Reproducible Method for 1-Methylcylopropene (1−MCP) Application and Quantitation for Post-Harvest Research

Abstract

Actively combating post-harvest food spoilage and waste can dramatically increase the efficiency of food utilization worldwide. In climacteric fruits, chemical treatments such as 1−MCP are an effective way of reducing post-harvest spoilage and waste by inhibiting the fruit’s ability to perceive ethylene. 1−MCP treatment is increasingly being used to explore the complex nature of ripening physiology at a fundamental level; however, differences in application and quantitation methods create difficulties in comparing conclusions across studies. Here, we report an effective and reproducible method for 1−MCP application and quantitation for small–medium-sized research applications. By use of surrogate alkene standards 1-butene and cis-2-butene, the highly volatile and elusive 1−MCP molecule can be identified and quantified by gas chromatography and subsequent standard curves may be developed. It is hoped that the methodology outlined here can help standardize consistent 1−MCP application for post-harvest research without excessive investment in specialized equipment.

1. Introduction

The Food and Agriculture Organization of the United Nations estimates that upwards of 1.3 billion metric tons of food is wasted after harvest every year, which is enough to feed more than 1.9 billion people or roughly ¼ of the world population [1]. This does not include the food loss that occurs prior to harvest. In fruits, early ripening, and associated processes such as flesh softening, and starch-to-sugar conversion are major causes of food wastage post-harvest. Crops with high water content or those susceptible to damage from changes in temperature or humidity such as fresh fruits and vegetables are far more likely to suffer postharvest decay and wastage than grains or tree nuts, for example.

Most susceptible of all are climacteric fruits. Climacteric fruit exhibit a burst in respiration (characterized by CO₂ evolution), with an accompanying increase in ethylene synthesis. These fruits continue to ripen through phytohormone-driven positive feedback loops even after they are picked from the mother plant. Maintenance of fruit constitution during and post harvest is a complex task involving coordination between growers, storage houses, distributors, cold-chain transporters, and the demands of the market. The complexity of this multi-faceted system results in wastage. Research investigating the specifics of ripening and potential post-harvest control mechanisms is seen as a viable approach to reducing the amount of food spoilage.

One means of combatting food wastage, especially in vulnerable crops such as climacteric fruit, is to delay the onset of ripening. Perhaps the most powerful tool for this effort is the gaseous four-carbon cyclical alkene 1-methylcyclopropene (1−MCP), which inhibits perception of the “ripening phytohormone” ethylene, patented as an inhibitor of ethylene action in 1996 [2]. 1−MCP competitively binds to ethylene receptors, thereby delaying ripening, preserving fruit flesh firmness, and extending storage life in climacteric fruits [2,3]. This compound has been used to great effect in apple (Malus domestica) post-harvest systems producing reliable and repeatable results. The use of 1−MCP in research settings has bolstered some understanding of ethylene action. For example, there seems to be a compounding ‘time × concentration’ relationship for 1−MCP in most fruit and vegetable crops, where higher concentrations or extended treatment times show greater inhibition ripening [4], a relationship in agreement with ethylene research data.

In some instances, rather than shed light on the complex patterns of ripening processes, research into the effects of 1−MCP treatments in various crops has revealed counter-intuitive and highly variable results. In pineapple, it was observed that ethylene levels remain elevated longer and decline slower in 1−MCP-treated fruits than in non-treated fruits [5]. Ethylene biosynthesis has even been shown to increase upon application of 1−MCP in figs [6], jujube [7], and mango [8]. Stone fruits such as apricots, peaches and plums have been reported to develop internal browning, flesh breakdown and flesh reddening when stored at low temperatures after 1−MCP treatment [9]. These studies highlight just how much of a knowledge gap still exists in the collective understanding of 1−MCP and its interactions with plants.

Observations of extreme variation and, in some cases, lack of reproducibility in 1−MCP treatment experiments point towards the role of physiological and genetic variability across different species. Recent research demonstrates a shift away from commercial application (i.e., physiology) research and a move towards molecular and biochemical studies of fruit interactions with 1−MCP [10]. Additionally, the inability to standardize consistent applications of 1−MCP has been problematic given it is a highly volatile molecule. Several studies have shown 1−MCP–fruit interactions are most likely due to inconsistent 1−MCP applications, as concentrations even in the parts per billion range are effective at eliciting ripening inhibition—for example, in European pear [11,12]. Experimentation at such an intricate biological level must minimize independent variable disparity to obtain results with a high level of confidence. Furthermore, interest in exploring 1−MCP response at an increasingly granular level must be paired with increased focus on how to apply 1−MCP in a quantifiable, replicable, and consistent manner.

To address the technical gap for 1−MCP application, we have developed a standardized and reproducible 1−MCP application and quantitation method to reduce variability in 1−MCP-related post-harvest experiments. Because such low concentrations of 1−MCP are effective at inhibiting ethylene related response in fruits, it is important that application methods be accurate and reproducible. The use of non-reactive acrylic chambers that are very effective at maintaining constant headspace concentrations can aid in the standardization process [13]. Adoption of this methodology is expected to allow for greater control of a crucial independent variable in post-harvest horticultural studies (i.e., 1−MCP application rate), thereby limiting observed variation in the biological realm and enabling additional research in the area of 1−MCP with its interactions in various plant systems to develop efficient methods for post-harvest waste reduction.

2. Materials and Methods

2.1. Materials and Reagent Acquisition

1−MCP-cyclodextrin inclusion complex 3.3% active ingredient was sourced from Chesen Biochem Co. Ltd. (Hefei, China). We sourced 110 L acrylic desiccator chambers (catalog number 1400-1-AB) from Cleatech Cleanroom and Laboratory Solutions (Orange, CA, USA). Cis-2 butene and 1-butene standards were sourced from Sigma Aldrich (St. Louis, MO, USA). Sodium hydroxide was sourced from Fisher Scientific (Hampton, NH, USA).

2.2. 1−MCP Theoretical Yield Calculations

Yield calculations were made assuming complete liberation of 1−MCP active ingredient from the cyclodextrin inclusion complex. Utilizing the ideal gas law (PV = nRT), corrections were made for elevation (Pullman WA, 717 m) and temperature (20 °C) to give the maximum theoretical yield of 1−MCP for the chamber headspace.

2.3. 1−MCP Application and Sampling

1−MCP compound was weighed and deposited into glass dishes containing a magnetic stir bar before being placed in the desiccation cabinets near one of the septum-lined injection ports; the chamber was sealed by closing the door and fastening the clasps. The chamber was elevated such that a magnetic stir plate could be placed beneath, facilitating agitation of the 1−MCP compound (Figure 1). With agitation, a 7.5% (w/v) sodium hydroxide solution was administered to the 1−MCP-containing plate from the outside of the chamber by needle and syringe through the septa. Only enough sodium hydroxide solution required to saturate the 1−MCP powder was administered. Agitation with a stir bar ensured that all the 1−MCP/cyclodextrin compound entered the solution and increases the probability of active ingredient liberation near or at theoretical yield for a given temperature and pressure. After roughly 10 min, when the 1−MCP–cyclodextrin complex was completely dissolved into the solution, a 500 µL headspace sample was taken from a septa-lined injection port opposite the sample Petri dish. Another sample was taken in the same manner 24 h after sample hydration to ensure chambers remained airtight.

2.4. Surrogate Standard Application and Sampling

Cis-2-butene [14] and 1-butene [15] surrogate standards were chosen for their similarity in chemical composition and structure to 1−MCP as well as their prior use in 1−MCP-related research. Dilutions of pure standards were prepared in a syringe and injected into the chambers. Surrogate standard application did not require the use of the sodium hydroxide hydration method as the gases were supplied in pure format. After 10 min of equilibration time, headspace samples were removed and analyzed in the same manner as 1−MCP. No 24 h samples were taken of surrogate standards.

2.5. The 1−MCP Detection and Quantification

Samples were analyzed via gas chromatography on an HP 5890 GC fitted with an Agilent HP Plot-Q-15 m × 0.5 mm column with 18 µm phase. Oven, injector, and detector temperatures were 120, 140, and 200 °C, respectively. Nitrogen was used as a carrier gas at a flow rate of 5 mL min⁻¹. Sample injection volume was 500 µL and all samples were analyzed in triplicate. 1−MCP peaks were distinct, with retention times always preceding cis-2-butene and succeeding 1-butene (Figure S3). Because of this unique peak profile, the 1−MCP peak could be clearly identified for every sample.

3. Results

A formulation of 3.3% active ingredient 1−MCP surrounded by a cyclic oligosaccharide was necessary to weigh and apply the compound, as 1−MCP at physiologically relevant temperatures is a gas. Concentrations ranging from 50 to 500 parts per billion 1−MCP were tested. The theoretical yield assuming complete liberation of active ingredient from the cyclodextrin inclusion complex was determined using the ideal gas law coefficients, where corrections were made for air pressure at elevation 717 m above sea level, 20 °C, and the formula weight of 1−MCP at 54.9 g mol ⁻¹. The linear regression relating mass of 1−MCP inclusion product to theoretical headspace concentration in the 110 L application chamber can be seen in Figure 2. 1−MCP liberation from inclusion complexes has traditionally been accomplished by complete hydration of the powdered complex for research purposes [16,17] and as advised by commercial chemical manufacturer AgriFresh [18]. However, recent research into the release kinetics of 1−MCP from inclusion complexes suggests decreased release due to inclusion complex collapse at relative humidity of greater than 60% [19]. The methods outlined within this document attempt to resolve the discrepancy between the two application processes by incorporating constant agitation via a magnetic stir bar.

Dilutions of surrogate standards were used to generate standard curves and determine appropriate integrator time scales (Supplementary File 1). Only then were 1−MCP dilutions run in parallel with blanks. The similarity in chemical makeup and structure between the surrogate standards and 1−MCP and the absence of peaks in blank samples could be leveraged to ensure accurate detection of 1−MCP without the use of a mass spectrometer. The use of surrogate standards was necessary as 1−MCP is typically supplied and used by industry as an impure compound (i.e., contained within an inclusion complex), making detection by chromatography difficult. By first running dilutions of pure surrogate standards, the retention time of the1−MCP peak could be estimated and observed carefully in later 1−MCP headspace measurements. Appropriate masses determined from the theoretical yield regression equation were used for 1−MCP standard curve development. Care was taken to maintain constant agitation by stirring of the inclusion complex during hydration to avoid aggregation of particles, as previously reported [19]. Remarkably, concentrations as low as 50 ppb were easily observed as distinct peaks. The relationship between headspace 1−MCP concentration based on theoretical yield and area under the curve remained linear throughout all concentrations tested (50–500 ppb; Figure 3).

4. Conclusions

The presented method attempts to circumvent use of costly mass spectrometry equipment in favor of gas chromatography machinery more widely available in post-harvest research laboratories. As such, adherence to the outlined methodology and use of surrogate standards has been sufficient in identification of the 1−MCP peak of interest. Should further validation of specific alkene peaks be necessary, the United States Department of Commerce National Institutes of Standards and Technology have published freely accessible electron ionization mass spectra for 1-methylcylopropene (Figure 4), as well as cis-2-butene [20] and 1-butene [21].

Molecular and biochemical studies exploring 1−MCP–fruit interactions need to be replicable. Variability in response to 1−MCP is often high, resulting in inconclusive outcomes [10]. If the observed variation is exclusively biological, then the role of 1−MCP in a given experiment can be elucidated. The method and approach reported here provide a framework for accurate 1−MCP application and quantitation for use in post-harvest research and aim to minimize experimental variation by standardizing 1−MCP application. By sourcing high-quality 1−MCP-cyclodextrin product and carefully accounting for pressure and temperature, theoretical yield estimations can be generated for the application volume. 1−MCP application in non-reactive gas-tight chambers minimizes leakage and ensures theoretical yield closely matches quantitative measurement. Furthermore, constant agitation of the 1−MCP/cyclodextrin during hydration by magnetic stir plate increases the probability of complete liberation of the active ingredient from inclusion complex by ensuring compound solubilization. 1−MCP liberation from inclusion complexes has traditionally been accomplished by complete hydration of the powdered complex for research purposes [16,17] and as advised by commercial chemical manufacturer AgroFresh [18]. However, as mentioned previously, recent research into the release kinetics of 1−MCP from inclusion complexes suggests decreased release due to inclusion complex collapse at a relative humidity of greater than 60% [19]. The method outlined in this report has attempted to improve upon the two aforementioned approaches by incorporating constant agitation via magnetic stir bar and thereby ensuring complete liberation of 1−MCP. Utilization of inexpensive, reliable surrogate standard gases such as 1-butene and cis-2 butene helps in identifying 1−MCP chromatogram peaks and validating the concentrations reliably. It is expected that adoption of these considerations and methodology will facilitate further understanding of 1−MCP–fruit interactions and help maximize experimental reproducibility and confidence in results.