Progress report for GNE24-331
Project Information
Microgreens nutritional quality declines rapidly post-harvest which could be problematic from both a food safety and extranutritional standpoint. Once microgreens are harvested, they rapidly exude nutrient-rich fluid, wilt and can quickly decay, potentially favouring enteric pathogen persistence (Turner et al., 2020). This project will attempt to address these shortfalls in microgreen production. The project will investigate the efficacy of regulated abiotic stress as an elicitor of plant secondary metabolites on plants grown under controlled environments to improve extranutritional properties, food safety and post-harvest quality (shelf-life). Using sustainable approaches, microgreens will be subjected to UV-exposure or cold stress with the aim of enhancing nutritional quality, safety and shelf-life.
The objectives of this project are:
- Improved nutrition and quality: Assess a) the effect of exposure to UV or cold stress on secondary metabolite accumulation in microgreens of leafy vegetables and herbs, and b) retention of nutritional quality post-harvest and packaging.
Utilizing controlled environments, I will expose seedlings to abiotic factors such as cold temperature and UV irradiation to improve microgreen phytochemical profiles and quality post-harvest. Secondary metabolites which are typically indicative of improved nutritional value such as total phenolics, total flavonoids, carotenoids, betalain alkaloids (red beet-leaf group only) anthocyanins (red varieties non-leaf beet group), glucosinolates (Brassica spp. only) and vitamin C will be measured. Rate of decline of major phytochemical groups will be measured over a one-week period post-packaging.
Progress Update:
We are currently in the planning stages for this objective and will be purchasing seeds in the new year to begin our experiments.
On September 3rd, I attended a training at a local farm to gain insights into their microgreen-growing techniques for market production. During the training, I learned about their specific methods and the types of seeds they utilize. Their microgreen operation uses organic purple stem radish, broccoli, and pea seeds sourced from a commercial seed supplier.
The farm employs a specialized germination process using a soil-like mix made of a 1:1 ratio of compost and hydrate. They fill trays (with drainage holes) with this soil mixture and place a clean tray filled with rocks on top of the soil to compress it before moistening. The soil is then watered using reverse osmosis-filtered municipal water, and seeds are directly sown onto the soil's surface.
After sowing, trays are stacked four to five high and weighed down. After one week, the trays are unstacked and moved to benches, where the seedlings are allowed to grow until their first two true leaves emerge. On harvest day, the microgreens are cut, washed in a bubbler, and dried using a large commercial salad spinner, packaged and sold at market within 2-3 days.
We are also in the design and planning stage for building a UV chamber to expose microgreens to UV light. The chamber will be constructed and operational in early 2025.
- Improved food safety: Evaluate the effect of phytochemical manipulation via abiotic stress application on foodborne pathogen association with microgreens.
Seeds of different microgreens will be inoculated with the foodborne pathogens Salmonella, Listeria monocytogenes and STEC, which will then be grown under conditions which shift phytochemical levels. I will measure the persistence and transfer of foodborne pathogens in the phyllosphere of microgreens at low concentrations, over a period of 5 days post-harvest. Enteric pathogens retrieved will be enumerated by spread plating or Most Probably Number (MPN) method.
Progress update:
Rifampicin resistant strains of Salmonella and STEC cultures are being maintained in preparation for their use in January 2025. In addition, we have acquired a Listeria strain from the FDA, originally isolated from leafy greens. I have successfully adapted the Listeria strain to rifampicin in preparation for experiments using this pathogen.
- Integrated food safety and sustainability: Assess the food safety implications of various growth media for microgreen production.
The effect of substate type on the persistence of foodborne pathogens will be evaluated. Inoculated seeds of microgreens will be grown as outlined in objective 2 on either a) soil mix, b) biodegradable Jute fibre, and c) non-biodegradable rockwool mats. The level and persistence of foodborne pathogens remaining in the substate will be evaluated for a period of 4 days.
Progress update:
Following the training I received in September, I will be purchasing supplies in the new year to begin experiments to address this objective.
The purpose of this project is to investigate methods to synergistically improve extranutritional properties, food safety and post-harvest quality of microgreens.
Microgreens are delicate young seedlings of edible plants and include the shoots, two cotyledons and small emerging true leaves which appear 7-14 days post-germination. Microgreens require a substrate to germinate on and many small scale farmers use a soil-based mix or a fibre-based mat (Du et al., 2022). There are two forms of microgreen products available, ‘living’ and ‘cut’. Living microgreens remain on the substrate mat, which provides a high degree of quality, while cut microgreens are harvested, washed with an approved sanitizer such as SaniDate 15.0 and sold as a ready-to-eat product (Turner et al., 2020). Revenue generated from microgreens sales for US farmers ranges from <$5,000 to > $50,000 per year (Hamilton et al., 2023). Despite the apparent economic benefit of branching out into microgreen production, farmers may be reluctant to embark on this high-end product due to the lack of specific training and barriers to risk management practices (Hamilton et al., 2023).
Microgreens offer a pop of colour on the plate and provide a relatively nutritious morsel, densely packed with vitamins (A, C, E and K) and minerals (K, P, Ca, Mg, Fe) (Dereje et al., 2023). Manipulation of their nutritional quality has been explored in recent years and studies have shown that the phytochemical profiles of microgreens shift when exposed to abiotic stressors. Broccoli microgreens exposed to cold temperatures had significantly increased antioxidant capacity, soluble sugars, total phenolics, tannins, and glucosinolates (Šola et al., 2024). Additionally, broccoli microgreens exposed to UV-B post-harvest boosted the glucosinolate levels (Lu et al., 2018). Although it is known that accumulation of bioactive compounds is beneficial for consumers’ health (Yao et al., 2004), the effects of these phytocompounds on associating microbiota is not fully explored.
Due to the short growing time and suitability for growing in controlled environments, microgreens offer a valuable product that can be brought to market quickly. However, microgreens can wilt and decay quickly, leak electrolytes post-packaging and exude nutrient rich fluid from the cut stems after harvest which reduces the nutritive and food quality post-harvest, and may render the product vulnerable to contamination with foodborne pathogens (Bulgari et al., 2021; Işık et al., 2024; Turner et al., 2020). Contamination of seeds, soil, water, worker hygiene, growing media, and harvesting tools are all possible routes of contamination (Işık et al., 2024). There is evidence of a food safety risk associated with microgreens as illustrated by the increasing number of food recalls. Since 2015, there have been ten recalls for microgreen products sold in the USA and Canada. Four of which were related to contamination with Listeria monocytogenes and the remaining six were due to Salmonella (Canadian Food Inspection Agency (CFIA), 2024; Turner et al., 2020; US Food and Drug Administration, 2024). The majority of recalls are for products containing broccoli, arugula or salad mix microgreens.
Xiao et al. (2015) showed a concerning level of Escherichia coli O157:H7 could be transferred from inoculated seeds to aerial parts of microgreens, with the highest levels found on hydroponically grown microgreens compared to those grown on soil based media (Xiao et al., 2015). Similarly, Işık et al. showed Salmonella, Shiga Toxin-producing Escherichia coli (STEC) O157:H7, and Listeria monocytogenes transferred to microgreens from inoculated perlite (as a soilless substrate) and seeds. They found the level of transfer was dependent on cultivar, which they attribute to the variance in the seed characteristics (seed weight and surface area). The authors suggest seeds with high surface area to volume (SA:V) ratio (e.g. mustards), were more susceptible to transfer of pathogens than seeds with lower SA:V ratios (e.g. pea) (Işık et al., 2024).
There is an opportunity to advance current knowledge by exploring the interaction between elicitation of secondary metabolites via abiotic stress in microgreens and the subsequent effect on foodborne pathogens when seeds are grown on various substrates. I hypothesize that microgreens grown under abiotic stress will produce more nutritious seedlings that are more resistant to colonization by foodborne pathogens when grown in soil compared to fibre mats and exhibit a prolonged shelf-life post-packaging.
The outcomes of this project will help inform current and future microgreen growers of the best management practices to boost nutrition and minimize contamination risk of their microgreen crop. Further, the outreach portion of this project will give growers the confidence to make decisions about their microgreen production methods to produce the best quality and safest product possible.
Research
Growing microgreens
For the purposes of this project, I will use single species rather than seed mixes. Seeds of popular microgreen varieties will be used: broccoli (Brassica oleracea), basil, ‘Genovese’ (Ocimum basilicum), beet ‘Bull’s blood’ (Beta vulgaris), kale ‘Red Russian’ (Brassica napus) and arugula (Eruca sativa) will be used for this study.
Following the seed package instructions for each microgreen cultivar, inoculated seeds (described below) will be sown in 225 cm2 shallow containers with one of the following substrates a) soil mix (using the recipe provided by our partner farmer), b) jute fibre or c) rockwool. The seeds will be moistened and grown for 7-14 days in a Percival incubation chamber (AR-41L3) with a light:dark schedule of 16 h:8 h illuminated with LED lights at 405 µmoles m-2 s-1 and kept at a constant day and night temperature of 16°C, with 65% relative humidity (control conditions).
Microgreens will be harvested 10-14 days after sowing by cutting the hypocotyl ~1 cm above the substate surface with a pair of sterilized scissors, and either used immediately for enumeration of bacteria and phytochemical analysis or processed for packaging and used to determine post-harvest food safety and quality measurements.
Abiotic treatments
UV exposure: From first emergence, each day for 5 consecutive days, microgreen seedlings will be transferred to a growth chamber equipped with a UV-B fluorescence light source (measured at 312 nm) to receive ~0.5 Wh m-2 UV-B exposure for 2 h at 16°C. After each daily exposure, seedlings will be transferred back to the Percival incubation chamber under conditions described above.
Cold stress: Five-day-old seedlings will be transferred to a growth chamber set to a day/night regime of 12°C/7°C and subjected to these temperatures for 3 days and nights. Lighting conditions and humidity levels will remain at 405 µmoles m-2 s-1 and 65% relative humidity.
Inoculation experiments
Inoculum preparation
A lettuce outbreak strain of Escherichia coli O157:H7 FDA#MD2019EC strain 2705C (2705C) (Chen et al., 2023), a river water isolate of Salmonella enterica serovar Typhimurium (Callahan et al., 2019) and a cantaloupe outbreak strain of Listeria monocytogenes (CDC, 2018), previously adapted to rifampicin (R) (MilliporeSigma, MA, USA), will be revived from frozen stock by streaking onto Trypticase Soy Agar (TSA, BD) plates amended with antibiotic and incubated for 18-20 h at 37°C or 32°C (Listeria). Single colonies grown overnight will be streaked onto fresh TSAR plates and incubated for 18 h. Inoculum of the single strain pathogens will be prepared by suspending fresh bacterial colonies in 5 mL sterile water and adjusted to 0.5 McFarland standard using a Sensititre Nephelometer (Intertek, Integrated Technologies). Appropriate dilutions will be prepared to obtain inoculation levels with the final level of pathogens on seeds to be 2-3 log CFU g-1.
Seed inoculation
Seeds will be inoculated following the protocol outlined by Işık et al. with slight modifications (Işık et al., 2024). Fifty grams of seed will be weighed and transferred into sterile 50 mL falcon tubes and 25 mL of prepared inoculum will be added. Tubes of seeds will be vortexed briefly and shaken for 5 min at room temperature. The supernatant will be discarded and seeds transferred to a biosafety cabinet where they will be spread onto aluminium foil and allowed to dry at room temperature for ~6 h with laminar airflow left on. Inoculated seeds will be sown as described above.
Pathogen quantification and persistence
Five g of harvested microgreens of elicited and control plants will be placed into sterile Whirl-Pak bags (Nasco, WI, USA) with mesh and homogenized in 5 mL 0.1% peptone water. One mL of the homogenate will be spread plated on TSAR in duplicate and incubated overnight. Viable colonies will be counted and converted into log CFU g-1.
To determine the presence of pathogens in the substrate, 5 g of substrate material will be placed into sterile stomacher bags and 25 mL 0.1% peptone water added and stomached for 2 min. Viable colonies will be enumerated by spread plating as outlined above.
Determining microgreen nutrient and phytochemical content
Plant tissue extracts of control and elicited seedlings pre- and post-inoculation experiments will be assessed for, total phenolics, flavonoids, anthocyanins (kale ‘Red Russian’ only), betalain alkaloids (beet only), glucosinolates (cruciferous vegetables only), vitamin C and sugars.
All spectrophotometric readings will be achieved using a Synergy HTX microplate reader (Biotek, VT).
Preparation of Leaf Tissue Extracts for Antioxidant Capacity, Total Phenolics, and Flavonoids
Ariel parts will be collected and placed in a sterile Whirl-Pak bag and immersed in liquid nitrogen to flash freeze the tissue. Samples will be immediately transferred to a HarvestRight Pharmaceutical Pro Freeze Dryer. Dried samples will be powdered and stored at -80°C.
Ten mg of dried tissue will be extracted in a 2 mL tube with 1.5 mL 70% aqueous methanol. Samples will be vortexed for 5 mins and transferred to a float and sonicated in a water bath at 35°C for 60 minutes (Sampaio et al., 2016). Extracts will be filtered through 0.45 µm nylon syringe filters and placed into a clean 1.5 mL centrifuge tube, either processed immediately or stored at -80°C for future analysis.
Total Phenolics
Total phenolics will be determined using the Fast Blue assay (Ravindranath et al., 2021). To perform the Fast Blue assay 200 µL aliquot sample extracts, gallic acid standards and blanks will be added to a clean 96-well microplate, then 20 µl of freshly prepared Fast Blue diazonium salt (FB) solution (0.01%) will be added in all wells. The plate will be incubated in the dark for 10 min, then 20 µl of 1 N potassium hydroxide will be added to all wells. The plate will be incubated for an additional 120 min to allow for the reaction to occur. Absorbance will be measured at 420 nm using a Synergy HTX microplate reader. Total phenolics will be calculated as gallic acid equivalents.
Total Flavonoids
Total flavonoid content of samples will be determined by the colorimetric method with some modifications (Rajabbeigi et al., 2013; Zhishen, Mengcheng, & Jianming, 1999). One hundred µl leaf extract, catechin standards or blanks will be added to a clean 96-well microplate, 30 µl NaNO2 will be added followed by 30 µl 10% AlCl3 5 m later. (Alfa Aesar, MA, USA) Finally, 100 µl 1 mol/L NaOH (VWR Chemicals, BDH) will be added to each well 1 m later. Absorption of the mixture will be measured after shaking for 30 s at 510. Total flavonoids content will be calculated as catechin equivalents.
Flavonoids will also be characterized via UV-Vis spectral scans using the Genesys 180 spectrophotometer. Methanolic extracts will be filtered and scanned using quartz cuvettes to obtain shifts in secondary metabolites indicated from the absorption of compounds within 210-400 nm. Peaks within this range are characteristic of Band I and Band II flavonoids.
Total Anthocyanins
Anthocyanins will be determined using the Association of Official Analytical Chemists (AOAC) standard protocol (Johnson et al., 2020). Fifty mg of dried powdered plant sample will be extracted in 500 µl 90% aqueous methanol, vortexed and transferred to a sonicating water bath and extracted twice and supernatants pooled. Total anthocyanins will be determined using a modification of the pH differential method. Aqueous buffer solutions at pH 1 and 4.5 will be prepared using 0.025 M potassium chloride and 0.4 M sodium acetate, respectively, and the pH adjusted with concentrated HCl. Sample extracts (200 µl) will be diluted with 1.8 mL of buffer and vortexed. After equilibrating at room temperature in the darkness for 15 min, the absorbance at 510, 520 and 700 nm will be read using DI water as the blank. Total monomeric anthocyanin concentration will be calculated using the formula provided in the protocol.
Betalain alkaloid determination
Major groups of betalain alkaloids will be determined spectrophotometrically as described by Von Elbe. Briefly, absorbance of tissue extracts will be measured at 528 and 476 nm and the concentrations for all minor betacyanins and betazanthins will be calculated respectively using the formula provided in the protocol (Von Elbe, 2001).
Estimated Glucosinolate Content
Glucosinolates of Brassica microgreens will be estimated using a method developed by Mawlong et al. 2017. One hundred mg of powdered leaf tissue will be extracted in 1.5 mL 80% methanol, vortexed and shaken at room temperature overnight. One hundred µl of tissue extract will be combined with 300 µl DI water and 3 mL of 2 mM sodium tetrachloropalladate (58.8 mg sodium tetrachloropalladate acidified with 170 μl concentrated HCl in 100 mL distilled water). After 1 h of incubation at room temperature, 200 µl of the extract and methanol blanks will be transferred to a 96 well-plate and the absorbance measured at 425 nm.
Glucosinolates estimates will be made using the authors predictive formula generated from regression models between glucosinolates obtained by HPLC and the absorbance at 425 nm spectrophotometrically (Mawlong et al., 2017).
Sugars
Sucrose, D-Fructose, D-Glucose will be estimated using a Neogen® Megazyme Assay Kit. The Sucrose/D-Fructose/D-Glucose test kit is suitable for the measurement and analysis of sucrose, D-glucose and D-fructose in plant and food products. The limit of detection for this assay is 1.38 mg/L.
Vitamin C
The vitamin C content will be estimated using a Neogen® Megazyme Ascorbic Acid Assay Kit (L-Ascorbate) with a limit of detection of 0.175 mg/L.
Quality measurements
Antioxidant Capacity
One hundred microliter samples, positive controls (50 mM ascorbic acid) or solvent blanks will be added to a clean 5 mL tube, 900 µl Tris HCL and 2 mL DPPH• (2,2-diphenyl-1-picrylhydrazyl; 0.1 mM DPPH) will be added to the reaction tube, vortexed and incubated for 60 minutes. Two hundred microliters of the reacted mixture will be transferred to a 96 well plate and the absorbance measured against the blank at 517 nm. The percent DPPH radical quenched will be calculated as follows: (Absblank – Abssample) / Absblank x 100 (Yu et al., 2002).
Electrolyte leakage
Total electrolyte leakage of microgreens post-harvest will be determined for 5 cumulative days of storage following the protocol described by Lu et al. Fresh microgreens (3 g) will be submerged in 150-mL sterile distilled water at 4 °C for 30 m. and the electrical conductivity will be measured with a conductivity meter. Samples will then be frozen at −20 °C for 24 h. to lyse the cells. Samples will be thawed at room temperature and a second conductivity measurement will be obtained (total electrolyte conductivity). Tissue electrolyte leakage will be expressed as a percentage of the total conductivity (Lu et al., 2018).
HPLC analysis of selected samples
Selected microgreen samples which are identified as the least supportive of inoculated bacteria on plant tissue will be explored further using Reversed Phase High Performance Liquid Chromatography (RP-HPLC) to produce compositional fingerprints of polyphenolic compounds. The identification and quantification of phenolic compounds of microgreen methanolic extracts will be determined following the protocol developed by Francisco and Resurreccion. Briefly, a C18 column fitted with diode array detection at 250 and 320, 280 and 370, and 306 nm for phenolic acids, flavonoids and stilbenes, respectively will be utilized. The mobile phases will consist of 0.1% formic acid in water and 0.1% formic acid in 100% acetonitrile (Francisco and Resurreccion, 2009). Phytocompounds will de identified by their elution time and the quantification will be determined by internal standards of chosen target compounds.
Statistical Analysis
All analyses will be conducted in JMP Pro 15.2 (SAS, Cary, NC).
In order to control for type I and type II errors, experiments will be repeated at least twice using a sample size between 4-8 to prevent variability within the system. Bacterial count data generated from microbiological assays will be log transformed and interpreted as log CFU g-1 reduction for inoculation experiments. One-way analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) or Student’s t-test for pairwise comparisons will be employed on control versus elicited data to determine treatment effects (abiotic stress elicitation) on microbiological persistence and physicochemical changes.
Multivariate statistical methods, such as Multidimensional Scaling (MDS) or Principal Component Analysis (PCA) will be employed to analyse phytochemical data to determine differences between treatments and secondary metabolite characteristics.
Education & Outreach Activities and Participation Summary
Participation Summary:
Examples of the outreach outlets I will contact to arrange opportunities to disseminate the findings of my project will include:
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Produce Bites Podcast
Michigan State University Extension Educators, Michigan Produce Safety Technicians, fruit and vegetable growers, and others in the industry talk about produce safety related issues. I will contact the podcast authors, Phil Tocco, Micah Hutchison and Amanda Kinchla, to discuss the opportunity for me to be a guest on their podcast to discuss the findings of my research generated from this project. -
Online media
I will reach out to the curators of Foodsafety Clearing House from the University of Vermont to provide resources to be uploaded to the curated collection of produce safety and preventive controls for human food related resources. This website provides searchable resources available to anyone to view free of charge. -
Newsletter article
Carroll Allen, food safety education extension agent creates and curates a newsletter as part of the offerings from The Plant Science Food Safety Group which goes out to professionals, growers, students and faculty across the Northeast Region. I will contact Carroll to discuss providing content for her newsletter. -
Presentations
I will present at one of the food safety trainings conducted in Maryland between February and April (organized by the Food Safety Education Team in my Department) or at other local workshop for farmers. I will work with UMD extension agents and growers in my network to discuss ways to cast a wider net among local and regional microgreen growers. -
Scientific outreach
I will present the findings from my project at the International Association for Food Protection annual meeting. This is the largest Food Safety Meeting in the United States and attracts academics, industry professionals and government officials who work in all aspects of food production, handling, processing and policy making. - Scientific publications
I will submit at least one manuscript to an open access peer reviewed journal such as Frontiers in Microbiology, Journal of Food Protection or Food Science & Nutrition to make sure my research reaches the scientific community.