99
5.76
5.79
0.889
C20:5n3 (EPA)
0
1.65x
1.71x
1.57
1.58
0.051
12
b0.94y
b1.17y
a1.52
a1.6
0.056
C22:6n3 (DHA)
0
1.36x
1.39x
1.41x
1.42x
0.036
12
b0.89y
b1.01y
a1.2y
a1.08y
0.037
Total omega-3
0
8.91
9.08
9.02
8.91
0.373
12
7.56
8.23
8.47
8.32
0.355
The effects of GSE on fatty acid values are presented Table 2. The concentrations of EPA was greater (P < 0.05) with the GSE or BHA than with the control. The concentrations of EPA and DHA for the 0.2% GSE were greater (P < 0.05) than the 0.1% GSE on day 12. Grape seed extract at 0.2% had greater (P < 0.05) EPA concentration compared with the BHA.
Fatty acids concentrations (mg/g food DM) for BHA and the different levels of grape seed extract (GSE) on days 0 and 12 a-c Means with different subscripts within a row are significantly different at P < 0.05 x-y Means with different subscripts within a column are significantly different at P < 0.05 EPA – eicosapentaenoic acid; DHA – docosahexaenoic acid; BHA – butylated hydroxyanisole; GSE – grape seed extract; SEM – standard error of the mean
Fatty acid
Day
Control
BHA, 0.02%
GSE, 0.1%
GSE, 0.2%
SEM
C18:2
0
9.67x
9.49x
9.61
9.77x
0.165
12
b8.49y
a9.27y
ab8.9
a9.2y
0.171
C18:3n3
0
5.99
6.05
6.09
5.97
0.097
12
b5.73
ab5.99
a6.11
a6.01
0.081
C20:5n3 (EPA)
0
1.65x
1.71x
1.62x
1.7
0.046
12
c0.94y
b1.17y
b1.23y
a1.51
0.048
C22:6n3 (DHA)
0
1.36x
1.39x
1.25x
1.38
0.035
12
b0.89y
ab1.01y
b0.85y
a1.23
0.038
Total omega-3
0
8.91x
9.08
8.91
9.08
0.317
12
b7.56y
ab8.23
ab8.26
a8.85
0.317
Different analytical methods can be used for measuring lipid oxidation in foods. The thiobarbituric acid reactive substances (TBARS) method is one of the methods used to measure oxidation of fat-containing foods. Lipid oxidation can also be estimated by quantitatively measuring the loss of initial substrates. In foods containing fats or oils, unsaturated fatty acids are the main reactants whose composition changes significantly during oxidation. Changes in fatty acid composition provide an indirect measure of the extent of lipid oxidation. In a modelling study, the fatty acid concentration of various fats and oils was shown to be highly correlated with the results of four different classical assays of oxidative stability measurements (18).
In the present study, TBARS values for samples increased during storage and the increase was greater in the control samples compared with the samples treated with natural antioxidants, except for açai berry. However, previous
The present results also suggested that pomegranate is also an effective reducer of lipid oxidation as its TBARS value increased by only 54% on day 12, compared with the 158% increase in TBARS value with the control. These results are consistent with Naveena
The data obtained also confirmed the antioxidant properties of cranberry extracts. Previous studies reported reduced TBARS values in meat products containing cranberry extracts (17, 22). However, Sampels
Although all of the natural antioxidants, except açai berry, tested in experiment one were effective in reducing the formation of TBARS relative to the control, only two natural antioxidants (curcumin and GSE) were selected for experiment two because of their higher availability and lower cost in comparison to the other antioxidants.
Curcumin (diferuloylmethane) is a phenolic component of
Commercial curcumin contains curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Jayaprakasha
Our results suggested that GSE is also an effective reducer of lipid oxidation. A study by Pazos
In conclusion, after 12 days of storage, GSE, curcumin, pomegranate, and cranberry were effective at slowing lipid oxidation. Açai berry was the only antioxidant that did not have a positive antioxidant effect on lipid oxidation. The stability of EPA and DHA in dog food also improved with the addition of GSE and curcumin. Future studies need to be aimed at finding a suitable effective dose of these supplements for inhibiting lipid oxidation in dog food.