Leonie
van 't Hag
a,
Jessica
Danthe
a,
Stephan
Handschin
a,
Gibson P.
Mutuli
b,
Duncan
Mbuge
b and
Raffaele
Mezzenga
*ac
aDepartment of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland. E-mail: raffaele.mezzenga@hest.ethz.ch
bDepartment of Environmental and Biosystems Engineering, University of Nairobi, P.O. Box 30196, 00100 G.P.O, Nairobi, Kenya
cDepartment of Materials, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland
First published on 2nd January 2020
The problem of malnutrition and nutrition deficiency, as well as droughts that lead to reduction in food supply and starvation, is well documented for Sub-Saharan Africa. Reducing post-harvest losses of five species of African leafy vegetables (ALVs) by preservation through drying is studied herein. Energy efficient gentle drying conditions using superabsorbent polymers and a temperature of 40 °C were shown to preserve most leaf structures and vitamins. The microbial safe moisture content of the ALVs was found to be ≤14% dry basis. Dried Slender Leaf and Nightshade leaves could be rehydrated to the equilibrium moisture content of fresh leaves upon dry storage, while it was not possible for Jute Mallow, Cowpea and Amaranthus. This was attributed to different palisade parenchyma cell lengths. An increased amount of starch granules as observed in the microstructure of Cowpea and Nightshade leaves is suggested to explain their fibrous texture upon cooking. These results show that the ALVs can be effectively preserved using the same drying method and that this can be used to fight micro-nutrient deficiencies during droughts.
ALVs thrive well in the rainy season but are highly liable to degradation once harvested. Microbial contamination and toxicity commonly lead to post-harvest losses.6 One of the main microorganisms responsible for the formation of food toxins is Aspergillus flavus (A. flavus) which is a pathogenic fungus. Its highly toxic secondary metabolite aflatoxin B1 is estimated to be responsible for up to 28% of the cases of hepatocellular carcinoma, the most common form of liver cancer.7 In Kenya, outbreaks of aflatoxin poisoning have been reported in 1978, 1982 and annually between 2001 and 2012, leading to loss of human lives and destruction of tons of contaminated food.8 Drying of ALVs can address post-harvest losses due to reduced microbial contamination.9 Importantly, it was demonstrated that the consumption of sun-dried Cowpea and Amaranthus leaves improved the level of β-carotene and consequently vitamin A levels in children.10 Traditional sun drying methods, however, often yield poor results.9 This has become particularly significant in recent years, since the weather has become more unpredictable and the rainy season often continues during the harvest time.7 A recent study by some of us showed that upon controlled drying at 40 °C the β-carotene levels (pro-vitamin A) in Cowpea and Jute Mallow leaves was only reduced by ∼10%, whereas drying at 100 °C caused a reduction of 35–50%.11 The more hydrophilic vitamins B2 and C were also preserved significantly better at 40 °C (loss of only 15–20% and 25–40%, respectively) compared to 100 °C (both loss of ∼50%).11 We will focus on controlled and gentle drying at 40 °C herein since especially there is widespread vitamin A deficiency in children.
The range of water activities at which pathogenic microorganisms cannot grow is crucial for determining safe preservation conditions of food.12 Water activity (aw) is the partial pressure of water in food (P) relative to the vapor pressure of pure water at the same temperature (P0).13 Pathogenic bacteria are not able to grow at aw < 0.86 while yeast and molds do not grow at aw < 0.62.14 For this reason, it is crucial to understand the moisture sorption characteristics of vegetables for effective preservation: this indicates the values of aw for different equilibrium moisture contents. Moisture sorption isotherms of fresh and pre-dried leaves at 40 °C were used to determine the monolayer moisture content and microbial safe moisture content (aw < 0.6) of five species of ALVs.
The five species of ALVs used in this study are Corchorus olitorius (Jute Mallow), Crotalaria ochroleuca (Slender Leaf), Vigna unguiculata (Cowpea), Solanum villosum (Nightshade) and Amaranthus blitum (Amaranthus). Jute Mallow and Slender Leaf break down into a pasty (slimy) mass when cooked while Cowpea, Nightshade and Amaranthus are highly fibrous. These characteristics are representative of other ALV species in the particular groups and the soft matter analysis can give an indication of the behavior of other ALVs with similar compositions. In this study, we explain the observed differences in moisture sorption characteristics of the five different species using their microstructure. Thin and ultra-thin cross-sections of the leaves were investigated using brightfield microscopy and transmission electron microscopy (TEM) to analyze their microstructure. These results provide guidelines for the safe storage of ALVs during droughts, providing a good source of micro-nutrients and helping in fighting hidden hunger.
Fig. 1 Photographs of six-week-old plants: (A) Jute Mallow, (B) Slender Leaf, (C) Cowpea, (D) Nightshade and (E) Amaranthus. The pots are 13 cm high and their diameter (∅) is 15 cm. |
Five different moisture sorption models (Table 1) were used to fit the DVS data with the non-linear least squares method (MATLAB, v. R2017a, MathWorks, Inc.). The models predict the equilibrium moisture content (Me) as a function of the water activity (aw) which is the relative partial pressure of water in the leaves (P) compared to the vapor pressure of pure water at the same temperature (P0) as shown on the x-axes in Fig. 2 and 3 in percent (%). Firstly, the Henderson equation is widely used to model sorption isotherms and the constants A and B depend on temperature and characterize the product.16 The other two-parameter model, the Caurie model, was derived to describe limited multilayer adsorption. M0 is the monolayer moisture content and C is the Caurie constant which is related to the heat of sorption.17,18 The other three models are three-parameter models. The modified Halsey equation provides an expression for multilayer condensation at a large distance from the surface. It takes the effect of temperature (T) into account and further consists of the constants A, B and C.19 The SIPS isotherm model was developed for predicting systems with heterogeneous adsorption and it uses the sorption capacity (KS), sorption intensity (N) and energy of adsorption (C).8 The Guggenheim–Anderson–de Boer (GAB) model is commonly used for food systems where M0, the Guggenheim constant (KG) related to the first layer of sorption and a constant (k) describing the sorption of the multilayers characterize the samples.13,20
Fig. 3 Dynamic vapor sorption analysis of superabsorbent polymer Luquafleece fabric at 40 °C. (A) Adsorption isotherm followed by (B) desorption isotherm. Data are shown with a fit using the GAB model and fitting parameters are reported in Table 4. |
The goodness of fit for the five different models was evaluated with the root mean square error (RMSE), coefficient of determination (R2), mean relative deviation modulus (E%) and chi-square test (χ2), Table 1. The RMSE determines the average error based on the sum of squared errors.21R2 indicates the percentage of variability in the dependent variable and adopts values from zero to one, where one represents a perfect fit.22E% is widely used to assess sorption model evaluation and indicates a tendency to over- or underestimate the experimental data. A value below 10% is indicative of a good fit and a value below 5% an excellent fit.23–25χ2 indicates the similarity between the predicted and measured data where small values indicate a good fit and large values represent significant variation.22
Ultrathin cross-sections of ∼50 nm thickness for TEM after chemical fixation and embedding were obtained from fresh and dried leaves upon rehydration for ∼24 hours followed by degassing in 0.1 M PBS buffer (pH 7) for 20 minutes under vacuum as described for the thin sections. For dried samples, the leaves were again dried for 24 hours (48 hours for Cowpea since the weight was not stable within 24 hours) at 40 °C and stored in a sealed Petri dish for two weeks. Sample processing for chemical fixation of the hydrated leaves was done in a PELCO BioWave, Pro+ microwave system (Ted Pella, USA), following a microwave-assisted fixation and dehydration procedure. Fixation was done in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M PBS buffer (pH 7) followed by post-fixation in 1% osmium tetroxide (OsO4) in 0.1 M PBS (pH 7). After washing in the same PBS buffer once and twice in dd-H2O post-fixation was done in 1% uranyl acetate (UA), followed by washing in dd-H2O three times. Dehydration was done in a graded series of ethanol (50%, 60%, 70%, 80%, 90%, 96 and 100%) on ice. For embedding, Spurr's resin at 25% in ethanol was used twice. The embedding in 50% (twice: 2 hours at room temperature), 75% (2 h at room temperature and then overnight at 4 °C) and 100% (three times: 2, 4, 2 hours) was performed without microwave-assistance. Polymerization was performed at 60 °C for 48 hours. Ultrathin sections (∼50 nm) were cut on a Leica Ultracut FC6 (Leica Microsystems, Vienna, Austria), transferred on formvar coated carbon-grids (Quantifoil, Germany) and post-stained with uranyl acetate and lead citrate. Imaging was performed in a Morgagni 268 TEM (Thermo Fisher Scientific, USA) operated at 100 kV. Slight shading of images in Fig. 5(A, B and D, fresh) was corrected in Photoshop.
MC (% d.b.) | MC (% w.b.) | |
---|---|---|
Jute Mallow | 282 ± 42 | 74 ± 3 |
Slender Leaf | 565 ± 130 | 85 ± 3 |
Cowpea | 509 ± 44 | 84 ± 1 |
Nightshade | 471 ± 99 | 82 ± 3 |
Amaranthus | 313 ± 29 | 76 ± 2 |
Dynamic vapor sorption analysis of the five species of ALV plants at 40 °C is shown in Fig. 2(A–E). For each of the species the desorption and adsorption isotherms of fresh leaves are shown, as well as the adsorption (rehydration) and desorption isotherms of pre-dried and stored leaves. All curves can be described as Type II, with a sigmoidal shape, according to the Brunauer classification which is based on the van der Waals adsorption of gases on solid substrates.26 This shape is typical of moisture sorption isotherms of food that is low in sugar and has also been observed for leaves of other plant species.13,27–29 At high relative humidities (P/P0) excess water is present in the leaves which can behave as bulk water; from P/P0 ∼ 20–70% water is present in multilayers weakly bound to the solid, and from P/P0 ∼ 0–20% water is strongly bound and represents adsorption of the monolayer of water which is not available for chemical and deteriorative reactions. Hysteresis is defined as the difference in equilibrium moisture contents between the adsorption and desorption isotherms, and is often observed as delayed desorption for food samples due to water trapped in capillaries.13 Hysteresis was not observed for the fresh and pre-dried samples of the five ALV species suggesting that dehydration to a significantly lower moisture content already occurs at high relative humidities. For quantification, the data fitting results in the next sub-sections are used.
For Slender Leaf (Fig. 2(B)) and Nightshade (Fig. 2(D)) the moisture sorption isotherms of pre-dried and stored leaves were equal to those of the fresh leaves indicating complete rehydration of leaves that were stored dry for 4–8 weeks. We have confirmed for Slender Leaf that dry storage for four weeks (Fig. 2(B)) and nine weeks resulted in identical moisture sorption isotherms. At high relative humidities the moisture content of pre-dried and stored Jute Mallow (Fig. 2(A)), Cowpea (Fig. 2(C)) and Amaranthus (Fig. 2(E)) leaves was lower than that for the fresh leaves. This suggests that physical (e.g. structural) or chemical changes occurred during storage resulting in a modified material and therefore no complete rehydration of these species. Differences between the five species are directly compared and shown in Fig. 2(F and G) for the desorption isotherms of fresh leaves and adsorption isotherms (rehydration) of pre-dried and stored leaves. No significant changes were observed in the desorption characteristics of the five species indicating that both fibrous and slimy species can be dried for preservation with the same protocol. Jute Mallow, Cowpea and Amaranthus, however, did not rehydrate to the same level after being stored dry. There was no correlation between the equilibrium moisture content of the five species as obtained with the moisture sorption isotherms and the moisture content of the fresh leaves (Table 2).
Five different empirical and semi-empirical models were used to model the moisture sorption isotherms (Table 1). The best fit with each of these models is shown for the desorption isotherm of a fresh Cowpea leaf in Fig. 2(H–J) as an example. The goodness of fit parameters for the four moisture sorption isotherms of Cowpea are shown in Table 3. The two-parameter (p1) Henderson and Caurie models did not fit the data as well as the three-parameter (p2) Halsey and GAB models. The F-test with p = 0.05 and also showed that the GAB model resulted in a significantly better fit for the Cowpea data (F = 11–80 which is larger than the critical F-value of 6.6) compared to the Henderson and Caurie models for the majority (6/8) of data sets shown in Table 3. The exceptions were the fits of the adsorption and desorption curves of dried Cowpea (F = −3 & 5) with the Caurie model. Additionally, the SIPS model did not result in a good fit for the dried Cowpea leaves and different combinations of the SIPS fitting parameters could lead to the same fitting result. For this reason, the obtained values for the sorption capacity (KS), sorption intensity (N) and energy of adsorption (C) could not be interpreted. Since the Halsey model uses fitting parameters with no physical meaning, the equally well performing GAB model is used herein to interpret the DVS data.
Cowpea | |||||
---|---|---|---|---|---|
Model | Parameters | Fresh ads. | Fresh des. | Dried ads. | Dried des. |
Henderson | RMSE | 2.10 | 2.10 | 2.05 | 2.15 |
R 2 | 0.989 | 0.989 | 0.989 | 0.989 | |
E (%) | 40.43 | 40.19 | 26.53 | 27.90 | |
χ 2 | 5.62 | 5.87 | 4.97 | 6.23 | |
Caurie | RMSE | 0.94 | 0.92 | 0.49 | 0.75 |
R 2 | 0.998 | 0.998 | 0.999 | 0.999 | |
E (%) | 18.85 | 19.53 | 6.61 | 10.20 | |
χ 2 | 1.04 | 1.24 | 0.30 | 1.01 | |
Halsey | RMSE | 0.66 | 0.60 | 0.62 | 0.30 |
R 2 | 0.999 | 0.999 | 0.999 | 1.000 | |
E (%) | 14.78 | 8.98 | 9.62 | 2.85 | |
χ 2 | 0.64 | 0.35 | 0.92 | 0.09 | |
SIPS | RMSE | 0.63 | 0.57 | 1.64 | 1.56 |
R 2 | 0.999 | 0.999 | 0.993 | 0.994 | |
E (%) | 12.52 | 7.82 | 17.28 | 19.27 | |
χ 2 | 0.49 | 0.30 | 2.44 | 2.92 | |
GAB | RMSE | 0.51 | 0.51 | 0.82 | 0.53 |
R 2 | 0.999 | 0.999 | 0.996 | 0.998 | |
E (%) | 10.46 | 11.61 | 6.40 | 4.67 | |
χ 2 | 0.37 | 0.53 | 0.47 | 0.22 |
The fitting parameters for the GAB model for each of the DVS curves in Fig. 2 are shown in Table 4. The monolayer moisture content M0 was determined to be 3.7–4.9% d.b. for all species. Similar values of M0 were obtained with the Caurie model (3.6–6.5% d.b.). This strongly bound moisture does not participate in deteriorative reactions. The Guggenheim constant (KG) is related to the first layer of sorption and determined from the low relative humidity (P/P0 < 20%) region of the moisture sorption isotherms. Differences in KG were significantly larger between sample treatments than between species. For pre-dried and stored leaves, values of KG are an order of magnitude (23–190) larger than those for fresh leaf samples (4–20). The increased moisture sorption at the lowest relative humidities for dried leaves can also be seen in Fig. 2(A–E). This is expected to be caused by chemical changes during storage. The constant describing water sorption of the multilayers (k) is lower for pre-dried leaves of Jute Mallow, Cowpea and Amaranthus than those for their fresh leaves. This is represented by the reduced moisture content for these samples at high relative humidities (Fig. 2(A, C and E)). This is again expected to be due to chemical and structural changes during storage as investigated and confirmed by using microscopy in the next sub-section. The microbial safe moisture content of the African leafy vegetables (aw < 0.6) was determined using the GAB model and found to be ≤14% d.b.
Fresh adsorption | Fresh desorption | Dried adsorption | Dried desorption | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
GAB model | M 0 | K G | k | M 0 | K G | k | M 0 | K G | k | M 0 | K G | k |
Jute Mallow | 0.043 | 7.46 | 0.96 | 0.043 | 8.06 | 0.96 | 0.040 | 47.8 | 0.93 | 0.037 | 58.6 | 0.93 |
Slender Leaf | 0.046 | 9.84 | 0.97 | 0.049 | 7.54 | 0.96 | 0.043 | 35.8 | 0.96 | 0.044 | 82.5 | 0.96 |
Cowpea | 0.043 | 3.89 | 1.01 | 0.043 | 3.81 | 1.01 | 0.045 | 50.2 | 0.92 | 0.041 | 108 | 0.93 |
Nightshade | 0.041 | 9.91 | 0.97 | 0.042 | 8.22 | 0.97 | 0.040 | 23.4 | 0.97 | 0.044 | 10.9 | 0.96 |
Amaranthus | 0.051 | 10.2 | 0.96 | 0.049 | 20.2 | 0.97 | 0.049 | 55.5 | 0.91 | 0.049 | 190 | 0.91 |
SAP | 0.210 | 1.27 | 0.86 | 0.179 | 1.71 | 0.88 |
The DVS data in Fig. 2 show that the ALVs already significantly increase their moisture content above 60% RH. Considering that the relative humidity in sub-Saharan Africa is in the range of 70–80% for most of the year, we suggest using superabsorbent polymers (SAP) to lower the moisture content of air in a standard convective dryer during dehydration at gentle temperatures (40 °C) and to use SAP to ensure storage of the ALVs below the microbial safe moisture content. DVS data of SAP Luquafleece fabric (BASF, Germany) are shown in Fig. 3 and it readily adsorbs moisture over the entire water activity range, including <60% RH (Table 4: M0 ∼ 20% d.b. and significantly higher than those for the ALVs). Additionally, the desorption curve in (B) shows that this process is completely reversible and that the SAP can be effectively reused for energy efficient drying. Efficient SAP-drying of an edible African crop has already been demonstrated by us in the case of maize.7
The microstructure of the rehydrated leaves shows significant differences in all five species. Palisade parenchyma and spongy parenchyma could still be distinguished. The shape of the cells, however, was distorted and the palisade cells were significantly shorter (Cowpea: 77 ± 9 μm and Jute Mallow, Slender Leaf and Nightshade: 36–55 μm). The final thickness of the rehydrated Cowpea and Jute Mallow leaves was reduced to ∼75% compared to that of the fresh leaves. For Slender Leaf, Nightshade and Amaranthus, however, the final thickness of the rehydrated leaves was similar to that of the fresh leaves. It is shown in Fig. 5 that the kinetics of dehydration and rehydration was also most significantly impacted (rate constants: R) for the species with the greatest parenchyma wall damage. Cell compartmentation was also partly lost through drying and individual round chloroplasts could not be observed in rehydrated leaves. The chlorophyll appears to be distributed throughout the cells as was also confirmed using confocal microscopy using chlorophyll auto-fluorescence (data not shown). The chlorophyll content and significantly altered chloroplast structures have also been reported for desiccant-tolerant plant species in the literature.32,33 For Jute Mallow, oxalate crystals were also observed in rehydrated leaves but the relative oxalate concentrations could not be quantified and compared.
To investigate the microstructure of the fresh and rehydrated cells of the ALV species more in detail we used TEM of ultra-thin cross-sections of ∼50 nm thickness from chemically fixed and embedded samples: Fig. 6 shows representative images. Cells of the fresh leaves showed well-organized peripheral chloroplast structures near the cell walls (cw) with grana (gt) and stroma (st) thylakoid membranes and plastoglobuli (p, lipid rich granules). Furthermore, they showed a large central vacuole (v). As an example, the plant cell organelles are indicated for cross-sections of the fresh Cowpea leaf shown in Fig. 6(C). For cross-sections of the fresh Amaranthus leaf lead crystals due to post-staining are visible in the images. Cowpea and Nightshade showed a significantly larger amount of starch granules (sg) in their microstructure, which may explain their fibrous structure upon cooking compared to the paste that is formed by Slender Leaf and Jute Mallow. Starch granules can gelatinize and swell at high temperatures (above ∼68 °C) and give these leaves a firmer texture with less free water upon cooking.34 For Amaranthus, the starch granules may be concentrated in the bundle sheath cells32 and are not visible in the images shown herein.
The microstructure of the rehydrated leaves again showed significant differences compared to the fresh leaves for all five species. Firstly, the chloroplasts appeared disordered and lost their peripheral structure. The thylakoid membranes, starch granules and lipids from the plastoglobuli were distributed throughout the central vacuole. Rehydrated leaves of Slender Leaf and Nightshade showed the most significant disruption of the chloroplasts and their grana thylakoid membranes could not be recognized in Fig. 6(B and D). For Jute Mallow, the chloroplast structures were also significantly disrupted while some regions of organized thylakoid membranes could still be observed (Fig. 6(A)). For Cowpea and Amaranthus, the chloroplasts also lost their peripheral orientation and well-defined shape but the grana and stroma thylakoid membranes and plastoglobuli could still be recognized (Fig. 6(C and E)).
Moisture sorption isotherms of Slender Leaf and Nightshade showed that these species could be rehydrated to the equilibrium moisture content of the fresh leaves upon dry storage. Light microscopy also confirmed rehydration to the same thickness as the fresh leaves and similar dehydration and rehydration kinetics for these species. While the palisade parenchyma cells were slightly shorter in the rehydrated leaves the spongy parenchyma region increased in thickness. The microstructure of the cells, however, was significantly disrupted for these species and the thylakoid membrane organization of the chloroplasts was lost. This suggests that the changes in the microstructure compensated for the rehydration capacity of the leaves. Nevertheless, the significantly increased amount of starch granules as was observed in the nanostructure of Cowpea and Nightshade leaves can explain their fibrous structure compared to the paste that is formed upon cooking of Jute Mallow and Slender Leaf (slimy). Whether the species were fibrous or slimy did not affect the moisture sorption characteristics.
Moisture sorption characteristics did not depend on the moisture content of the fresh leaves. The microbial safe moisture content of the five species of African leafy vegetables was determined using the GAB model and found to be ≤14% d.b. The monolayer moisture content was determined to be ∼4.5% d.b. for all species and this moisture does not participate in any deteriorative reactions. These results indicate that the same drying technology can be used for the effective preservation of a wide range of ALV species. Additionally, using superabsorbent polymers improves the energy efficient gentle drying conditions and ensures drying and storage of all species below their microbial safe moisture content. Combined with the high nutritional value for leaves dried at 40 °C,11 this shows that drying under controlled conditions is an effective method to preserve African leafy vegetables and fight hidden hunger and micro-nutrient deficiency in sub-Saharan Africa.
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