Oil Content and Fatty Acid Composition of the Seeds of 16 Avocado (Persea americana) Accessions Collected From Southern China and Their Application in a Soap Bar

The avocado (Persea americana), an edible fruit, is one of the main agricultural products in many tropical regions. Avocado fruit is rich in fat, and commercialized for fresh consumption and industrially processed leaving seed as a major residue. Avocado seed from the industry is worthy of attention for certain industrial applications and feasibility. Transforming avocado seed lipids into ecologically friendly or sustainable materials suitable for the cosmetic industry is promising from the perspective of green and environmental protection. The oil contents and fatty acid compositions of the seeds of 16 avocado accessions collected from southern China were investigated, revealing significant differences among most of the accessions. Seventeen fatty acids were identified and quantified by gas chromatography-mass spectrometry in the seeds of all 16 avocado accessions. Linoleic (40.14%), palmitic (23.54%), and oleic acids (16.23%) were the major fatty acids in the seeds, and the total contents of unsaturated fatty acids in the seeds were all higher than those of saturated fatty acids. The biochemical properties of the avocado seed oils relevant to their application in industrial practice were examined [e.g., the acid (3.74 mg KOH/g oil), iodine (124.09 g I2/100 g oil), peroxide (49.83 meq H2O2), and saponification (167.98 mg KOH/g oil) values]. Furthermore, the bar soap containing avocado seed oil was made, and its physicochemical properties (pH and foamability) were evaluated.


Introduction
Avocado (Persea americana Mill.), a member of the family Lauraceae of the order Laurales, is a plant native to Central America, South America, and Mexico (Schaffer et al., 2012).The avocado is among the most economically important subtropical/tropical fruit crops in the world, and production and consumption levels having increased dramatically during the last 150 years (Schaffer et al., 2012).In the world, the total area of avocado cultivation has reached 563,916 hectares, with almost ten tons per hectare in the annual production of avocado in 2016 (FAOSTAT, 2018).The consumption of avocado in the whole world has rapidly increased from 3,426,294 tons in 20083,426,294 tons in to 5,567,044 tons in 20163,426,294 tons in (FAOSTAT, 2018)).One factor contributing to this marked increase was the constant expansion of avocado products into new markets in parts of the world where avocado was previously unknown or scarcely available (Schaffer et al., 2012).In China, the total area of avocado cultivation has reached over 20,000 hectares, with over six tons per hectare in the annual production of avocado in 2016 (FAOSTAT, 2018).The consumption of avocado in China has slowly increased from over 95,000 tons in 200895,000 tons in to 122,942 tons in 201695,000 tons in (FAOSTAT, 2018)).Nowadays, there are many avocado-planting regions in provinces across southern China cultivating foreign cultivars with superior quality (e.g., Fuerte, Hass, and Shepard) along with native Chinese cultivars and accessions (Ge et al., 2017a).
In recent years, there has been growing interest in identifying phytochemical alternatives to the synthetic substances commonly used in the food, pharmaceutical, and cosmetic industries.This effort was supported by consumer concerns over the safety of products containing synthetic chemicals, which were believed to cause or enhance negative health effects and pollute the environment (Vinha et al., 2013;Galvão et al., 2014).Although vegetable oils have traditionally been used primarily in the food industry, they are gaining applications in, for example, the cosmetic industry (Lourith et al., 2014(Lourith et al., , 2016)).In terms of oil content in the fruit, avocado fruit was exceeded only by the fruits of the palm and olive trees (Knothe, 2013).Remarkably, the lipid content in avocado could reach 5% to 30% of the fruit fresh weight, depending on the cultivar, seasonality, and planting conditions (Rodríguez-Carpena et al., 2011;Vinha et al., 2013;Galvão et al., 2014).Avocado fruit lipids contained 50% to 60% monounsaturated fatty acids and 10% to 15% polyunsaturated linoleic and linolenic acids, which were beneficial to human cardiovascular health (Dreher & Davenport, 2013).Furthermore, avocado fruit lipids could be used in non-food industries, for example, as an alternative biodiesel source instead of the conventional petroleum-based diesel fuel (Giraldoa & Moreno-Piraján, 2012;Knothe, 2013).In addition, the high non-saturated content of avocado fruit lipids provided superior skin permeability and sunscreen performance, and could be used in sunscreen cream as the emulsifier (Santo et al., 2014).
Given the increasing demand for vegetable oils in the cosmetic industry (Lourith et al., 2014(Lourith et al., , 2016;;Wu et al., 2015), transforming avocado seed lipids into ecologically friendly or sustainable materials suitable for the cosmetic industry, in particular for personal health products, is promising.In this context, we firstly evaluated and compared the yields and fatty acid compositions of oils from avocado seed.To explore the potential values of the major residue (seed) of avocado fruit, we then determined the physicochemical properties of avocado seed oil to evaluate the oil's applicability in cosmetic applications.In addition, a soap containing avocado seed oil was made and the physicochemical properties of the soap were assessed with respect to possible cosmetic applications.

Plant Material, Reagents, and Sample Preparation
The 16 avocado accessions used in the present study were obtained from Ge et al. (2018).Six ten-year old trees were collected from each accession.Two homogeneous trees were used as a unit with three biological repetitions for each accession.The samples of 18 mature fruits of each biological repetition in each accession were randomly collected when the mesocarp dry matter was ≥ 22% based on the criterion according to Medina-Carrillo et al. (2017), and immediately transported to the laboratory in standard foam boxes used for export packaging.The fruit samples were maintained at 5 °C to 6 °C.The seeds were separated from the fruits, homogenized using a domestic blender, and then stored at 4 °C for a maximum of one week before analysis.

Determination of Total Lipid Contents
Oil content was evaluated using the method of Ge et al. (2017b).Eighteen seeds of each biological repetition in each accession were mixed.The total lipid content was expressed as g/100 g on a fresh weight (FW) basis.The experiments were performed in three biological replicates for each accession.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis and Fatty Acid Identification
The fatty acid profiles were determined as described by Ge et al. (2017b).Eighteen seeds of each biological repetition in each accession were mixed.The analyses were performed using an Agilent7890B-7000B GC-MS (Santa Clara, CA, USA) equipped with a DB-5MS column (60 m × 0.25 mm i.d., 0.25-m film thickness) using helium (1.2 mL/min) as the carrier gas.The fatty acid methyl esters (FAMEs) were identified by comparing the retention times of the peaks with those of commercial standards and comparing the respective ion chromatograms with those reported in the NIST 2011 library.The FAMEs were quantified against methyl nonadecanoate, which was added as an internal standard and then quantified using the calibration curves of the respective FAMEs (R 2 ≥ 0.995).The contents of the FAMEs were expressed as mg/100 g FW.The experiments were performed in three biological replicates for each accession.

Biochemical Properties of Avocado Seed Oil
The acid, iodine, peroxide, and saponification values were determined from the mixed avocado seed oil (a mixture of the seed oils of 16 avocado accessions), and the standard AOAC methods (Association of Official Analytical Chemists, 2000, Method number Cd 3a-63, Cd 1-25, Cd 8-53 and Cd 3-25, respectively) were used.All determinations were carried out in triplicate.

Formulation of the Bar Soap
The self-made bar soap containing the mixed avocado seed oil was made via saponification with NaOH.To prepare the soap, 14.11 g NaOH was dissolved in 47 mL purified water and kept at 40 °C.The oil mixture (86.21 g) was heated to 80 °C and added dropwise to the NaOH solution.Subsequently, the mixture was added to a rotary evaporator to allow oil saponification at 50 °C for 1 h, after which a pasty liquid appeared.The liquid was poured gradually into a mold and kept at 25 °C for two weeks.

Physicochemical Properties of the Bar Soap
The pH values of the self-made bar soap solutions (10%) in deionized water were measured (S20K, Mettler Toledo Ltd., Shanghai, China) in triplicate after the bar soaps were stored for 15 days under normal conditions and under ultraviolet (UV) irradiation (10 W, 60 lux, 365 nm).A commercial soap (Nice, Shanghai, China) stored under normal conditions was tested in the same way.The avocado seed oil bar soap (1.0 g) was added to a 100-mL glass measuring cylinder containing 50 mL deionized water and shaken vigorously for 2 min to generate foam.The height of the foam generated was determined immediately following shaking and after 10, 20, 30, 40, 50, and 60 min.The foamability tests were repeated in triplicate under normal conditions and under UV irradiation (10 W, 60 lux, 365 nm).A commercial soap (Nice, Shanghai, China) was tested in the same way under normal conditions.

Statistical Analysis
All determinations were carried out in three measurements and the results are given as means±standard deviation.The data were analyzed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA).Significant differences among the fruit characteristics, oil contents, and fatty acid compositions of the 16 avocado accessions were verified by one-way analysis of variance, and Duncan's multiple comparison test was used to determine the statistical significance of differences between means at a 95% confidence level.

Total Lipid Contents and Fatty Acid Compositions of Avocado Seed Identification
The total lipid contents of the seeds of the 16 avocado accessions are presented in Table 1, indicating significant differences between most of the accessions (p < 0.05).Hass had the highest total seed lipid content (4.75 g/100 g FW), followed by DL-2 (2.05 g/100 g FW).The seed lipid contents of the remaining 14 avocado accessions were lower, ranging from 0.99 to 1.78 g/100 g FW.The seed lipid contents determined in the present study were in accordance with the results of Galvão et al. (2014) and Rodríguez-Carpena et al. (2011), but the Hass seed lipid content measured herein was much lower than that determined by Vinha et al. (2013) (14.7 g/100 g FW).The variation of the total lipid levels of the seeds of the avocado cultivars from around the world could be due to the different cultivation conditions, varieties, or mature degrees of fruits.Note.Different letters within the column are significantly different (p < 0.05).
The fatty acid compositions of the seeds of the 16 avocado accessions are provided in Table 2, 3.The same 17 fatty acids were detected in the seeds of all 16 avocado accessions, although significant differences in composition were observed among most of the accessions (p < 0.05).The major fatty acids (≥ 15%, the percentage of the individual fatty acid out of the total fatty acid content) quantified in avocado seed oil in the present study were linoleic acid (40.14%), palmitic acid (23.54%), and oleic acid (16.23%).Intermediate amounts (1% to 15%, the percentage of the individual fatty acid out of the total fatty acid content) of palmitoleic acid (5.78%), linolenic acid (4.63%), stearic acid (3.01%), and myristic acid (1.76%) were detected, and small amounts (≤ 1%, the percentage of the individual fatty acid out of the total fatty acid content) of the remaining 10 fatty acids were found.These results were in accordance with the previous study showing that linoleic, palmitic, and oleic acids were the predominant fatty acids in avocado seed oil (Galvão et al., 2014).The content of linoleic acid was slightly higher than those of palmitic acid and oleic acid in this study, which was basically in agreement with the results of Galvão et al. (2014).In the present study, 69% of TFA were unsaturated, while the remaining 31% were saturated ( Note.Different letters within the same row are significantly different (p < 0.05); ΣSFA = total saturated fatty acids; ΣUFA = total unsaturated fatty acids; TFA = total fatty acids.
Table 3.The small amounts of fatty acids compositions (mean value±standard deviation, mg/100 g FW, n = 3) in the seeds of 16 avocado accessions grown in southern China  Note.Different letters within the same row are significantly different (p < 0.05); ΣSFA = total saturated fatty acids; ΣUFA = total unsaturated fatty acids; TFA = total fatty acids.

Confirmation of Biochemical Properties of Avocado Seed Oil
The biochemical properties of avocado seed oil were evaluated and compared with those of unconventional seed bio-oils (Table 5).These properties were vital for the application of crude extracted oil in cosmetics (Lourith et al., 2014).A lower acid value corresponds to a better quality of bio-oil (Wu et al., 2015).The acid value of avocado seed oil was higher than that of avocado and mango seed oil determined by Galvão et al. ( 2014) and Wu et al. (2015), but lower than those of other two seed bio-oils (Lourith et al., 2016;Warra, 2016).The iodine value of avocado seed oil detected in the present study exceeds those of other seed bio-oils except for that of pumpkin seed oil (Table 5).This high iodine value implies an enhanced degree of oxidation or hydrogenation resulting from the degree of UFAs (Wu et al., 2015), when the oil was stored in the sealed state.That displayed that the seeds of avocado contained higher unsaturated fatty acid compositions than other seed bio-oils.Similarly, the peroxide value also has the positive correlation with unsaturated fatty acid content (Lourith et al., 2014;Warra, 2016).The avocado seed oil in the present study had the same peroxide value as Para rubber and Neocarya macrophylla seed oils (Lourith et al., 2014;Warra, 2016).The saponification value of the avocado seed oil was lower than most of the other seed bio-oils (Table 5).The saponification value in the present study indicated that the proportion of long-chain or great-molecular-weight fatty acids in the avocado seed oil had more than twice than those of the majority of seed bio-oils (Table 5).This saponification value was in accordance with the fatty acid profiles shown in Table 2, 3, which presented that the major fatty acids quantified in avocado seed oil were long-chain fatty acids (linoleic acid, palmitic acid, and oleic acid).
Different letters within the same row are significantly different (p < 0.05); ΣSFA = total saturated fatty acids; ΣUFA = total unsaturated fatty acids; TFA = total fatty acids.

Table 1 .
Total lipid contents (mean value±standard deviation, g/100 g FW, n = 3) of the seeds of 16 avocado accessions grown in southern China

Table 5 .
Physicochemical properties of avocado seed oil and other crops seed oils (mean value±standard deviation, n = 3) Note.AV: acid value, IV: iodine value, PV: peroxide value, SV: saponification value.