Moving toward long-duration missions in Space, it will be vital to grow healthy plant food intended for astronaut consumption and to regenerate resources during spaceflights and extraterrestrial colonization (Dueck et al., 2016; De Pascale et al., 2021). Even more, considering that pre-packaged food has been shown to not meet the shelf-life requirements of long-duration scenarios (Cooper et al., 2017; Carillo et al., 2020), it is mandatory to develop Bio-regenerative Life-Support Systems (BLSS) that will ultimately replace also the need for food resupply from Earth (Dueck et al., 2016). MELiSSA (Micro-Ecological Life Support System Alternative), the European Space Agency program, seeks to develop BLSSs with plant compartments to support human life for long crewed Space missions (Carillo et al., 2020). BLSSs implementation starts with small plant growth units (i.e., salad machines) to produce some fresh foods to supplement stowed food for astronauts onboard the International Space Station or during early lunar missions. The cultivation area will then be expanded for longer duration lunar missions, which will also provide an opportunity to test systems and procedures for Mars missions, where BLSSs would play a more crucial role (Wheeler, 2017). In the last decades, various plant cultivation systems in Space have been developed such as Veggie and the Advanced Plant Habitat (APH) (Zabel et al., 2016). Specifically, Veggie is a small plant growth chamber designed by NASA to produce fresh vegetables onboard the International Space Station (ISS) and is currently providing fresh food as supplement for astronaut diet (Massa et al., 2016). APH is also currently working on the ISS with a higher technological level compared to Veggie, for purposes more related to research studies on plant growth in Space conditions. In this framework, the Italian Space Agency (ASI), in collaboration with the University of Naples Federico II, the University of Tor Vegata, the Italian National Research Council (CNR) and the “Agenzia nazionale per le nuove tecnologie’’ (ENEA), is currently developing novel systems and technologies intended for microgreens production in Space.

Although much effort has been devoted to the design and development of cultivation systems in Space, only a few studies provide a selection of the best species/cultivars to be used in flight experiments or cultivations in Space. For example, De Micco et al. (2012) developed an objective and repeatable methodology for the selection of soybean cultivars to be used in BLSSs. A specific algorithm considering the relevance of the different plant traits was elaborated to rank the soybean cultivars and to identify the best ones for subsequent cultivation trials. Similarly, Massa et al. (2015) reported a selection of leafy greens vegetables as supplement astronaut diet to be grown in the Veggie growth chamber onboard the ISS by using a ranking method based on growth, nutrition, and organoleptic parameters. Dueck et al. (2016) also reported a selection methodology for choosing Space crops using literature data and information provided by growers on horticultural aspects, human nutrition, and psychology, and precluding the selection of crops that are not ready to eat. A further study by Aronne et al. (2020) was aimed to select the best species among 50 candidate crops to conduct an experiment onboard the ISS using refurbished hardware. Authors applied subsequent inclusion/exclusion criteria to select the biological system that best fit to the hardware and timing expected during the prelaunch, launch and flight operations (Aronne et al., 2020).

Overall, the selection of plant species for cultivation in Space is effort demanding since there are specific constraints and Space factors to be addressed in the different mission scenarios. Microgreens are functional food crops recently valued for their flavor and phytonutrient content and have been proposed to produce fresh food to supplement stowed food for astronauts during spaceflight or onboard orbital platforms since they can be harvested and eaten fresh by crew members (Kyriacou et al., 2016; Kyriacou et al., 2017). As candidate crops for plant production system in Space, microgreens represent a source of vitamins, minerals and antioxidant which are often lacking in non-perishable or frozen food but fundamental to maintain a healthy status of the crew members and counteract negative effects due to the spaceflight environment (Gómez et al., 2021). Microgreens may be considered a resilient phytochemical factory with the potential to provide a significant nutritional addition in just a small serving size so they can be used to meet dietary needs of the astronauts by reaching targeted daily intakes of specific nutraceuticals and reducing the need for large storage volumes and the related costs. Overall, the plant species to be used for microgreens production is the first factor influencing the availability and the content level of specific phytonutrients (Teng et al., 2022). Candidate genotypes of microgreens mostly include species from the Brassicaceae, Asteraceae, Chenopodiaceae, Lamiaceae, Apiaceae, Amarillydaceae, Amaranthaceae, and Cucurbitaceae families (Caracciolo et al., 2020; Ebert, 2022).

As explained by Kyriacou et al. (2021), microgreens other than being a gastronomic novelty, they gather appreciable characteristics such as compact shape, fast production, and confined space requirement. Moreover, microgreens easily fit the criteria cited for Space farming such as plant size, harvest index, light requirement, nutritional value (Dueck et al., 2016; Carillo et al., 2020), volume/energy efficiency, disease resistance, and handling time (Dueck et al., 2016). As reported by Carillo et al. (2020), the choice of the crop should consider its amount of biologically active compounds that contribute to psychological wellbeing and health promotion of astronauts. However, to the authors’ knowledge no previous studies have reported a selection of microgreens to be used as functional Space food, and there is a need to identify those species/cultivars with satisfactory yield and food quality. In this context, we developed a selection method for microgreens species, based on an objective comparison of numerous genotypes aiming to identify those with the best growth and nutritional characteristics for cultivation in Space. Herein we describe the algorithm used to generate the ranking list by adopting a two-level selection based on growth and nutritional parameters, and their prioritization.

Microgreens generally have a higher content of phytonutrients and lower nitrates compared to their mature-leaf counterparts, thus resulting in high quality functional food (Xiao et al., 2012; Pinto et al., 2015). Indeed, microgreens provide a significant quantity of phytonutrients that can help astronauts in coping with the stressful conditions of the Space environment (Kyriacou et al., 2016; Kyriacou et al., 2017).

Since there are many species that have been proposed for microgreens production (Kyriacou et al., 2016), we developed a method based on an algorithm with the aim of objectively select those microgreens species most suitable for cultivation in Space according to productivity and phytonutrient profile criteria. The selection method consisted of two subsequent rankings elaborated using first literature data and, successively, using data obtained with cultivation tests specifically conducted for this study. The first ranking list included 39 plant species commonly used for the production of microgreens according to the reference literature. Most of the species herein considered belong to the family Brassicaceae (26 out of 39) whereas the remaining species belong to Amaranthaceae (1), Apiaceae (2), Chenopodiaceae (3), Fabaceae (1), Lamiaceae (2), Malvaceae (1), Poaceae (1), Polygonaceae (2). In this regard, although there is a growing interest in microgreens and in the use of new microgreens varieties, no studies presented a comparison between species for the selection of microgreens genotypes based on productivity and phytonutrient profile criteria. Indeed, most studies on microgreens investigated the effects of environmental conditions or cultivation techniques using one or a few species. For this study, in the first selection phase, we focused on documents providing a comparison between different microgreens species grown under standard conditions. With this approach we gathered literature data on horticultural and nutritional parameters to be included in the working matrix for a comparative analysis between the different species based on the prioritization of the different parameters.

A similar approach based on parameter prioritization has been used by De Micco et al. (2012) and Massa et al. (2015), but with differences from our study mostly due to the specific aims of the selection. Indeed, prioritization of parameters can have a large effect on the final scores of the different species. Nevertheless, the method herein reported provides the possibility of modeling species selection by adjusting the prioritization of productivity and phytonutrient parameters according to specific objectives. In our case, the selection process aimed to ensure that the selected plant food had elevated nutraceutical value.

After the first selection phase we identified six top species with the highest score that were then used for germination and cultivation tests. This restricted list of microgreens allowed us to study each species with more details introducing new parameters to be measured and implemented in the working matrix for the elaboration of the ranking list. For this second selection phase, we chose to include in the working matrix data of “Phytonutrients” parameters expressed as the daily production per m². This approach allowed us to better identify those species with higher production efficiency of phytonutrients, considering there are constraints regarding volume available for cultivation in Space. Furthermore, data on daily production of phytonutrients per m² are useful to estimate the surface to be cultivated according to the growth period of each species and the astronauts’ daily dietary requirements.

Ghoora et al. (2020) investigated the contribution of nutrients (NC), compared to the nutrient claims of FDA (U.S. Food and Drug Administration), of ten culinary microgreens with a consumption of 85g (USDA reference amount customarily consumed of green leafy vegetables) of fresh biomass. Authors pointed out that the vitamin’s NC ranged between 28.2–116.4%, 28.5–332% and 24.3–71.8% respectively for ascorbic acid (vitamin C), α-tocopherol (vitamin E) and β-carotene (pro-vitamin A). Since microgreens can efficiently fulfill the daily intakes of vitamins, along with a great contribution in antioxidants, the prioritization of different categories favored the “Phytonutrients” category. However, growth characteristics that describe the general productivity efficiency of the crop, such as yield, dry weight, and growth period, cannot be understated since they impact the overall size of the cropping system, thereby affecting key aspects of Space facilities as total resources used and mass at launch. For these reasons, we assigned the second highest priority level to the “Growth” category. Pinto et al. (2015) investigated the potential contribution of lettuce microgreens to supply the recommended daily intake of minerals. In the study by Pinto et al. (2015), the Ca, P, Mg, K, and Na Estimated Daily Intake (EDI), based on a consumption of 25 g of microgreens, respectively represented only the 3.48, 3.10, 1.76, 1.75, 0.83% of the EFSA RDAs (recommended dietary allowances) and AIs (adequate intakes) mean for men and women. In the study by Ghoora et al. (2020) the NC of macro-elements such as Ca, Mg, K, Na reached respectively 6.2%, 14.4%, 8.7% and 8.4%. In our view, producing food rich in minerals via microgreens is less relevant than producing bioactive molecules, since minerals are stable during conservation. Furthermore, even if microgreens can contribute to the mineral supply, the achievement of the recommended daily intake solely through the consumption of microgreens is difficult, while it can be easily obtained using other food sources. Therefore, we assigned to “Elements” category the lowest priority level.

Maintaining proper antioxidant health is a priority for astronauts during spaceflight. Several factors including ionizing radiation, microgravity, exercise and even diet are a source of ROS (Powers and Jackson, 2008; Azzam et al., 2012; Zwart et al., 2013; Ran et al., 2016). The role of phytochemicals such as vitamins and polyphenols in maintaining antioxidant health have been reviewed by several authors (Smith et al., 2014; Heer et al., 2020; Gómez et al., 2021; Kulkarni et al., 2022). The protective effect of Vitamin A, C, and E against γ-rays was investigated in vitro and the potential to reduce DNA damages before and after irradiation was shown (Konopacka and Rzeszowska-Wolny, 2001). In addition, the radioprotective potential of both vitamin A and C were successfully tested in mice (Mortazavi et al., 2015; Changizi et al., 2019). These compounds also have a synergistic effect by enhancing the antioxidant potential when administered together (Tauler et al., 2002). Moreover, vitamin A, C and E are involved in several fundamental physiological processes in the human body such as immune function, bone health, eye health, and are related to the body’s response to the space environment (Smith et al., 2014).

In order to give priority to a specific nutraceutical component useful for the astronauts’ diet, it is necessary to evaluate the deficiencies that may occur during the spaceflight. Smith et al. (2005) analyzed the blood levels of vitamins and minerals in 11 astronauts before and after 128-195 day of spaceflight on board of the International Space Station. They pointed out that vitamin E was affected by the spaceflight since γ-tocopherol significantly decreased after landing. However, no losses were detected for β-carotene, whereas the ascorbate status has not yet been investigated. As regards the stability of the nutraceuticals in Space-stored food, while vitamin A declines but with sufficient concentrations kept after 3 years of storage, the vitamin C content in most fruit products degrades between 32 and 83% (Cooper et al., 2017). Moreover, Vitamin E has been found to be the most sensitive to irradiation during storage (Watkins et al., 2022). According to the aforementioned criteria, the highest priority among the parameters of the “Phytonutrient” category, in the first selection phase, was assigned to ascorbic acid, tocopherols, β-carotene and polyphenols.

Bone loss is a significant health concern associated with long-duration Space missions and the microgravity environment (Leblanc et al., 2007). The rate of bone mineral loss during spaceflight is estimated to be between 0.5-1% per month (Weaver, 2000). Net rate of bone calcium loss in Mir and Skylab missions resulted in approximately 250 mg every day. Adequate intake of calcium-rich foods or supplements will play a significant role in ensuring the skeletal health of astronauts during prolonged Space missions (Smith et al., 2005). In this regard, the highest priority level for parameters included in the “Element” category was assigned to calcium.

As regards iron and nitrate parameters included in the “Element” category, data were inverted in order to valorize microgreens genotypes with low accumulation. Low iron and nitrate accumulator species are desirable when selecting crops for BLSSs. The International Space Station (ISS) food system has high levels of dietary iron. The current iron U.S. Dietary Reference Intake (DRI) is 8-10 mg d^-1 and crew members on long-duration Space missions have been observed to consume excessive amounts of iron (20-25 mg iron d^-1) (Smith and Zwart, 2008). In addition, physiological changes during spaceflight led to an accumulation of iron in storage tissues which can act as an oxidizing agent inducing damages at DNA level (Glei et al., 2002). In addition, high amounts of nitrate can cause serious health problems for humans. The accumulation of nitrate in leafy crops has been largely reported (Maynard et al., 1976; Colla et al., 2018). The European Commission Regulation No 1258/2011 establishes the limits for nitrate content in vegetable productions based on species and growing conditions. Importantly, despite microgreens are young plantlets with low incidence of nitrate accumulation (Pinto et al., 2015), the cultivation practices could influence the content of this compound (Bulgari et al., 2021; Kyriacou et al., 2021).

Overall, according to parameters prioritization discussed above and the algorithm calculation, radish and savoy cabbage are the most suitable microgreens genotypes to be used for cultivation in Space. Nevertheless, with the possibility to adjust the algorithm calculation based on productivity and phytonutrient profile criteria, further research deepening the effects of microgravity on human’s metabolism, and the related countermeasures based on specific diets, can provide data and insights to optimize the selection of plant food species intended for cultivation in Space. Horticultural research, on the other hand, can provide strategies for maximizing the production of certain characteristics.

The selection method of microgreens developed in this study uses estimated scores based on prioritization of parameters and is preferred over a simple listing of plants since it has an argued basis for the given priorities. The method herein reported consists of two subsequent rankings elaborated using first literature data and, successively, using data obtained with cultivation tests specifically conducted for this study. The method also provides the possibility to adjust the algorithm calculation and can be repeated for further selection including new crops or new data. Furthermore, the approach of grouping the different parameters into different categories allow to easily assign higher or lower priorities related to other parameters included in the same group, instead of prioritizing parameters all together. So far, the flexibility of this selection method provides potential adaptability for selecting new cultivars and/or including other criteria relevant to support astronauts’ diet for different aims such as growth chambers on orbital platforms, long-duration travels, or extraterrestrial colonization.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

LI and GA conceived the paper. All authors contributed to general planning and data analyses, LI, CN and GP developed a first draft of the manuscript. All authors provided constructive discussions, revised, and approved the submitted version of the article.