ROBERTA DA SILVEIRA

Título da Tese: Avaliação de adulteração em fontes lipídicas com emprego de infusão direta ESI-MS

Orientador: Prof. Dr. Jesuí Vergílio Visentainer

Data da Defesa:28/05/2021

INTRODUCTION.

The pet food industry is continuously developing and seeking new ingredients for greater animal welfare and health. The feed must be balanced in order to meet all the animal nutritional requirements (NRC, 2006). Dog food is classified according to the ingredients quality and its cost, being divided in standard, premium and super-premium (Carciofi et al., 2009). Generally, in feed composition are present flours of meat, bone and chicken viscera, as well as phosphates, cereal flours and bran, and food additives such as acidulants, antioxidants and flavorings (Goes et al., 2013). The lipid sources used in feed are usually chicken fat, bovine tallow, swine lard, fish and vegetable oils (ABINPET, 2016). Aiming the animal health, the nutritional supplementation with omega-3 (n-3) and omega-6 (n-6) is widely used. The incorporation of essential fatty acids (FAs), such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, is related to lower rate of behavioral changes, as well as to increase learning capacity and visual acuity in pups (Heinemann et al., 2005). Eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) FAs are part of the n-3 fatty acid family. EPA is involved in the eicosanoids synthesis, particularly prostaglandins, leukotrienes, and thromboxanes, competing with arachidonic acid (20:4n-6, AA) for cyclooxygenase and 5-lipoxygenase enzymes, leading to increased production of anti-inflammatory eicosanoids, rather than pro-inflammatory eicosanoids derived from AA (Vaughn et al., 1994; Bispo et al., 2014). DHA is essential for the neurological system development and it is present in the retinal membrane. Furthermore, EPA and DHA have beneficial effects on the immune and inflammatory systems, assist in the protection of cardiac and renal functions (anti-inflammatory and antihypertensive actions) and stimulate learning ability (Zeng et al., 2011; Kralovec et al. 2012; Bispo et al., 2014).
AIMS. Observing the crescent number of adulterated products with the addition of low cost vegetable oils is crucial to evaluate the authenticity of the lipid source used in the manufacture of dog food. Therefore, diverse feed brands and classifications were analyzed in order to verify fraud existence regarding the FA composition, mainly EPA and DHA. Consequently, fatty acid composition and lipid profile of each feed were obtained by gas chromatographic techniques with flame ionization detector (GC-FID) and mass spectrometry with electrospray ionization (ESI-MS), respectively.
MATERIAL AND METHODS. Dog feed considered n-3 and n-6 sources (composition on the label) from different classifications and brands available in the Brazilian market were purchased in the city of Maringá, Paraná - Brazil (23°25'31"S51°56.18°C).
For the FAs analysis by GC-FID, Figueiredo's (2016) direct methylation method was employed in order to extract and prepare the fatty acid methyl esters (FAMEs). FAMEs were separated on a Thermo GC, trace ultra 3300 model, equipped with FID, automatic injector and fused silica capillary column CP-7420 (Select FAME, 100 m long, 0.25 mm internal diameter and 0.25 μm cyanopropyl). Gas flow as follows: 1.2 mL min-1of H2, 30 mL min-1 of N2, 35 and 300 mL min-1 of H2 and synthetic air, respectively, to the detector flame. The injected volume was 1.0 μL, using a sample split of 40:1, with injector and detector temperatures being 250 and 230 °C, respectively. Heating ramp was applied in the column, initiating the temperature at 165 °C for 18 min and raised to 235 °C with heating rate of 4 °C min-1, remaining for 20 min (Silveira et al., 2017). FAMEs were identified by comparing its retention times with standards (FAME Mix, C4-C24, Sigma-Aldrich) and the results were expressed as mg g-1 of total lipids, determined automatically by integration of peak areas through ChromquestTM 5.0 software.
For the lipid profile analysis by ESI-MS, the lipid fraction was extracted according to Figueiredo (2016). Then, 50 μL of the extracted oil was diluted in chloroform (950 μL). 1.0 mL of methanol/chloroform (9:1, v/v) was added in 5.0 μL of this solution and then 20 μL of ammonium formate (0.10 mol L-1 in methanol) were added (Youzbachi et al., 2015). The final solution was infused directly into a triple-quadrupole Xevo-TQD MS equipped with ESI Z spray™ ionization source (Waters, Milford, MA, USA). The lipid profiles were obtained in the ratio range of 100 to 1200 m/z and extracted in positive (ESI+) mode. ESI source parameters were as follows: source temperature of 150 °C, desolvation temperature of 200 °C, capillary voltage of 3.0 kV and cone voltage of 20.0 V. High purity nitrogen was produced by nitrogen generator (NM32LA, Peak Scientific®, Renfrewshire, Scotland) and it was used as desolvation gas with flow rate of 500 L h-1. The sample solutions were injected with a continuous flow of 10.0 μL min-1. Data were processed using MassLynxTM software.
The results obtained from the FA composition analysis by GC-FID were submitted to analysis of variance (ANOVA), the means were compared using the Tukey test, with significance level of 95%, and the results obtained by ESI-MS were analyzed by principal component analysis (PCA), through the R Studio software (R Studio Team, 2015).
RESULTS AND DISCUSSION. Feed manufacturers must present the guarantee levels on its products labels (MAPA, 2007). These levels establish the product nutritional quality offered to the consumer, demonstrating a quality standard that is dependent on the adequate quality control during and after the production process, as well as the raw material used. Among the information presented in the guarantee levels are: FAs levels such as n-3, n-6, EPA and DHA. The FA composition analysis was carried out with the objective of comparing the FAs amount of the samples with the respective guarantee levels declared by the manufacturers. Oleic acid (18:1n-9) was the major FA in all samples analyzed, followed by linoleic (18:2n-6) and palmitic (16:0) acids. Myristic acid (14:0) was found in the range of 0.19 to 0.51%, 16:0 acid in the range of 0.61 to 1.23%, stearic acid (18:0) in the range of 5.83 to 9.85%, 18:1n-9 acid in the range of 31.98 to 39.48%, 18:2n-6 acid in the range of 23.62 to 28.96 % and α-linolenic acid (18:3n-3) in the range of 1.10 to 2.65%.
In all samples analyzed, there is the addition of chicken oil. This information is presented in the labels of each sample and was confirmed with FA composition analysis by GC-FID comparing the feed and the chicken oil samples (Figueiredo et al., 2016), becoming clear that all analyzed samples have the same FA composition, predominating the FAs 18:1n-9, 18:2n-6 and 16:0. On the labels of samples 1, 2, 4, 7 and 9, there is the information that fish oil is added, these being the main sources of EPA and DHA (Lowe et al., 2008; Bispo et al., 2014). However, the information on the amount of EPA+DHA, presented on the feed sample labels, does not agree with the analysis results.
According to the lipid profiles Figures, it can be observed that all samples presented its individual characteristic and also some similarity among it, also the FAs present in the feeds are in the (triacylglycerol) TAG form, due to the most abundant region of ions, predominantly in m/z 800 to 1000.
The lipid profiles in the TAG region of all feed samples were similar to the profile presented by Cajka et al. (2013) for chicken oil. Hence, to confirm this similarity, chicken oil extraction was carried out by Bligh and Dyer (1959) and ESI(+)-MS direct infusion analysis was performed. Thus, it is possible to observe the similarity between the profiles of all the feed samples (1-10) with the chicken oil profile, proving the presence of it in all feed samples (1-10). The major TAG encountered were 52:3 (m/z 874), followed by 52:2, 54:4, 54:5, 52:4, and 54:3. These results are according to Porcari et al. (2016) and Cajka et al. (2013) for chicken oil lipid profile. One of the possible TAGs found for m/z 874 is PLO, consisting of the FAs 16:0/18:2n-6/18:1n-9. These FAs were also found in greater amounts in the analysis performed by GC-FID.
The feed manufactures of samples 1, 2, 4, 7, and 9 reported the presence of EPA and DHA at the guarantee levels on its labels. These two FAs come predominantly from fish oil, such as sardines and salmon (Bispo et al., 2014). Therefore, to verify and compare the lipid profile of the sardine and salmon oils with the lipid profiles presented by the feed, both oils were extracted by Bligh and Dyer (1959) and the direct infusion analysis by ESI(+)-MS was carried out. It can be observed that none of the feed samples presented lipid profiles similar to the profiles analyzed for sardine or salmon. Moreover, the profiles obtained for fish oils were also compared in this work with the lipid profiles of fish oils present in omega-3 capsules analyzed by direct infusion using ESI(+)-MS with ionization [TAG+NH4]+, and it was observed that the capsules have profiles similar to those obtained for sardine oil (Galuch et al., 2018).
The results obtained by ESI(+)-MS confirm the data obtained by GC-FID, showing the presence of chicken oil in the feed and absence of EPA and DHA, since no lipid profiles were found similar to fish oils or TAGs containing these two FAs.
PCA was performed to clarify the results contribution obtained by ESI-MS. PC1 (65.1%) and PC2 (21.2%) explained 86.3% of the total variance. A separation was observed in two distinct groups, in PC1 negative quadrant one group was formed by samples 1, 2, 3 and 10 due to the closer signal strength m/z, and the samples 1 and 2 contributed positively to PC2 and samples 3 and 10 contributed negatively to this separation. In PC1 positive quadrant another group was formed by samples 5,6,7 and 8 due to the fact previously exposed. For this group only sample 6 contributed positively to PC2, while samples 5,7 and 8 contributed negatively to PC2.
CONCLUSIONS. The FA composition obtained was compared to the dog food labels samples, it was observed that the omega 3 and omega 6 amounts are within the limit determined by each manufacturer, however, the labeling information on the EPA and DHA concentrations are not in accordance with the results obtained by GC-FID nor by ESI-MS. PCA analysis revealed that PC1 and PC2 explained 86.3% of the total variance.
Consequently, the information displayed on the labels are in disagreement with the results obtained for the fatty acid composition analysis by GC-FID and for the lipid profile analysis by ESI(+)-MS.
Keywords: dog feed; fatty acid composition; product quality; GC-FID; ESI-MS; lipid profile.

Artigos Publicados Vinculados a Tese:

https://www.scielo.br/j/jbchs/a/7Wc4YbfPtB7ZtNNFtFMZHpq/abstract/?lang=en