RESULTS AND DISCUSSION
A shelf-life study is an objective, methodical means to determine the amount of time a food product can reasonably be expected to remain fresh without appreciable changes to quality/safety (Galic, 2009). For oils/fats and the food products that contain them, shelf-life is contingent upon lipid oxidation and hydrolysis during the time of processing and storage (Hu and Jacobsen, 2016; Kilcast and Subramaniam, 2000). Lipid oxidation is a series of chemical reactions involving oxygen which can be described in terms of oxidative rancidity, or deterioration of oils/fat causing undesirable changes in color, taste, odor, and palatability. This also threatens the quality of nutrients and can lead to the formation of toxic compounds (Miller, 2010). Oxidation rates in lipids are largely a function of the structure of the fatty acid chains (Kilcast and Subramaniam, 2000; Osawa et al., 2008). Poultry fats contain high percentages of unsaturated fatty acids (ranging 57%-75%) with linoleic acid (C18:2) accounting for as much as 20% of the total fatty acid profile, much higher than that of beef tallow and pork lard (Hu and Jacobsen, 2016). Unsaturated fatty acids contain reactive double bonds between some of their carbon atoms, this results in higher susceptibility to oxidation. Lipid oxidation is complex and involves a multiple number of reactions (Osawa et al., 2008). Numerous analytical methods exist for the measurement of lipid oxidation, each with their own desired value and limitations (Hu and Jacobsen, 2016; Gray, 2015).
The FFA value measures the concentration of free fatty acids cleaved from the triacylglyceride molecules by hydrolytic breaking of the ester bonds between the fatty acids and the glycerol backbone (Miller, 2010). The percentage FFA in this study was expressed as a percentage (in weight) of oleic acid based on titration with a standard solution of KOH using phenolphthalein as the indicator (AOCS, 2009). The free fatty acid concentration of the rendered chicken fat used in this study was approximately 6.88% on initiation of the study and remained consistent through week 5; however, on week 6, FFA increased slightly to 8.53% (P<0.05) (Fig. 1). The hydrolysis of fat can occur due to the enzymatic (lipase) hydrolysis before rendering, or through acid or steam hydrolysis after rendering. The increase in the FFA over time, especially in the LA added chicken fat could be due to the acid hydrolysis of fat by lactic acids leading to more free fatty acid production. This finding is also related to the finding by Tadesse et al. (Tadesse et al., 2017) who also reported a constant increase in the FFA over the incubation time with no regular pattern of increase. Osawa et al. (2008) reported that FFA level in dry pet food samples increased during the storage period which is indicative of hydrolytic rancidity. They reported a range of 4.6 to 28.0% FFA (as oleic acid equivalents) in the pet food samples. The FFA percentage as well as the range in our study was smaller compared to their finding which could be dependent on the fat source and storage time before analysis. Both the main effects (time and treatments) were significant (P < 0.05) whereas, the interactions were not.
Peroxide values measure the primary oxidative products of fat. In the control fat PV was in the range of 0-0.67 meqv/100 gm fat throughout the storage period (Fig. 2). The values were in a range of 0.56-0.67 meqv/100 gm fat for the first week before it declined. This decline could be due to primary oxidation products breaking down to form secondary oxidation product as the storage time increased. The PV value of chicken fat stored for 7 days at 4°C was measured at 0.215 meqv/100 gm of fat in a study reported by Shantha and Decker (1994). This lower PV value compared to our study is likely due to the lower storage temperature compared to ours (40°C). The PV values in the SBS treated fat were also in a close range of 0.11-0.39 meqv/100 gm of fat, with a higher value (<0.05) on day 5 and week 4 of the sampling. The reason for these elevated values is not immediately obvious. The continuous increase in the PV value up to day 5 in control and SBS treated fat in our study may be similar to the findings of Tadesse et al. (2017), wherein the PV value in animal butter continuously increased during storage period up to 72 hours when stored at 25°C and 65°C. However, in the LA added chicken fat the PV values increased over the storage period until 5 weeks, and then declined on 6thweek. The highest PV value of 2.53 meqv/100 gm of fat was recorded on 5th week. The increase in the PV values before it started falling on 6th week may be the effect of the acidulant (lactic acid) causing more lipid oxidation to form peroxides before they start breaking down to secondary oxidation products. The main effects and interactions of treatments and days were higher (P < 0.05). The PV values in the LA treated and control fat increased linearly (P < 0.05) over time.
The second stage of oxidation occurs as hydroperoxides are cleaved to form carbonyl compounds such as aldehydes, which can be measured as non-volatile secondary oxidation products such as AV. These secondary compounds can impact the sensory quality of foods, producing off-tastes and off-smells (Hu and Jacobsen, 2016). It is important to note that initially the peroxide values increased during lipid oxidation but as the secondary compounds formed the primary values dropped, so it is essential to use these tests in tandem to achieve meaningful information about what is occurring at the time of measurement. The AV values for the control fat ranged between 2.11 to 6.68. The LA treated fat had AV values between 1.89 on day 0 to 8.18 on week 2; whereas, the SBS treated fat had AV values of 2.00 on day 0 to 10.28 on the week 5 (Fig. 3). For a good quality fat or oil, the AV values should be lower than 10 (List et al., 1974). In our study, both the acidulant treated fats, as well as control fat had AV values at or below this threshold of 10, with the single exception of 10.28 for SBS treated fat on week 5. The higher AV value indicates lower oxidative stability of the fats and oils. Tadesse et al. (2017) reported that the AV values of animal butter increased during storage at 65°C which was similar to our findings at 40°C storage. Christensen and Holmer (1996) reported that the increase in the AV value of fats and oils were a function of storage time and temperature. The time effect and the interaction of the main effects were significant (P < 0.05). Both the acidulant treated samples as well as the control showed a linear (P < 0.05 increase in the AV value over the time.
In the fats and oil industry, the TOTOX value has been proposed as a mean to combine the anisidine value and the peroxide value (Shahidi et al., 2002; O’ Keefe et al., 2010). The TOTOX value provides a broad accounting for the history of an oil or fat, but it “does not have any sound scientific basis because it combines variables with different dimensions” (Shahidi et al., 2002). The TOTOX value is calculated by adding the AV value with twice the PV value. There was a linear increase (P<0.05) in TOTOX values over time for all treatments. Individually, the TOTOX values of control fat after 1 week of storage remained constant over the storage period. Whereas, the TOTOX values for the acidulant treated chicken fats increased throughout the storage time with a maximum TOTOX of 10.67 on week 5 for SBS treated fat and 11.36 on week 5 for LA treated fat (Fig. 4) and differed (P<0.05) from the control (TOTOX of 6.0).
In conclusion, this study demonstrated that the FFA of fat rose slightly over the 6 weeks regardless of the acidulant treatments. The addition of LA increased the PV of the chicken fat and SBS led to a slight increase in AV at the last two weeks of the study. Taken in combination the acidulant treatment each led to a rise in TOTOX values for the fat by weeks 5 and 6. This would indicate that use of acidulants for pathogen control are stable to oxidation for at least 4 weeks and that LA may have greater impact on the primary oxidation product (PV), whereas SBS more impact on secondary oxidation products (AV) beyond 4 weeks of storage. There may be limitations to these findings because the experiment was conducted in a “bulk oil” model and the results might be amplified if applied in a thin layer to pet foods where exposure to air would be greater. Future research should evaluate the effects on shelf life on dry pet food kibbles coated with rendered chicken fat treated with acidulants. In conclusion, while changes were observed over time due to acidulant use for 6 weeks, the oxidation products measured remained within acceptable levels for fat used in the production of the pet foods.