Nutritional value of duckweeds (Lemnaceae) as human food

Food Chemistry 217 (2017) 266–273

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Nutritional value of duckweeds (Lemnaceae) as human food Klaus-J. Appenroth a,⇑, K. Sowjanya Sree b, Volker Böhm c, Simon Hammann d, Walter Vetter d, Matthias Leiterer e, Gerhard Jahreis c a

University of Jena, Institute of General Botany and Plant Physiology, 07743 Jena, Germany Central University of Kerala, Department of Environmental Science, RSTC, Padannakad, Kerala 671314, India c University of Jena, Institute of Nutrition, Jena, Germany d University of Hohenheim, Institute of Food Chemistry, Stuttgart, Germany e Thuringian State Institute of Agriculture, Jena, Germany b

a r t i c l e

i n f o

Article history: Received 13 June 2016 Received in revised form 23 August 2016 Accepted 28 August 2016 Available online 29 August 2016 Keywords: duckweed Landoltia Lemna Spirodela Wolffia Wolffiella Proteins Fatty acids

a b s t r a c t Duckweeds have been consumed as human food since long. Species of the duckweed genera, Spirodela, Landoltia, Lemna, Wolffiella and Wolffia were analysed for protein, fat, and starch contents as well as their amino acid and fatty acid distribution. Protein content spanned from 20% to 35%, fat from 4% to 7%, and starch from 4% to 10% per dry weight. Interestingly, the amino acid distributions are close to the WHO recommendations, having e.g. 4.8% Lys, 2.7% Met + Cys, and 7.7% Phe + Tyr. The content of polyunsaturated fatty acids was between 48 and 71% and the high content of n3 fatty acids resulted in a favourable n6/n3 ratio of 0.5 or less. The phytosterol content in the fastest growing angiosperm, W. microscopica, was 50 mg g 1 lipid. However, the content of trace elements can be adjusted by cultivation conditions. Accordingly, W. hyalina and W. microscopica are recommended for human nutrition. Ó 2016 Elsevier Ltd. All rights reserved.

0. Introduction Bhanthumnavin and McGarry emphasized already decades ago (Bhanthumnavin & McGarry, 1971) that ‘‘Khai-nam”, literally meaning ‘‘eggs of the water” (duckweed) in Thai language, is ‘‘a possible source of inexpensive protein” that can be used as human Abbreviations: AA, amino acids; AAS, amino acid score; Ala, alanine; ALA, a-linolenic acid; Arg, arginine; Asp, aspartic acid; Cys, cysteine; DW, dry weight; EAA, essential amino acids; EAAI, essential amino acid index; essential AA/non-essential AA, EAA/NEAA; EPA, eicosapentaenoic acid; FA, fatty acids; FAO, Food and Agriculture Organization of the United Nations; FAME, fatty acid methyl esters; Glu, glutamic acid; Gly, glycine; Ile, isoleucine; LA, linoleic acid; LCFA, long-chain fatty acids; LC-PUFA, long-chain polyunsaturated fatty acids; Leu, leucine; Lys, lysine; MCFA, medium chain fatty acids; Met, methionine; MUFA, monounsaturated fatty acids; N, nitrogen; NPN, non-protein nitrogen; Phe, phenylalanine; PUFA, polyunsaturated fatty acids; SCFA, short-chain fatty acids; SD, standard deviation; SDA, stearidonic acid; SFA, saturated fatty acids; Thr, threonine; Trp, tryptophan; Val, valine; WHO, World Health Organization. ⇑ Corresponding author at: Institute of General Botany and Plant Physiology, University of Jena, Dornburger Str. 159, 07743 Jena, Germany. E-mail addresses: [email protected] (K.-J. Appenroth), ksowsree@ gmail.com, [email protected] (K.S. Sree), [email protected] (V. Böhm), [email protected] (W. Vetter), matthias.leiterer@tll. thueringen.de (M. Leiterer), [email protected] (G. Jahreis). http://dx.doi.org/10.1016/j.foodchem.2016.08.116 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

food. They identified the species as Wolffia arrhiza and stressed on their high growth rate. This species is rather rare in Thailand (Landolt, 1986). We investigated three samples of the duckweeds sold in the market for human consumption from Northern Thailand in 2016, and identified all of them as W. globosa, which is in accordance with Landolt and Kandeler (1987) suggesting that these authors dealt with the more common species W. globosa. Bhanthumnavin and McGarry (1971) measured the protein, carbohydrate, and fat content of these plants and reported that ‘‘Khai-nam” was used as food of poor people for many generations in Laos, Thailand and Burma (now Myanmar). In many other South-Asian countries like India, Bangladesh and Pakistan, food is rich in carbohydrates but poor in proteins. Thus, protein-rich duckweed would be a perfect supplement to the rice-based staple food in these countries. Moreover, duckweed might add on to the protein content of the vegetarian or vegan diet as this life style becomes more and more popular in the developed countries. In their landmark paper, Rusoff, Blakeney, and Culley (1980) investigated four species of duckweeds, Spirodela polyrhiza, Landoltia punctata (termed as Spirodela punctata), Lemna gibba and Wolffia columbiana, with respect to the protein and fat contents, and the amino acid composition. They already mentioned

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the favourable amino acid composition of duckweed proteins as per the FAO recommendations. Later, a detailed investigation of the amino acid composition of the proteins in W. arrhiza, clone 9528 was performed by Appenroth, Augsten, Liebermann, and Feist (1982). Yan et al. (2013) investigated 30 out of the 37 duckweed species (cf. Sree, Bog, & Appenroth, 2016) and reported the presence of 11 saturated and unsaturated fatty acids. Recently, Tang, Li, Ma, and Cheng (2015) reported the starch and fatty acid content in four duckweed species (S. polyrhiza, L. punctata, L. aequinoctialis, and W. globosa), all from the lake Chao, China. In the present study, we aimed to give an overview of the nutritional quality of different duckweed species for their possible use in human nutrition. However, as stressed by van der Spiegel, Noordam, and Fels-Klerx (2013), the concerned legal issues need to be considered before it can be commercially marketed as human food. As a step forward, it would be important to investigate the nutritive composition of duckweeds to prevent any unwanted effects on humans and also to make it more acceptable to the general public of the countries that do not have the tradition of consuming duckweeds. With our present report, we want to contribute to this issue. To have a broader view, we investigated six species representing all five genera, Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Lemna minor, Wolffiella hyalina, and the recently rediscovered species Wolffia microscopica (Sree, Maheshwari et al., 2015). Initially, we investigated dry weight, protein, fat, and starch content as well as their amino acid and fatty acid distributions. As a part of the detailed investigation, we focused on W. microscopica investigating other components having relevance to human consumption, i.e. content of minerals, antioxidants (carotenoids and tocopherols), phytosterols, fibre and ash content.

1. Material and methods 1.1. Plant material and cultivation Plant material was taken from the collection of duckweed ecotypes, or clones, of the Institute of Plant Physiology, University of Jena, Germany. The duckweeds in this stock collection, most of which stem from the collection of E. Landolt, ETH, Zürich, Switzerland, were maintained under axenic conditions on agar medium as described by Appenroth, Teller, and Horn (1996). Six species encompassing all five duckweed genera were selected: Spirodela polyrhiza 7498 (USA, NC), Landoltia punctata 9589 (India, Delhi), Lemna minor 9441 (Germany, Marburg), Lemna gibba 7742 (Italy, Sicilia), Wolffiella hyalina 9525 (India, Telangana), and Wolffia microscopica 2005 (India, Gujarat). Duckweeds were pre-cultivated under axenic conditions at 25 ± 1 °C in 300 mL Erlenmeyer flasks containing 180 mL nutrient medium. They were exposed to continuous white light at 100 lmol m 2 s 1 (photosynthetically active radiation) from fluorescence tubes TLD 36 W/86 (Philips, Eindhoven, Netherlands) following the ISO 20079 protocol (Naumann, Eberius, & Appenroth, 2007). Accordingly, the plants were conditioned to the nutrient medium for four weeks during this pre-cultivation phase in order to ensure reproducible results. The nutrient medium was replenished every week to prevent nutrient limitation. In place of the Steinberg medium, specified by the ISO 20079 protocol, however, a modified Schenk-Hildebrandt medium was employed with the following composition: CaCl2
2H2O 0.68 mM, KNO3 12.4 mM, MgSO4
7H2O 0.81 mM, (NH4)H2PO4 1.3 mM, MnSO4
H2O 30 lM, H3BO3 40 lM, ZnSO4
7H2O 1.74 lM, KI 3.0 lM, CuSO4
5H2O 0.4 lM, Na2MoO4
2H2O 0.21 lM, CoCl2
6H2O 0.21 lM, FeNaEDTA 27.0 lM, and Na2EDTA
2H2O 2.74 lM. The pH of the medium was adjusted to 5.5.

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The main phase of cultivation used for determining the biomass composition employed the same conditions as described for the pre-cultivation, except that plastic trays (area 60  40 cm) filled with 15 L Schenk-Hildebrandt medium and covered with glass plates were used. The main cultivation phase lasted for 14 days, during which the size of the inoculum ensured that the fronds did not completely cover the surface of the medium and thus avoiding a stress response. Thereafter, biomass was harvested and lyophilized for further analysis. 1.2. Analytical methods Fresh duckweeds were analysed for dry weight (DW). After freeze-drying plants were finely ground and homogenised with a laboratory mill for small amounts for further use. 1.2.1. Lipid extraction and fatty acid separation Total lipids were extracted from 1.0 g (or less) of the homogenised samples with methanol/chloroform/water in the ratio 1:2:1 (v/v/v). These lipid extracts were transmethylated using a combination of 0.5 N methanolic sodium hydroxide (Merck) and 10% (w/w, Supelco) boron trifluoride-methanol (at 100 °C for 5 min each). Subsequently, fatty acid methyl esters (FAME) were purified by TLC and dissolved in n-hexane for analysis. The separation of FAMEs was performed by GC (GC-17 V3, Shimadzu, Japan) equipped with a cooled auto sampler and a flame ionization detector. A fused-silica capillary column of medium polarity was used (DB-225MS/ 60 m  0.25 mm i.d. with a 0.25 mm film thickness; Agilent Technologies, USA). The initial oven temperature was maintained at 70 °C for 2 min, then increased by 10 °C/min to 180 °C, further increased by 2 °C/min to 220 °C and held at this temperature for 5 min. Finally, it was increased by 2 °C/min to 230 °C and held for 15 min. The injector and detector temperatures were maintained at 260 °C. Hydrogen was used as carrier gas. The amount of the separated FAME was expressed as % of the total FAME. Various reference standards were used as FAME mix to identify fatty acid peaks/: No. 463, 674, (NU-CHEK PREP; INC., US), BR2, BR4, ME 93 (Larodan; Sweden), Supelco137 Component FAME Mix, PUFA No. 3. Lab Solutions software for GC (GC solution; Shimadzu, Japan) was used for peak integration. 1.2.2. Protein/amino acids (AA) The protein content of freeze-dried duckweeds was calculated via the Kjeldahl procedure using the N factor 6.25. The quantitative determination of AA in duckweed samples was based on the chemical properties of the proteinogenic AA. Majority of the proteinogenic AA was determined after subjecting the samples to acid hydrolysis with phenolic hydrochloric acid. For the sulphurcontaining AA, methionine and cysteine, an oxidation was performed before acid hydrolysis. The separation of AA occurred by means of ion exchange chromatography (Biochrom 30, Laborservice Onken, Gründau, Germany) and post column derivatization was carried out with ninhydrin. 1.2.3. Minerals After acid digestion of the ash under pressure, macro and trace elements were determined using the following techniques: calcium, phosphorus, magnesium, sodium, potassium, iron, copper, zinc, and manganese with ICP-AES; arsenic, selenium with hydride-AAS, mercury with cold vapour AAS by direct mercury analyser, cadmium, lead with graphite furnace AAS and iodine after ammoniac extraction with ICP-MS. 1.2.4. Carotenoids/tocopherols To determine the contents of carotenoids and vitamin E, the freeze dried duckweed samples were extracted three times under

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subdued light with methanol/tetrahydrofuran (1 + 1, v/v) by using an ultra turrax. Prior to solvent extraction, the plant material was softened with water for 5 min. The combined supernatants were concentrated in a rotary evaporator and the dry residue was re-dissolved in methanol/tetrahydrofuran (1 + 1, v/v) for further analysis. HPLC analysis of carotenoids was performed with a C30 column (column temperature: 13 ± 1 °C) using a gradient (1.0 mL/min) of methanol and methyl tert-butyl ether as mobile phase (Arnold, Schwarzenbolz, & Böhm, 2014). For analysis of vitamin E, an aliquot of the solution extracted with methanol/ tetrahydrofuran was evaporated under nitrogen stream and re-dissolved in n-hexane/methyl tert-butyl ether (98 + 2, v/m). HPLC analysis was performed with a diol column (column temperature: 35 ± 1 °C) using 1.5 mL/min n-hexane/methyl tert-butyl ether (98 + 2, v/m) as mobile phase (Franke, Fröhlich, Werner, Böhm, & Schöne 2010). 1.2.5. Sterols Freeze-dried duckweeds were used to extract lipids by accelerated solvent extraction (ASE) according to Hauff and Vetter (2009). Aliquots of this lipid extract were taken in 6 mL test tubes to which, the internal standard cholestanol-D2 was added, and the solvent was removed by a gentle stream of nitrogen. After addition of 0.9 mL ethanol and 0.1 mL aqueous KOH solution, the tube was sealed and heated to 80 °C for 1 h. The sample was cooled to room temperature, 1 mL of both distilled water and n-hexane was added and the tube was vigorously shaken. After phase separation, 500 lL of the organic phase was transferred into a screw cap vial, the solvent was removed and 25 lL pyridine and 50 lL BSTFA/TMCS (99:1) were added for silylation (60 °C, 30 min). The silylating agent was removed by a gentle stream of nitrogen and the residue was re-dissolved in 1 mL n-hexane. Then, the second internal standard 5a-cholestane was added and the solution was analysed by GC/MS. Silylated sterols were analysed with a 6890/5973 GC/MS system equipped with a 7683 auto sampler (Agilent) and an on-column inlet. Samples (1 lL) were injected into a guard column (2 m, 0.53 mm i.d.) which was press-fit connected to a 15 m, 0.25 mm i.d., 0.1 lm film thickness ZB-1 column (Phenomenex, Torrance, CA, USA). Helium (99.999% purity) was used as the carrier gas at a flow rate of 1.0 mL/min. The GC oven program was started at 60 °C which was held for 1 min. Then, the temperature was raised at 10 °C/min to 320 °C (hold time 10 min). Temperatures of the transfer line, ion source and quadrupole were set to 300 °C, 230 °C and 150 °C, respectively. Data were recorded from m/z 50–800 after a solvent delay of 6 min. 2. Results 2.1. Overview of nutritional quality over the five genera of Lemnaceae As a first step, representatives from all five genera of duckweeds (Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Lemna minor, Wolffiella hyalina and Wolffia microscopica) were investigated concerning their dry weight and the contents of protein, fat and starch (Fig. 1A–D). Additionally, the amino acid composition of the proteins and the fatty acid distribution of total fat were also investigated in all six duckweed species (Tables 1 and 2). Dry weight content of the six investigated species was between approximately 4 and 8% (Fig. 1A) of the fresh weight. All the following measures have been recorded on a dry weight basis. The lowest protein content was found in L. punctata with ca. 20% and the highest in W. hyalina with ca. 35% (Fig. 1B). The fat content was between ca. 4% and almost 7% (Fig. 1C). Also, the starch content showed larger differences between ca. 4% in L. minor and almost 10% in L. punctata (Fig. 1D).

The amino acid composition of total protein in all six species has been made available in Table 1. The contents of the 17 tested amino acids have been found to be the highest in Wolffiella hyalina and Wolffia microscopica, both belonging to the subfamily Wolffioideae. A comparison of the amino acid composition of duckweeds, given as an average of all six presently investigated species, with earlier results of four duckweed species (Rusoff et al., 1980), as well as with the amino acid composition of soya and chickpea flour (Jahreis, Brese, Leiterer, Schaefer, & Böhm, 2016) (Table S1) indicates that the amino acid composition in duckweeds is comparable with that of other plant proteins and it should be stressed that in no case the content of critical amino acids in duckweeds was lower than those recommended by WHO (2007). The content of cysteine + methionine was 22% higher than that of the recommendation. In addition, the content of threonine (in average 83% higher than that of the recommendation), phenylalanine plus tyrosine (102%) and leucine (25%) are above the recommended limits. For convenience of comparison, this table also provides the recommended amino acid composition for human nutrition i.e., the ‘‘adult indispensable amino acid requirement” (WHO, 2007). The distribution of fatty acids was investigated in the same samples as the proteins. Saturated fatty acids (SFA) were between 25 and 46% of the total fatty acid content (Table 2). Palmitic acid represented the most frequently available SFA, whereas the content of monounsaturated fatty acids (MUFA; mainly oleic acid, Table 2) was very low, with the maximum being 5.65% in L. punctata. In contrast, the content of polyunsaturated fatty acids (PUFA) was very high ranging from 48% in L. punctata to 71% in W. microscopica. Thus, the content of PUFA was in general close to or more than half of the total fatty acid content. In all the species investigated, the content of n3 fatty acids was surprisingly higher than the content of n6 fatty acids. This was mainly caused by the high content of a-linolenic acid, which was between 11 and 25% whereas c-linolenic acid was present only in small amounts or even close to the detectable limits. Stearidonic acid was only detected at low levels in L. gibba, L. minor and W. microscopica and in all cases, it was below 5%. As a consequence, the n6/n3 ratio of fatty acids was less than 0.3 in three of the duckweed species (S. polyrhiza, L. gibba, W. hyalina), below 0.5 in two of the species (L. punctata and L. minor) and in W. microscopica it was 0.61 (Table 2). Short chain fatty acids (SCFA) and medium chain fatty acids (MCFA), together contributing to less than 1%, play hardly any role on a quantitative scale. 2.2. In-depth investigations of Wolffia microscopica clone In order to evaluate the nutritional value of duckweeds besides protein, fatty acids and starch content, a detailed analysis including the composition of minerals, antioxidants (carotenoids and tocopherols), phytosterols, and fibre content was performed in one of the species, W. microscopica. This species was selected based on its promising results concerning amino acid and fatty acid distributions. Macro elements were characterized by very low contents of Na+ and high contents of K+ (Table 3). Microelements contained very high amounts of Cu2+, Fe2+/3+, Mn2+ and Zn2+. As discussed in the following section, the content of microelements depends to a large degree on the nutrient medium used for the cultivation of duckweeds. The ash content was detected to be 16.5% of dry weight (Table 3) and the fibre content was 28.6 ± 0.3% of dry weight. The main components of carotenoids were (all-E)-lutein with ca. 70 mg per 100 g dry weight (Fig. 2), followed by (all-E)violaxanthin with (46 mg/100 g DW) and (all-E)-b-carotene (28 mg/ 100 g DW). (all-E)-Zeaxanthin was present at much lower level (4.3 mg/100 g DW) but it might be of special importance

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Fig. 1. Main components in six species of duckweed. (A) Dry weight, (B) Total protein content, (C) Fatty acid content, (D) Starch content. Data (B–D) are related to dry weight. Means ± standard deviations are given.

Table 1 Amino acid composition of proteins from different duckweed species [g/ 100 g protein]. Amino acid

Spirodela polyrhiza

Landoltia punctata

Lemna minor

Lemna gibba

Wolffiella hyalina

Wolffia microscopica

CYS MET ASP THR SER GLU GLY ALA VAL ILEU LEU TYR PHE LYS HIS ARG PRO

0.8 1.6 7.8 4.2 4.1 9.6 4.3 5.4 4.4 3.3 6.8 3.1 3.97 4.2 1.6 4.7 3.5

1.1 1.6 8.1 4.1 4.0 9.5 4.5 5.3 4.6 3.5 7.3 3.1 4.5 4.1 1.6 4.7 4.1

0.9 1.6 8.2 4.0 4.1 9.8 4.6 5.1 4.6 3.7 7.3 3.1 4.4 5.0 1.5 4.8 3.8

0.9 1.6 10.6 4.0 4.2 10.3 4.6 6.0 4.5 3.4 7.2 3.2 4.3 4.2 1.6 4.9 3.9

1.0 2.0 7.3 4.2 4.3 10.5 5.0 6.0 4.8 3.9 8.0 3.8 5.1 5.8 1.7 4.7 3.7

1.2 1.6 10.4 4.7 4.7 10.9 4.7 7.8 4.9 3.7 7.7 3.3 4.2 5.7 1.7 5.2 3.6

concerning human consumption. a-Tocopherol (9 mg/ 100 g DW) and c-tocopherol (13 mg/100 g DW) were detected as additional antioxidants (Fig. 2) as investigated in W. microscopica. Because sterols play an important role in regulating membrane fluidity and permeability (Luthria, Lu, & John, 2015), and are

known to lower the serum cholesterol in humans (Kritchevsky & Chen, 2005), we also investigated phytosterols in W. microscopica. The total content was approximately 50 mg per g lipid. The main components were sitosterol, campesterol and stigmasterol followed by D5-avenasterol (Table 4). Traces of cycloartenol

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Table 2 Fatty acid distribution in lipids of different duckweed speciesa,b

a b

Fatty acids

S. polyrhiza

L. punctata

L. minor

L. gibba

W. hyalina

W. microscopica

SFA MUFA PUFA Sum n3 Sum n6 n6/n3 ratio SC-FA (C-4 > C-10) MC-FA (C-11 > C-14)

39.85 ± 0.01 4.40 ± 0.02 55.75 ± 0.01 44.26 ± 0.24 10.85 ± 0.02 0.25 ± 0.00 0.02 ± 0.00 0.60 ± 0.02

46.23 ± 0.57 5.65 ± 0.46 48.12 ± 1.03 32.66 ± 0.51 14.89 ± 0.52 0.46 ± 0.01 0.01 ± 0.00 0.41 ± 0.01

27.99 ± 0.35 4.63 ± 0.04 67.38 ± 0.31 46.00 ± 0.06 20.89 ± 0.12 0.45 ± 0.00 0.00 ± 0.01 0.48 ± 0.03

28.25 ± 0.11 4.13 ± 0.03 67.62 ± 0.07 53.08 ± 0.01 14.23 ± 0.09 0.27 ± 0.00 0.10 ± 0.00 0.62 ± 0.03

32.20 ± 0.05 5.32 ± 0.06 62.48 ± 0.01 47.99 ± 0.11 14.12 ± 0.01 0.29 ± 0.00 0.00 ± 0.01 0.61 ± 0.01

25.07 ± 0.14 3.77 ± 0.09 71.16 ± 0.23 44.22 ± 0.18 26.77 ± 0.04 0.61 ± 0.00 0.00 ± 0.00 0.14 ± 0.00

Single FAME > 1% C-16:0 C-18:1c9 C-18:2c9,c12 cC-18:3c6,c9,c12 aC-18:3c9,c12,c15 C-18:4c6,c9,c12,c15 C-22:0 C-24:0 C-26:0

29.38 ± 0.04 1.71 ± 0.02 10.59 ± 0.01 0.01 ± 0.00 43.86 ± 0.24 0.00 ± 0.01 0.42 ± 0.02 3.01 ± 0.19 0.56 ± 0.06

32.59 ± 0.16 3.09 ± 0.39 14.36 ± 0.14 0.01 ± 0.00 32.40 ± 0.54 0.00 ± 0.00 0.52 ± 0.01 3.83 ± 0.02 2.30 ± 0.31

21.33 ± 0.25 1.67 ± 0.00 20.11 ± 0.12 0.52 ± 0.00 44.47 ± 0.10 1.16 ± 0.04 0.37 ± 0.01 1.32 ± 0.05 0.46 ± 0.02

22.08 ± 0.23 0.69 ± 0.01 12.99 ± 0.08 1.02 ± 0.01 48.60 ± 0.10 3.91 ± 0.12 0.54 ± 0.02 1.61 ± 0.09 0.26 ± 0.02

22.03 ± 0.11 1.08 ± 0.01 13.75 ± 0.01 0.02 ± 0.01 47.85 ± 0.12 0.00 ± 0.00 2.57 ± 0.03 1.81 ± 0.03 0.93 ± 0.02

20.23 ± 0.13 1.52 ± 0.00 25.42 ± 0.01 1.20 ± 0.01 39.58 ± 0.04 4.57 ± 0.13 0.90 ± 0.00 0.96 ± 0.00 0.33 ± 0.01

Fatty acids were separated gas chromatographically as corresponding fatty acid methyl esters (FAME). Data are expressed as mean ± SD of % total FAME.

Table 3 Macro and micro elements in Wolffia microscopica. Macro elements [g/kg DW]

Micro elements [mg/kg DW]

Heavy metals [mg/kg DW]

Ca

6.0

Fe

240

Cd

0.40

K Mg Na P

83 3.1 0.30 7.04

Mn Cu Zn I Se

755 3.52 30.8 0.75 0.06

Pb Hg As

0.24 0.04 0.05

Ash content: 165.1 ± 0.6 g/kg DW

Table 4 Pattern of phytosterols in Wolffia microscopica (Total content of sterols: 50 mg/g lipid extract). Peak number

Sterol

Retention time [min]

Sterol composition

1 2 3 4 5

Campesterol Stigmasterol Sitosterol D5-Avenasterol D5,24(25)Stigmastadienol* Cycloartenol

20.2 20.4 20.7 20.8 20.9

18% 15% 53% 11% 1%

21.0

2%

6 *

Tentatively identified by (relative) GC retention time and GC/MS data.

(2% of total sterol content) and an isomer of avenasterol (most probably D5,24(25)-stigmastadienol based on GC/MS data) were also detected in the samples. 3. Discussion 3.1. Protein and amino acid composition

Fig. 2. Content of carotenoids and tocopherols in the duckweed Wolffia microscopica. Means ± standard deviations are given.

The total protein content of duckweeds as investigated in the present project was between 20 and 35% per dry weight for the different species. As also were several other reports with the protein content on an average ranging from 18.9% to 36.5% (Bhanthumnavin & McGarry, 1971; Bergmann, Cheng, Classen, & Stomp, 2000; Rusoff et al., 1980). Most important, however, is

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the observation that the protein content depends on the cultivation conditions of the plants. Xu, Cui, Cheng, and Stomp (2011) observed that the duckweed biomass transferred from artificial swine medium to clean source water or salty water showed a decrease in the protein content but at the same time an increase of the starch content. The balance between these two components under the influence of nitrate and phosphate deficiency was investigated by Zhao et al. (2015). It is noteworthy that already in 1987 Landolt & Kandeler stressed the very high impact of cultivation conditions viz. high light intensity and high nitrate concentrations on the protein content (ranging from 6.8% to 45% of dry weight) in different duckweed species. It can be concluded that the protein content of duckweeds can be easily manipulated by optimizing the cultivation conditions of different duckweed species and clones with the protein content reaching close to 40% of the dry weight. Yu et al. (2011) investigated thoroughly the energetic properties of duckweed (S. polyrhiza) protein. Owing to the amino acid composition, the total protein of duckweeds qualifies as a high quality protein source for human nutrition. It should be stressed that in no case the content of critical amino acids in duckweeds was lower than the WHO recommendations (2007). Concerning the six species investigated, W. microscopica and W. hyalina (Table 1) showed the highest contents of amino acids indispensable for human nutrition. Here, the high contents of lysine should be mentioned that are above the levels in common flours of wheat, corn and rice (Edelman & Colt, 2016). The amino acid composition of duckweeds can successfully be compared with that of flours from legumes like chickpea, lupine, or pea (Table S1, supplementary material), which have been recommended for vegetarian or vegan nutrition (Jahreis et al., 2016). The amino acid composition seemed to be mainly determined by the species under investigation as observed from the investigations on S. polyrhiza, L. punctata, and L. gibba by Rusoff et al. (1980) where the duckweed were cultivated in a cattle waste lagoon, and the present project which included laboratory grown cultures. It was observed that the cultivation conditions like the light conditions did not have any impact on the amino acid composition of duckweeds (Appenroth et al., 1982). Out of the presently investigated six species, W. hyalina and W. microscopica had the highest levels of lysine, cysteine + methionine, and tyrosine + phenylalanine. The protein contents and protein quality of these two species were not investigated in earlier reports but these two species may have high potential for human nutrition as they have a very high potential in terms of biomass production with W. microscopica being the fastest growing angiosperm known till date (Sree, Sudakaran, & Appenroth, 2016; Ziegler, Adelmann, Zimmer, Schmidt, & Appenroth, 2015). 3.2. Fat and fatty acid distribution The fat content of the six investigated species was not very different and between approximately 4 and 6% per dry weight. It should be stressed, however, that this content is related to the whole plant biomass and not only to e.g. seeds as in most crop plants. The highest content was found, as in case of protein content, in the species W. hyalina and W. microscopica. Yan et al. (2013) investigated 30 out of the 37 known duckweed species under cultivation conditions different from that in the present project, i.e. at 22 °C and in Schenk-Hildebrand medium supplemented with glucose (i.e. mixotrophic cultivation conditions) and the fatty acid content ranged between approximately 4 and 14% dry weight. Comparing the contents in species that were investigated in both projects it turns out that these values obtained by Yan et al. (2013) were approximately double of that in the present project. This holds true also for S. polyrhiza and L. gibba where even the same clones were investigated. We assume that this significant

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difference was caused by the mixotrophic cultivation conditions in Yan et al. (2013). We decided to cultivate under autotrophic conditions because mixotrophic conditions are not useful when large-scale production of biomass would be required. Bhanthumnavin and McGarry (1971) reported 5.0% total fatty acid for a Wolffia species, where also autotrophic conditions were maintained. Total fat content in flours from chickpea and lupine was comparable to that in the whole plants of duckweeds but was found to be much higher in soya bean (Table S2, supplementary material; Jahreis et al., 2016). For the intended use of duckweed for human nutrition, the fatty acid distribution is very important. The content of saturated fatty acids (mainly palmitic acid, followed by lignoceric acid) was relatively high, that of monounsaturated fatty acids (mainly based on oleic acid) and of SCFA and MCFA was very low. Interestingly, the content of polyunsaturated fatty acids (PUFA) was very high comparable or even higher than in legume flours (Table S2, supplementary material). In comparison to common plant oils (Table S2), except palm and coconut oil, the fraction of SFA was higher and the fraction of MUFA was much lower. The main PUFA were the n6 fatty acids linoleic acid and c-linolenic acid, and the n3 fatty acids a-linolenic acid and stearidonic acid. The ratio between omega-6 to omega-3 (n6/n3) ranged between 0.25 in S. polyrhiza and 0.61 in W. microscopica based on the very high content of the n3 fatty acid a-linolenic acid. Yan et al. (2013) presented a very extensive survey of 30 species of duckweed investigating the fatty acid distribution. We calculated the n6/n3 ratio from the data given by Yan et al. (2013) in their Table 2. The highest ratios were obtained for W. borealis (0.75) and values of 0.1 or lower were obtained for S. polyrhiza, S. intermedia, L. punctata, and W. neotropica and the average of all 30 data was 0.36 ± 0.16 (mean ± standard deviation). The species W. globosa (Tang et al., 2015; Yan et al., 2013) should be mentioned here because it is the dominating Wolffia species in many Asian countries, which have a tradition of using duckweed as human food. The concentration of PUFA was given as 61–64% TFA and the ratio n6/n3 could be calculated from their data as 0.23–0.29. In legume flours the n6/n3 ratio was determined to be as high as 4.6 (lupine), 5.7 (green pea), and 21.8 (chickpea) and in common edible plant oils, the n6/n3 ratio showed a high variation from 0.28 in flax seed and 2.2 in rapeseed up to 15 in olive oil and 150 in sunflower (Table S2, supplementary material; Jahreis & Schaefer, 2011). However, in palm, coconut and peanut oil, hardly any n6 fatty acid could be detected. According to the recommendations of FAO (2010), the n6/n3 ratio should not be higher than 5. This is not fulfilled with at least some of the legume flours. Simoupolos (2006) summarized in his review that ‘‘studies at the molecular level indicate that human beings evolved on a diet with an n6/n3 ratio of essential fatty acids (EFA) of 1 whereas in Western diets the ratio is 15:1–16.7:1. A high n6/n3 ratio, as is found in today’s Western diets, promotes pathogenesis of many diseases, including cardiovascular disease, cancer, osteoporosis, and inflammatory and autoimmune diseases”. Russo (2009) also stressed the clinical implications of n6 and n3 PUFA. Duckweed total fat is very rich in the n3 a-linoIenic acid, which results in very low n6/n3 ratios. Addition of duckweed plants to human food would therefore correct the unfavourable n6/n3 ratio of fatty acids, which is especially present in the Western diet. 3.3. Starch and fibre content The starch content in the six investigated duckweed species (Fig. 1D) was between 4% (L. minor) and 10% (L. punctata). Tang et al. (2015) reported approximately 11% starch for four investigated species. This means that the starch content is much lower than the protein content in duckweeds grown under favourable cultivation conditions. This is interesting with regards to the

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attempts of de-energization of human food in industrial countries. In some of the Asian countries, additional feed times rich in starch are not of much interest considering the use of starch-rich staple food, rice. The food in these countries is rich in starch but poor in protein. As discussed in Section 3.2, the starch content in duckweeds is strongly affected by the cultivation conditions (Cui & Cheng, 2015; Xu et al., 2011). It has been shown that salty nutrient media can dramatically increase the starch content to >40% on dry weight basis (Sree, Adelmann, Garcia, Lam, & Appenroth, 2015). Also lack of some nutrients (like phosphate or nitrate) or the presence of some heavy metals increases the starch content as demonstrated in several duckweed species, e.g. L. minor (Zhao et al., 2015). Reid and Bieleski (1970) reported the starch content in L. punctata (in that time described as Spirodela oligorrhiza) to be 75%. However, these plants were cultivated in the presence of glucose, which is impractical from a biotechnological point of view. Without added glucose these authors reported starch content to be only 29%. Although high starch content is important for biofuel production (Xu, Cui, Cheng, & Stomp, 2012), in countries with rice-based staple food high starch content is not required for food and in industrial countries it would be against the interest of low energy food. The low starch content detected in fast growing duckweed populations, i.e. under optimal growth conditions, would represent a favourable food item for human nutrition both in developing and industrial countries. 3.4. Further investigations in Wolffia microscopica Water, protein, fat and starch contents were investigated in all six duckweed species. Additional investigations, important for the use as human food, were carried out in W. microscopica, which showed very good performance concerning the above mentioned parameter. The mineral composition of duckweeds also revealed some very interesting aspects. In the present investigation, W. microscopica showed a very high K+/Na+ ratio of 276 and an Mg2+/Ca2+ ratio of 0.5. This was of course caused by the composition of the nutrient medium. Thus, food poor in Na+ and rich in Mg2+ is easily available through duckweed. The trace element analysis revealed high contents of Mn2+, Fe2+/3+, Zn2+, and Cu2+. This reflected the content of these trace elements in the Schenk-Hildebrand medium. It can be expected that other nutrient media would change the composition. This is confirmed by phytotoxicity investigations in duckweed by applying heavy metals like cobalt (Sree, Keresztes et al., 2015), and zinc (Lahive, O’Callaghan, Jansen, & O’Halloran, 2011). Thus, by selecting the required concentration in the nutrient medium, almost any concentration of trace elements in the plant material can be adjusted to the requirement for specific human nutrition. As an example, production of selenium-rich food should be mentioned here (Lo, Elphick, Bailey, Baker, & Kennedy, 2015). Landolt and Kandeler (1987) summarized a larger number of older reports of duckweed species cultivated in different nutrient media with the limits for K+/Na+ as 0.67–37 and the ratio of Mg2+/Ca2+ as 0.05–20, and reported the Zn2+ content to range from 40 to 1400 mg/kg dry weight. Recently, Edelman (2016) reported ratios of K+/Na+ = 40 and Mg2+/Ca2+ = 0.4 and Zn2+ content to be >150 mg/kg dry weight in one species of Wolffia. Unfortunately, the composition of the nutrient medium was not reported. The content of antioxidants showed very high concentrations of lutein and also of zeaxanthin in comparison to other vegetarian foods. This makes duckweeds interesting with respect to prevention of age-related macular degeneration, especially as the content of omega-3 fatty acid and the n6/n3 ratio supports very much the effects of xanthophylls (Chew et al., 2013). The high content of tocopherols adds to the useful properties of this plant material.

Also the phytosterol content (50 mg g 1 fat) of W. microscopica is worth to be stressed because this concentration was at least 5-fold higher than in most other plant oils (typically, 1–10 mg g 1 fat; Phillips, Ruggio, Toivo, Swank, & Simpkins 2002). Sitosterol (>50%), campesterol (20%) and stigmasterol (15%) dominated, which is comparable with other plant oils (Table 4). The increasing nutritional interest in phytosterols derives from the fact that phytosterols have the capacity to lower plasma cholesterol and LDL cholesterol (Luthria et al., 2015). Duckweeds seem to be one of the interesting plants having high content of phytosterols. 3.5. Yield of nutritional components The yield (for definition see Ziegler et al., 2015) of dry weight, protein, fatty acid and starch were given for all six duckweed species investigated here (Table S3, supplementary material). The high growth rates of W. hyalina and W. microscopica (Sree, Sudakaran et al., 2016) makes these rarely used plants an excellent material for human nutrition, even in comparison with other duckweed species. This is in accordance with their properties concerning the amino acid composition and the fatty acid distribution. 3.6. Conclusion and future perspectives Duckweeds, especially species belonging to Wolffia, have been traditionally eaten in some parts of the world as a component of the nutritious human diet. Duckweeds are eaten as salad, mixed in soups or curries or in omelettes. Both vegetarian and nonvegetarian meals are possible (for recipes, form of uses and market price cf. Sree & Appenroth, 2016). Its present day acceptance as human food source needs thorough investigations with respect to its nutritive value, large-scale yield, economical and sustainable market supply. Our present report provides an in-sight into the nutritional composition of at least one representative duckweed species from each of the five genera, highlighting the beneficial aspects of duckweed as human food item. It has been already reported that members of Wolffioideae (the present study included two of them, Wolffiella hyalina and Wolffia microscopica), have oxalate in the free form unlike the other duckweed genera which form oxalate crystals (Landolt & Kandeler, 1987) and this feature favours the use of Wolffioideae members as human food. More investigations in this direction of analysing the anti-nutritive components of duckweeds, if any, would be of importance to its widespread acceptance as nutritive human food source. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. 08.116. References Appenroth, K.-J., Augsten, H., Liebermann, B., & Feist, H. (1982). Effects of light quality on amino acid composition of proteins in Wolffia arrhiza (L.) Wimm. using a specially modified Bradford method. Biochemie und Physiologie der Pflanzen, 177, 251–258. Appenroth, K.-J., Teller, S., & Horn, M. (1996). Photophysiology of turion formation and germination in Spirodela polyrhiza. Biologia Plantarum, 38, 95–106. Arnold, C., Schwarzenbolz, U., & Böhm, V. (2014). Carotenoids and chlorophylls in processed xanthophyll-rich food. LWT – Food Science and Technology, 57, 442–445. Bhanthumnavin, K., & McGarry, M. G. (1971). Wolffia arrhiza as a possible source of inexpensive protein. Nature, 232, 495. Bergmann, B. A., Cheng, J., Classen, J., & Stomp, A.-M. (2000). In vitro selection of duckweed geographical isolates for potential use in swine lagoon effluent renovation. Bioresource Technology, 73, 13–20. Chew, E. Y., Clemons, T. E., SanGiovanni, J. P., Danis, R., Ferris, F. L., Elman, M., et al. (2013). Lutein + Zeaxanthin and Omega-3 fatty acids for age-related macular

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