Source: STIXFRESH USA, INC. submitted to
DOSE RESPONSE CURVES OF ESSENTIAL OIL COMPOUNDS IN POSTHARVEST SPOILAGE PREVENTION OF FRESH PRODUCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1025750
Grant No.
2021-33530-34365
Cumulative Award Amt.
$97,697.00
Proposal No.
2021-00850
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Feb 28, 2023
Grant Year
2021
Program Code
[8.5]- Food Science & Nutrition
Recipient Organization
STIXFRESH USA, INC.
300 DESCHUTES WAY SW STE 304
TUMWATER,WA 985017719
Performing Department
(N/A)
Non Technical Summary
Post-harvest losses (PHLs) of agricultural produce during storage, distribution, and retail is a cause of significant economic and environmental burden. Microbial causes of PHLs, which includes fungal spoilage, is an open challenge that is largely lacking safe and effective solutions. A conservative estimate puts the economic burden from microbial spoilage at more than $10B/year worldwide. Compared to the synthetic antimicrobial agents which can have harmful and irreversible side effects, various essential oils (EO) of plant origin are generally a safe and natural alternative for suppressing microbial spoilage. Their antimicrobial activity has been well established throughout scientific literature. Their potential for extending shelf life of produce has also been published by independent researchers and demonstrated by our internal testing. Motivated by the scale of PHLs and the importance of this problem, our aim is to develop and commercialize safe, effective technologies and products to reduce the PHLs of fresh produce based on natural EO compounds of plant origin.Our focus for this Phase I effort is on the development of EO-based formulations and the characterization of their intrinsic dose response curves in their vapor phase. The proposed research project will focus on the following objectives: 1) to define, develop and validate the experimental set up and techniques to control and measure the EOC concentrations, 2) to perform in-vitro and in-vivo testing on fruits, and 3) to analyze and document the results. The results of this research will be used as primary inputs for the follow-on product development activities for Phase II.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5031129100025%
5031099100025%
5031199100025%
5030999100025%
Knowledge Area
503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
1129 - Berries and cane fruits, general/other; 1099 - Tropical/subtropical fruit, general/other; 1199 - Deciduous and small fruits, general/other; 0999 - Citrus, general/other;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
WP 1: Method development, validation, and test planningThe objective for WP1 is to define, develop and validate the experimental set up and techniques. This WP will involve development of the test chambers, methods to control and measure the EOC concentrations in the gas phase, methods to monitor and measure the growth and spread of B. cinerea infections on fruits as well as various quality attributes of the fruits.Thus, the tasks included in this WP are:Definition of the EOCs and selection of their target gas-phase concentrations to be tested. This portion of the plan will be informed by the EOC screening and characterization experiments that are being carried out separately by our bioactive formulation development team within StixFresh. The EOCs will be chosen from a subset that are shown to be effective against B. cinerea and we are most likely to commercialize as determined at the time of selection.Detailed definition of the use case to be simulated and definition of the initial conditions of the produce. For example, in an effort to standardize the starting conditions, produce may first be sanitized in a bleach solution and then dipped in solutions containing known concentrations of the target spoilage organism. In Phase I, we plan to test the effectiveness of EOCs against Botrytis cinereaDefinition and development of the experimental techniques that will be required to support the testing phase in WP2.Development and validation of techniques for "volatilizing" the target EOCs quantitatively so as to approximate the target gas-phase concentrations in the test environments. This effort will be informed by the thermodynamic and physicochemical properties of the EOCs (for example vapor pressure as a function of temperature) when possible. Whenever possible, estalished or previously published techniques such as quantitative injection with a syringe combined with heat evaporation will be employed.Development and/or adaptation of techniques measuring the head-space EOC concentrations in the gas phase with GC-MSMicrobiological methods to characterize the in-vitro growth, and extent of infection and spoilage of produce in-vivoMethods for characterizing the relevant quality attributes of the produce such as %Brix or total soluble solids and firmness.Design and build of any test chambers, fixtures etcPreliminary experiments as needed to validate the methods chosen and verify the workflows.Development, review, and approval of the detailed test plan.WP 2: Detailed testingThe objective for WP2 is to obtain the gas-phase dose response curves (DRC) of candidate EOCs under the select use case conditions both in-vitro and on fruits by executing the test plan developed in WP1. This objective will involve testing the effect of EOCs across a range of gas phase concentrations at 5 concentration levels on the suppression of growth and spread of B. cinerea inoculated in growth media (in-vitro), as well as on fruits (in-vivo). Potential target produces of interest include bananas, strawberries, blueberries, cherries, grapes, kiwi fruit, mangos, peaches, oranges, and avocados. The test systems chosen include a selection from this set, to be determined at that time based on the most relevant fruit(s) we are likely to target with our first product to market.WP 3: Results analysis, reporting, and recommendationsThe objective for WP3 is to analyze, synthesize and capture the learnings from the project. This work package will focus on detailed analysis and interpretations of the results obtained in WP 2. Results will culminate in a narrative for recommendations for further R&D as well as design guidelines for the follow-on product development activities in Phase II.
Project Methods
The basic approach will be performing testing to characterize the effect of the gas phase concentration of our active compounds on suppressing microbial spoilage of fresh produce.The above approach requires a number of capabilities and methods to be developed as a prerequisite. These include- ability to control gas phase concentration- ability to measure the gas phase concentration- ability to also control and monitor other parameters of interest in the test setting such as temperature- ability to measure and quantify "responses" such as spoilage, microbial growth, physical and sensory/organoleptical changes in produce

Progress 07/01/21 to 02/28/23

Outputs
Target Audience:The protocols and technology developed in this project and the resulting measurements will be used by Ryp Labs to prepare to commercialize their produce shelf-life extension technology. The target audience is the internal staff scientists and engineers at Ryp Labs. Based on the results of this project Ryp Labs has developed the tools necessary for measuring and optimizing the dose of essential oil compounds needed to extend the shelf-life of produce. This improvement in measurement capability and knowledge of optimized doses will be the foundation for a USDA SBIR Phase II project and a product development process. The result of this follow on work will be a minimum viable product (MVP) that will be commercialized and marketed to customers. Ryp Labs' shelf-life extension technology, which when applied at scale, has the potential to significantly reduce food waste and the associated carbon footprint at all levels of food production including producers, distributors, retailers, and consumers. Ryp Labs' products will also have the potential to increase the range of distribution of many producers and distributors, opening new markets for produce traditionally only locally available. Finally, Ryp Labs' products have great potential in regions that don't have access to complete cold-chain infrastructure, which could beneficially impact many agricultural communities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Experimental setups for testing fruit in controlled headspaces of volatile compounds were developed and validated over the course of this project. Protocols for setting and measuring the concentrations of volatile compounds in the air space in the experimental setups were developed. These methods are crucial for accurately determining the optimum amount of volatile compounds needed to inhibit pathogens and increase produce shelf life. Protocols for measuring fruit quality and pathogen growth rates were developed and validated over the course of this project. These methods provide a quantitative measurement of the efficacy of volatile compounds in preserving produce, allowing for optimization and engineering design of improved delivery methods. The development and validation of these experimental setups and protocols were the output of work package 1, accomplishing all of its goals. The experimental setups and measurement protocols that were developed allowed Ryp Labs to perform detailed testing of strawberries and measure the minimum amount of volatile compounds required to inhibit mold and increase their shelf life. This will be the starting point for a Phase II project and product development process to produce a product that can be commercialized. The experimental setups and measurement protocols that were developed also allowed Ryp Labs to measure the minimum amount of volatile compounds that result in adverse quality effects in produce. This quantitative measurement gives Ryp Labs a key understanding of the maximum amount of compound that can be applied before the benefits of increasing shelf life are outweighed by decreasing fruit quality. The detailed testing and measurements were the output of work package 2, accomplishing all of its goals. The measurements of mold inhibition were analyzed to determine a dose response curve of a volatile compound against strawberry mold pathogens. This analysis was the output of work package 3, accomplishing all of its goals.

Publications


    Progress 07/01/21 to 06/30/22

    Outputs
    Target Audience: Nothing Reported Changes/Problems:One of the original goals was to correlate in vitro (petri dish) experiments with in vivo (on produce) experiments. We have found that the experimental systems and conditions required for these cases are so different that correlations are extremely difficult and don't seem to provide easily applicable insight. Instead, we have chosen to focus primarily on in vivo (on produce) experiments, and include varying the applied compound concentration over time in our project scope. What opportunities for training and professional development has the project provided?This grant has provided two junior scientists with the opportunity for training on a variety of specialized analytical equipment. The grant has also provided these scientists the opportunity to develop experience with method development, experimental design, and project management. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?- Testing of fresh produce under controlled conditions with known concentrations of natural compounds will be performed. - Monitoring of compound concentrationsand produce quality and response over timewill be performed. - Applying compounds at varying concentrations over time will also be studied.

    Impacts
    What was accomplished under these goals? Our company is focused on reducingfood waste at all levels of the distribution chain. Food waste results in significant financial losses for farmers, distributors, and consumers as well as harming the environment and contributing to global warming. A significant factor that causes food waste is produce infection by pathogens like mold or bacteria, which are typically treated with synthetic pesticides that are often expensive, environmentally harmful, and sometimes dangerous for users. Our company reduces food wasteby inhibiting pathogens withnatural and safevolatile compounds derived from plants that have evolved to resist pathogen infection.The goal of this project is to develop the methods our company needs to determine the most efficient amount of natural volatile compounds necessary to prevent pathogens from making the produce unsalable or inedible. Accomplishments so far: - Short list of natural volatile compoundsfor further investigation have been defined. - Experimental setup foruse-case simulation like produce packaging, storage temperatureand humidity have been defined. Experimental techniques forenvironmental controls like temperature and air quality, along withvariable monitoring have been defined and developed. - Experimental techniques forapplying the candidate compounds at desired concentrations has been developed and validated. - Experimental techniques for measuring volatile compound concentrations, both qualitatively and quantitatively, have been developed and validated. - Experimental techniques for measuringproduce conditions or responses to treatment, such as color, texture, and degree of spoilage, have been developed. - Test chambers and systems have been designed, built, and validated. - Preliminary experiments have been performed using all ofthe developed experimental techniques to validate methods and workflows.

    Publications