Microgreens nutrient content

Vegetables are important in the diet to prevent and control diseases in humans (Vauzour et al., Reference Vauzour, Rodriguez-Mateos, Corona, Oruna-Concha and Spencer2010; Sedani et al., Reference Sedani, Pardeshi, Bhad and Nimkarde2018; Gonzali and Perata, Reference Gonzali and Perata2020; Rani et al., Reference Rani, Arfat, Aziz, Ali, Ahmed, Asim, Rashid and Hocart2021). Millions of people around the globe suffer from chronic diseases such as diabetes and cardiovascular diseases (Choe et al., Reference Choe, Yu and Wang2018; Powell-Wiley et al., Reference Powell-Wiley, Poirier, Burke, Després, Gordon-Larsen, Lavie, Lear, Ndumele, Neeland, Sanders and St-Onge2021). According to WHO (2003a), insufficient fruit and vegetable intake was estimated to cause about 2.7 million deaths annually and was among the top 10 risk factors contributing to the yearly death rate. Consequently, the WHO asked countries to carry out targeted campaigns to increase the consumption of fruits and vegetables (WHO, 2003b). Furthermore, statistics from the Health Service Executive (HSE) (2016), approximately 37% of people in Ireland have normal body weight, 37% are overweight and a further 23% are obese. Therefore, the HSE Healthy Eating Guidelines for Irish people over the age of five years recommends that Irish people should eat up to seven servings of fruits and vegetables a day (HSE, 2016), and the WHO and FAO suggested that the global vegetable intake be increased to five servings a day (WHO, 2003b). Consequently, Microgreens have been suggested as a vegetable alternative that would help reduce global food insecurity, and complications arising from malnutrition (see Fig. 3 below)

Fig. 3. Microgreens, the Answer to global food insecurity, malnutrition and food safety.

Although the nutrient levels found in some mature vegetables are less than the adult recommended daily allowance (RDA) [Table 1 (Choe et al., Reference Choe, Yu and Wang2018)], many studies have reported that some microgreens contain more of these compounds and other health-promoting micronutrients than their mature counterparts (Johnson et al., Reference Johnson, Prenni, Heuberger, Isweiri, Chaparro, Newman, Uchanski, Omerigic, Michell, Bunning and Foster2021; Teng et al., Reference Teng, Liao and Wang2021). However, the exact quantity per microgreen serving is not clearly stated. It will be important to ascertain how much of this compound is contained in a serving of microgreens.

Table 1. Five mature vegetables and their microgreens counterparts were assessed for vitamin concentrations (Choe et al., Reference Choe, Yu and Wang2018)

Measurements are in micrograms/100 g fresh weight (FW) for Vitamin K and milligrams/100 grams fresh weight (FW) for all other vitamins. Microgreens show higher concentrations than their mature counterparts.

The following vitamins and minerals have been identified in some microgreens.

Phylloquinone

Phylloquinone is vital for blood coagulation and bone restoration (Palermo et al., Reference Palermo, Tuccinardi, D'Onofrio, Watanabe, Maggi, Maurizi, Greto, Buzzetti, Napoli, Pozzilli and Manfrini2017; Beato et al., Reference Beato, Toledo-Solís and Fernández2020), and has been identified in some microgreen species (Xiao et al., Reference Xiao, Rausch, Luo, Sun, Yu, Wang, Chen, Yu and Stommel2019). A study from the USDA National Nutrient Database (2011), reported that red cabbage microgreens had a 6-fold higher concentration of vitamin C than previously published data for mature red cabbage (24.4 mg 100 g FW⁻ ) 17 and 2.6 times greater (57.0 mg 100 g FW⁻¹) than that recorded in the USDA National Nutrient Database for Standard Reference, Release 24. In addition, garnet amaranth had a much higher total ascorbic acid (TAA) concentration than their mature counterparts, (131.6 mg 100 g FW⁻¹ compared to 11.6–45.3 mg 100 g FW⁻¹) (Xiao et al., Reference Xiao, Lester, Luo and Wang2012). Vitamin E in the α and γ forms were also found to be more abundant in the green daikon radish, cilantro, opal radish and pepper cress microgreens than in their mature counterparts in Xiao et al. (Reference Xiao, Lester, Luo and Wang2012).

Carotenoids

The antioxidant β-carotene, known for its critical roles including vision development (Sies and Stahl, Reference Sies and Stahl1995), abounds in some species of microgreens (Kyriacou et al., Reference Kyriacou, Rouphael, Di Gioia, Kyratzis, Serio, Renna, De Pascale and Santamaria2016). Twenty-five cabbage microgreens were studied, in which wide ranges of β-carotene concentrations were detected and compared with that of their mature counterpart (Xiao et al., Reference Xiao, Lester, Luo and Wang2012). Red cabbage microgreens had approximately 260—fold more β-carotene concentration than mature red cabbage (Xiao et al., Reference Xiao, Lester, Luo and Wang2012). Another study by Xiao et al. (Reference Xiao, Rausch, Luo, Sun, Yu, Wang, Chen, Yu and Stommel2019) reported that broccoli and cauliflower microgreens had higher β-carotene concentrations (6—fold) than the mature florets.

Furthermore, it has been documented that, two of the seven carotenoids found in human, lutein and zeaxanthin found in the blood (Watson and Preedy, Reference Watson and Preedy2010), and the only carotenoids present in the retina and lens of the eye that protects the eye from the UV rays and works as an antioxidant (Bone et al., Reference Bone, Landrum, Friedes, Gomez, Kilburn, Menendez, Vidal and Wang1997), abound in microgreens (Xiao et al., Reference Xiao, Rausch, Luo, Sun, Yu, Wang, Chen, Yu and Stommel2019). In the latter study, which examined the concentration of lutein and zeaxanthin in some microgreens and their mature counterparts, microgreens had 11.2-fold and 28.6-fold higher lutein/zeaxanthin concentrations than the mature plants. In contrast, Klopsch et al. (Reference Klopsch, Baldermann, Voss, Rohn, Schreiner and Neugart2018) reported that mature leaves of pea and lupin had higher carotenoid concentrations than pea and lupin microgreens. In another study, Kale and mustard microgreens were noted to have lower ascorbic acid than their mature counterparts (de la Fuente et al., Reference de la Fuente, López-García, Máñez, Alegría, Barberá and Cilla2019). These contrasting results suggest that the abundance of bioactive compounds in a specific plant life stage is species dependent.

Polyphenol and GSL

Polyphenol and glucosinolates (GSL) which reduce the risk of certain cancer developments (Odongo et al., Reference Odongo, Schlotz, Herz, Hanschen, Baldermann, Neugart, Trierweiler, Frommherz, Franz, Ngwene and Luvonga2017) have recently been reported in kale and broccoli microgreens (Liu et al., Reference Liu, Shi, Wan, Pham, Zhang, Sun, Yu, Luo, Wang and Chen2021). The protection offered by brassica vegetables against cancer is partly related to their relatively high content of GSL (Odongo et al., Reference Odongo, Schlotz, Herz, Hanschen, Baldermann, Neugart, Trierweiler, Frommherz, Franz, Ngwene and Luvonga2017). However, Liu et al. (Reference Liu, Shi, Wan, Pham, Zhang, Sun, Yu, Luo, Wang and Chen2021) did not compare the polyphenol and GSL content of kale and broccoli microgreens to their mature counterpart. Though these studies show that some microgreens contain more of certain nutrients than their mature counterparts, it is not documented whether the environmental growing conditions of the microgreens or the nutrient extraction methods were taken into consideration. According to Giménez et al. (Reference Giménez, Martínez-Ballesta, Egea-Gilabert, Gómez, Artés-Hernández, Pennisi, Orsini, Crepaldi and Fernández2021) and El-Nakhel et al. (Reference El-Nakhel, Pannico, Graziani, Kyriacou, Gaspari, Ritieni, De Pascale and Rouphael2021), environmental conditions, as well as cultivation methods (Liu et al., Reference Liu, Shi, Wan, Pham, Zhang, Sun, Yu, Luo, Wang and Chen2021), play a pivotal role in nutrient availability in plants including microgreens. In the latter study, the phytochemical contents of kale and broccoli microgreens grown under windowsill conditions were significantly lower compared to those grown under chamber conditions.

Health benefits of microgreens

Foods are necessary to sustain human growth, development and survival by supplying necessary nutrients to the body including vitamins and minerals to enable it to ward off diseases (De Filippo et al., Reference de Filippo, Meldrum, Samuel, Tuyet, Kennedy, Adeyemi, Ngothiha, Wertheim-Heck, Talsma, Shittu and Do2021). Many people suffer from chronic diseases such as diabetes, high blood pressure and arteritis (Anderson and Durstine, Reference Anderson and Durstine2019). A plethora of studies including Choe et al. (Reference Choe, Yu and Wang2018) and Martinon et al. (Reference Martinon, Fraticelli, Giboreau, Dussart, Bourgeois and Carrouel2021) suggest that the consumption of vegetables can significantly reduce the risk of many chronic diseases. However, the nutrients most influential in improving human health are not always adequately found in regular mature vegetables (Choe et al., Reference Choe, Yu and Wang2018). Providentially, many microgreens have been found to contain an abundance of these nutrients and health-promoting micronutrients compared to their mature counterparts (Sies and Stahl, Reference Sies and Stahl1995; Kyriacou et al., Reference Kyriacou, Rouphael, Di Gioia, Kyratzis, Serio, Renna, De Pascale and Santamaria2016; Choe et al., Reference Choe, Yu and Wang2018; Johnson et al., Reference Johnson, Prenni, Heuberger, Isweiri, Chaparro, Newman, Uchanski, Omerigic, Michell, Bunning and Foster2021; Teng et al., Reference Teng, Liao and Wang2021).

Even though research on microgreens is still in its infancy, their health benefits have been published through research in microgreen nutrition. The consumption of microgreens can prevent or alleviate some health problems including inflammation of the immune system pathways, obesity (Castelão-Baptista et al., Reference Castelão-Baptista, Barros, Martins, Rosa and Sardão2021), cardiovascular disease, type 2 diabetes, cancers and modulation of the gut microbiome (Machlin and Bendich, Reference Machlin and Bendich1987). Other studies have reported that their abundance in vitamins C, E, K, carotenoids and polyphenols, has the potential to regulate cellular pathways, modulating inflammation (Choe et al., Reference Choe, Yu and Wang2018; Begum et al., Reference Begum, Howlader, Mamun-Or-Rashid, Rafiquzzaman, Ashraf, Albadrani, Sayed, Peluso, Abdel-Daim and Uddin2021; Sirajunnisa et al., Reference Sirajunnisa, Surendhiran, Kozani, Kozani, Hamidi, Cabrera-Barjas and Delattre2021), as well as the generation and scavenging of reactive oxygen species (Finley et al., Reference Finley, Kong, Hintze, Jeffery, Ji and Lei2011).

Exhibits indicate that microgreen supplementation of 0.4 mL 20 g (kg body weight)^−k broccoli microgreens juice to male rats that had been fed a high-fat diet could reduce weight gain associated with a high-fat diet (Li et al., Reference Li, Lalk and Bi2021a and Reference Li, Tian, Wang, Liu, Wang and Lu2021b). In an earlier study, microgreen supplementation significantly lowered low-density lipoprotein cholesterol levels in animals fed with a high-fat diet and reduced hepatic cholesterol ester, triacylglycerol levels and expression of inflammatory cytokines in the liver (Huang et al., Reference Huang, Jiang, Xiao, Yu, Pham, Sun, Chen, Yokoyama, Yu, Luo and Wang2016). It is therefore suggested that microgreens may reduce weight gain and cholesterol metabolism, thereby preventing hypercholesterolemia and subsequent cardiovascular diseases in humans (Huang et al., Reference Huang, Jiang, Xiao, Yu, Pham, Sun, Chen, Yokoyama, Yu, Luo and Wang2016). More recent studies also found that microgreens' flavonoid content, such as quercetin, modulates weight gain to prevent obesity and other complications (Rayalam et al., Reference Rayalam, Dellafera and Baile2008; Castelão-Baptista et al., Reference Castelão-Baptista, Barros, Martins, Rosa and Sardão2021).

Another facet of the health benefit of microgreens is their ability to aid in the prevention of certain cancers (Maina et al., Reference Maina, Ryu, Cho, Jung, Park, Nho, Bakari, Misinzo, Jung, Yang and Kim2021; Truzzi et al., Reference Truzzi, Whittaker, Roncuzzi, Saltari, Levesque and Dinelli2021). It was estimated that over 43,000 people were diagnosed with cancer in Ireland in 2019 (Irish cancer society, 2020). Compared with other diseases, the cause of cancer is not yet well understood and there are few effective treatments (Irish cancer society, 2020). Therefore, research into cancer prevention would be of vital importance. It is suggested that a third of all cancers could be prevented by eating a diet rich in fruits and vegetables and low in fat and calories (Donaldson, Reference Donaldson2004; Bertoia et al., Reference Bertoia, Mukamal, Cahill, Hou, Ludwig, Mozaffarian, Willett, Hu and Rimm2016).

Although the preventative mechanism is not yet clear (Liu, Reference Liu2004), studies suggest that bioactive compounds may have protective effects against a variety of cancers, such as breast (Liu and Lv, Reference Liu and Lv2013), prostate (Liu et al., Reference Liu, Mao, Cao and Xie2012) and colon cancers (Wu et al., Reference Wu, Yang, Wang, Han and Xiang2013). According to Kopsell and Sams (Reference Kopsell and Sams2013) and Poiroux-Gonord et al. (Reference Poiroux-Gonord, Bidel, Fanciullino, Gautier, Lauri-Lopez and Urban2010), brassica vegetables (such as broccoli, cabbage and radish) contain high levels of cancer-fighting glycosylates, carotenoids, phytochemicals, vitamins and minerals. Bradfield and Bjeldanes (Reference Bradfield and Bjeldanes1987) and Minich and Bland, Reference Minich and Bland2007 also found that cruciferous microgreens are rich in indole-3-carbinol (I3C), indole-3-acetonitrile (IAN) and 3,3′-diindolylmethane (133′) which possess anticarcinogenic properties. They further explained that the activation of Phase I, and II xenobiotic 14 metabolizing enzymes may be a potential mechanism by which microgreens protect against cancers. Microgreens may also aid in the regulation of the sex-steroid hormone-mediated pathway by protecting against hormone-dependent cancers such as breast and prostate cancer (Bradfield and Bjeldanes, Reference Bradfield and Bjeldanes1987).

According to Ni et al. (Reference Ni, Li and Panagiotou2015) and Li et al. (Reference Li, Lalk and Bi2021a and Reference Li, Tian, Wang, Liu, Wang and Lu2021b), plants contain 400 compounds, including kaempferol, quercetin and apigenin, which are associated with 609 microbial targets in the gut suggesting that the consumption of microgreens can also modulate the gut microbiome. All these health benefits have been associated with the consumption of microgreens, but because the benefits are still species-dependent and, in addition, the gut pH and the impact of other external factors can reduce their effects (Kong and Singh, Reference Kong and Singh2008; Li et al., Reference Li, Hu and McClements2011), a study to ascertain the synergetic effects of the above parameters on the bioavailability of these nutrients would be of importance.

Furthermore, some nutrients may not be bioavailable due to the presence in microgreens of antinutrient factors such as phytates, tannins, trypsin inhibitors and lectins, which have been reported in chickpea, legumes, cotton seeds and leaves and seeds of other species (Francis et al., Reference Francis, Makkar and Becker2001) and amaranth species (Nana et al., Reference Nana, Hilou, Millogo and Nacoulma2012). Moreover, these anti-nutrient chemicals that have been reported to be harmful to some organisms, including humans (Friedman and Brandon, Reference Friedman and Brandon2001; Gilani et al., Reference Gilani, Xiao and Cockell2012), have not yet been fully profiled in microgreens. This notwithstanding studies on how to decrease these antinutrient factors in microgreens are being conducted. Yang et al. (Reference Yang, Guo, Jin, Shen, Zhou and Gu2015) in their study used zinc sulfate (ZnSO₄) as a sulfur (S)-source and found that it stimulated the sulforaphane formation in broccoli microgreens by enhancing myrosinase activity and gene expression related to glucoraphanin biosynthesis. More research into technologies to enhance nutrient availability in microgreens is very important.

Most grown as microgreens species

Although these young vegetables are nutritive (Ghoora et al., Reference Ghoora, Babu and Srividya2020; Teng et al., Reference Teng, Liao and Wang2021) not all species should be eaten as it has been reported that some species such as potatoes, tomatoes, eggplants, peppers and potentially rhubarb are toxic if eaten when immature (Jagatheeswari, Reference Jagatheeswari2014). The consumption of these plants has been associated with symptoms including diarrhea, fever or hypothermia, headache, breathing disorders, abdominal pain, vomiting and nervous problems (Jagatheeswari, Reference Jagatheeswari2014). This area of research in microgreens should be considered to identify those phytochemicals capable of causing allergenic reactions in some consumers. Fortunately, several species have been identified to be safe for consumption as microgreens (Table 2).

Table 2. Some commercially grown microgreens (Xiao et al., Reference Xiao, Lester, Luo and Wang2012)

Fast-growing vegetables (7 to 14 days): cabbage, corn, cress, kale, kohlrabi, mustard, radish Slow-growing vegetables (15 to 25 days): amaranth, arugula, beet, carrot, Swiss chard, scallion Slow-growing herbs (15 to 30 days): anise, basil, cilantro, dill, fennel, parsley, saltwort, shisho and sorrel.

Microgreens production

Because of their high nutritional content, microgreens are a highly sought-after and highly-priced commodity (Folta, Reference Folta2019). This together with their short production cycle (Xiao et al., Reference Xiao, Lester, Luo and Wang2012; Folta, Reference Folta2019) is attracting many growers (Kyriacou et al., Reference Kyriacou, Rouphael, Di Gioia, Kyratzis, Serio, Renna, De Pascale and Santamaria2016). They can be produced indoors in a protected environment, such as a greenhouse or high tunnel (Singh, Reference Singh2018) using the vertical agricultural technique which is a significant part of the global push towards climate-controlled agriculture (Benke and Tomkins, Reference Benke and Tomkins2017) provided the growing conditions are right and the facility meets the local standards and regulations for food safety as stipulated in the factsheet for Commercial Microgreens: Food Safety and Third-party Certifications, Agdex 268/089-1 [Alberta Ag-Info Center, 268/18-1 (2018)].

Most commercial growers according to Alberta Ag-Info Center, 268/18-1 (2018), use either of the following methods to grow microgreens:

• • Soil-based (garden beds, raised planters).

• • Various forms of hydroponic crop production; substrate culture (a pH-neutral media) solution culture, (nutrient solutions) and Nutrient Film Technique, (where the roots are grown in a constant flow of nutrient-rich water).

• • Aeroponics.

Considering the various factors that may affect microgreens production, a good knowledge of the environment and requirements is important to understand what the seeds need to germinate, and what seedlings to grow to maturity. Consequently, optimizing the environmental factors necessary for microgreens production and in addition, those which would discourage the growth of food borne-disease-causing pathogens, and improve the self-life of microgreens bound for the markets is pivotal

Fertilizer application rate

Generally, microgreens do not require fertilizers (Treadwell et al., Reference Treadwell, Hochmuth, Landrum and Laughlin2020). Nevertheless, to improve yield and to stimulate growth in slow-growing species, seedlings can be fertigated with water containing 20N-8.7P-16.6 K (Peters® Professional 20-20-20 General Purpose; ICL Specialty Fertilizers containing (wt/wt) 0.05% Mg, 0.05% Fe, 0.025% Mn, 0.013% boron (B), 0.013% copper (Cu), 0.005%; 16 molybdenum (Mo) and 0.025% Zn,) at a rate of 100 mg L⁻¹ N four days after planting. Li et al. (Reference Li, Lalk and Bi2021a and Reference Li, Tian, Wang, Liu, Wang and Lu2021b) and Kou et al. (Reference Kou, Yang, Luo, Liu, Huang and Codling2014) also documented the increase of microgreen biomass, and calcium content, and reduced microbial growth during storage by spraying the crop with calcium chloride preharvest. Microorganisms have also been used for the same purpose. For example, in a study carried out by Briatia et al. (Reference Briatia, Jomduang, Park, Lumyong, Kanpiengjai and Khanongnuch2017), where seed and soil were inoculated with Herbaspirillum sp. ST-B2, sprouts, and microgreen buckwheat species yields were increased. In a more recent study by Dembele (Reference Dembele2021), bio-stimulants were shown to increase the biomass of some microgreen species.

However, the use of fertilizer has been linked to bacterial colonization of microgreens as well as soils (Jechalke et al., Reference Jechalke, Schierstaedt, Becker, Flemer, Grosch, Smalla and Schikora2019). Another study by Reed et al. (Reference Reed, Ferreira, Bell, Brown and Zheng2018) also noted that high levels of E. coli were heavily persistent in manure-fertilized soils for over three months. As such, microgreens grown in fertilized media are at a higher risk of microbial contamination compared to those grown in non-fertilized media. As a preventative measure, the FAO recommends the harvesting of organic crops whose edible portions are exposed to soil be done at least 120 days post-application of non-composted manure (Reed et al., Reference Reed, Ferreira, Bell, Brown and Zheng2018).

Photoperiods

In the past, growers supplemented natural lights with gas-discharged lamps (GDL) but these days, light-emitting diodes (LEDs) are becoming crucial in the horticulture industry as technologies to replace natural light are advancing (Ajdanian et al., Reference Ajdanian, Babaei and Aroiee2019). The LED light is a better option because it gives a more uniform light distribution, is more flexible, and is more eco-friendly than GDLs (Morrow, Reference Morrow2008).

The quality of light affects various aspects of the plant's growth and phytochemical content (Brazaitytė et al., Reference Brazaitytė, Vaštakaitė, Viršilė, Jankauskienė, Samuolienė, Sakalauskienė, Novičkovas, Miliauskienė and Duchovskis2018; Ajdanian et al., Reference Ajdanian, Babaei and Aroiee2019; Misra, Reference Misra2020). Studies have shown that red/blue light may have a better effect on certain crops. For example, Ajdanian et al. (Reference Ajdanian, Babaei and Aroiee2019) found that cress exposed to red/blue light yielded more that the plants exposed to natural light. Red/blue light has also been reported to promote elongation for cabbage, kale, arugula and mustard without affecting crops' yield and quality (Kong, and Zheng, Reference Kong and Zheng2019). Further, Lim and Harrison (Reference Lim and Harrison2016) and Alrifai et al. (Reference Alrifai, Hao, Marcone and Tsao2019), reported that red/blue and combined spectrums of light are better than white light in aiding photosynthesis and plant metabolism regulation.

In the research by Alrifai et al. (Reference Alrifai, Hao, Marcone and Tsao2019), it was noted that different wavelengths had varying effects on the antioxidant components of the product which concurs with the findings of a study by Misra, (Reference Misra2020). The latter found that higher light conditions increased photosynthetic capacity which was enabled by increased photosynthesis and electron transport; while low light conditions, on the other hand, resulted in the plant leaves experiencing harvesting complexities and weaker thylakoid membranes which shifted the xanthophyll pigments to improve photosynthesis. This can result in easier Salmonella and E. Coli spread (Misra, Reference Misra2020). Moreover, the study by Lim and Harrison (Reference Lim and Harrison2016), in which UV light's efficacy in reducing salmonella on food contact surfaces was evaluated, found that coupons that were exposed to UV light had greater salmonella population reduction compared to control coupons. This points to light as a factor that influences microbial colonization of plants including microgreens. Therefore, studies to optimize the photoperiodism of growing microgreens void of disease-causing pathogens are important.

Relative humidity, pH and air circulation

There is a dearth of literature identifying the optimal levels of relative humidity and pH for microgreens production. A recent study reported that most microgreens flourish with humidity levels between 40 and 60% (Li et al., Reference Li, Lalk and Bi2021a and Reference Li, Tian, Wang, Liu, Wang and Lu2021b). In addition, the growth of bacteria is affected by the pH of the environment. Bacteria are divided into three groups according to their response to pH. The acidophiles are acidic lovers, some can thrive in pH as low as pH 1 e.g., Thiobacillus thiooxidans, (Booth, Reference Booth1985). The neutrophils (Escherichia coli, Rhizobium and Bradyrhizobium) grow best at pH 6.5–7, and the alkalophilic, e.g., Bacillus alkalophilus grows at pH 10.5or 0.15 mS cm⁻¹ (Li et al., Reference Li, Lalk and Bi2021a and Reference Li, Tian, Wang, Liu, Wang and Lu2021b). The optimal conditions for microgreen growth are a pH between 6.56 and 7.54, and electrical conductivity of 0.41 mS cm⁻¹ (Kyriacou et al., Reference Kyriacou, El-Nakhel, Pannico, Graziani, Soteriou, Giordano, Palladino, Ritieni, De Pascale and Rouphael2020).

Good air circulation is vital for plant growth and disease prevention (Sharma et al., Reference Sharma, War, Pathania, Sharma, Akbar and Munghate2016). Air circulation regulates the temperature and humidity throughout the growing area (Chakraborty et al., Reference Chakraborty, Bhaduri, Uprety and Patra2014). This is usually achieved using horizontal airflow fans along with forced air or natural air vents to mix and exchange air within the growing space (Wolverton et al., Reference Wolverton, McDonald and Watkins1984). This affects the overall temperature of the growing area (Wolverton et al., Reference Wolverton, McDonald and Watkins1984). Studies to optimize air circulation in growth chambers will be vital to the microgreens industry in that mold-causing microorganisms on red maranta and other mildew-prone species could be prevented.

Temperature

The optimal environment for microgreen production can be variety-specific, but a favorable temperature range of 18–25°C has been documented, and temperatures above this have been shown to encourage microbial growth (Li et al., Reference Li, Lalk and Bi2021a and Reference Li, Tian, Wang, Liu, Wang and Lu2021b). Several studies have reported the growth of foodborne bacteria on plant extracts during the production process including work by Posada-Izquierdo et al. (Reference Posada-Izquierdo, Del Rosal, Valero, Zurera, Sant'Ana, Alvarenga and Pérez- Rodríguez2016); the growth potential for E.coli O157:H7 has been evaluated in water, where maximum growth rates in plant extracts were significantly influenced by temperature (Vital et al., Reference Vital, Stucki, Egli and Hammes2010; Merget et al., Reference Merget, Forbes, Brennan, McAteer, Shepherd, Strachan and Holden2019). Other factors were also influential including the plant tissue type and species, as well as the plant age, defense response, growth conditions and associated microbiomes (Merget et al., Reference Merget, Forbes, Brennan, McAteer, Shepherd, Strachan and Holden2019).

Effect of growing medium on bacterial colonization

Soil temperature and humidity alone do not influence microbial colonization of plants; the type of medium used also contributes to the overall environmental factors that influence the growth of pathogens and the colonization of microgreens as well as the nutrient content (Fig. 3). Peat-based mixes and synthetic mats are the main growing media used for microgreens production (Işik et al., Reference Işık, Topalcengiz, Güner and Aksoy2020; Le et al., Reference Le, Chiu and Hsieh2020). However, both are expensive and non-renewable (Di Gioia et al., Reference Di Gioia, De Bellis, Mininni, Santamaria and Serio2017). Recycled textile-fiber (polyester, cotton and polyurethane traces) and jute-kenaf-fiber have been proposed as low-cost and renewable alternative substrates; in addition, they both deliver a good yield, low nitrate content, and a similar microbiological quality (Di Gioia et al., Reference Di Gioia, De Bellis, Mininni, Santamaria and Serio2017).

Similarly, Misra and Gibson (Reference Misra and Gibson2020) pointed out that a growing medium with the highest carbon and micronutrient content would support bacterial growth and persistence. In their study, Biostrate and hemp growing mats, coconut coir and peat were evaluated, and BioStrate® and hemp growing mats were found to have supported the growth of S. javiana and L. monocytogenes R2–574, while coconut coir and peat did not. This also ties in with reports from Di Gioia et al. (Reference Di Gioia, De Bellis, Mininni, Santamaria and Serio2017) and Reed et al. (Reference Reed, Ferreira, Bell, Brown and Zheng2018). Although growing media have been identified as one of the means through which bacteria contaminate and colonize microgreens, many other contamination routes have been documented.

Contamination routes

The seed sprouting process presents an environment for microorganisms (Gu et al., Reference Gu, Strawn, Oryang, Zheng, Reed, Ottesen, Bell, Chen, Duret, Ingram, Reiter, Pfuntner, Brown and Rideout2018). Fertilization is also a contamination route (Reed et al., Reference Reed, Ferreira, Bell, Brown and Zheng2018; Jechalke et al., Reference Jechalke, Schierstaedt, Becker, Flemer, Grosch, Smalla and Schikora2019) as well as a growing medium (Di Gioia et al., Reference Di Gioia, De Bellis, Mininni, Santamaria and Serio2017; Misra and Gibson, Reference Misra and Gibson2020). Contamination risks from pre-or post-harvest are also presented in the harvesting process. The most significant limitation to the growth of the microgreens industry is the short shelf-life (Kaiser and Ernst, Reference Kaiser and Ernst2018). Microgreens are hard to store because their quality deteriorates very quickly after harvest Turner et al. (Reference Turner, Luo and Buchanan2020). Kaiser and Ernst, (Reference Kaiser and Ernst2018) suggested the product be used as soon as possible after harvest to maintain quality. Turner et al. (Reference Turner, Luo and Buchanan2020) also highlighted that post-harvest decay and transpiration make the product leak nutrients and its tissues become damaged by bacterial contamination and subsequent colonization. As a result, advances are being made to combat these limitations by exploring post-harvest treatments that can increase the shelf lives of microgreens.

Post-harvest

After harvesting, microgreens will last longer if they are maintained cold in a bag or container devoid of humidity which ensures that they do not wilt or dry during transport or storage (Turner et al., Reference Turner, Luo and Buchanan2020). Various technologies are being researched to prolong the shelf life of microgreens.

Pathogenicity of microgreens

Most pathogens especially those that are pathogenic to humans such as E. coli, Salmonella and Listeria can adapt and thrive in a wide variety of environments (Gandhi and Chikindas, Reference Gandhi and Chikindas2007). About 15% of cases of Salmonellosis in humans have been connected to the consumption of Salmonella-contaminated vegetables and/or fruits (Batz et al., Reference Batz, Hoffman and Morris2011). Listeria was responsible for the deaths of 10 people in 2010 and 30 people due to contaminated melon in 2011 (Gaul et al., Reference Gaul, Farag, Shim, Kingsley and Silk2010) and 14 people were reported dead from the infection of enterohaemorrhagic E. coli (EHEC) from consuming romaine cabbage in 2011 (Buchholz et al., Reference Buchholz, Bernard, Werber, Böhmer, Remschmidt, Wilking, Deleré, an der Heiden, Adlhoch, Dreesman and Ehlers2011). In addition, the CDC has recently announced a recall and disposal of romaine salads due to an outbreak of E. coli in 5 states in America (CDC, 2020).

From the literature, controlling the growing environmental conditions and postharvest storage of microgreens has been aimed mostly at pathogen prevention. However, the pathways or factors influencing the survival and proliferation of pathogens on plants including microgreens have not been well documented. The persistence of pathogens on fresh produce is influenced by the organism, species and pre-/ post-harvest environmental conditions (Harris et al., Reference Harris, Farber, Beuchat, Parish, Suslow, Garrett and Busta2003). Pathogenic organisms may survive but not persist on the uninjured outer surface of fresh fruits or vegetables, partly because of the plant's protective structures (Harris et al., Reference Harris, Farber, Beuchat, Parish, Suslow, Garrett and Busta2003). Unfortunately, these structures are underdeveloped in young plants (Warriner et al., Reference Warriner, Spaniolas, Dickinson, Wright and Waites2003). This suggests that young plants such as microgreens are more vulnerable to bacterial internalization than mature plants. They further stated that bacteria present in seeds can become part of the endophytic microflora. Many other studies have also reported the internalization of pathogens in young plants including Dong et al. (Reference Dong, Iniguez, Ahmer and Triplett2003) who reported significant pathogen colonization of the interiors of 6–9-day old seedlings of Medicago sativa (alfalfa).

Plant pathogens have developed many ways to get nutrients from their host, and plants in turn have also developed physical and chemical properties to fight against pathogen attacks (Zhang et al., Reference Zhang, Lubberstedt and Xu2013). Studies have reported that Salmonella strains not having SPI-1, SPI-2, 97 SPI-3, SPI-4 and SPI-5 can colonize tomato and cantaloupe fruits (de Moraes et al., Reference de Moraes, Desai, Porwollik, Canals, Perez, Chu, McClelland and Teplitski2017); suggesting Salmonella may not only infect plants by the translocation of SPI-encoded T3SS infectors. This raises the question of understanding the requirement of the corresponding genes during plant-host interaction which influences plant colonization and in addition the genetic functions with which these pathogens colonize microgreens. Therefore, a study on the phenotypic characterization for the persistence of these pathogens within microgreens in relation to their growing environmental conditions; particularly, pH, temperature and relative humidity is imperative.

Foodborne contamination associated with microgreens consumption

Vegetables that are eaten raw such as microgreens carry foodborne diseases (Riggio et al., Reference Riggio, Wang, Kniel and Gibson2019). Various outbreaks have been reported in Europe and North America with the increased production of microgreen products.

Microgreens are defined as the first 2 or 3 inches of shoots from germinating vegetable seeds mostly grown in greenhouses in soil mixes, hydroponic growth mediums and peat mixes as well as recycled fiber mats (Jechalke et al., Reference Jechalke, Schierstaedt, Becker, Flemer, Grosch, Smalla and Schikora2019). Since microgreens are harvested very close to the soil matrices, it is very easy to get them contaminated with microorganisms from their growing matrices. Furthermore, a study by Wang and Kniel (Reference Wang and Kniel2015) indicated that, microgreens can become contaminated by microorganism from poorly disinfected recirculated water.

Their growing and environmental conditions are also the optimum conditions for growth for most pathogens to thrive (Gu et al., Reference Gu, Strawn, Oryang, Zheng, Reed, Ottesen, Bell, Chen, Duret, Ingram, Reiter, Pfuntner, Brown and Rideout2018).

Even though various contamination routes are documented, contamination risks from pre-or post-harvest are present from the farm to the marketing process; where many attempts including postharvest treatments (Wells and Butterfield, Reference Wells and Butterfield1997; Berba and Uchanski, Reference Berba and Uchanski2012; Garrido et al., Reference Garrido, Tudela, Hernández and Gil2016; Wright and Holden, Reference Wright and Holden2018) have been made to minimize the risk of contamination and consequently prolong the shelf-life of these farm produces. These risks in association with microgreens have not been adequately evaluated.