Characterization of the chemical and structural properties of native and acetylated starches from avocado (Persea americana Mill.) seeds

ABSTRACT

Avocado seeds are discarded as by-products during pulp consumption or processing despite their high starch content. This study aimed to characterize the chemical, morphological, and functional properties of the native and acetylated starches extracted from avocado seeds. The granules of native avocado seed starch exhibited an oval shape and A-type crystallinity pattern, while the acetylated form obtained with 0.07 degree of substitution (DS) yielded granules with a rounded bell shape and C-type pattern. The solubility and swelling power (SP) of the native and acetylated forms of avocado seed starch increased with increasing temperature; however, at 55°C and 65°C the acetylated form exhibited a higher solubility index (SI). The acetylated avocado seed starch showed reduced breakdown and synergy during freezing, as well as greater oil absorption, compared to the native form. These results showed that acetylation improved the functional properties of avocado seed starch, thereby increasing its potential for use in food products.

KEYWORDS:

• Starch
• Viscosity
• Gelatinization
• DSC analysis
• Functional properties

Introduction

Avocado (Persea americana Mill.) is widely distributed in tropical countries such as Mexico, Guatemala, and Brazil. Avocado pulp is usually eaten fresh, and its consumption has been linked to health benefits such as combating hypertension and diabetes.^[1] The avocado fruit, also known as the alligator pear or butter pear, is characterized by the presence of only one seed, which is encased in a hard shell and corresponds to 16% of the fruit weight.^[2] Some studies have shown that avocado seeds can be used as raw material for oil and pigment production;^[3] however, they are usually discarded in the environment as by-products.

The starch content of an avocado seed can reach up to 74.47% of its dry weight, depending on the cultivar.^[4] In Brazil, the “Manteiga” avocado cultivar is widely distributed and readily identified by its yellow butter and consistent pulp.^[5] Starch is a natural biopolymer that is composed of amylose (up to 30%) and amylopectin (up to 80%), associated by hydrogen bonds.^[6] The arrangements of these macromolecules in starch granules (i.e., the molecular structures) have been thoroughly characterized and are directly related to the functional properties of the starches extracted from different sources.^[7,8,9]

Starch is one of the most important polymers in the food industry because it can provide desirable texture and consistency in food formulations. The market for starch has been growing during the last few years, and there is a high demand for new starches due to the enormous pressure on the few commonly known sources^[7,9,10]. Additionally, the application of starch could be limited by some properties of the native form, such as low solubility in cold water, limited emulsification capability, retrogradation tendency, and low stability under refrigeration^[11]. Consequently, chemical modifications have been applied to enhance the technological functionality of starches that are extracted from distinct botanical sources and to expand the use of these starches in food^[12].

Starch modifications can be made by chemical, enzymatic, and physical methods and can promote specific functional properties^[12,13,14]. The acetylation reaction occurs by the esterification of starch hydroxyl groups, replacing these hydroxyl moieties with acetyl groups. This structural modification reduces the interactions between the outer chains of amylopectin and the amylose chains^[12]. Thus, acetylated starches possess higher stability, greater resistance to retrogradation, and the ability to form a paste with a lower gelatinization temperature^[13,15]. However, the functional properties of acetylated starch depend on the starch source, the degree of substitution (DS), and the distribution of the acetyl groups, which may be related to the events associated with the gelatinization degree and the swelling power (SP) of the starch granule^[16].

Starches modified by acetylation are used mainly as thickeners, stabilizers, and gelling agents in sauces, mayonnaise, cakes, instantaneous puddings, fillings, and toppings^[17,18]. Generally, the acetylated forms are obtained from the native starches extracted from legumes, fruit seeds, cereals, roots, and tubers^{[14,16,19,20]}. Despite knowledge about the abundance of starch in avocado seeds, studies focusing on the characterization of native or modified avocado seed starch are still scarce. Thus, the aim of the present study was to characterize the native and acetylated forms of starch extracted from avocado seeds (cultivar Manteiga), focusing on the physicochemical, morphological, and functional properties to determine the potential applicability of this material in the food industry.

Materials and methods

Extraction of starch from avocado seeds

Avocado seeds (Persea americana Mill., cultivar Manteiga) were obtained from fruits of the same maturation stage that were purchased from the farmer’s market in the city of Juazeiro, Bahia, Brazil. Starch was extracted from the avocado seeds as previously described^[19]. The seeds were peeled, cut into eight pieces, and immersed in a sodium metabisulphite solution (0.2%) for 24 h. The raw material mixed with sodium metabisulphite was ground in a regular blender at low speed for 30 min. After homogenization, the mixture was filtered through a 200-mesh sieve (0.074 mm). The material obtained was decanted for 24 h and centrifuged at 5000 rpm for 15 min. The supernatant was discarded, and the precipitate was resuspended in sodium metabisulphite for a second decantation procedure. The final precipitate (white starch) was lyophilized at −45°C, packaged in a polyurethane bag, and maintained under cold storage ^[20].

Acetylation and determination of the substitution degree

Starch acetylation was performed according to procedures previously described^[21], with modifications. Briefly, 100 g of starch extracted from avocado seeds and dispersed in 500 mL of distilled water was added to 10.2 g of acetic anhydride and stirred for 1 h (pH 8.0–8.5, adjusted with 1 M NaOH). Then, the pH was adjusted to 4.5 (with 0.5 M HCl), and the dispersion was filtered, washed (4x) with distilled water, and dried under air circulation at 30 ± 2°C for 48 h. To determine the acetyl group content (expressed as a percentage of the dry weight) and the substitution degree, 5 g of acetylated starch diluted in 50 mL of distilled water was titrated with 0.1 M sodium hydroxide solution using phenolphthalein as an indicator. Then, 25 mL of 0.45 М hydroxide was added to the suspension. The samples were stirred vigorously for 30 min, saponified, and titrated with standard 0.2 M HCl solution^[22]. Native starch was titrated under the same conditions (to establish a baseline value). The acetyl group content (A%) and substitution degree (DS) were calculated according to Eqs. (1)–(2).

Equation (1)

where: V_a = titration volume of sample (mL); V_b = titration volume of blank (mL); m = acid molarity; PA = sample weight (dry weight).

Equation (2)

where: DS = degree of substitution; 162 = molecular weight of a glucose unit; A% = acetyl group content.

Morphological characteristics of avocado seed starch granules

Shape and size

The shapes of the starches granules were verified using a digital scanning electron microscope (LEO1430; Cambridge, England). Starch dispersions (2 g/100 mL) were placed on double-sided tape and coated with gold (via sputtering). The mean particle size was determined using a Zeiss microscope (model Axiovert25; Oxford, England). A total of 10 granules were photographed and measured in 30 fields randomly selected (total of 300 granules).

X-ray diffraction

The diffractogram was performed with powder starch containing 10% moisture. The interval of 2θ angles ranged from 4° to 60° in the X-Ray Diffractometer (Model D5000, São Paulo, Brazil), at a rate of 1.2°/min and operating at a power of 40 KV/20 mA. The diffractogram patterns obtained were analysed as previously described by Zobel^[23].

Functional properties of native and acetylated starch from avocado seed

The solubility and SP were measured according to the method described elsewhere^[24]. An aliquot of 0.1 g of starch was added to 10 mL of distilled water and the obtained suspension was stirred and maintained in a water bath at temperatures ranging from 55°C to 95°C. The temperature was increased by 10 degrees each 10 min and the samples were centrifuged during 15 min at 3400 g. The supernatant was collected in petri dishes and dried at 105°C for 24 h. The SP and solubility index (SI) were determined as follows (Eqs. 3–4):

Equation (3)

where: SP = swelling power (g.g⁻¹); WP = weight of the petri dish (g); RAC= residue after centrifugation (g); DWP + S = dry weight of petri dish plus sample(g); WS = weight of sample (g).

Equation (4)

where: S = solubility index (%); WPE = weight of the petri dish with sample after evaporation; WP = weight of the petri dish.

Viscosity

Viscosity was determined using a Viscometer RVA-4, equipped with the Software Thermocline for Windows 2.3 (Newport Scientific Pty. Ltd). The analyses were performed in accordance with the Number 162 methodology of the ICC. Briefly, 3.5 g of starch on dry weight basis was diluted in 25 mL of water and the temperature of the system was 50°C, and it remained constant for 1 min. Initially, the temperature of the system was 50°C, and it remained constant for 1 min. Then the sample was heated for 7.5 min from 50°C to 92°C, and then held at a constant temperature of 92°C for 5 min. The samples were cooled down to 50°C in 7.5 min, and finally this temperature was kept constant for 2 min. The total time for the test was 23 min. The operation conditions were: vessel diameter 0.037 m and height 0.068 m; impeller diameter 0.034 m and height 0.013 m, the frequency of the system was 160 rpm. The measured parameters were expressed in Rapid Visco Units (RVU) or centipoise (cP)^[25].

Paste transparency

The transparency of the paste obtained with avocado seed starches was determined as described by Craig et al.^[26]. The paste transparency was determined using a starch suspension (3% w/v) in deionized water. Transmittance (% T) was measured at 650 nm using a Spectrophotometer Coleman 33D. The samples were maintained at 4°C for 17 days, and the transmittance was read each 24 h to observe retrogradation.

Gelatinization

The parameters of the starch gelatinization were set by Differential Scanning Calorimetry (DSC). The analyses were performed using a calorimeter Shimadzu DSC 60 at a flow rate of 50 mL/min in a nitrogen atmosphere. Samples were prepared using 2 mg of starch and 6 μL of sterile distilled water. Aluminium crucibles were sealed and maintained at room temperature for 24 h before analysis. The range of the scanning temperature was 30°C–150°C at a heating rate of 10°C/min^[22].

Oil and water absorption capacity

The method of Beuchat^[27] was employed to determine the oil and water absorption capacities of the starch. Briefly, 10 mL of distilled water or oil (Bunge, São Paulo, Brazil) was added to 1 g of sample. The sample was mixed for 30 s and allowed to stand for 30 min. Then, the supernatant volume was recorded. The mass of oil or water absorbed was expressed as g/g starch on a dry weight basis.

Statistical analysis

All experiments were performed in triplicate on three different occasions. The results were expressed as the means ± standard errors of the mean, and the statistical analysis was performed using analysis of variance (ANOVA) and the Tukey test, with a statistical significance cut-off value of p < 0.05. The statistical analysis was carried out using the Evaluation Edition for Windows Statistical Software – 14.0 (SPSS Inc., Chicago, IL, USA).

Results and discussion

Acetylation and determination of the substitution degree

Average values of 0.22% acetyl group content and 0.07 ± 0.01 DS were observed for the modified avocado seed starch. A previous study reported similar or higher DS values (0.05 and 0.11) and higher acetyl group content values (1.54% and 2.92%) in modified oat starch, depending on the concentration of acetic anhydride used during the acetylation procedure^[28]. The DS obtained for starches modified by acetylation is influenced by the botanical source and granule size. The reaction conditions, such as the rate of acetic anhydride addition, the homogeneity in stirring, and the reaction time, can also affect the acetylation results^[12,28]. A low DS in starch may be attributed to a lack of either granular surface pores or large inner channels, both of which facilitate the physical access of acetic anhydride to the interior of the granule. It was reported that the acetyl content of acetylated starch with large granules was slightly higher than that of small granules^[29]. Overall, it has been reported that acetylated starches extracted from oat and corn with a DS value of 0.05 exhibit enhanced solubility and swelling compared with native starches^[16,28].

Morphological characterization of avocado seed starch granules

The scanning electron microscopy analysis revealed an oval shape for the native avocado starch granules (Fig. 1A) and a round bell shape for the acetylated form (Fig. 1B). The native granules exhibited a smooth surface similar to that previously described for tamarind (Tamarindus indica L.) Kernel starch^[10], whereas the granules of acetylated starch exhibited surfaces with some grooves and sharp deformations. Similarly, a previous study reported changes after chemical modification for native starch granules extracted from different sources^[30,31].

Figure 1. SEM micrographs of native avocado seed starch (A) and acetylated avocado seed starch (B) at 1000 × resolution.

The shape differences observed between the granules of acetylated and native avocado seed starch probably occurred because the molecular structure of the native starch changed upon substitution of the hydroxyl groups with acetyl groups during the acetylation process^[13]. The average sizes of the starch granules analysed by the optical microscope were 26–37 µm for native and 26–36 µm for acetylated starch granules, with no differences in granule size (p > 0.05). The maintenance of the same granular size after acetylation was previously reported^[12,16,22]. The shape (round, oval, or polyhedral) and particle size (2–100 µm) of starch granules have been shown to vary with the biological origin^[16,32,33]. Studies characterizing starch granules extracted from the seeds of distinct botanical sources have reported huge variations in granule size and shape. Madruga et al.^[19] reported round or bell shapes and sizes ranging from 6 to 13 µm for native starch granules of the soft jackfruit seed (Artocarpus heterophyllus L), while Li et al.^[33] observed granules of round, triangle, and elliptical forms with sizes ranging from 3.3 to 126.2 µm for the native starch granules of acorn seeds (Quercus glandulifera BL). Additionally, it is known that the functional properties of native and modified starches are related to the molecular structures and arrangements of amylose and amylopectin in the granules rather than to granule shape and size^[34].

The X-ray diffractogram for native starch, shown in Fig. 2, indicates an A-type crystallinity pattern with three main peaks around the diffraction angles of 14.8°, 17.2°, and 23.1° at 2θ. These results are in accordance with those previously described for starch obtained from acorn seeds^[33], soft jackfruit seeds^[19], and avocado seeds^[35].

Figure 2. X-ray diffraction patterns of avocado seed starches: native and modified by acetylation.

The acetylated avocado seed starch exhibited a profile similar to that of the native form but with a new peak at approximately 5.6°, suggesting a C-type pattern. This C-type pattern was also indicated by the strong signals around the diffraction angles of 5.6°, 15.0°, 17.0°, and 23.5° at 2θ^[36]. The modification of the crystalline pattern by acetylation was also reported for corn starch granules and is a result of the new structure of the acetylated starch^[37]. Considering the retrogradation tendency and the limited solubility of A-type crystals, typically described for cereal starches, this chemical modification might be beneficial in improving these characteristics ^[17,37].

Functional properties of native and acetylated starches from avocado seed

The SP and SI as a function of temperature are presented in Figs. 3A and 3B, respectively, for the native and acetylated avocado seed starches. The native starch showed a higher SP than its acetylated form (p < 0.05) until reaching temperatures near 75°C; above this temperature, no differences were observed between the native and acetylated starches, and both exhibited an increased SP. Similar results were previously reported for native and acetylated oat starches^[28]. The SP is influenced by the intermolecular bond strength^[29]. It is likely that the acetylation-induced changes in the molecular structure of avocado seed starch affected the SP at temperatures below the temperature of gelatinization, limiting the water absorption by restricting the mobility of starch chains in the amorphous region^[19,38].

Figure 3. Swelling power (g.g⁻¹) (A) and solubility index (%) (B) of native and modified starches from avocado seed cv. “Manteiga”.

The SI values of both the native and acetylated avocado seed starches were directly related to temperature; however, the acetylated form exhibited a higher SI at temperatures of 55°C and 65°C compared with the native starch (p < 0.05). No differences were observed between the SI values of the native and acetylated avocado seed starches at the higher temperatures tested (p > 0.05). Similarly, Choi et al.^[39] observed an increased SI after the acetylation of native corn starch. The increase in SI at mild temperatures is a desirable property for modified starches because this increases the possibilities for applications in foods, particularly as thickener, stabilizer, or gelling agents^[17,18].

The viscosity curve represents the behaviour of the starch during heating and allows for the evaluation of both the characteristics of the paste formed and the tendency for retrogradation to occur during cooling^[40]. Obtained using a Rapid Visco Analyser (RVA), the viscoamylograph curves for the native and acetylated avocado seed starches showed that increasing temperatures led to starch gelatinization, which increased the viscosity due to the swelling of the starch granules. The pasting temperature (i.e., the initial gelatinization temperature at which the viscosity curve begins) was higher for the native avocado seed starch (80.22°C) than for its acetylated form (79.25°C). However, acetylation increased the peak viscosity of the native starch (Table 1); the maximum viscosity achieved for the acetylated starch was higher (321.92 cP) than that for the native starch (284.34 cP).

Table 1. Main viscosity parameters of native and acetylated avocado seed starches, as calculated from the curves measured by the Rapid Viscosity Analyzer.

Parameters	Native	Acetylated
Paste temperature (°C)	80.22	79.25
Peak viscosity (cP)	284.34	321.92
Minimum viscosity (cP)	163.34	205.92
Breakdown (RVU)	121.00	116.00
Final viscosity (cP)	467.42	509.50
Retrogradation trend (RVU)	304.08	303.58
Peak time (min)	5.00	4.67

During the period of constant temperature (95°C) while stirring, the granules begin to dissociate, and the solubilization of amylose molecules causes a decrease in viscosity. The difference between the maximum and minimum viscosities is called “breakdown,” which represents the resistance of starch to mechanical agitation. During this resistance period, it is possible to evaluate the starch stability at high temperatures by examining which granules are broken under mechanical stirring^[41]. Acetylated avocado seed starch showed a lower breakdown value (116 cP) than native starch (121 cP). Thus, the acetylated avocado seed starch can be considered more stable (i.e., resistant to heating) and exhibits reduced breakdown when compared to the native form. The final viscosities of the starches under study were 467.42 cP (native form) and 590.50 cP (acetylated form). The setback (tendency for retrogradation) for the acetylated avocado seed starch was significantly lower (303.58 cP) compared to that of the native avocado seed starch (304.08 cP). These results are in accordance with the previously published avocado seed starch viscoamylograph^[36], as well as with reports that acetylation increases the viscosity and stability of breakdown^[28,42]. Additionally, acetylation decreased the tendency for retrogradation, one of the most common problems that occur during the storage of food products made with native starch^[42].

The transparency of the native starch paste (2.20 ± 0.06%) did not differ from that of the acetylated avocado seed starch (2.23 ± 0.02%). Both starch forms yielded an opaque paste, suggesting that these avocado seed starches may be interesting to use in formulations that do not require transparency, such as soups, sauces, and creams^[19].

Subsequently, the effects of a seven-day refrigerated storage period on gel transparency were assessed. The transmittance values on the first day of storage (after 24 h) were 0.40 ± 0.01% and 0.70 ± 0.02% for the native and modified starches, respectively. After 7 days, the values of 0.20 ± 0.00% and 0.30 ± 0.00% transmittance were obtained for the native and modified starches, respectively. It is well known that the reduction in transparency of starch pastes stored under refrigeration is mainly related to starch retrogradation^[19]. This could explain the lower reduction in paste clarity observed for the native starch paste, reinforcing the viscoamylograph results and clearly showing the higher retrogradation tendency for the native avocado seed starch.

Water and oil absorption capacity

Modification by acetylation decreased the tendency of native starch to absorb water (from 79.88 g H₂O.100 g⁻¹ starch for the native form to 68.08 g H₂O.100 g⁻¹ starch for the acetylated form); this may have occurred because the acetyl group limits the penetration of water molecules into the granule^[42]. The hydrophobicity of native starch (82.71 g oil.100 starch g⁻¹) was enhanced by acetylation (105.38 g oil.100 g⁻¹ starch). These are interesting results because they indicate a higher efficacy for the modified avocado seed starch as a thickener in comparison to the native form.

Gelatinization

The thermal properties of the native and modified starch samples, which represent the amount of thermal energy involved in the gelatinization process, are presented in Table 2. The acetylation did not change the initial, peak, and final temperatures; however, acetylation did increase the enthalpy involved in gelatinization (p < 0.05). Lacerda et al.^[35] reported different values for the temperatures and enthalpies involved in the gelatinization of avocado seed starch but for an avocado cultivar distinct from that studied here as well as different extraction methods. The increase in gelatinization enthalpy could be attributed to the distinct crystallinity pattern acquired by the avocado seed starch after acetylation.

Table 2. Temperature and enthalpy associated with the gelatinization of native and acetylated starches from avocado seed cv. “Manteiga”.

	Temperatures (°C)
Starch	Ti	Tp	Tf	Tf-Ti	∆H (J.g^−1)
Native	43.17	88.57	94.65	51.48	666.34
Acetylated	41.46	88.69	94.68	53.22	797.47

Ti = initial temperature; Tp = Peak temperature; Tf = final temperature; ∆H = gelatinization enthalpy.

Stability to freezing and thawing

The results for the release of water from starch pastes when submitted to freezing and thawing cycles are expressed in terms of the percentage of released water in relation to the initial paste mass (Table 3). The percentage of water released by the native starch in the first cycle was lower than the value found for the starch modified by acetylation (p < 0.05). In the second cycle, the percentage of water released by the acetylated starch was lower than that found in the first cycle, but the native starch continued to release water. In the third and fourth cycles, there was a reduction in the percentage of water released by both the native and modified starches. The percentage of released water (syneresis) is a very important parameter for evaluating the potential applicability of acetylated starches^[16]. Because acetylation improves the water retention, food products formulated with acetylated starches show lower syneresis during the storage period and consequently greater shelf life^[12].

Table 3. Percentage of water released by native and modified avocado seed starch pastes in four freezing and thawing cycles.

Starch	1st cycle (%)	2nd cycle (%)	3rd cycle (%)	4th cycle (%)
Native	7.05^b ± 0.87	12.89^a ± 1.34	2.06^a ± 0.22	1.12^a ± 0.85
Acetylated	51.03^a ± 1.77	1.86^b ± 0.31	1.48^a ± 0.18	1.44^a ± 0.25

Means followed by different letters in the same column indicate that there was a statistical difference at 5% significance by the Tukey test.

Conclusion

Native avocado (cv. “Manteiga”) seed starch showed functional properties that are interesting for applications in food systems. Furthermore, modification by acetylation improved the technological functionality of this material. The acetylated avocado seed starch exhibited an increased SI, reduced breakdown, and decreased syneresis during freezing, suggesting great potential for applications in instant puddings, desserts, and frozen products. In addition, the acetylated form of avocado seed starch showed an enhanced oil absorption capability.

Acknowledgements

The authors thank CAPES-Brazil for the scholarship from the authors Silva, IR and Batista, KS.