Vegetables and fruits are essential foods in our diet and also have many compounds that are beneficial for health due to minor components. These minor components include phenolic substances [1
]. These are secondary metabolites of plants. They have an aromatic ring with one or more hydroxyl groups. Their complexity may be high, as for example quercetin, which is one flavone with several aromatic rings. The properties depend on the arrangement and/or structure of the molecule [2
In recent times, many plants have been studied in order to characterize them depending on the amount of polyphenols they have and on their potential use [3
The polyphenols are associated with the potential prevention of diseases which are due to the presence of free radicals, such as cardiovascular insufficiency, hypertension, inflammatory conditions, asthma, diabetes and Alzheimer’s [4
], thanks to their antiradical power. For this reason, they are very useful in food products, since they prevent lipid peroxidation due to the attack of free radicals [5
]. They also protect against oxidation, direct or indirect, caused by metal cations [6
]. These cations stimulate the creation of reactive oxygen species (ROS), which are harmful to the health. In some cases, polyphenols have been used as preservatives, protecting against microorganisms [7
The process of food, especially for IV and V gamma products, produces many byproducts and waste. This type of waste has a significant environmental impact due to the organic charge. It also has associated handling, transport and storage costs, among others. Therefore, more and more alternative uses for these residues are sought, as for instance animal feed and fertilizers, among others. In the present case, it is interesting to obtain, through an optimized extraction process, harmless substances with high antioxidant power. Thus, what was a waste becomes a “high value-added” product [8
]. Previous examples already studied [10
] are the orange juice industry, where a large amount of skin and seeds are produced with a high content of polyphenols and the industry of processed apple, pear and peach, with a significant amount of skin byproduct. There is evidence that the skin may even have a greater amount of polyphenols than the flesh [13
]. Also, the waste from wine and beer production includes phenolic compounds [9
]. Other studies have focused on the shells of nuts, rice and wheat in which large amounts of polyphenols are found [14
In the avocado industry the pulp is used, while the skin and the seeds are discarded as waste. These residues are rich in polyphenols with antioxidant and antimicrobial power [15
]. Among the polyphenols the (+)-catechin and (−)-epicatechin [16
] and chlorogenic and protocatechuic acid, are included [14
]. Previous studies on this residue have been applied to pork burgers and have been shown to be effective in preventing oxidation and microbial growth [15
Given the above, it can be concluded that polyphenols obtained from these industrial wastes can be potent antioxidants and, in some cases, they are better than synthetic antioxidants such as BHA or BHT which in high doses can become toxic [17
In order to optimize the extraction process, response surface methodology (RSM) has been used. Phenolic compounds extraction optimization from strawberries [18
], apple pulp [9
] and residues of chestnuts [19
], are examples of this. This method establishes a multivariable mathematic model to obtain the relationship between responses and independent variables [20
] with the use of a minimal number of experiments.
This paper consists of two main objectives. First, a mathematical model was obtained to predict the best conditions of extraction of polyphenols from dried avocado seed. Second, an extract using these conditions was obtained and the effect of lyophilized powder in the delay oxidation in oil-in-water (O/W) emulsions and beef meat burgers analyzed.
2. Experimental Section
2,2′-Azo-bis(2-amidinopropane) dihydrochloride (AAPH), was used as peroxyl radical source. Trolox (6-hydroxy-2,5,8-tetramethylchroman-2-carboxylic acid), ethanol, fluorescein, AAPH, BHA, egg albumin, p-anisidine (4-amino-anisole; 4-methoxy-aniline), isooctane, potassium persulfate, acetic acid (glacial) and 2-thiobarbituric acid were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK). Folin-Ciocalteu reagent, sodium carbonate and 1,6-diaminohexane were supplied by Merck (Darmstadt, Germany). Trichloroacetic acid, hydrochloric acid and Tween® 20 (Panreac Química S.L.U, Barcelona, Spain) were acquired from Panreac Química S.L.U. (Barcelona, Spain). Refined sunflower oil, with no added antioxidants, was purchased from a local retail outlet. All compounds were of reagent grade.
2.2. Avocado Preparation
The avocado (Persea americana) was obtained in the local market; the seeds were separated from other edible parts. This waste was homogenized and frozen at −80 °C for lyophilization. Then the seeds were ground into a powder by using a Moulinex mill (A5052HF, Moulinex, Lyon, France). The particle size was standardized with a number 40 mesh sieve. Finally, the powder was stored in a dark bottle in a desiccator until use.
2.4. Total Phenolic Content (TPC)
TPC was determined spectrophotometrically following the Folin-Ciocalteu colorimetric method [22
]. A sample diluted 1:4 with milli-Q water was stirred in triplicate. The final concentration in the well (96 wells plate was used) was: 7.7% v/v sample, 4% v/v Folin-Ciocalteu’s reagent, 4% saturated sodium carbonate solution and 84.3% of milli-Q water, all mixed. The solution was allowed to react for 1 h in the dark and the absorbance was measured at 765 nm using a Fluostar Omega (BMG Labtech, Ortenberg, Germany). The total phenolic content was expressed as mg Gallic Acid Equivalents (GAE)/g dry weight.
Experimental design and responses for extraction.
Experimental design and responses for extraction.
|Temperature (°C)||Ethanol Concentration (%)||Time (min)||TPC (mg GAE/g dw)||ORAC (mg TE/g dw)|
|60.00||60.00||25.00||41.00 ± 0.97||104.16 ± 2.13|
|60.00||93.63||25.00||35.10 ± 0.24||116.12 ± 1.03|
|80.00||80.00||5.00||46.78 ± 0.59||153.17 ± 3.84|
|26.36||60.00||25.00||40.78 ± 0.17||70.54 ± 0.97|
|60.00||60.00||25.00||41.10 ± 0.57||106.10 ± 2.40|
|40.00||40.00||45.00||43.24 ± 0.76||104.01 ± 2.35|
|80.00||80.00||45.00||45.43 ± 0.49||144.94 ± 2.84|
|80.00||40.00||45.00||45.37 ± 1.39||130.08 ± 2.65|
|80.00||40.00||5.00||43.70 ± 0.66||150.03 ± 1.73|
|60.00||60.00||25.00||40.90 ± 0.47||104.28 ± 1.03|
|60.00||60.00||55.22||42.87 ± 0.70||158.77 ± 1.33|
|40.00||40.00||5.00||41.19 ± 0.55||99.17 ± 1.81|
|60.00||60.00||2.77||42.92 ± 1.13||155.44 ± 2.71|
|93.64||60.00||25.00||46.95 ± 0.09||126.23 ± 3.35|
|60.00||26.36||25.00||42.33 ± 0.10||129.78 ± 3.84|
|40.00||80.00||45.00||38.98 ± 0.45||100.72 ± 3.27|
|40.00||80.00||5.00||35.48 ± 0.55||91.01 ± 3.51|
2.5. ORAC Assay
Antioxidant activities of avocado seeds extracts were determined by the ORAC assay, as reported by Casettari et al.
]. The assay was carried out using a Fluostar Omega equipped with a temperature-controlled incubation chamber. The incubator temperature was set to 37 °C. The extract samples were diluted 1:20 with milli-Q water. The assay was performed as follows: 20% of sample was mixed with Fluorescein 0.01 mM, and an initial reading was taken with excitation wavelength, 485 nm and emission wavelength, 520 nm. Then, AAPH (0.3 M) was added, measurements were continued for 2 h every 5 min. This method includes the time and decrease of fluorescence. The area under the curve (AUC) was calculated. A calibration curve was made each time with the standard Trolox (500, 400, 250, 200, 100, 50 mM). The blank was 0.01 M phosphate buffered saline (pH 7.4). ORAC values were expressed as mg Trolox Equivalents (TE)/g of dry weight.
2.6. Statistical Analysis
RSM was used to determine the optimal conditions of polyphenol extraction. A central composite design (CCD) was used to investigate the effects of three independent variables with two levels (solvent concentration, extraction temperature, and extraction time) with the dependent variables (TPC, ORAC activity). CCD uses the method of least-squares regression to fit the data to a quadratic model.
The adequacy of the model was determined by evaluating the lack of fit, coefficient of determination (R2
) obtained from the analysis of variance (ANOVA) that was generated by the software. Statistical significance of the model and model variables were determined at the 5% probability level (α = 0.05). The software uses the quadratic model equation shown above to build response surfaces. Three-dimensional response surface plots and contour plots were generated by keeping one response variable at its optimal level and plotting that against two factors (independent variables). Response surface plots were determined for each response variable. The coded values of the experimental factors and factor levels used in the response surface analysis are shown in Table 1
. The graphics and the RSM analysis were made by software Matlab version R2013b (The MathWorks Inc., Natick, MA, USA, 2013). All responses were determined in triplicate and are expressed as average ± standard deviation. The answers have a percentage deviation less than 10%.
2.7. Water-Oil Emulsions
Oil-in-water emulsions (20.2 g) were prepared by dissolving Tween-20 (1%) in acetate buffer (0.1 M, pH 5.4), either with or without protein, namely egg albumin (0.2% w/w) and avocado seeds extracts (0.45% w/w, 0.225% w/w, 0.1125% w/w). The emulsion was prepared by the dropwise addition of oil (sunflower oil) to the water phase, cooling it in an ice bath with continuous sonication with a Vibracell sonicator (Sonics and Materials, Newtown, CT, USA) for 4 min. All emulsions were stored in triplicate in 60 mL glass beakers in the dark (inside an oven) at 30 °C in an incubator. Two aliquots of each emulsion (0.005–0.1 g, depending on the extent of oxidation) were removed periodically for determination of peroxide value (PV).
2.8. Peroxide Value (PV)
PV was determined by the ferric thiocyanate method [24
] (after calibrating the procedure with a series of oxidized oil samples analyzed using the AOCS Official Method Cd 8-53). Data from the PV measurements were plotted against time.
2.9. Meat Preparation
Fresh beef meat was purchased from a local processor 96 h postmortem. All subcutaneous and inter-muscular fat and visible connective tissue were removed from the fresh beef muscle. Lean meat was ground through Ø-4 mm plate using a meat grinder (PM-70, Mainca, Barcelona, Spain). The ground meat was divided into six portions for each experiment prior to the addition of the sodium chloride or different concentration of powder (freeze-dried extract of powder of avocado). The lyophilized avocado and the powder of direct avocado were mixed with the salt final concentration of 1.5% (w/w). Each portion of beef meat was mixed manually with each solid. Each mixed sample was divided into nine smaller portions (about 10 g each) and allocated onto trays. The meat was packed under MAP (20% CO2 and 80% O2) in polystyrene/EVOH/polyethylene trays, heat sealed with laminated barrier film and stored at 4 ± 1 °C for 8 days. Patties were evaluated for lipid oxidation.
2.10. Thiobarbituric Reactive Substances
Fat meat oxidation was determined by the concentration of thiobarbituric acid-reactive substances (TBARS) using the method described by Domenech Asensi (2013) [25
] with some modifications. In the dark, 1 g of burger patty was dispensed in tubes and 1 mL of EDTA was added. The samples were homogenized for 5 min sin an Ultra-Turrax (Ika®
-Werke, Staufen, Germany) with 5 mL of TBARS reactive (Trichloroacetic acid, 9.2%; Hydrochloric acid, 2%; Thiobarbituric acid, 0.22%, all w/w final). During homogenization, the tubes were placed in an ice bath to minimize the development of oxidative reactions. The sample tubes were heated at 90 °C in a boiling water bath for 20 min and then left to cool. Two milliliters of slurry was centrifuged (10,000 rpm for 10 min). The absorbance was measured at 531 nm in a Spectrophotometer Zuzi model 4201/20 (AUXILAB, SL, Navarra, Spain). The result is expressed in mg of MDA/kg sample.
RSM was used to identify the best conditions for the extraction of compounds with an antioxidant activity from an organic residue: the avocado pit. The reduced model obtained provides parameters that fit with those of the TPC (with a 3.13% error when compared to the experimental value).
The lyophilized extract was used as protection from the oxidation of oils (oil-in-water emulsions) and fat (beef burgers) with excellent results, especially in meat, in which the durability of the burger meat is significantly increased relative to oxidation.
These studies should encourage further exploration in this area of study in order to obtain a byproduct of the natural antioxidants that currently as waste are worthless.