Changes in the expression of genes encoding xanthophyl acyltransferases during the postharvest ripening of avocado (Persea americana) fruit.

BACKGROUND: Avocado fruit is rich in xanthophylls, which have been related to positive effects on human health. Xanthophyl acetyltransferases (XATs) are enzymes catalyzing the esterification of carboxylic acids to the hydroxyl group of the xanthophyll molecule. This esterification is thought to increase the lipophilic nature of the xanthophyll and its stability in a lipophilic environment. Studies on XATs in fruits are very scarce, and no studies had been carried out in avocado fruit during postharvest. The objective of this work was to investigate the changes in the expression of genes encoding XAT, during avocado fruit ripening. RESULTS: Avocado fruits were obtained from a local market and stored at 15 °C for 8 days. The fruit respiration rate, ethylene production, and fruit peel's color space parameters (L*, a*, b*) were measured during storage. Fruit mesocarp samples were taken after 1, 3, 5, and 7 days of storage and frozen with liquid nitrogen. Total RNA was extracted from fruit mesocarp, and the quantification of the two genes designated as COGE_ID: 936743791 and COGE_ID: 936800185 encoding XATs was performed with real-time quantitative reverse transcription polymerase chain reaction using actin as a reference gene. The presence of a climacteric peak and large changes in color were recorded during postharvest. The two genes studied showed a large expression after 3 days of fruit storage. CONCLUSIONS: We conclude that during the last stages of ripening in avocado fruit there was an active esterification of xanthophylls with carboxylic acids, which suggests the presence of esterified xanthophylls in the fruit mesocarp. © 2024 Society of Chemical Industry.

In vitro and in vivo inhibition of Hass avocado polyphenol oxidase enzymatic browning by paeonol, ß-cyclodextrin, and paeonol:ß-cyclodextrin inclusion complex.

Polyphenol oxidase (PPO) was extracted from Hass avocados and its physicochemical properties were analyzed. The optimum pH and temperature of the enzyme were pH 7.5 and 20°C. This PPO showed a high thermal stability, since 26% of the initial activity was retained by the enzyme after heating at 60°C for 40 min. Inhibition studies were performed using different chemical reagents, and the order in the inhibition efficiency was paeonol > 4-hydroxybenzaldehyde > ß-cyclodextrin (ß-CD). The first two inhibitors presented a non-competitive mechanism while the inhibition by ß-CD results from a mixed type mechanism. Since the aqueous solubility of paeonol (a natural compound) is very low, the inclusion complex between this drug and ß-CD was obtained in solution and solid state. The stoichiometry of the paeonol:ß-CD complex was 1:1 and its ΔG° of formation was -26 kJ/mol. The complexation of paeonol by ß-CD not only enhances the aqueous solubility and thermal stability of the drug, but also improves the in vitro inhibition efficiency against PPO. Colorimetric analysis on avocados pulp (in vivo) showed that the inclusion complex does not increase the inhibitory effect of paeonol, remaining practically unchanged. However, the formulation of paeonol:ß-CD inclusion complex allows employing this compound as PPO inhibitor in aqueous solutions.

Control of anthracnose disease via increased activity of defence related enzymes in 'Hass' avocado fruit treated with methyl jasmonate and methyl salicylate.

Development of anthracnose disease caused by Colletotrichum gloeosporioides Penz. is one of the major issues within the avocado supply chain. Exposure to methyl jasmonate (MeJA) and methyl salicylate (MeSA) vapours at 10 and 100µmoll-1 was investigated as an alternative solution to commercial fungicide - prochloraz® that is currently being used by the industry. The incidence of anthracnose disease was found to be significantly reduced in 'Hass' avocado fruit treated with MeJA or MeSA vapours, especially at 100µmoll-1. The mechanism involved enhanced activity of defence related enzymes, i.e. chitinase, ß-1,3-glucanase and PAL, and higher content of epicatechin.

Enzymatic browning in avocado (Persea americana) revisited: History, advances, and future perspectives.

Considering nearly 80 years of research regarding one of the enzymes responsible for catalyzing the formation of pigments in higher animals, plants, fungi and bacteria, this review will focus on collecting and categorizing the existing information about polyphenol oxidase (PPO) in fruits, with particular emphasis on the information in relation to avocado, which is one of the hardiest species in terms of inactivation, has documented dual activity (EC 1.14.18.1/EC 1.10.3.1), and represents one of the oldest challenges for food science research and fruit processors. It is expected that this review will contribute to the further development of the field by highlighting the questions that have arisen during the characterization of PPO, the progress that has been made and the questions that remain today, in addition to new methodologies that are being applied to study this system. Holistic methodologies offer unexplored potential for advancing our understanding of the complex phenomena that govern PPO activity in fruits, because these methodologies will enable the characterization of this family of enzymes in all of its complexity. Subsequently, it will be possible to develop better techniques for controlling enzymatic browning in this valuable fruit.

The effect of ultrasound on particle size, color, viscosity and polyphenol oxidase activity of diluted avocado puree.

The effect of ultrasound treatment on particle size, color, viscosity, polyphenol oxidase (PPO) activity and microstructure in diluted avocado puree was investigated. The treatments were carried out at 20 kHz (375 W/cm(2)) for 0-10 min. The surface mean diameter (D[3,2]) was reduced to 13.44 µm from an original value of 52.31 µm by ultrasound after 1 min. A higher L(∗) value, ΔE value and lower a(∗) value was observed in ultrasound treated samples. The avocado puree dilution followed pseudoplastic flow behavior, and the viscosity of diluted avocado puree (at 100 s(-1)) after ultrasound treatment for 1 min was 6.0 and 74.4 times higher than the control samples for dilution levels of 1:2 and 1:9, respectively. PPO activity greatly increased under all treatment conditions. A maximum increase of 25.1%, 36.9% and 187.8% in PPO activity was found in samples with dilution ratios of 1:2, 1:5 and 1:9, respectively. The increase in viscosity and measured PPO activity might be related to the decrease in particle size. The microscopy images further confirmed that ultrasound treatment induced disruption of avocado puree structure.

Self-assembled films containing crude extract of avocado as a source of tyrosinase for monophenol detection.

This paper reports on the use of the crude extract of avocado (CEA) fruit (Persea americana) as a source of tyrosinase enzyme. CEA was immobilized via layer by layer (LbL) technique onto indium tin oxide (ITO) substrates and applied in the detection of monophenol using a potentiometric biosensor. Poly(propylene imine) dendrimer of generation 3 (PPI-G3) was used as a counter ion in the layer by layer process due to its highly porous structure and functional groups suitable for enzyme linkage. After the immobilization of the crude CEA as multilayered films, standard samples of monophenol were detected in the 0.25-4.00 mM linear range with approximately 28 mV mM(-1) of sensitivity. This sensitivity is 14 times higher than the values found in the literature for a similar system. The results show that it is possible to obtain efficient and low-cost biosensors for monophenol detection using potentiometric transducers and alternative sources of enzymes without purification.

Thyme oil vapour and modified atmosphere packaging reduce anthracnose incidence and maintain fruit quality in avocado.

BACKGROUND: Postharvest application of prochloraz fungicide is commercially practiced to control anthracnose, a postharvest disease in avocado. Increasing consumer concern regarding food safety and demand for organically produced fruits make it necessary to search for natural environmentally friendly alternative products and processes for the fruit industry. RESULTS: A combination of modified atmosphere packaging (MAP; ∼8% CO2, 2% O2) plus thyme oil (TO) was evaluated on the incidence and severity of anthracnose, physiological disorders (grey pulp, vascular browning), fruit quality parameters (L*, h°, firmness, weight loss) and sensory parameters (taste, texture, flavour and overall acceptance), phenylalanine ammonia-lyase (PAL) enzyme activity, total phenolic compounds, flavonoid contents and antioxidant activity in avocados ('Fuerte' and 'Hass' cultivars) held at 10 °C cold storage for 18 days and thereafter, ripened at 25 °C for 5-10 days. Stand-alone MAP, commercial treatment (prochloraz 0.05%) and untreated (control) fruit were included for comparison. MAP + TO treatment significantly (P < 0.05) reduced the incidence and severity of anthracnose, grey pulp, vascular browning, weight loss and loss of fruit firmness, and showed acceptable taste, flavour, texture and higher overall acceptance, increased PAL activity, total phenolic compounds, flavonoid contents and antioxidant activity, after ripening at 25 °C followed by cold storage at 10 °C. CONCLUSION: This investigation recommends MAP + TO combination treatment as a suitable alternative to the currently adopted prochloraz application.

Photosystem II fluorescence lifetime imaging in avocado leaves: contributions of the lutein-epoxide and violaxanthin cycles to fluorescence quenching.

Lifetime-resolved imaging measurements of chlorophyll a fluorescence were made on leaves of avocado plants to study whether rapidly reversible ΔpH-dependent (transthylakoid H(+) concentration gradient) thermal energy dissipation (qE) and slowly reversible ΔpH-independent fluorescence quenching (qI) are modulated by lutein-epoxide and violaxanthin cycles operating in parallel. Under normal conditions (without inhibitors), analysis of the chlorophyll a fluorescence lifetime data revealed two major lifetime pools (1.5 and 0.5 ns) for photosystem II during the ΔpH build-up under illumination. Formation of the 0.5-ns pool upon illumination was correlated with dark-retention of antheraxanthin and photo-converted lutein in leaves. Interconversion between the 1.5- and 0.5-ns lifetime pools took place during the slow part of the chlorophyll a fluorescence transient: first from 1.5 ns to 0.5 ns in the P-to-S phase, then back from 0.5 ns to 1.5 ns in the S-to-M phase. When linear electron transport and the resulting ΔpH build-up were inhibited by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the major fluorescence intensity was due to a 2.2-ns lifetime pool with a minor faster contribution of approximately 0.7 ns. In the presence of DCMU, neither the intensity nor the lifetimes of fluorescence were affected by antheraxanthin and photo-converted lutein. Thus, we conclude that both antheraxanthin and photo-converted lutein are able to enhance ΔpH-dependent qE processes that are associated with the 0.5-ns lifetime pool. However, unlike zeaxanthin, retention of antheraxanthin and photo-converted lutein may not by itself stabilize quenching or cause qI.

Dimethyl isotope labeling assisted de novo peptide sequencing.

Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.

Biochemical changes during the storage of high hydrostatic pressure processed avocado paste.

High hydrostatic pressure (HHP) processing improves the shelf life of avocado paste without a significant impact on flavor; however, scarce information is available on biochemical modifications during its extended storage period. The present study focused on the changes in oxidative enzyme activities of pressurized avocado paste (600 MPa for 3 min) during refrigerated storage (45 d at 4 degrees C). Aerobic plate counts (APC), lactic acid bacteria counts (LAB), pH, and instrumental color were also evaluated during storage. Processing with HHP caused a decrease in polyphenol oxidase (PPO) and lipoxygenase (LOX) activities, resulting in residual enzyme levels of 50.72% and 55.16%, respectively. Although instrumental color values didn't change significantly during the evaluated storage period, both enzymes (PPO and LOX) recuperated their activities at 10 to 15 d of storage, reached the original values observed in the fresh paste, and then started a declining phase until the end of the storage period. Pulp pH presented a consistent decline during the first 20 d of storage. LAB counts were very low during storage, discarding lactic acid production as responsible for the observed pH decline. Enzyme reactivation, cell disruption, and a gradual migration of intracellular components such as organic acids are herein proposed as the main mechanisms for the deterioration of HHP treated avocado paste during its refrigerated storage. Practical Application: At the present, HHP is the most effective commercial nonthermal technology to process avocado paste when compared to thermal and chemical alternatives. Although it has proven to be an excellent product-technology match, little information is known on the biochemical changes that take place in the product during its refrigerated shelf life. Biochemical reactions during storage are important, since they can influence avocado paste nutritional and flavor qualities at the time of product consumption. The present study reports for the first time the re-activation of PPO and LOX during storage of avocado paste under commercial and economically feasible processing conditions (600 MPa and 3 min). The reactivation of oxidative enzymes observed in the present study is relevant for future studies on the HHP stability of food systems in general, and it is considered an important finding for the food industry and researchers seeking to deliver products with superior nutritional and flavor characteristics.

Immobilization of avocado phytase on epoxy-activated Sepabead EC-EP and its application in soymilk phytate hydrolysis.

There is a great demand for using phytases to reduce phytate content in animal feed stuffs and food for human consumption. Industrial application of phytase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to the free enzyme. In the present study, phytase was immobilized on Sepabead EC-EP and then used in the biodegradation of soymilk phytate. The immobilized enzyme exhibited an activity of 0.1 U per g of carrier and activity yield of 70.83%. Optimum temperature and pH for the immobilized enzyme were 55 degrees C and 5.5, respectively. Both the enzymes were stable between pH 3.0-8.0 and below 70 degrees C. Kinetic parameters(K(m) and V(max)) and the usability of the immobilized enzyme were determined. The immobilized enzyme hydrolyzed 65% of soymilk phytate in 8 h at 60 degrees C, as compared with 56% hydrolysis observed for the native enzyme over the same period of time.

The role of the embryo and ethylene in avocado fruit mesocarp discoloration.

Chilling injury (CI) symptoms in avocado (Persea americana Mill.) fruit, expressed as mesocarp discoloration, were found to be associated with embryo growth and ethylene production during cold storage. In cvs Ettinger and Arad most mesocarp discoloration was located close to the base of the seed and was induced by ethylene treatment in seeded avocado fruit. However, ethylene did not increase mesocarp discoloration in seedless fruit stored at 5 degrees C. Application of ethylene to whole fruit induced embryo development inside the seed. It also induced seedling elongation when seeds were imbibed separately. Persea americana ethylene receptor (PaETR) gene expression and polyphenol oxidase activity were highest close to the base of the seed and decreased gradually toward the blossom end. By contrast, expressions of PaETR transcript and polyphenol oxidase activity in seedless avocado fruit were similar throughout the pulp at the base of the fruit. Application of the ethylene inhibitor, 1-methylcyclopropene, decreased mesocarp browning, embryo development, seedling growth, and ion leakage, and down-regulated polyphenol oxidase activity. The results demonstrate that ethylene-mediated embryo growth in whole fruit is involved in the mesocarp response to ethylene perception and the development of CI disorders.

Biochemical analysis of reactive oxygen species production and antioxidative responses in unripe avocado (Persea americana Mill var Hass) fruits in response to wounding.

We analyzed the production of reactive oxygen species (ROS) and of detoxifying enzymes and enzymes of the ascorbate (ASC) acid cycle in avocado fruit (Pesea Americana Mill cv Hass) in response to wounding. The levels of superoxide anion (O(2-), hydroxyl radicals (OH.) and hydrogen peroxide (H(2)O(2)) increased at 15 min and 2 and 15 h post-wounding. Peroxidase (POD) activity had increased to high levels 24 h after wounding; in contrast, catalase and superoxide dismutase (SOD) levels hat decreased significantly at 24 h post-treatment. Basic POD was the major POD form induced, and the levels of at least three apoplastic POD isozymes -increased following wounding. Using specific inhibitors, we characterized one MnSOD and two CuZnSOD isozymes. CuZnSOD activities decreased notably 12 h after treatment. The activities of dehydroascorbate reductase and glutathione reductase increased dramatically following the wounding treatment, possibly as a means to compensate for the redox changes due to ROS production.

Methyl de-esterification as a major factor regulating the extent of pectin depolymerization during fruit ripening: a comparison of the action of avocado (Persea americana) and tomato (Lycopersicon esculentum) polygalacturonases.

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction.

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.