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Physicochemical, functional and emulsion properties of edible protein from avocado (Persea americana Mill.) oil processing by-products
Accepted Manuscript Physicochemical, functional and emulsion properties of edible protein from avocado (Persea americana Mill.) oil processing by-products Jia-Shui Wang, An-Bang Wang, Xiao-Ping Zang, Lin Tan, Bi-Yu Xu, Hai-Hong Chen, Zhi-Qiang Jin, Wei-Hong Ma PII: DOI: Reference:
S0308-8146(19)30421-2 https://doi.org/10.1016/j.foodchem.2019.02.098 FOCH 24409
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
5 January 2019 18 February 2019 19 February 2019
Please cite this article as: Wang, J-S., Wang, A-B., Zang, X-P., Tan, L., Xu, B-Y., Chen, H-H., Jin, Z-Q., Ma, WH., Physicochemical, functional and emulsion properties of edible protein from avocado (Persea americana Mill.) oil processing by-products, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.02.098
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Physicochemical, functional and emulsion properties of edible protein from avocado (Persea americana Mill.) oil processing by-products
Jia-Shui Wanga, An-Bang Wanga, Xiao-Ping Zanga, Lin Tana, Bi-Yu Xub, Hai-Hong Chenc, ZhiQiang Jina, b*, Wei-Hong Maa*
a
Key Laboratory of Genetic Improvement of Bananas, Hainan province, Haikou Experimental
Station, China Academy of Tropical Agricultural Sciences, Haikou 570102, PR China b
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical
Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China c
Guangxi Vocational and Technical College, Nanning 530226, PR China.
Running title: Edible protein from avocado oil processing by-products
* Corresponding author: Tel.: +86 89866765936; fax: +86 89866710152. E-mail address: [email protected] (Z.-Q. Jin) and [email protected] (W.-H. Ma)
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ABSTRACT: Avocado (Persea americana) is a tropical fruit that has drawn great interest its oil for foods and cosmetic industries; however, avocado oil processing by-product is a potential source of edible protein. Herein, edible protein was prepared from defatted avocado meal, and it’s physicochemical, functional and emulsion properties were investigated. The avocado protein showed U-shaped exhibiting strong effect of pH, and a minimum solubility being observed at pH 4.5, confirming the isoelectric point of avocado protein. Nutritionally, the avocado protein contains all the essential amino acids. Avocado protein provided higher water and oil absorption capacities, higher radical scavenging capacity but lower in-vitro digestibility compared with soy protein. Furthermore, the avocado protein as emulsifier afforded a stability oil-in-water emulsion system, resulting in a greater emulsifying stability than that of soy protein. The present results highlight the potential source of edible protein from avocado oil processing by-products for functional food ingredients.
KEYWORDS: avocado; protein; physicochemical properties; functional properties; emulsion
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1. Introduction World population is currently (2018) growing at a rate of around 1.09% per year and will projected to reach 11.2 billion by 2100, leading to many concerns about food security issues (Population Pyramids of the World from 1950 to 2100 (2018), https://www.populationpyramid.net/). Currently, more special attention has been paid to revalorization of by-products from food processing, peculiarities of their alternatives to the usage of crops for food industry (Ayala-Zavala et al., 2011). Among them, plant residues from food industries represent a major disposal problem, but they are also promising sources as low cost material for the production of edible proteins, especially from oil processing. Avocado (Persea Americana Mill.), or also called alligator pears, is a tropical and subtropical fruit that is very popular in vegetarian cuisine due to its high fat content. Apart from that, avocados are also considered as one of the healthiest foods because they enriched in excess of 25 essential nutrients, including vitamins, minerals, proteins and fibers, with a unique composition of lipids (Calderón-Oliver et al., 2016; Rodríguez-Carpena, et al., 2011). The cultivation and consumption of avocado have increased rapidly over the last few years (up to 5.6 million tonnes/year) owing to its high nutritional value and health-benefits. Although avocado is primarily consumed fresh or as a directly substitute for meats in sandwiches and salads, a consumption drastic increase (growing at a CAGR of 2.8% over the forecast period from 2017 to 2025) in avocado oil through various applications duo to an uniquely high oil content and nutritional values (Rodríguez-Carpena et al., 2011). Especially, avocado oil’s high levels of omega fatty acids make it health benefits supporting cardiovascular health and boosts immune system with great interest for food, cosmetic and pharmaceutical industries. However, avocado oil manufacturing was results in a huge amount of solid residue, which is rich in polyphenols, protein and dietary fiber (López-Cobo et al., 2016). 3
Additionally, increasing environmental and sustainable development concerns were resulted in a drive to strategically utilize the by-products (Ayala-Zavala, Rosas-Domínguez, Vega-Vega, & González-Aguilar, 2010). However, to our best, there is no literature focused on the preparation of protein from avocado oil processing by-products. Avocado is generally believed to be a concentrated source of lipids (~50% of the dry matter) and proteins (~10% of the dry matter) to human diets. Avocado oil processing industries are generating substantial quantities of by-products, which are usually considered a nuisance from the processing point of view. The by-product could be a valuable natural source of protein to be employed as ingredients. Moreover, valorisation of by-products, Ayala-Zavala et al. (2011) pointed out that it is important to utilize the entire fruit for improving profitability by decreasing by-product treatment costs and producing natural food-grade functional ingredients. A number of by-products from the fruit and vegetable processing have been found as protein sources (Gómez & Martinez, 2017; Thaiphanit & Anprung, 2016; Xu et al., 2017). For example, Rodsamran & Sothornvit (2018) found that the protein form coconut milk cake and coconut oil cake exhibited good performance on foaming capacity and emulsifying activity although has a lower protein content (52.06%). Xu et al., (2017) reported that functional leaf protein can be effective recyclable from cauliflower by-products. In fact, it is highly recommended that this makes avocado oil processing by-products to be a potential source for natural protein with high health beneficial. Several studies have been conducted that it is necessary to investigate the physicochemical, functional and emulsifying properties of protein, because of these properties affect the products in manufacturing and storage. Moreover, based on our knowledge, there are no studies on the development of protein from avocado oil processing by-product and its physicochemical 4
characteristics. Therefore, the present study was aimed to prepare protein from avocado oil processing by-product, and then it’s solubility, water and oil adsorption capacities, in vitro digestibility and emulsifying properties were evaluated, providing useful information to utilize as functional protein ingredients and producing value-added foods for improving human health. 2. Materials and methods 2.1. Materials Defatted avocado meal from Hass avocado oil extraction was purchase from local suppliers in Hainan of China. The pepsin, pancreatin and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were bought from Sigma-Aldrich Co. LLC (U.S.). All other chemicals used are of analytical grade. 2.2. Preparation of avocado protein Avocado protein (AP) was prepared from defatted avocado meal (DAM) was prepared according to an alkaline-soluble acid-precipitation strategy method described by Chel-Guerrero, Perez-Flores, Betancur-Ancona, & Davila-Ortiz, (2002), with minor modifications. After passed through an 80 mesh screen, defatted avocado flour was mixed with 15-fold (w/v) deionized water and adjusted to pH 8.5 with 1.0 mol/L NaOH. After soaking for 3 h, the dispersion was centrifuged at 8,000g for 30 min. The supernatant was adjusted to an isoelectric point (pI, seen Figure 2B) of 4.5 with 1.0 mol/L HCl. And then, the precipitate was collected by centrifugation (8,000g, 10 min). The precipitated protein was collected and redispersed in deionized water and dialyzed at 4°C for 72 h. Finally, the dispersion freeze-dried to produce the loose AP products. As a control, soybean protein (SP) was also prepared by the same method. 2.3. Chemical analysis Protein content was determined with a Kjeltec System (Tecator, Sweden) with a conversion 5
factor of 6.25. Lipids, ash and moisture contents were determined according to official AOAC procedures (AOAC, 2000). The total polyphenols content (TPC) was measured according to Naczk and Shahidi (2004), where the results were expressed as gallic acid equivalent (mg GAE/g sample), according to a calibration curve (75-650 μg/mL, R2=0.9932). 2.4. Characterization of proteins The particle size distribution, z-average diameter and ζ-potential of proteins were determined by dynamic light scattering (Malvern, Worcestershire, Malvern-UK) as described in our previous work (Wang et al., 2018). The protein morphology was observed using an atomic force microscopy (AFM). After the complete dissolution, 10 μL of 20 μg/mL protein solution was deposited onto a freshly mica surface and air-dried overnight. Then, the microscope was examined using Bruker Multimode 8 atomic force microscopy (Bruker Corporation, Germany) in noncontact mode. 2.5. Free sulfhydryl (SH) and surface hydrophobicity (H0) The free sulfhydryl (SH) contents were determined using Ellman’s reagent (DTNB) according to the method described by Beveridge, Toma, and Nakai (1974), with a few modifications. Protein powder (100 mg) was dispersed in 1.5 mL of 0.086 mol/L Tris-glycine buffer (pH 8.0), agitated and extracted for 1 h at room temperature. After that, the suspension was centrifuged at 10,000g for 15 min to remove the particulate. 0.03 mL of 4 mg/mL Ellman’s reagent solution was added to 3 mL supernatant and reacted for 15 min at room temperature, then the absorbance was measured at 412 nm using a spectrophotometer (Alpha-102, Shanghai PUYUAN Instrument Co., Ltd, China). For the measurement of surface hydrophobicity (H0), 0.05-0.2 mg/mL protein dispersions were dispersed in phosphate buffer (0.01 mol/L, pH 7.0). Then, 20 μL of 8 mmol/L ANS was added and left to react for 10 min, and the fluorescence intensity was determined using a F4500 fluorometer 6
(Hitachi Co., Japan) at a wavelength 370 nm and measured at 490 nm. The initial slope of the linear regression equation was taken as an index of average protein surface hydrophobicity. 2.6. Protein solubility (PS) Aqueous solutions (1%, w/v) of the protein were prepared in 20 mL of distilled water adjusted adjusted to an appropriate pH (from 2.0 to 12.0) with either 1.0 mol/L NaOH or HCl, and stirred for 30 min at room temperature. Then, the dispersions were centrifuged at 4,320g for 30 min and following filtered into tubes. The protein contents of the filtrate were taken by a Bradford method with a standard of BSA. Solubility was defined as the percentage of the protein concentration in the supernatant over that of total protein. 2.7. Amino Acid Analysis The amino acid profile was determined using an L-8900 automatic Amino Acid Analyzer (Hitachi, Japan). Samples (500 mg) were placed in an ampoule, and hydrolyzed with 6 mol/L HCl under a nitrogen atmosphere for 24 h at 110 °C. The samples were then subjected to reversed-phase highperformance liquid chromatography (Agilent 1100) analysis. The amino acid composition was reported as g/100 g protein. 2.8. Water and oil holding capacity Both water-holding capacity (WHC) and oil-holding capacity (OHC) were measured using the method described by Timilsena et al. (2016). Briefly, 1.0 g of protein was vigorously mixed with 10 g distilled water or soybean oil for 20 s with a vortex, respectively. After incubation at 25 °C for 30 min, the mixture was centrifuged at 5,000g for 30 min and the free water or oil was decanted. WHC and OHC were expressed as grams of water or oil trapped per gram of protein, respectively. 2.9. Antioxidant capacity determined by radical cation (ABTS) 7
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity assay was performed according to the method described by Re et al. (1999) with slight modifications. ABTS radical cation (ABTS+) was produced from the reaction of 7 mmol/L ABTS solution with 4.9 mmol/L K2S2O8 solution, and allowed the mixture stand in the dark at room temperature for 12 h. Then, the ABTS working solution was diluted with ethyl alcohol to an absorbance of 0.70 ± 0.02 at 734 nm. After that, an aliquot of 100 μL tested samples was added to 1.9 mL of ABTS working solution and measured at ten minute of reaction at 734 nm. Results were expressed as trolox equivalent antioxidant capacity (TEAC). 2.10. In vitro simulated gastrointestinal digestion The in vitro digestibility method used was a modification of that of Yu et al., (2017). Briefly, 0.5 g proteins were dissolved into 20 mL of 0.1 mol/L HCl containing 10 mg pepsin, followed by reacting in a 37 °C water bath for 60 min. After that, the pepsin digests was immediately neutralized with 1.0 mol/L NaOH. Then, 10 mL of phosphate buffer (20 mmol/L, pH 7.2) containing 20 mg of pancreatin was added the mixture and incubated at 37 °C for another 120 min in a water bath shaker. After that, the digestive juice was heated in boiling water for 5 min to inactivate enzyme and centrifuged at 8,000g for 20 min to precipitate the undigested protein. The nitrogen release during the digestion was calculated by Iwami et al. (1986): percentage of nitrogen release = (N0Nt)/N0×100%, where t is the digestion time (min), Nt (mg) is the TCA-insoluble nitrogen after digestion for time (min), N0 (mg) is the TCA-insoluble nitrogen in the protein sample, and Ntot (mg) is the total nitrogen of the protein sample. 2.11. Emulsion properties 2.11.1. Surface and interfacial tension. A contact angle and surface tension meter (Krüss Force 8
Tensiometer K20, Germany) was used to examine the interfacial tension of protein at the oil-water interface. Protein dispersions were prepared by mixing proteins into PBS (10 mmol/L phosphate buffer, pH 7.0) with stirring at 300 rpm for 2 h at room temperature, and left overnight at 4°C to fully hydrate. The tensiometer operated in a volume-controlled regime, via continuous measurement of the drop area and volume, determined through the Young-Laplace profile. 2.11.2. Emulsion preparation. Emulsions were prepared at three different protein concentrations (0.5, 1.0, 1.5, and 2.0 wt%) that were dispersed in 10 mL of 20 mmol/L phosphate buffer of pH 7.0 and mixed with 1.0 mL aliquots soybean oil. The mixture was homogenized in a Polytron PT 3100 homogenizer (Kinematica AG, Lucerne, Switzerland) at 6,000 rpm first for 5 min, and then secondly for another minute after a 2-second pause at 500 bar using a high-pressure homogenization (SRH 6070, Shanghai shen deer homogenizer co., LTD, China). 2.11.3. Mean particle size and microstructure of emulsions. The mean particle size and droplet size distributions of the emulsion were measured with a Mastersizer 2000 static light-scattering analyser (Malvern Instruments Ltd., Malvern, U.K.). The refractive indices used for oil droplet and water were 1.467 and 1.330, respectively. The mean droplet size was characterized as the volumeweighted mean diameter d4,3 = ∑nidi4/∑nidi3, where ni is the number of droplets corresponding to the diameter di. The creaming index (CI) was measured by macroscopic observations of emulsions assessed during 14 days of storage. Ten milliliter of each emulsion was filled into a glass test tube (1.5 cm × 12 cm), and the creaming index was reported as the following equation: creaming index (%) = (Hs/Ht)×100%, where Hs is the serum height and Ht is the total height of the emulsion. 2.12. Statistical analyses Data are reported as mean value ± standard deviation. Statistical analysis of the results (reported 9
here are the average of at least three measurements carried out on two replicated experiments) was performed with SPSS 21 (SPSS Inc., U.S.A.) and a one-way variance (ANOVA) was employed. Values were considered significant when p < 0.05.
3. Results and discussion 3.1. Proximate compositions After extraction of oil from avocado (Persea americana), a significant quantity of solid residue are generated. These by-products may be still ideal raw materials for food due to their high nutritional value, e.g., protein, polyphenol, and dietary fiber. As shown in Table 1, the defatted avocado meal from the oil processing by-products was content 15.05±0.35% protein. After protein precipitation processes, the protein contents of avocado protein (AP) powders was found to be 69.34±1.55%, which is 4.7-folds higher than raw materials. The purified soy protein (SP) had slightly higher protein (90.20%) and lower lipid (1.08%), moisture (4.94%) and ash (2.38%) contents. The low protein may be due to the fact that the non-protein components (e.g., polyphenols, dietary fiber) reserved during the fractionation step. Although, no statistically significant differences (p > 0.05) between the avocado protein and soy protein were observed in the results of moisture, lipids and ash, the slightly higher others of avocado protein compared with that of the soy protein. However, avocado protein was found to have a higher deep color. The results obtained revealed significant variation (p < 0.05) in the total polyphenolics content (TPC) among defatted avocado meal, avocado protein and soy protein (Table 1). Avocado protein variety contained more phenolic compounds at 15.85 mg CE/g more 2-folds than that of defatted avocado meal (8.33 mg CE/g), and more 30-folds than that of soy protein (0.54 mg CE/g). Consistently, Lopez-Cobo et al. (2016) has 10
been reported previously that Hass avocado has exceptionally high TPC (e.g., caffeic, chlorogenic, coumaric, ferulic, gallic and hydroxybenzoic, etc.), and its byproduct also showed higher phenolic content than other fruits and vegetables (Rodríguez-Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011). 3.2. Physicochemical properties of avocado protein 3.2.1. Characterization of avocado protein The z-average diameter (Dz) and ζ-potential of avocado protein as a function of pH are summarized using DLS technique, as shown in Fig. 1A. The z-average diameter of avocado protein at pH 7.0 was about 82.17±0.63 nm, which was evidently larger than that of soy protein (54.61 ± 0.65 nm). The reduced ζ-potential of avocado protein with increasing of pH was progressively decreased from 18.1 mV to -26.3 mV, suggesting surface group ionization (Shevkani, Singh, Kaur, & Rana, 2015). Meanwhile, a zero surface charge was showed at near pH of 4.2. At the zero surface charge, the protein was aggregated state in the dispersions with a larger size of 280 nm. Considering high amounts of polyphenols of avocado protein, the interaction between protein and polyphenols may be interact each other associating into larger sizes (Calderón-Oliver et al., 2016; López-Cobo et al., 2016). To direct observant the morphology, we evaluated the proteins morphology using AFM, as shown in Fig. 1B and Fig. 1C. Quite different morphology with particle size ranging from several to 100 nm was exhibited in the different samples. For soy protein as displayed in Fig. 1C, it is confirmed the nature of soy protein that the height and the contour was about 1-8 nm and (30-50) × (40-60) nm, respectively (Utsumi, Matsumura, & Mori, 1997). However, the morphology of avocado protein was seems less regular and much larger (Fig. 1B), which is well agreed with the DLS data 11
(inset of Fig. 1B and 1C). The particle size distribution of avocado protein was calculated to be around 82 nm at pH 7.0. 3.2.2. Free sulfhydryl (SH) content and surface hydrophobicity (H0) The free SH and H0 are shown in Fig. 2A, which indicate obviously differences in avocado protein and soy protein. As expected, the free SH value of avocado protein (1.0 × 10-6 mol/g of protein) is significantly (p < 0.05) lower than that of soy protein (1.5 × 10-6 mol/g of protein). The significantly higher SH value for avocado protein is consistent with the relative cystine and methionine contents in avocado protein and soy protein as demonstrated in Table 2. Traditionally, protein molecules with their high SH value are more likely to associate with one another and form aggregates (Yu et al., 2017), while the result was inconsistent with AFM and DLS data (Fig. 1). The difference might be largely due to the high loading of polyphenols in avocado protein (Table 1). Similar phenomenon has been observed in the literature that polyphenols (e.g., tea polyphenols) were interacting each other’s resulting in larger sizes (Rodríguez, von Staszewski, & Pilosof, 2015). As shown in Fig. 2A, the surface hydrophobicity (H0) of avocado protein (1685.3) was also significantly higher than that its value of control soy protein (1111.4) (p < 0.05), suggesting more hydrophobic groups exposure out. This difference may be mainly attributed to the distinct structural conformations from various initial courses. Eventually, the avocado protein with higher surface hydrophobicity was tending to exhibit higher surface activity, which probably suitable for specific product applications. 3.2.3. Protein solubility Protein solubility is believed to be an important factor for functional application in the food industry. The solubility profile of avocado protein as a function of pH is presented in Fig. 2B, 12
compared to soy protein. In general, the solubility curves were nearly U-shaped as the function of pH. A minimal solubility at pH 4.5 was observed as a result of the proximity to the protein isoelectric point (pI). Moreover, the solubility (4.2%) was similar to that reported for the minimal protein solubility in Phaseolus calcaratus (5.1%), Dolichos lablab (5.08%), coconut (1%) and leftover granules (2.5%) (Thaiphanit & Anprung, 2016). However, greater solubility was seen at higher pH (pH 12) and lower pH (pH 2) attributing to an increase in net protein charge. The results showed in the protein solubility of avocado protein are closely related to the data of zeta potential (Fig. 1A). This could be due to the isoelectric precipitation by electrostatic attractions. Away from the isoelectric point, the high solubility of proteins was mainly attributed to the electrostatic repulsion and ionic hydration among protein molecules (Damodaran, 2017; Utsumi, Matsumura, & Mori, 1997). Similar results were observed that there is a positive correlation between protein solubility and surface charge for various protein sources (e.g., soy, peanut and cashew nut) (Lam & Nickerson, 2013). However, a lower solubility of avocado protein was observed than that of soy protein within this pH range. The lower solubility may be probably due to the higher degree of polyphenols in avocado protein, which would form nanoparticles with proteins resulting in lower protein solubility (Table 1, Fig. 1). 3.3. Functional properties of avocado protein 3.3.1. Amino acid analysis and chemical score Compositions of amino acids of the avocado protein are presented in Table 2, compared to soy protein. It was observed that the avocado protein is found to be rich in sulfur acid, aspartic acid and glutamic amino acids, which was in contrast with the findings of Kahn (1985). The amino acid compositions showed a similar to that of soy protein, indicating that the avocado protein is 13
considered to be a good source of amino acids. Compared overview of the essential amino acids (EAA) with the WHO/FAO/UNO requirements pattern, both avocado protein and soy protein have high levels of Thr, Val, Ile, Leu, Phe+Tyr, Lys and His, which than the levels suggested by WHO/FAO/UNO recommendations for both infants and adults. In addition, for avocado protein, the ratio of essential amino acid to total amino acids (as shown in Table 2) was approximately 49%, significantly higher than that of soybean (40.80%) and safflower (38.1%). This current result was an important aspect in favor of the functionality of avocado protein. 3.3.2. In-vitro digestibility In vitro digestibility was investigated with pepsin-pancreatin, as shown in Table 3. The in vitro digestibility of the avocado protein (68.9 ± 0.9%) was slightly higher than that of maize (66.6%), rice (59.4%), sorghum (59.1%), and wheat (52.7%), and slightly lower than that of soy protein (78.1 ± 2.7%) (Joshi, Liu, & Sathe, 2015). Protein composition, enzyme type and impurities were responded to the digestibility (Dufour et al., 2018; Huang, Kwok, & Liang, 2004). However, there is no report of anti-nutritive factors in avocado, ruling out the presence of protease inhibitors that could retard the in-vitro digestibility. It is suggested that the polyphenols may affect the in vitro digestibility (Dufour et al., 2018; Ren et al., 2018). Many previous studies have reported that polyphenols in proteins showed the significantly lower trypsin inhibitor activity and resulted a lower digestibility (Huang, Kwok, & Liang, 2004). Ma and co-author (2011) also concluded that the protein digestibility was negatively correlated with polyphenol (e.g., tannin) contents. Furthermore, Ren and co-author (2018) also found that the formation of soy protein/polyphenol complexes inhibited protein digestion in vitro. However, the antioxidant activity (ABTS) of avocado protein was significantly higher (p<0.05) than soy protein, as shown in Table 3. This result corresponds to the polyphenol content reported 14
above (Table 1). Therefore, the results highlight that the avocado protein is exhibiting good candidate for functional foods. 3.3.3. Water and oil holding capacities Water-holding capacity (WHC) and oil-holding capacity (OHC) are represents the ability to immobilize water and oil, respectively, and plays an important role in improving the stability, texture and flavor of food products. Interestingly, both WHC (6.60±0.25 g/g) and OHC (5.53±0.09 g/g) of avocado protein were higher than (p < 0.05) those of soy protein (Table 3). Furthermore, the WHC and OHC values of avocado protein determined in this study were higher than that of coconut protein (Physicochemical and functional properties of protein concentrate from by-product of coconut processing) and peanut protein (Comparative studies on the functional properties of various protein concentrate preparations of peanut protein). The results may be explained by the difference in the raw material diversity and the protein solubility as confirmed by the report of Kaur et al., (2005) and Kinsella (1976). They showed that several water-loving components (e.g., dietary fiber) generally have high WHC, while non-polar components (e.g., polyphenols) are contributed to a higher OHC. 3.4. Emulsion properties of avocado protein 3.4.1. Surface and interfacial behavior In this study, emulsion properties were further studied in order to enhance the utilization of avocado protein in food industry. Interfacial tension plays an important role in emulsification because of an emulsifier reduces the interfacial tension resulting in the formation of an emulsion (Thaiphanit & Anprung, 2016). With respect to the ability of avocado protein to act as an oil-inwater emulsifier in relation to protein concentration, the results are summarized in Fig. 3A. The 15
surface tension (π) showed consistent protein concentration reduction, which reflects that increase in protein has a beneficial effect on reducing the surface tension at the oil-water interface. At the same content, soy protein has shown a better effectiveness in the decreasing of π than that of avocado protein. The observation is consistent with the general viewpoint in the colloid and interface fields that the diffusion and adsorption of particles with larger sizes is slower than that of smaller sizes (Dickinson, 2010). For avocado protein, the aggregated insolubilizers were unable to unfold at interfaces, and may not be beneficial to reduce surface tension. However, it may be stable the interface by a Pickering mechanism. In the series of studies of Tang (2017) and Binks (2018), they were reported that many native globular proteins (e.g., soy protein, pea protein, whey protein and peanut protein) can be as outstanding Pickering nanostabilizer to stabilize emulsions. 3.4.2. Emulsification performance Emulsification performance of the avocado protein was then evaluated at varying protein concentrations of 0.5-2.0% (w/v). And, the droplet diameters (d4,3) and size profiles of these emulsions were measured as shown in Fig. 3B and 3C, respectively. It was shown that the oil droplet sizes stabilized by avocado protein were a little higher than those of soy protein at the same protein concentration, and progressively increased with decreasing the protein concentration (Fig. 3B). From the typical droplet size distribution profiles (Fig. 3C), all the emulsions exhibited a profile of bimodal or trimodal droplet size distributions. It also can be seen that a major size distribution peak (at 1-10 μm) observed at higher protein concentration, confirming more proteins were favor to form and stabilize the interface (Lam & Nickerson 2013). Moreover, similar result in average oil droplet size decreases steadily with increasing protein concentration was also observed in the light microscope images of all these emulsions (Fig. S). 16
Creaming behavior of the emulsions stabilized by avocado protein at different concentrations was also evaluated over a storage for up to 2 weeks, as shown in Fig. 3D. As expected, the creaming index (CI) of all the emulsions progressively increased over time. As we all known that emulsions with higher degree of flocculation tended to have higher degree of CI. In the case, the flocculation was prevented with increasing protein concentration, as shown in Fig. 3D that the creaming behavior was steadily decreasing with increasing to the avocado protein concentrations. This is in accordance with the droplet size (Fig. 3C). However, we can interestingly see that the avocado protein emulsions at the relevant concentrations showed much larger droplet size but exhibited much higher creaming stability compared with soy protein (Fig. 3D and Fig. S). The emulsions stabilized by avocado protein have significantly stability upon 10 days storage. Particularly, there was no distinct creaming for the emulsion at 2.0% avocado protein after storage for 14 days (see inset of Fig. 3D). Considering the protein size, avocado protein may be play a Pickering particle-like role in the stabilization of emulsions from the fact that the dimension of monomeric avocado protein as shown in Fig. 1C. In that case, the avocado protein aggregates would firmly adsorb at the oil-water interface form a more packed protein layer. On the other hand, we observed that a high surface hydrophobicity of avocado protein possibly attributed to their native polyphenols in avocado fruit. It is generally known that polyphenols could improve the emulsion system by the intermolecular between protein and polyphenol (Dickinson, 2010; Wang et al., 2016). 4. Conclusions This study highlighted that the avocado (Persea americana Mill.) oil processing by-products could be good source to produce edible protein with functional and emulsion properties. The minimum solubility was occurred at the pI of pH 4.5. Nutritionally, avocado protein contains all the 17
essential amino acids and similar to that of soy protein. Both WHC and OHC of avocado protein were observed at 6.60 and 5.53 g/g, respectively, which were much higher than those of soy protein (5.53 and 3.91 g/g, respectively). Avocado protein was also provided a high radical scavenging capacity compared with soy protein. After pepsin-trypsin digestion, the percentage of nitrogen release of avocado protein reached up to 68.9%. The presence of avocado protein as an emulsifier enhanced the stability of oil-in-water emulsion system. Furthermore, avocado protein showed a greater emulsifying stability than soy protein, even with a lower interfacial tension and emulsifying activity. This work demonstrates that the avocado oil processing by-products might provide a potential protein source as functional ingredients in food systems.
Conflict of interest The authors declare no conflicts of interest. Acknowledgements This work is supported by the Key Research and Development Program of Hainan Province (ZDYF2018058), the Agricultural Wild Plant Resource Protection Fund in Ministry of Agriculture of China (1251416305010), and the Cross-Strait Agricultural Cooperation Program of China.
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Tables Table 1. Proximate analyses of defatted avocado flour, avocado protein and soy protein.a TPC Protein
Moisture
Lipids
Samples
Others Ash (%)
(%)
(%)
(mg CE/g
(%)
(%) DW)
Defatted avocado
15.05±0.35
7.70±0.40a
3.48±0.15a 23
7.25±0.35a
8.33 ± 0.14b
66.52
flour
c
69.34±1.55
15.85 ± 4.02±0.33b
Avocado protein
2.17±0.22b
3.20±0.10b
b
21.27 0.24a
90.20±0.70 4.94±0.40b
Soy protein
1.08±0.14b
2.38±0.20b
0.54±0.14c
1.40
a
a
All data were based on the wet basis. Each value was the mean and standard deviation of triplicate measurements. The letters (a−c)
indicate significant (p < 0.05) difference within the same column. TPC, total polyphenolics content.
24
Table 2. Comparison of amino acid content of avocado protein and soy protein, and WHO/FAO recommendations for children and adult. Amino acids (mg/g protein)b Amino acids Avocado protein
Soy protein
Asp Thr a Ser Glu Gly Ala Val a Met+Cys a Ile a Leu a Phe+Tyr a Lys a His a Arg Pro
78.93±2.18 32.46±1.53 44.01±1.54 101.87±2.59 40.98±1.24 43.43±1.34 51.37±0.85 13.84±0.81 42.56±2.54 70.27±0.17 79.36±2.04 59.88±1.56 19.48±0.52 44.15±1.84 36.79±1.54
109.19±3.28 28.81±1.66 53.92±3.77 197.53±1.36 36.32±2.87 32.62±3.32 47.30±0.92 20.84±1.35 44.73±0.94 72.19±2.31 76.90±2.68 64.23±1.67 23.87±0.89 74.32±1.35 45.85±2.02
EAA/TAAd (%)
48.62±1.24
40.80±1.02
aEssential
FAO/WHO suggested Requirementsc 1-2 years old
3-10 years old
Adult
27
25
23
42 26 31 63 46 52 18
40 24 31 61 41 48 16
39 22 30 59 38 45 15
amino acids. bAmino acid content/total amino acid content. cAmino acid requirements/protein requirements for the selected age groups. d EAA/TAA = Essential Amino Acid/Total Amino Acid. Values represent the mean of duplicate measurements.
25
Table 3. In vitro digestibility, radical scavenging capacity, water absorption capacity and oil absorption capacity of avocado protein compered to soy protein. a
a
Samples
Digestibility (%)
ABTS
WAC (g/g)b
OAC (g/g) c
Avocado protein
68.9 ± 0.9 b
8486 ± 111 a
6.60±0.25 a
5.53±0.09 a
Soy protein
78.1 ± 2.7 a
1412 ±25 b
4.69±0.38 b
3.91±0.24 b
Values are the mean ± SD of three determinations. Different letters for the same protein types indicate significant differences (p <
0.05) in means. bWAC, water absorption capacity. cOAC, oil absorption capacity.
Figure Captions Fig. 1. (A) The z-average diameter (Dz) and ζ-potential of avocado protein and soy protein as a function of pH; Typical size distribution profiles and 3D AFM height images of (B) avocado protein and soy protein (C) dispersions in pH 7.0.
Fig. 2. (A) Free SH contents of avocado protein (AP) and soy protein (SP) at pH 7.0. (B) Nitrogen solubility of avocado protein and soy protein at different pH values. Between avocado varieties: *, p < 0.05.
Fig. 3. (A) Surface tension (π) as a function of time for different avocado protein (AP) concentrations (0.01−0.5%) and soy protein (SP) at the oil-water interface; (B) Mean emulsion droplet diameter and (C) particle size distributions of fresh emulsions stabilized by avocado protein and soy protein; (D) Creaming index of emulsions at four protein concentrations of 0.5, 1.0, 1.5 and 2.0% during quiescent storage of 14 days. The inlet figure shows the image of the 2.0% avocado protein emulsions after the 14d storage, wherein the arrows indicate bottom border of the cream 26
layer.
27
Figure 1.
28
Figure 2.
29
Figure 3.
30
Graphical abstract
Highlights Edible protein was prepared from avocado oil processing by-products. Physicochemical and functional properties of avocado protein were investigated. Oil-in-water emulsions stabilized by avocado protein. Avocado oil processing by-products is a sustainable protein resource.
31
S0308-8146(19)30421-2 https://doi.org/10.1016/j.foodchem.2019.02.098 FOCH 24409
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
5 January 2019 18 February 2019 19 February 2019
Please cite this article as: Wang, J-S., Wang, A-B., Zang, X-P., Tan, L., Xu, B-Y., Chen, H-H., Jin, Z-Q., Ma, WH., Physicochemical, functional and emulsion properties of edible protein from avocado (Persea americana Mill.) oil processing by-products, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.02.098
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Physicochemical, functional and emulsion properties of edible protein from avocado (Persea americana Mill.) oil processing by-products
Jia-Shui Wanga, An-Bang Wanga, Xiao-Ping Zanga, Lin Tana, Bi-Yu Xub, Hai-Hong Chenc, ZhiQiang Jina, b*, Wei-Hong Maa*
a
Key Laboratory of Genetic Improvement of Bananas, Hainan province, Haikou Experimental
Station, China Academy of Tropical Agricultural Sciences, Haikou 570102, PR China b
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical
Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China c
Guangxi Vocational and Technical College, Nanning 530226, PR China.
Running title: Edible protein from avocado oil processing by-products
* Corresponding author: Tel.: +86 89866765936; fax: +86 89866710152. E-mail address: [email protected] (Z.-Q. Jin) and [email protected] (W.-H. Ma)
1
ABSTRACT: Avocado (Persea americana) is a tropical fruit that has drawn great interest its oil for foods and cosmetic industries; however, avocado oil processing by-product is a potential source of edible protein. Herein, edible protein was prepared from defatted avocado meal, and it’s physicochemical, functional and emulsion properties were investigated. The avocado protein showed U-shaped exhibiting strong effect of pH, and a minimum solubility being observed at pH 4.5, confirming the isoelectric point of avocado protein. Nutritionally, the avocado protein contains all the essential amino acids. Avocado protein provided higher water and oil absorption capacities, higher radical scavenging capacity but lower in-vitro digestibility compared with soy protein. Furthermore, the avocado protein as emulsifier afforded a stability oil-in-water emulsion system, resulting in a greater emulsifying stability than that of soy protein. The present results highlight the potential source of edible protein from avocado oil processing by-products for functional food ingredients.
KEYWORDS: avocado; protein; physicochemical properties; functional properties; emulsion
2
1. Introduction World population is currently (2018) growing at a rate of around 1.09% per year and will projected to reach 11.2 billion by 2100, leading to many concerns about food security issues (Population Pyramids of the World from 1950 to 2100 (2018), https://www.populationpyramid.net/). Currently, more special attention has been paid to revalorization of by-products from food processing, peculiarities of their alternatives to the usage of crops for food industry (Ayala-Zavala et al., 2011). Among them, plant residues from food industries represent a major disposal problem, but they are also promising sources as low cost material for the production of edible proteins, especially from oil processing. Avocado (Persea Americana Mill.), or also called alligator pears, is a tropical and subtropical fruit that is very popular in vegetarian cuisine due to its high fat content. Apart from that, avocados are also considered as one of the healthiest foods because they enriched in excess of 25 essential nutrients, including vitamins, minerals, proteins and fibers, with a unique composition of lipids (Calderón-Oliver et al., 2016; Rodríguez-Carpena, et al., 2011). The cultivation and consumption of avocado have increased rapidly over the last few years (up to 5.6 million tonnes/year) owing to its high nutritional value and health-benefits. Although avocado is primarily consumed fresh or as a directly substitute for meats in sandwiches and salads, a consumption drastic increase (growing at a CAGR of 2.8% over the forecast period from 2017 to 2025) in avocado oil through various applications duo to an uniquely high oil content and nutritional values (Rodríguez-Carpena et al., 2011). Especially, avocado oil’s high levels of omega fatty acids make it health benefits supporting cardiovascular health and boosts immune system with great interest for food, cosmetic and pharmaceutical industries. However, avocado oil manufacturing was results in a huge amount of solid residue, which is rich in polyphenols, protein and dietary fiber (López-Cobo et al., 2016). 3
Additionally, increasing environmental and sustainable development concerns were resulted in a drive to strategically utilize the by-products (Ayala-Zavala, Rosas-Domínguez, Vega-Vega, & González-Aguilar, 2010). However, to our best, there is no literature focused on the preparation of protein from avocado oil processing by-products. Avocado is generally believed to be a concentrated source of lipids (~50% of the dry matter) and proteins (~10% of the dry matter) to human diets. Avocado oil processing industries are generating substantial quantities of by-products, which are usually considered a nuisance from the processing point of view. The by-product could be a valuable natural source of protein to be employed as ingredients. Moreover, valorisation of by-products, Ayala-Zavala et al. (2011) pointed out that it is important to utilize the entire fruit for improving profitability by decreasing by-product treatment costs and producing natural food-grade functional ingredients. A number of by-products from the fruit and vegetable processing have been found as protein sources (Gómez & Martinez, 2017; Thaiphanit & Anprung, 2016; Xu et al., 2017). For example, Rodsamran & Sothornvit (2018) found that the protein form coconut milk cake and coconut oil cake exhibited good performance on foaming capacity and emulsifying activity although has a lower protein content (52.06%). Xu et al., (2017) reported that functional leaf protein can be effective recyclable from cauliflower by-products. In fact, it is highly recommended that this makes avocado oil processing by-products to be a potential source for natural protein with high health beneficial. Several studies have been conducted that it is necessary to investigate the physicochemical, functional and emulsifying properties of protein, because of these properties affect the products in manufacturing and storage. Moreover, based on our knowledge, there are no studies on the development of protein from avocado oil processing by-product and its physicochemical 4
characteristics. Therefore, the present study was aimed to prepare protein from avocado oil processing by-product, and then it’s solubility, water and oil adsorption capacities, in vitro digestibility and emulsifying properties were evaluated, providing useful information to utilize as functional protein ingredients and producing value-added foods for improving human health. 2. Materials and methods 2.1. Materials Defatted avocado meal from Hass avocado oil extraction was purchase from local suppliers in Hainan of China. The pepsin, pancreatin and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were bought from Sigma-Aldrich Co. LLC (U.S.). All other chemicals used are of analytical grade. 2.2. Preparation of avocado protein Avocado protein (AP) was prepared from defatted avocado meal (DAM) was prepared according to an alkaline-soluble acid-precipitation strategy method described by Chel-Guerrero, Perez-Flores, Betancur-Ancona, & Davila-Ortiz, (2002), with minor modifications. After passed through an 80 mesh screen, defatted avocado flour was mixed with 15-fold (w/v) deionized water and adjusted to pH 8.5 with 1.0 mol/L NaOH. After soaking for 3 h, the dispersion was centrifuged at 8,000g for 30 min. The supernatant was adjusted to an isoelectric point (pI, seen Figure 2B) of 4.5 with 1.0 mol/L HCl. And then, the precipitate was collected by centrifugation (8,000g, 10 min). The precipitated protein was collected and redispersed in deionized water and dialyzed at 4°C for 72 h. Finally, the dispersion freeze-dried to produce the loose AP products. As a control, soybean protein (SP) was also prepared by the same method. 2.3. Chemical analysis Protein content was determined with a Kjeltec System (Tecator, Sweden) with a conversion 5
factor of 6.25. Lipids, ash and moisture contents were determined according to official AOAC procedures (AOAC, 2000). The total polyphenols content (TPC) was measured according to Naczk and Shahidi (2004), where the results were expressed as gallic acid equivalent (mg GAE/g sample), according to a calibration curve (75-650 μg/mL, R2=0.9932). 2.4. Characterization of proteins The particle size distribution, z-average diameter and ζ-potential of proteins were determined by dynamic light scattering (Malvern, Worcestershire, Malvern-UK) as described in our previous work (Wang et al., 2018). The protein morphology was observed using an atomic force microscopy (AFM). After the complete dissolution, 10 μL of 20 μg/mL protein solution was deposited onto a freshly mica surface and air-dried overnight. Then, the microscope was examined using Bruker Multimode 8 atomic force microscopy (Bruker Corporation, Germany) in noncontact mode. 2.5. Free sulfhydryl (SH) and surface hydrophobicity (H0) The free sulfhydryl (SH) contents were determined using Ellman’s reagent (DTNB) according to the method described by Beveridge, Toma, and Nakai (1974), with a few modifications. Protein powder (100 mg) was dispersed in 1.5 mL of 0.086 mol/L Tris-glycine buffer (pH 8.0), agitated and extracted for 1 h at room temperature. After that, the suspension was centrifuged at 10,000g for 15 min to remove the particulate. 0.03 mL of 4 mg/mL Ellman’s reagent solution was added to 3 mL supernatant and reacted for 15 min at room temperature, then the absorbance was measured at 412 nm using a spectrophotometer (Alpha-102, Shanghai PUYUAN Instrument Co., Ltd, China). For the measurement of surface hydrophobicity (H0), 0.05-0.2 mg/mL protein dispersions were dispersed in phosphate buffer (0.01 mol/L, pH 7.0). Then, 20 μL of 8 mmol/L ANS was added and left to react for 10 min, and the fluorescence intensity was determined using a F4500 fluorometer 6
(Hitachi Co., Japan) at a wavelength 370 nm and measured at 490 nm. The initial slope of the linear regression equation was taken as an index of average protein surface hydrophobicity. 2.6. Protein solubility (PS) Aqueous solutions (1%, w/v) of the protein were prepared in 20 mL of distilled water adjusted adjusted to an appropriate pH (from 2.0 to 12.0) with either 1.0 mol/L NaOH or HCl, and stirred for 30 min at room temperature. Then, the dispersions were centrifuged at 4,320g for 30 min and following filtered into tubes. The protein contents of the filtrate were taken by a Bradford method with a standard of BSA. Solubility was defined as the percentage of the protein concentration in the supernatant over that of total protein. 2.7. Amino Acid Analysis The amino acid profile was determined using an L-8900 automatic Amino Acid Analyzer (Hitachi, Japan). Samples (500 mg) were placed in an ampoule, and hydrolyzed with 6 mol/L HCl under a nitrogen atmosphere for 24 h at 110 °C. The samples were then subjected to reversed-phase highperformance liquid chromatography (Agilent 1100) analysis. The amino acid composition was reported as g/100 g protein. 2.8. Water and oil holding capacity Both water-holding capacity (WHC) and oil-holding capacity (OHC) were measured using the method described by Timilsena et al. (2016). Briefly, 1.0 g of protein was vigorously mixed with 10 g distilled water or soybean oil for 20 s with a vortex, respectively. After incubation at 25 °C for 30 min, the mixture was centrifuged at 5,000g for 30 min and the free water or oil was decanted. WHC and OHC were expressed as grams of water or oil trapped per gram of protein, respectively. 2.9. Antioxidant capacity determined by radical cation (ABTS) 7
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity assay was performed according to the method described by Re et al. (1999) with slight modifications. ABTS radical cation (ABTS+) was produced from the reaction of 7 mmol/L ABTS solution with 4.9 mmol/L K2S2O8 solution, and allowed the mixture stand in the dark at room temperature for 12 h. Then, the ABTS working solution was diluted with ethyl alcohol to an absorbance of 0.70 ± 0.02 at 734 nm. After that, an aliquot of 100 μL tested samples was added to 1.9 mL of ABTS working solution and measured at ten minute of reaction at 734 nm. Results were expressed as trolox equivalent antioxidant capacity (TEAC). 2.10. In vitro simulated gastrointestinal digestion The in vitro digestibility method used was a modification of that of Yu et al., (2017). Briefly, 0.5 g proteins were dissolved into 20 mL of 0.1 mol/L HCl containing 10 mg pepsin, followed by reacting in a 37 °C water bath for 60 min. After that, the pepsin digests was immediately neutralized with 1.0 mol/L NaOH. Then, 10 mL of phosphate buffer (20 mmol/L, pH 7.2) containing 20 mg of pancreatin was added the mixture and incubated at 37 °C for another 120 min in a water bath shaker. After that, the digestive juice was heated in boiling water for 5 min to inactivate enzyme and centrifuged at 8,000g for 20 min to precipitate the undigested protein. The nitrogen release during the digestion was calculated by Iwami et al. (1986): percentage of nitrogen release = (N0Nt)/N0×100%, where t is the digestion time (min), Nt (mg) is the TCA-insoluble nitrogen after digestion for time (min), N0 (mg) is the TCA-insoluble nitrogen in the protein sample, and Ntot (mg) is the total nitrogen of the protein sample. 2.11. Emulsion properties 2.11.1. Surface and interfacial tension. A contact angle and surface tension meter (Krüss Force 8
Tensiometer K20, Germany) was used to examine the interfacial tension of protein at the oil-water interface. Protein dispersions were prepared by mixing proteins into PBS (10 mmol/L phosphate buffer, pH 7.0) with stirring at 300 rpm for 2 h at room temperature, and left overnight at 4°C to fully hydrate. The tensiometer operated in a volume-controlled regime, via continuous measurement of the drop area and volume, determined through the Young-Laplace profile. 2.11.2. Emulsion preparation. Emulsions were prepared at three different protein concentrations (0.5, 1.0, 1.5, and 2.0 wt%) that were dispersed in 10 mL of 20 mmol/L phosphate buffer of pH 7.0 and mixed with 1.0 mL aliquots soybean oil. The mixture was homogenized in a Polytron PT 3100 homogenizer (Kinematica AG, Lucerne, Switzerland) at 6,000 rpm first for 5 min, and then secondly for another minute after a 2-second pause at 500 bar using a high-pressure homogenization (SRH 6070, Shanghai shen deer homogenizer co., LTD, China). 2.11.3. Mean particle size and microstructure of emulsions. The mean particle size and droplet size distributions of the emulsion were measured with a Mastersizer 2000 static light-scattering analyser (Malvern Instruments Ltd., Malvern, U.K.). The refractive indices used for oil droplet and water were 1.467 and 1.330, respectively. The mean droplet size was characterized as the volumeweighted mean diameter d4,3 = ∑nidi4/∑nidi3, where ni is the number of droplets corresponding to the diameter di. The creaming index (CI) was measured by macroscopic observations of emulsions assessed during 14 days of storage. Ten milliliter of each emulsion was filled into a glass test tube (1.5 cm × 12 cm), and the creaming index was reported as the following equation: creaming index (%) = (Hs/Ht)×100%, where Hs is the serum height and Ht is the total height of the emulsion. 2.12. Statistical analyses Data are reported as mean value ± standard deviation. Statistical analysis of the results (reported 9
here are the average of at least three measurements carried out on two replicated experiments) was performed with SPSS 21 (SPSS Inc., U.S.A.) and a one-way variance (ANOVA) was employed. Values were considered significant when p < 0.05.
3. Results and discussion 3.1. Proximate compositions After extraction of oil from avocado (Persea americana), a significant quantity of solid residue are generated. These by-products may be still ideal raw materials for food due to their high nutritional value, e.g., protein, polyphenol, and dietary fiber. As shown in Table 1, the defatted avocado meal from the oil processing by-products was content 15.05±0.35% protein. After protein precipitation processes, the protein contents of avocado protein (AP) powders was found to be 69.34±1.55%, which is 4.7-folds higher than raw materials. The purified soy protein (SP) had slightly higher protein (90.20%) and lower lipid (1.08%), moisture (4.94%) and ash (2.38%) contents. The low protein may be due to the fact that the non-protein components (e.g., polyphenols, dietary fiber) reserved during the fractionation step. Although, no statistically significant differences (p > 0.05) between the avocado protein and soy protein were observed in the results of moisture, lipids and ash, the slightly higher others of avocado protein compared with that of the soy protein. However, avocado protein was found to have a higher deep color. The results obtained revealed significant variation (p < 0.05) in the total polyphenolics content (TPC) among defatted avocado meal, avocado protein and soy protein (Table 1). Avocado protein variety contained more phenolic compounds at 15.85 mg CE/g more 2-folds than that of defatted avocado meal (8.33 mg CE/g), and more 30-folds than that of soy protein (0.54 mg CE/g). Consistently, Lopez-Cobo et al. (2016) has 10
been reported previously that Hass avocado has exceptionally high TPC (e.g., caffeic, chlorogenic, coumaric, ferulic, gallic and hydroxybenzoic, etc.), and its byproduct also showed higher phenolic content than other fruits and vegetables (Rodríguez-Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011). 3.2. Physicochemical properties of avocado protein 3.2.1. Characterization of avocado protein The z-average diameter (Dz) and ζ-potential of avocado protein as a function of pH are summarized using DLS technique, as shown in Fig. 1A. The z-average diameter of avocado protein at pH 7.0 was about 82.17±0.63 nm, which was evidently larger than that of soy protein (54.61 ± 0.65 nm). The reduced ζ-potential of avocado protein with increasing of pH was progressively decreased from 18.1 mV to -26.3 mV, suggesting surface group ionization (Shevkani, Singh, Kaur, & Rana, 2015). Meanwhile, a zero surface charge was showed at near pH of 4.2. At the zero surface charge, the protein was aggregated state in the dispersions with a larger size of 280 nm. Considering high amounts of polyphenols of avocado protein, the interaction between protein and polyphenols may be interact each other associating into larger sizes (Calderón-Oliver et al., 2016; López-Cobo et al., 2016). To direct observant the morphology, we evaluated the proteins morphology using AFM, as shown in Fig. 1B and Fig. 1C. Quite different morphology with particle size ranging from several to 100 nm was exhibited in the different samples. For soy protein as displayed in Fig. 1C, it is confirmed the nature of soy protein that the height and the contour was about 1-8 nm and (30-50) × (40-60) nm, respectively (Utsumi, Matsumura, & Mori, 1997). However, the morphology of avocado protein was seems less regular and much larger (Fig. 1B), which is well agreed with the DLS data 11
(inset of Fig. 1B and 1C). The particle size distribution of avocado protein was calculated to be around 82 nm at pH 7.0. 3.2.2. Free sulfhydryl (SH) content and surface hydrophobicity (H0) The free SH and H0 are shown in Fig. 2A, which indicate obviously differences in avocado protein and soy protein. As expected, the free SH value of avocado protein (1.0 × 10-6 mol/g of protein) is significantly (p < 0.05) lower than that of soy protein (1.5 × 10-6 mol/g of protein). The significantly higher SH value for avocado protein is consistent with the relative cystine and methionine contents in avocado protein and soy protein as demonstrated in Table 2. Traditionally, protein molecules with their high SH value are more likely to associate with one another and form aggregates (Yu et al., 2017), while the result was inconsistent with AFM and DLS data (Fig. 1). The difference might be largely due to the high loading of polyphenols in avocado protein (Table 1). Similar phenomenon has been observed in the literature that polyphenols (e.g., tea polyphenols) were interacting each other’s resulting in larger sizes (Rodríguez, von Staszewski, & Pilosof, 2015). As shown in Fig. 2A, the surface hydrophobicity (H0) of avocado protein (1685.3) was also significantly higher than that its value of control soy protein (1111.4) (p < 0.05), suggesting more hydrophobic groups exposure out. This difference may be mainly attributed to the distinct structural conformations from various initial courses. Eventually, the avocado protein with higher surface hydrophobicity was tending to exhibit higher surface activity, which probably suitable for specific product applications. 3.2.3. Protein solubility Protein solubility is believed to be an important factor for functional application in the food industry. The solubility profile of avocado protein as a function of pH is presented in Fig. 2B, 12
compared to soy protein. In general, the solubility curves were nearly U-shaped as the function of pH. A minimal solubility at pH 4.5 was observed as a result of the proximity to the protein isoelectric point (pI). Moreover, the solubility (4.2%) was similar to that reported for the minimal protein solubility in Phaseolus calcaratus (5.1%), Dolichos lablab (5.08%), coconut (1%) and leftover granules (2.5%) (Thaiphanit & Anprung, 2016). However, greater solubility was seen at higher pH (pH 12) and lower pH (pH 2) attributing to an increase in net protein charge. The results showed in the protein solubility of avocado protein are closely related to the data of zeta potential (Fig. 1A). This could be due to the isoelectric precipitation by electrostatic attractions. Away from the isoelectric point, the high solubility of proteins was mainly attributed to the electrostatic repulsion and ionic hydration among protein molecules (Damodaran, 2017; Utsumi, Matsumura, & Mori, 1997). Similar results were observed that there is a positive correlation between protein solubility and surface charge for various protein sources (e.g., soy, peanut and cashew nut) (Lam & Nickerson, 2013). However, a lower solubility of avocado protein was observed than that of soy protein within this pH range. The lower solubility may be probably due to the higher degree of polyphenols in avocado protein, which would form nanoparticles with proteins resulting in lower protein solubility (Table 1, Fig. 1). 3.3. Functional properties of avocado protein 3.3.1. Amino acid analysis and chemical score Compositions of amino acids of the avocado protein are presented in Table 2, compared to soy protein. It was observed that the avocado protein is found to be rich in sulfur acid, aspartic acid and glutamic amino acids, which was in contrast with the findings of Kahn (1985). The amino acid compositions showed a similar to that of soy protein, indicating that the avocado protein is 13
considered to be a good source of amino acids. Compared overview of the essential amino acids (EAA) with the WHO/FAO/UNO requirements pattern, both avocado protein and soy protein have high levels of Thr, Val, Ile, Leu, Phe+Tyr, Lys and His, which than the levels suggested by WHO/FAO/UNO recommendations for both infants and adults. In addition, for avocado protein, the ratio of essential amino acid to total amino acids (as shown in Table 2) was approximately 49%, significantly higher than that of soybean (40.80%) and safflower (38.1%). This current result was an important aspect in favor of the functionality of avocado protein. 3.3.2. In-vitro digestibility In vitro digestibility was investigated with pepsin-pancreatin, as shown in Table 3. The in vitro digestibility of the avocado protein (68.9 ± 0.9%) was slightly higher than that of maize (66.6%), rice (59.4%), sorghum (59.1%), and wheat (52.7%), and slightly lower than that of soy protein (78.1 ± 2.7%) (Joshi, Liu, & Sathe, 2015). Protein composition, enzyme type and impurities were responded to the digestibility (Dufour et al., 2018; Huang, Kwok, & Liang, 2004). However, there is no report of anti-nutritive factors in avocado, ruling out the presence of protease inhibitors that could retard the in-vitro digestibility. It is suggested that the polyphenols may affect the in vitro digestibility (Dufour et al., 2018; Ren et al., 2018). Many previous studies have reported that polyphenols in proteins showed the significantly lower trypsin inhibitor activity and resulted a lower digestibility (Huang, Kwok, & Liang, 2004). Ma and co-author (2011) also concluded that the protein digestibility was negatively correlated with polyphenol (e.g., tannin) contents. Furthermore, Ren and co-author (2018) also found that the formation of soy protein/polyphenol complexes inhibited protein digestion in vitro. However, the antioxidant activity (ABTS) of avocado protein was significantly higher (p<0.05) than soy protein, as shown in Table 3. This result corresponds to the polyphenol content reported 14
above (Table 1). Therefore, the results highlight that the avocado protein is exhibiting good candidate for functional foods. 3.3.3. Water and oil holding capacities Water-holding capacity (WHC) and oil-holding capacity (OHC) are represents the ability to immobilize water and oil, respectively, and plays an important role in improving the stability, texture and flavor of food products. Interestingly, both WHC (6.60±0.25 g/g) and OHC (5.53±0.09 g/g) of avocado protein were higher than (p < 0.05) those of soy protein (Table 3). Furthermore, the WHC and OHC values of avocado protein determined in this study were higher than that of coconut protein (Physicochemical and functional properties of protein concentrate from by-product of coconut processing) and peanut protein (Comparative studies on the functional properties of various protein concentrate preparations of peanut protein). The results may be explained by the difference in the raw material diversity and the protein solubility as confirmed by the report of Kaur et al., (2005) and Kinsella (1976). They showed that several water-loving components (e.g., dietary fiber) generally have high WHC, while non-polar components (e.g., polyphenols) are contributed to a higher OHC. 3.4. Emulsion properties of avocado protein 3.4.1. Surface and interfacial behavior In this study, emulsion properties were further studied in order to enhance the utilization of avocado protein in food industry. Interfacial tension plays an important role in emulsification because of an emulsifier reduces the interfacial tension resulting in the formation of an emulsion (Thaiphanit & Anprung, 2016). With respect to the ability of avocado protein to act as an oil-inwater emulsifier in relation to protein concentration, the results are summarized in Fig. 3A. The 15
surface tension (π) showed consistent protein concentration reduction, which reflects that increase in protein has a beneficial effect on reducing the surface tension at the oil-water interface. At the same content, soy protein has shown a better effectiveness in the decreasing of π than that of avocado protein. The observation is consistent with the general viewpoint in the colloid and interface fields that the diffusion and adsorption of particles with larger sizes is slower than that of smaller sizes (Dickinson, 2010). For avocado protein, the aggregated insolubilizers were unable to unfold at interfaces, and may not be beneficial to reduce surface tension. However, it may be stable the interface by a Pickering mechanism. In the series of studies of Tang (2017) and Binks (2018), they were reported that many native globular proteins (e.g., soy protein, pea protein, whey protein and peanut protein) can be as outstanding Pickering nanostabilizer to stabilize emulsions. 3.4.2. Emulsification performance Emulsification performance of the avocado protein was then evaluated at varying protein concentrations of 0.5-2.0% (w/v). And, the droplet diameters (d4,3) and size profiles of these emulsions were measured as shown in Fig. 3B and 3C, respectively. It was shown that the oil droplet sizes stabilized by avocado protein were a little higher than those of soy protein at the same protein concentration, and progressively increased with decreasing the protein concentration (Fig. 3B). From the typical droplet size distribution profiles (Fig. 3C), all the emulsions exhibited a profile of bimodal or trimodal droplet size distributions. It also can be seen that a major size distribution peak (at 1-10 μm) observed at higher protein concentration, confirming more proteins were favor to form and stabilize the interface (Lam & Nickerson 2013). Moreover, similar result in average oil droplet size decreases steadily with increasing protein concentration was also observed in the light microscope images of all these emulsions (Fig. S). 16
Creaming behavior of the emulsions stabilized by avocado protein at different concentrations was also evaluated over a storage for up to 2 weeks, as shown in Fig. 3D. As expected, the creaming index (CI) of all the emulsions progressively increased over time. As we all known that emulsions with higher degree of flocculation tended to have higher degree of CI. In the case, the flocculation was prevented with increasing protein concentration, as shown in Fig. 3D that the creaming behavior was steadily decreasing with increasing to the avocado protein concentrations. This is in accordance with the droplet size (Fig. 3C). However, we can interestingly see that the avocado protein emulsions at the relevant concentrations showed much larger droplet size but exhibited much higher creaming stability compared with soy protein (Fig. 3D and Fig. S). The emulsions stabilized by avocado protein have significantly stability upon 10 days storage. Particularly, there was no distinct creaming for the emulsion at 2.0% avocado protein after storage for 14 days (see inset of Fig. 3D). Considering the protein size, avocado protein may be play a Pickering particle-like role in the stabilization of emulsions from the fact that the dimension of monomeric avocado protein as shown in Fig. 1C. In that case, the avocado protein aggregates would firmly adsorb at the oil-water interface form a more packed protein layer. On the other hand, we observed that a high surface hydrophobicity of avocado protein possibly attributed to their native polyphenols in avocado fruit. It is generally known that polyphenols could improve the emulsion system by the intermolecular between protein and polyphenol (Dickinson, 2010; Wang et al., 2016). 4. Conclusions This study highlighted that the avocado (Persea americana Mill.) oil processing by-products could be good source to produce edible protein with functional and emulsion properties. The minimum solubility was occurred at the pI of pH 4.5. Nutritionally, avocado protein contains all the 17
essential amino acids and similar to that of soy protein. Both WHC and OHC of avocado protein were observed at 6.60 and 5.53 g/g, respectively, which were much higher than those of soy protein (5.53 and 3.91 g/g, respectively). Avocado protein was also provided a high radical scavenging capacity compared with soy protein. After pepsin-trypsin digestion, the percentage of nitrogen release of avocado protein reached up to 68.9%. The presence of avocado protein as an emulsifier enhanced the stability of oil-in-water emulsion system. Furthermore, avocado protein showed a greater emulsifying stability than soy protein, even with a lower interfacial tension and emulsifying activity. This work demonstrates that the avocado oil processing by-products might provide a potential protein source as functional ingredients in food systems.
Conflict of interest The authors declare no conflicts of interest. Acknowledgements This work is supported by the Key Research and Development Program of Hainan Province (ZDYF2018058), the Agricultural Wild Plant Resource Protection Fund in Ministry of Agriculture of China (1251416305010), and the Cross-Strait Agricultural Cooperation Program of China.
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Tables Table 1. Proximate analyses of defatted avocado flour, avocado protein and soy protein.a TPC Protein
Moisture
Lipids
Samples
Others Ash (%)
(%)
(%)
(mg CE/g
(%)
(%) DW)
Defatted avocado
15.05±0.35
7.70±0.40a
3.48±0.15a 23
7.25±0.35a
8.33 ± 0.14b
66.52
flour
c
69.34±1.55
15.85 ± 4.02±0.33b
Avocado protein
2.17±0.22b
3.20±0.10b
b
21.27 0.24a
90.20±0.70 4.94±0.40b
Soy protein
1.08±0.14b
2.38±0.20b
0.54±0.14c
1.40
a
a
All data were based on the wet basis. Each value was the mean and standard deviation of triplicate measurements. The letters (a−c)
indicate significant (p < 0.05) difference within the same column. TPC, total polyphenolics content.
24
Table 2. Comparison of amino acid content of avocado protein and soy protein, and WHO/FAO recommendations for children and adult. Amino acids (mg/g protein)b Amino acids Avocado protein
Soy protein
Asp Thr a Ser Glu Gly Ala Val a Met+Cys a Ile a Leu a Phe+Tyr a Lys a His a Arg Pro
78.93±2.18 32.46±1.53 44.01±1.54 101.87±2.59 40.98±1.24 43.43±1.34 51.37±0.85 13.84±0.81 42.56±2.54 70.27±0.17 79.36±2.04 59.88±1.56 19.48±0.52 44.15±1.84 36.79±1.54
109.19±3.28 28.81±1.66 53.92±3.77 197.53±1.36 36.32±2.87 32.62±3.32 47.30±0.92 20.84±1.35 44.73±0.94 72.19±2.31 76.90±2.68 64.23±1.67 23.87±0.89 74.32±1.35 45.85±2.02
EAA/TAAd (%)
48.62±1.24
40.80±1.02
aEssential
FAO/WHO suggested Requirementsc 1-2 years old
3-10 years old
Adult
27
25
23
42 26 31 63 46 52 18
40 24 31 61 41 48 16
39 22 30 59 38 45 15
amino acids. bAmino acid content/total amino acid content. cAmino acid requirements/protein requirements for the selected age groups. d EAA/TAA = Essential Amino Acid/Total Amino Acid. Values represent the mean of duplicate measurements.
25
Table 3. In vitro digestibility, radical scavenging capacity, water absorption capacity and oil absorption capacity of avocado protein compered to soy protein. a
a
Samples
Digestibility (%)
ABTS
WAC (g/g)b
OAC (g/g) c
Avocado protein
68.9 ± 0.9 b
8486 ± 111 a
6.60±0.25 a
5.53±0.09 a
Soy protein
78.1 ± 2.7 a
1412 ±25 b
4.69±0.38 b
3.91±0.24 b
Values are the mean ± SD of three determinations. Different letters for the same protein types indicate significant differences (p <
0.05) in means. bWAC, water absorption capacity. cOAC, oil absorption capacity.
Figure Captions Fig. 1. (A) The z-average diameter (Dz) and ζ-potential of avocado protein and soy protein as a function of pH; Typical size distribution profiles and 3D AFM height images of (B) avocado protein and soy protein (C) dispersions in pH 7.0.
Fig. 2. (A) Free SH contents of avocado protein (AP) and soy protein (SP) at pH 7.0. (B) Nitrogen solubility of avocado protein and soy protein at different pH values. Between avocado varieties: *, p < 0.05.
Fig. 3. (A) Surface tension (π) as a function of time for different avocado protein (AP) concentrations (0.01−0.5%) and soy protein (SP) at the oil-water interface; (B) Mean emulsion droplet diameter and (C) particle size distributions of fresh emulsions stabilized by avocado protein and soy protein; (D) Creaming index of emulsions at four protein concentrations of 0.5, 1.0, 1.5 and 2.0% during quiescent storage of 14 days. The inlet figure shows the image of the 2.0% avocado protein emulsions after the 14d storage, wherein the arrows indicate bottom border of the cream 26
layer.
27
Figure 1.
28
Figure 2.
29
Figure 3.
30
Graphical abstract
Highlights Edible protein was prepared from avocado oil processing by-products. Physicochemical and functional properties of avocado protein were investigated. Oil-in-water emulsions stabilized by avocado protein. Avocado oil processing by-products is a sustainable protein resource.
31