Ceylon Journal of Science 48(1) 2019: 91-96 DOI: hitp://doi.org/10,4038%ejs.v48i1,7593 ESE Germination, harvesting stage, antioxidant activity and consumer acceptance of ten microgreens Gayathree I, Senevirathne!, N. S. Gama-Arachchige'?,* and Anjani M. Karunaratne!* “Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka "Postgraduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka Received: 17/10/2018 ; Accepted: 28/01/2019 Abstract: Microgreens which are tender immature ‘vegetables and herbs are known for their health beneficial effects. Tae concept of microgreens is generally less popular in many countries including Sri Lanka, Ten species were rested to obtain data on seed germination, height gain, leat area expansion, and ‘consumer acceptance. Ofthese, three species of which the seeds are ‘commonly consumed in Sri Lanka were analyzed for antioxidaet activites as seeds, sprouts, and micragreens. Seed germination of ‘most of the species was>75% with the time taken to reach 75% germination (G.,) varying from 1 to >14 days. A strong positive correlation between seedling and leaf area was observed (R’=0.). Lttaceand carrot were found tobe the most preferred mierogroens followed by green peas, red amaranth and finger millet. For the three selected species where respective seeds, sprouts, and jcrogreens were compared, higher antioxidant act recorded in finger millet seeds and sesame microgreens; IC,, 697 ugiml. and IC,,772 gil. respectively with the later recording ‘high total phe! (4873 mg/100g dry weight). Green pea _mjerogreens showed higher total phenol content than its seeds and sprouts (1871 mg/100 g dry weight). The information generated Will be of value to introduce mictogreens to countries where consumers are unfamiliar with this product ties were con Keywords: Antioxidants, consumer acceptance, harvesting stage, mierogreens, seed germination, INTRODUCTION Microgreens have gained popularity worldwide for their micronutrient complement and health promoting phytochemicals (Xiao et al, 2016). Their bioactives include higher levels of antioxidant compounds such as polyphenols, fb carotene, and ascorbic acid, than their ‘mature plants thus qualifying as functional foods (Xiao et al, 2012; Mir ef al., 2017). With non-communicable diseases causing major health concems worldwide (WHO, 2017), seeking for functional foods has accelerated in recent years In the absence of a legal definition, the terms “microgreens’ and ‘baby greens’ are used to distinguish these miniature plants (Treadwell et al., 2010). A widely accepted definition of microgreens is “Tender immature greens produced from seeds of vegetables and herbs having. fully developed cotyledons with or without the emergence of a rudimentary pair of first true leaves” (Xiao et al., 2012), Microgreens are larger than sprouts, but are smaller than baby greens and therefore their harvesting stages are also in between those two stages (Xiao er al, 2012; Ebert, 2012), Baby greens are the oldest (usually 7-10 em tall) and ‘microgreens (usually 2.5-6 cm tall) are more differentiated than sprouts (Treadwell et a, 2010; Xiao et al, 2012) There is a scarcity of information on types of seeds suitable to be developed as microgreens. The definition of Xiao ef al, (2012) suggests the necessity to use epigea germination seeds. However, it would be informative to know how hypogeal germination plants would contribute as, functional foods, As ideal conditions for seed germination varies (Baskin and Baskin, 2014), meeting growth requirements without elaborate equipment is helpful for commercial microgreen growers, This preliminary study secks direction on the introduction of microgreens. MATERIALS AND METHODS ‘Seed sources and experimental conditions Seeds used for planting and for consumption were obtained from retailers in Kandy, Sri Lanka. Seeds for cultivation were pre-treated with fungicides, Throughout, the parameters defined by Xiao et al, (2012) were used as the specified standards for microgreens, Germination determined in light/dark treatments and in total darkness Seeds were germinated in an incubator (model: MQC 4508P) providing a daily 12 h/I2 bh light/dark period (temperature 25 °C, light phase photon irradiance was approx. 115 pmol photons ms, 400-700 nm, supplied by ‘coo! white fluorescent tubes). Surface stetilized seeds (1% sodium hypochlorite) were germinated in petri-dishes lined with moist paper towels, When testing the effect of total darkness (24 h), petri-dishes were covered with aluminum, foil, For light/dark treatment, the number of germinated seeds was counted daily for 14 days and for total darkness, counting was done after 7 and 14 days (separate petri- dishes). which permits unrestricted use, distr “Corsponding batho Ema papa © be roridorgoonsn0r-7225740 This ancl is published under the Creative Commons Attribution License (htps!creativecommons.onglicenses/by4.0), ston, and reproduction in any medium, provided the original work is properly cite.92 Growing microgreens Microgreens were grown in perforated plastic trays (20x15 cm) filled with autoclaved (120 °C, 120 Pa pressure, 90 min.) mixture of commercially processed compost and coir dust (I:1iv:v). Until cotyledons developed, overhead irrigation was done and after hypocotyls emerged, bottom inrigation (300 ml per day) was done (Kopsell el, 2012), Dark germinating seeds were covered with a sprinkling of the growth medium sufficient to prevent exposure to light. The trays were kept in the same incubator Harvesting stage and correlation between seedling height and leaf area ‘The height and leaf area (digital photographs with Image software (ver. 1.47) were measured every two days (from, day 2 to 14 depending on the species). Leaf areas were determined after cotyledons (epigeal germination) or true leaves (hypogeal germination) appeared until they exceeded the maximum specified height (6 em) for microgreens ‘Consumer acceptance of mierogreens All microgreen species were evaluated for consumer acceptance using a 9-point hedonic scale (Lawless and Hayman, 2010); 9-like extremely and I=dislike extremely where leaves of popularly consumed Asian pennywort (Centella asiatica) served as a control. — Monoculture salads (single microgreen type with pepper, salt and lime juice) (Figure 1) were prepared and the species identities including the control were not divulged and the purpose of testing was not explained. Ceylon Journal of Science 48(1) 2019:91-96 Phytochemical properties of seeds, sprouts and microgreens For determination of antioxidant activity and total phenol, content, three species of commonly consumed seeds (green peas, sesame, and finger millet) were selected. Batches of G 5) dry sceds, sprouts (germinated in the incubator) and microgreens were dried at 38 °C, ground, sieved (0.5, ‘mm) and extracted with 100% methanol (Sigma-Aldrich, 'MO,USA) on a mechanical shaker (2 days) and evaporated to a thick residue which was stored at -18 °C. 2,2-Diphenyl pierylhydrazil (DPPH) (Sigma-Aldrich, MO, USA) radical scavenging antioxidant activity was determined (Brand- Williams, 1995) using DPPH (3 mL) and methanolic sample (I mL) after incubating (30 min) in the dark “Absorbance was measured (UV-Visible spectraphotometer, Spec M02, Spectronic Camspee Ltd, UK) at $17 nm and for each sample 50% inhibitory concentration (IC,,) was calculated. Percentage Inkibition — (Absorbance of control Absorbance of Sample). 4g Absorbance of control The same methanolic extract was used to determine total phenols (ISO, 2005), Fotin Ciacalteu reagent (5.0 ml) (Loba Chemie, India) (10% viv) was added to the aqueous sample (30% methanol) (1.0 mL) and after mixing well (3-8 ‘min), 7.5 % Na,CO, (4.0 mL) was added, and incubated (60 min). Absorbance was measured at 765 nm. Total phenolics were expressed as gallic acid (Sigma-Aldrich, MO, USA) equivalents per 100 g dry weight basis of the sample Figure 1: Steps involved in preparation of microgreen salads, A: microgreen at harvesting stage, B:_ harvesting microgreens, C ‘washing to remave plant debris an sed coat, D: Mierogreen salad mix,Gayathree I. Senevirathne et al 98 lettuce, mustard and sesame (<2 days); moderate ‘in Chinese kale and green peas (2-3 days); slow in carrot Germination tess were performed in four replicates (25. (7 days); very slow in finger millet and red amaranth (14 seeds per batch), the measurements of height and leaf area ays), Arnon [actors that atibuted 10 poor germination were obtained in 3 replicates (15 seedlings per replicate) sre unfavourable temperature, quality and quantity of ight, using complete randomized design and all phytochemical jack of moisture and inherent factors (Baskin and Baskin, analyses were conducted from three repeated trials for each 2614), ttigh germination in Chinese kale, green radish sample, Pereentage germination data were normalized and lettace may indicate high seed vigor and seed coat by arcsine transformation prior to analysis. Consumer treatments like fungicide applications as these are meant acceptance test was conducted involving 50 untrained for planting. The finger millet seeds used were for human volunteers between 22-50 years of age, Date were analyzed consumption. Their poor germination may be attributed to using (ests forthe comparison of two samples and onc premature harvesting and they were susceptible to fungal ay analysis of variance (ANOVA) when comparing more tack despite disinfection, The red amaranth seeds were than (wo. The significance for all statistical analyses waS for culkivation and the label indicated ~80% germination set at P= 0.05 The difficulty of rowing red amaranth seeds is mentioned RESULTS AND DISCUSSION by Lee and Pill (2005) and recommended seed priming and break dormancy. Experimental design and statistical analysis application of plant growth regulators % ‘The absence of readily available, uniformly produced seeds was a constraint and we depended on seed stores, ‘meant for planting and food stores, Seeds meant for consumption did not give information on variety name, expiry dates, expected germination rates and chemical/ fungicide treatment, while all these data were available for the seeds meant for planting. As harvesting of microgreens, ‘commences at day 7 (Xiao et a, 2012) germination at day 7 was determined (Table 1), Seedling emergence occurred within 4 days in most species, bt carot and red amaranth took 6 days, with time required to reach harvesting height ranging fom ~6 t0 1M days after sowing (Figure 2A), Green radish exceeded the maximum height limit (6 cm) within 6 days and true leaves emerged on day 8. Lettuce, mustard, and sesame have shown moderate rates of growth and did not execed the maximum height within 14 days, however tue leaves ‘merged within 8-10 days, Chinese kale, green peas and ‘The germination of sesame, green peas, finger millet, — genugreck excceded the specified height range within 8 days green peas and sesame slowed down significantly under and true leaves emerged in the latter two on day 8. Carrot, {otal darkness (P values 0,002, 0,006 nd 0,000respectively) finger millet, and red amaranth showed comparatively slow while light regime didnot influence that of other seeds. ed growth and exceeded the maximum height within 14 days, amaranth showed poor germination (< 10%) irespective but tue leaves emerged on day 16 and 14 in carot and red of the light regime, The G,, values (Table 1) indicate amaranth respectively that germination rates were fastest in fenugreek, green Table {ean germination percentage and time taken to reach 75% germination (G,,) of 10 mierogreens ‘Mean germination percentage after 7 days Gyn light? Common ataneal name Vanity tigheare SPR? Germination darkness (12 es/t2 (Days) ‘ype es) Caan carot Karola Gian Woe asa gal «Brassica oleracea var . Kae Brusca ChineseKale 950819" 940K 2.7802 epigeal Femugreek zona oem Nia sau oO 18801 epigcl Finger millet Efewine oracona Wi a ET iypogal Green poss Pisum satin NA 2edelo) skis SP 2.6207 ypogsl Re Raphanus sativus apanese round 98.021.2" 9801.2" 0920.0 epigeal tsuce* acca si Green coral 9G WOLDS pigs Muslsd Brac juncea NA EROEI2 AGED 10.6 epigel ‘Amara? Amaranthus Red Amaraah —10e1.0 ae i pigs Sesame Sesamum indica NA 20s. 19a epigel * Sign indicates fungicide treated seeds for growing purposes, Values are expressed as mean= standard error (n=4). Different letters indicate a significant difference (p < 0.05) within the same species, NIA, variety names not available94 Seating hg nen eaten afr) Tme(Dey) Figure 2: (A) Seedling height vs, time of harvest of seedings ofthe ten species, The rangs Ceylon Journal of Science 48(1) 2019:91-96 3 ed amar Sete Tee Daye) ighlightod in grey (60 -25 mm) indicates acceptable height at harvest of microgrvens; () Cotyledon or leaf area vs. time oF harvest of seedlings of the ten species. Green radish showed the highest cotyledon area followed by Chinese kale, while comparatively low values. were exhibited by carrot, finger millet fenugreek, lettuce, mustard, red amaranth and sesame (Figure 2B), Even finger millet and green peas showed low rate of tue leaf. expansion (Figure 2B). In fenugreek, lettuce, mustard and sesame, rates of increase of cotyledon area appeared to decrease after true leaves emerged. The contribution of Ieaves in their role of functional foods is significant (Kopsell et ai., 2012), hence having a larger leaf area is, beneficial not only for the health of the growing plant but, also for the nutrition and health of the consumer. While the definition for microgreens (Xiao et al, 2012) does not take the leaf area into account, in the present study, it was noted that there is a strong positive correlation between seedling height and leaf area, where R? was 0.80. Therefore, the usage of the height in the definition of microgreens which, is more convenient than measuring leaf area is justified. Interestingly, days 6-8 are suitable for harvesting fenugreek, ‘green peas, mustard, lettuce, and sesame based on their leaf areas which remain constant while their heights are still, ‘within the prescribed range. Considering the height and leaf area, 6-8 days is more suitable for harvesting green radish and Chinese kale, As finger millet did not show a considerable increase in height and leaf atea after 16 days, harvesting can be between 12-16 days. Carrot and red amaranth did not exceed the maximum specified height even after 16 days but true leaves emerged between 14-16 days, which indicates that itis the suitable period for their harvesting, Although, all the species were grown at 25 °C, ideally they will flourish at optimum temperature conditions Cool season crops (kale, radish, carrots and lettuce) show optimum growth at 15- 20°C. In spite of that, all plants have performed well at 25 °C considering their G., values, leaf area and height (Figure 2), which is encouraging as ‘maintaining diverse conditions will incur higher production, cost on the grower, On sensory evaluation (Table 2), among the ten microgreen species, lettuce has received the highest preference (average hedonic value of 7.90 - like very much), while fenugreek and green radish have received least preference (average hedonic value of 3.90 and 3.72 respectively ~ dislike slightly). Lettuce and carrot can bbe considered as the most preferred microgreens along with Asian pennywort leaves (control). Green peas, red amaranth, and finger millet microgreens are next in the consumer preference followed by kale, sesame, and ‘mustard, Since the panelists did nol understand the concept of microgreens it was necessary to use lime juice, salt and pepper, to make them familiar to what they consume locally as leaf salads. Mustard is neither liked nor disliked and groen radish is disliked slightly. Similarly, in a previous study (Xiao et al, 2015), Dijon mustard has received @ generally intermediate score and Chinese rose radish received a low score in overall eating quality. While red ‘amaranth is liked slightly in the present study, itis reported to have a higher rank (Xiao et al., 2015) duc to its appealing red colour and acceptable flavour, The acceptability based on organoleptics is heavily dependent on acquired attributes and this may be an important consideration in the initial stages of commercializing microgreens. Ironically, unacceptable flavours such as bittemess may be due 10 various biochemicals which may be beneficial for health, Radish and mustard are known to be rich in glucosinolates ‘which are bitter (Drewnowski and Gomez-Carneros, 2000) and phenolics generally contribute to a not so appealing astringent flavour (Tomds-Barberdn and Espin, 2001), With appropriate education of the consumer of the benefits of microgreens, their acceptance may increase despite not so superior taste attributes, The control, Asian pennywort, although its identity was not revealed, ‘was recognized by all panelists by the taste, Despite its familiarity and popularity, it was only liked moderately (Table 2). This is an observation that not so superior ast attributes will get underplayed in consumer acceptance if the health benefits are known, As microgreens are consumed in mixed salads, the consumer can incorporat many varieties in combined salads giving vivid colour, flavor, texture and odour, Reducing the proportions of theGayathree I. Senevirathne et al. Table 2: Consumer acceptance rating of ten mierogreens, 95 ‘Mean hedonic Microgreen species acceptance value Hedonic scale description Special comments of panelists ia pamper ono Tae Tie moderately z Cant 730r Tike moderately Smear Chinese Fae 90 neither ike or dike Fenugteck 3.90" ise slightly aa Finger mle ke sighy Earthyfgrasy mouth el Green peas Tike sity : Green Rash Gapanse round) ise slightly Cipieaan doar Late (Green cor Like very much Savoury crunchy Favour Mustard neither like nor dislike —_~ Red amaranth Tike slighly 7 Sesame neither like nor dislike Slight bitter taste Different liters indicate a signifiant difference (p= 0.05) indifferent species compared to the contro Table 3: DPPH (Diphenyl pyerylhydrazl) radical scavenging antioxidant activity expressed as (50% Inhibitory concentration) and {otal phenols content and moisture content of the selected three species of which seeds are consumed popularly in Sri Lanka, DPPH Antioxidant acti Total phenols (Galle acid equivalents of mgi100 g dry ‘Moisture Content (%) Species (ug! mL) weight) Seed Sprout Seed Sprout Microgreen Seed Sprout Microgreen Finger millet 697213" _ 30 TOs ISO SISOS Green peas S2S7=31* 2519599" IANO 46S GRD” —CRTIGH RSMAS Sesame B19 895238 772 ToL 3351 ASNT 2808S Al values are expressed as mean species rot so acceptable species in the salad mixture is another ‘way to gain health benefits without compromising the taste Of the ten species, three popularly consumed seeds ‘were singled out to analyse antioxidants at three stages; seeds, sprouts, and microgreens. Absence of fungicide or chemical treatments, and not being investigated before in other studies, as microgreens are other criteria to choose these three. The microgreens of green peas and sesame show the highest antioxidant activity (based on lower IC,,) as compared to respective seeds and sprouts (Table 3). Health beneficial substances may be formed in diverse ‘ways depending on the species during growth from seed to sprout to microgreens. Therefore, future studies should aim, at determining relative levels of such substances with time of germination, The sesame microgreens exceeded total phenol content (4873 mg Ga equivalents/100 g dry weight) when compared to all seeds, sprouts and microgreens. Additionally, in sesame, IC,, decreased from seed to ‘microgreen production indicating that the best method of consuming sesame is as mierogreens. ‘Also, despite its hypogeal germination, green pea microgreens show higher antioxidant activity and higher total phenol value. Finger millet, the other hypogeal standard error (a3). Different letters indicate a significant difference (p < 0.05) wit the same germinating seed tested, showed a decrease in total phenols from seeds to sprouts, and again an increase from sprouts to microgreens with seeds showing the highest levels. Finger millet seeds also recorded a lower IC,, value showing that the best way to consume them is as seeds, The presence of carotenoids, tocopherol and phenolic compounds such as luteolin in finger millet seeds result in high antioxidant activity (Mathanghi and Sudha, 2012). The seed coat of green peas is rich in phenolic compounds such as glycosides of quercetin, futeolin and apigenin and the cotyledons are known to be rich in hydroxyl benzoic acid and hydroxyl cinnamic acid (Duenas et al, 2004). As different parts of the plants such as. seeds, cotyledons and leaves may have different health promoting properties, the ideal time of consumption to benefit by their phytochemicals vary, which shows the importance of determining antioxidant activity at different stages. Seed germination is known to increase the levels of nutrients such as antioxidants and reduce the levels of antinutrients such as phytic acids (Seneviratne et al, 2012). Also, in spite of the fact that the microgreen market concentrates on seeds with epigeal germination,96 the positive results obtained for mictogreens of peas in the present study is a cue to test for other seeds with hypogeal germination as microgreens, To start a lucrative business in microgreens, itis important to have a ready supply of seeds with high viability, fast germination and free of agrochemicals. CONCLUSION With the simple sctup used, it was possible to grow microgreens satisfactorily, Use of seedling height as f harvesting index can be recommended, as it can be determined easily. However, results from the study showed that leaf area can also be used as a harvesting stage index. As green pea microgreens showed higher total phenol content than its seeds, the advantage of using microgreens from hypogeal germinated species too should be determined. Microgreen consumption should be popularized by educating the consumers of their benefits, as taste alone is not a good etiterion to judge the health promoting properties. As the highest antioxidant activity, ‘was shown by finger millet seeds, rather than its sprouts or icrogreens, itis not possible to assume that microgreens, of any species are healthier than the seeds. Therefore more information is required on species that are suitable to be developed as microgreens rather than consuming as seeds alone, Conflicts of interest ‘The authors declare that they have no conflicts of interest pertaining to this study. REFERENCES Baskin, ©. C. and Baskin, J. M. 2014), Seeds: ecology; biogeography, and evolution of dormancy and ‘germination. (2ed.). Elsevier. Brand-Williams, W., Cuvelier, MIE, and Berset, C. (1995) Use of a free radical method to evaluate antioxidant activity. Lebensmittel Wisseenchaft Technologie 28: 25- 30. Duenas, M., Estrella, I. and Hemandez, T. (2004). Occurrence of phenolic compounds inthe seed coat and the cotyledon of peas (Pisum sativum L.). European Food Research Technology 219: 116-123 Drewnowski, A. and Gomez-Cameros, C., (2000). Bitter taste, phytonutrients, and the consumer: a review. American Journal of Clinical Nutrition 72: 1424-1435. Bbert, A. (2012). Sprouts, microgreens, and edible lowers: the potential for high value specialty produce in Asia Proceedings SEAVEG2012. Regional Symposium, Chiang Mai, Thailand, Pp. 216-227. 180 (2005).Content of total polyphenols in tea-colorimetric method using folinciocalteu reagent-international standards-ISO 14502-1 Kopsell, D.A., Pantanizopoulos, N.I, Sams, CE. and Kopsell, D.E, (2012). Shoot tissue pigment levels increase in ‘Florida Broadleaf” mustard (Brassica juncea L.) microgreens following high light treatment. Scientia Horticulturae 140: 96-98. Lawless, H.T. and Heyman, H. Sensory Evaluation of Food. (2%ed,) Springer, New York. (Chapter 14), Ceylon Journal of Science 48(1) 2019:91-96 Lee, JS. and Pill, W.G. (2005),Advaneing greenhouse «establishment of radish, kale, and amaranth mierogreens through seed treatments. Horticulture Environment and Biotechnology 46: 363-368 Mathenghi, SK. and Sudha, K. (2012). A review: Functional and phytochemical properties of finger rillet (Blewsine coracana I.) for health, International Journal of Pharmaceutical, Chemical and Biological Seiences 2: 421-838, Mit, S.A, Shah, M.A. and Mir, M.M. (2017). Microgreens: Production, shelf life, and bioactive components. Critical Reviews in Food Seience and [Nitrition, $7, 2730-2736, Seneviratne, M., Gunasinghe, WK.R., Gama-Arachchige, NS., Dissanayake, N. and Karunaratne, A. (2012). Mineral bioavailability in three locally consumed pulses processed using popular methods: interpreted using molar ratios with phytic acid, Ceylon Journal of Science (Biological Sciences) 41: 19-26. Tomas-Barberin, F. and Espin, J.C. (2001). Phenolic compounds and related enzymes as determinants of quality in fits and vegetables. Journal of Science of Food and Agriculture 81: 853-816. ‘Treadwell, D. D., Hochmuth, R, Landrum, L. and Laughlin, ‘W. (2010). Microgreens: a new specialty crop. Institute ‘of Food and Ageiculture Sciences, University of Florida, (UFIFAS Extension Service). hip:/ledis.ifas.uf.edw hs1164 / (16.01.2017). World Health Organization. (2017). The 10 leading causes ‘of death in the world, 2000 and 2012.htp:/Avww.who. int/mediacentre/factsheets/fs3 10/en/(02.02.2018) Xiao, Z., Lester, G.E, Luo, Y. and Wang, Q. (2012), ‘Assessment of vitamin and carotenoid concentrations ‘of emerging food products: edible microgreens. Journal of Agricultural and Food Chemistry 60: 7644-7651 Xiao, Z., Lester, GE, Park, E., Safiner, R.A., Lou, Y. and Wang, Q. (2015). Evaluation and correlation of sensory attributes and chemical compositions of emerging fresh produce: microgreens. Postharvest Biology and Technology 10: 140-148. Xiao, Z., Codling, E.E., Luo, ¥., Nou, X., Lester, GE. and ‘Wang, Q. (2016). Microgreens of Brassicaceae: Mineral composition and content of 30 varieties, Jounal of Food Composition and Analysis 49: 87-93.