Like protective agents against lipid oxidation in oleine and beef


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Like protective agents against lipid oxidation in oleine and beef

Materials and methods
Results and discussion


In the present study was evaluated the antioxidant activity (AA) of extracts obtained of agro-industrial by-product of avocado (Persea americana) var. Hass. Extracts of different polarity were obtained from epicarp and seeds of avocado by soxhlet extraction under reduced pressure, using as solvents hexane, ethyl acetate and ethanol in successfully way. For all the extracts obtained, total phenol content (TPC), total flavonoid content (TFC) and the ability to reduce oxidative deterioration of beef and palm olein (OP), were determined. Multivariate statistical techniques were used to analyse the AA, which was performed by quantification of linoleic acid hydroperoxides (LAH) and thiobarbituric acid reactive species (TBARS) generated over time by subjecting the beef or palm olein to a process of accelerated oxidation. The extracts obtained from the seeds showed higher antioxidant activity in palm olein and beef than those obtained from epicarp, presenting similar activity to some commercial antioxidants currently used in food preservation. There was good correlation (Pearson -0,78) between antioxidant activity and TPC for OP. The results of this study allow us to propose to Hass avocado seeds as a promising source of extracts with antioxidant activity which could replace the synthetic antioxidants used in the food industry.
Key words: Persea americana Mill. var. Hass, lipid oxidation, palm olein, beef, antioxidant activity.


Many edible tropical fruits are processed into juices, concentrates, jellies or extracts, generating by-products such as epicarp and seeds (Kumar et al., 2012), without considering that these can be source of components applicable in industry (Ayala-Zavala et al., 2011; Benchikh et al., 2014) instead of being used as animal feed (Hernández-López et al., 2016). In recent years the food industry has focused its interest in using plant and fruit extracts as additives (Jaswir et al., 2000; Tril et al., 2014) in order to retard oxidative degradation of lipids (Balboa et al., 2014) and improve the quality and nutritional value of food, as indicated by studies carried out in palm olein (Bansal et al., 2010; Muik et al., 2005), beef (Farouk et al., 2014; Ghasemi Pirbalouti et al., 2013) and poultry products (Karre et al., 2013). Recently, it was found that extracts obtained from Mexican Hass avocado by-products protect the pork paté from lipid peroxidation (J. G. Rodríguez-Carpena et al., 2011; J.-G. Rodríguez-Carpena et al., 2011).
The Hass avocado is very appreciated because its sensory characteristics and high nutritional value, it has health benefits , as anticancer activity (Ding et al., 2007), hypercholesterolemia, hypertension, inflammatory affections and diabetes. Additionally Hass avocado by-products present antimicrobial activity (Guil-Guerrero et al., 2016) besides antioxidant activity in vitro (Calderón-Oliver et al., 2016).
Despite the foregoing, avocado seeds and epicarp are matter of concern because by-products generation for avocado processors worldwide (Segovia et al., 2016). This study aims to assess the impact of addition of extracts from Hass avocado by-products on the oxidative stability of lipids present in palm olein (PO) and beef.

Materials and methods

2.1 Materials
Avocados were acquired in Sonsón-Antioquia (Colombia), manually separating the seed (S), pulp and epicarp (E). Both epicarp and seeds, separately were cut into smaller pieces and dried in vacuum oven (35 °C, 15 mm Hg) for 48 hours, and ground with dry ice using a grain mill. Finally, each of the dried and ground materials with particle size 0.2-0.5 mm, were stored at 4 ° C until further extractios. The solvents used were analytical grade and previously distilled. The OP (refined, bleached, deodorized and antioxidants free) was supplied by Grasco S.A. and the beef without additives was purchased at a local supermarket.
2.2 Extract obtention
Each of the dried and ground samples (10 g. aprox.), separately, were extracted under reduced pressure using a soxhlet extractor, taking care that the solvent temperature did not exceed 35 °C. Extracting each material, epicarp (E) or seeds (S), has a duration of 8 hours using hexane, ethyl acetate or ethanol (15 mL solvent /g dry sample), successively one after the other, in order of polarity starting with hexane. Other extractions were performed without hexane (starting with ethyl acetate) and finally some extractions were realized employing only ethanol.
Extracting every by-product directly with each respective solvent, extracts were obtained: hexane (1), ethyl acetate (3), ethanol (6). Extracting each by-product successively employing 2 or 3 solvents, extracts were obtained: ethyl acetate extract degreasing previously with hexane (2), ethanoic extract after being extracted with hexane and ethyl acetate (4), ethanoic extract previously extracted with ethyl acetate (5). Therefore, from every by-product six extracts were obtained (1E, 2E, 3E, 4E, 5E, 6E, 1S, 2S, 3S, 4S, 5S, 6S), the first number indicates the solvent or the sequence of solvents used, and the last letter refers to the type of by-product. All extracts were concentrated to dryness using a rotary evaporator at a temperature of 308 K in order to evaluate the extraction yield. Then, they were reconstituted with ethanol (or ethyl acetate considering their solubility) as solvent to a final concentration of 4 mg/L, and used to determine TPC, TFC and AA.
2.3 Total phenolic content (TPC)
Total phenolic content (TPC) in the by-products extracts were determined by the Follin-Ciocalteu method (Berker et al., 2013) with some modifications. An aliquot of 150 &µL of extract (10 mg/L) was mixed with150 &µL of Folin-Ciocalteu reagent without dilution and 1.55 mL NaOH 0.45 M. After incubation at room temperature and dark for 20 min., the asorbance was recorded at 765 nm against a reagent blank prepared with the same procedure using ethanol or ethyl acetate instead of sample solution. A calibration curve was prepared with gallic acid (GA) solutions (0.002-0.10 mg/mL),TPC values were expressed as mg GA in100 g of dryed by-product.
2.4 Total flavonoid content (TFC)
Quantification of flavonoids was performed by the colorimetric method of aluminum chloride (Chander et al., 2014) with some modifications. An aliquot of 250 &µL of extract dilution (2.0 mg/mL) in ethyl acetate/ethanol (1:1 v/v) were mixed separately with 750 &µL etanol (96%), 50 &µL AlCl3 (10%), 50 &µL potassium acetate solution (1 M) and 1.4 mL distilled water. After 30 min the absorbance was read at 415 nm. A calibration curve was prepared with quercetin (Q) solutions (25 -100 &µg/mL), TFC values were expressed as &µg Q in 100 g of dryed by-product.
2.5 Lipidic oxidation
2.5.1 Palm olein (PO)
Each obtained extract was mixed separately with PO to a concentration of 200 ppm (Codex Committee on Fats and Oils, 1999) and ferrous chloride solution (aprox. 0.1% w/v) in ethanol, to a final concentration of 3.5 ppm. A control (no antioxidants) and samples of PO with one commercial antioxidant (gallic acid-AG, BHA or BHT) were prepared separately at 200 ppm as well. The system was subjected for 15 days to oxidation (60°C) by air bubbling every 24 hours (two minutes of bubbling) and sporadic stirring in dark conditions (Hernández-Acosta et al., 2011; Huang & Frankel, 1997). As products of oxidation were determined linoleic hydroperoxides (LHP) (Frankel et al., 1994) and tiobarbituric acid reactive species (TBARS) (Ohkawa et al., 1979), formed during 0, 3, 6, 9, 12 and 15 days of essay.
2.5.2 Beef
The beef was ground in a blender at high speed using 6 cycles of 20 s, to a homogeneous mass (Mariem et al., 2014). Portions of 20 grams of homogeneous beef were placed in amber glass bottles (50 mL) and mixed in vortex for 2 min. with each extract or commercial antioxidant (BHA, BHT or GA) at a final concentration of 100 ppm (Codex Committee on Fats and Oli, 1981). Beef samples: control sample (no antioxidants), beef with extract solutions and beef with commercial antioxidants remained cold (4°C) for nine days (Brettonnet et al., 2010; Juntachote et al., 2006). HPL and TBARS measures were performed on days 0, 3, 6 and 9 days.
2.6 Statistical analysis
Each extraction and the respective measurements (yield, TPC, TFC, LHP and TBARS) were realized by triplicate, expressing the data as the average ± standard deviation, determining significant differences at level of 5% using one-way ANOVA and Tuckey test. To compare AA of extract in OP and beef, Principal Component Analysis (PCA) was employed with extraction of variables based on eigenvalue> 1, Varimax rotation and Kaiser normalization (previous KMO test and Bartlett sphericity realized). For the correlation analysis between AA essays lasting15 days (normal essay) and those with 3 days (accelerated essay), Pearson correlation coefficient (r) was determined using R software version 3.2.3.

3. Results and discussion

Values of extraction yield for each of the twelve extracts are shown in Table 1. It was found that higher yields correspond to the epicarp extracts obtained only with a first and unique extraction solvent, either hexane (13.6%), ethyl acetate (13.7%) or ethanol (13.9%), while the higher seed yield was 5.8% obtained with ethanol. Fat avocado seed has been obtained using soxhlet extraction with hexane and supercritical CO2 extraction, yielding 3.1% (García-Fajardo et al., 1999) , this is because the composition of avocado seed is essentially carbohydrates and fiber that provide food for the further development of the plantula.
TPC are shown in table 2, it is noted that both types of by-products (epicarp or seed) and the extraction solvent, influence this values: more polar extracts from seeds have a higher TPC, exceeding 1.182 mg Gallic Acid (GA) by mg extract, values within the range reported by other authors (Ayala-Zavala et al., 2011; Kosinska et al., 2012) .
Recently, it has been found that by macerating, TPC of Hass waste increases significantly when applying highly polar solvent extraction like methanol:water (Kosinska et al., 2012), or acetone:water (Kosinska et al., 2012; J.-G. Rodríguez-Carpena et al., 2012), reaching values of 9.51± 0.161 mg Gallic acid by gram dry by-product; these extracts also have hypocholesterolemic activity (Pahua-Ramos et al., 2012).
Concerning TFC (table 3), it was observed hexane extracts and ethyl acetate extracts (1E, 1S, 2E, 2S, 3E, 3S) presented similar values compared with TFC, indicating that is probably to be the same substances which are being detected by the two methods.
Table 4 shows the results of quantification of hydroperoxides for 3,6,9,12 and 15 days of oxidation. When the essay started, the PO presented evidence of oxidation (zero day 6,51 ± 0,05 mmol HPL/kg) and the values were increased with time. This is because hidroperoxides are initial reaction products in the early stages of the oxidations process and PO begins its oxidation with oxygen from the atmosphere immediately upon contact, although initially the reaction kinetics is very slow. The most active extracts against the formation of hydroperoxides were 2E and 5S, and in less extension 4E and 4S.
Table 5 shows the results of quantification of TBARS for 3,6,9, 12 and 15 days of oxidation. When the essay started, the PO presented a very low value of 0.01 ± 0.01 mg MDA/kg within the error parameters of the measuring instrument. TBARS values were increased with time as it was expected. This is because TBARS are related to final oxidation products like aldehydes, ketones and carboxylic acids, oxidized compounds that depending on the molecular size will be released to the volatile phase, providing characteristic tones of rancidity, rejection criterion of a product. The extract 5S, and in less extension extracts 4E, 4S and 6S protects the PO facing the formation of TBARS, indicating the importance of the polarity: being less polar extract, it refers to the importance of the solubility in the food. Similar results were obtained in the protection of pig meat extracts obtained from avocado seeds (Hernández-López et al., 2016; J. G. Rodríguez-Carpena et al., 2011).
A PCA was realized in order to analyze the results as a whole process instead of consider each day separately. KMO test and Bartlett’s sphericity test evaluate the applicability of factor analysis to the model (KMO>0.75, p-value 1.06×10-51). From the first two principal components, the total variance was explained as follows: CP1, 56% and CP2, 15%.
Table 6 shows the loading values for CP1 and CP2 matrix (Varimax rotation and Kaiser Normalization). CP1 correlates with TBARS (loadings 0.538 – 0.907) while CP2 correlates with LHP (loadings 0.655 – 0.881), explaining the need to perform the two measurements to conclude on the efficiency of an extract and / or antioxidant against lipid oxidation.
Figure 1 represents the scatter diagram obtained for the PCA, in general, all extracts showed antioxidant activity compared to the control. 5S extract is located at the bottom left of the diagram, presenting similar AA to commercial antioxidants like GA and BHT, therefore this extract is proposed as the best extract to protect PO from lipidic oxidation. Correlation study between TPC and CP1 proved to be significant (p-value<0.05, Pearson -0.78), the negative value means that a higher TPC results in a lower concentration of TBARS or higher AA.
In the case of beef, on day zero values were 0.45 ± 0,01 mmol HPL/kg beef, and 0.02 ± 0.01 mg MDA/kg beef. The results for the other days are shown in table 7 and 8. The content of oxidation products increases as time passes, although in smaller proportion compared with the kinetics of PO oxidation. The PCA allowed to select the extract with the greatest potential as a source of antioxidants to protect the beef of lipid peroxidation, comparing the efficacy of the extracts with respect commercial antioxidants (BHT, BHA and GA) and CONTROL. According to PCA from the first two principal components, total variance explained was 71%, with contributions of 41% CP1 and 30% CP2. As in PO oxidation, CP1 correlates with TBARS, and CP2 with far LHP. As PO oxidation, CP1 correlates with TBARS and CP2 with LHP values. The more promising extract in beef corresponds to 2S, with level of protection similar to GA (figure 2).
From the above results, the avocado seed is promising source of extracts used to protect PO and beef from lipid oxidation, in some cases their action is greater than the protection afforded by synthetic antioxidants currently used in food industry.

4. Conclusions

From Hass avocado seds and epicarp (P. Americana Mill. var. Hass) is possible to obtain extracts that inhibit or delay lipid oxidation in palm olein (PO) and beef. Ethanoic extract obtained from Hass avocado seeds with after extraction with ethyl acetate (5S extract) presented added protection from lipid oxidation, beating commercial antioxidants like BHT, BHA and GA, commonly used in the industry of fats and oils. The extract obtained in ethyl acetate from the seed, prior extraction with hexane (2S extract) revealed better protection in beef.

5. Recognitions

This study was supported by Red Nacional para la Bioprospección de Frutas Tropicales-RIFRUTBIO (project 550854332012 COLCIENCIAS) and Vicerrectory Research-Directorate, Bogotá, Universidad Nacional de Colombia (project 201010021420).

6. References

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