Iron-enrichment of kale via combination moderate electric field processing enhances iron delivery in vitro

Abstract

Combination-moderate electric field (MEF) processing successfully increases the iron content of kale by ~ 160 × as compared to untreated kale, but it is unknown whether this kale better delivers iron. It is also not known how combination-MEF treatments impact other nutritionally relevant bioactive kale compounds. This study aimed to (1) determine intestinal delivery of iron from the iron-enriched kale leaf following in vitro digestion and (2) assess ascorbic acid (AA), carotenoid, and α-tocopherol (i.e., vitamin E) concentrations in the combination-MEF treated kale. AA, carotenoids and α-tocopherol were separated and quantitated from non-polar kale extracts using high-performance liquid chromatography-diode-array detection (HPLC–DAD). Iron-enriched kale, hemoglobin, and FeSO₄ were digested in vitro, and the digesta was centrifuged, filtered, diluted 16x, and applied to confluent Caco-2 cells. The combination-MEF treated kale increased cell ferritin 4.4 × and 1.8 × relative to FeSO₄ and hemoglobin, respectively. The combination-MEF treatment decreased β-carotene and lutein by 3–4 ×, produced no change in α-tocopherol, and increased AA 3 ×, as compared to untreated kale. In conclusion, combination-MEF treated kale is a potentially excellent source of dietary iron for those at risk of iron deficiency, and the combination-MEF treatment merits further investigation for its ability to enrich other vegetables with iron.

Introduction

Iron deficiency remains the most pervasive nutritional deficiency globally, affecting two billion people worldwide,¹ despite decades of widespread food fortification to ameliorate this burden.^2–4 The persistence of iron deficiency is primarily attributed to inadequate dietary intake and poor bioavailability of non-heme iron from plant foods.⁵ Animal based sources provide more bioavailable iron, but growing concerns around climate change,⁶ and chronic disease risk have led some health conscious consumers to limit red-meat intakes.⁷ The majority of individuals would prefer to consume iron-rich vegetables over supplements, and are willing to pay more for such foods.⁸ In response to this challenge, our previous work demonstrated that the application of novel moderate electric field (MEF, 1–1000 V/cm, arbitrary waveform, enhancement in electropermeabilization with or without ohmic heating) combination technology for food treatments dramatically enhance kale leaf iron content up to 160x, compared to raw kale.⁹ MEF treatment in food and biological materials enables microbial inactivation, enzyme deactivation, and enhanced mass transfer at lower temperatures, making MEF-based combinations promising for preserving nutritional and sensory qualities.¹⁰ It is possible that ascorbic acid concentrations also increased, as it was added to keep the iron in the treatment solution throughout processing. Ascorbic acid is particularly relevant, as co-consumption with inorganic iron has been shown to increase uptake in healthy adults by 56–350%.^11,12 Building on these findings, the present study sought to determine if this iron-enriched kale would better deliver iron to human intestinal epithelial cells, as compared to iron commonly prescribed for supplementation. We also quantitated the retention of key micronutrients (i.e., ascorbic acid (AA), provitamin A carotenoids, α-tocopherol) and non-provitamin A carotenoids (i.e., lutein) following combination-MEF treatments, given their sensitivity to heat and oxidation.¹³.

Results

The iron-enriched kale selected for digestion and Caco-2 cell experiments was treated as previously described,⁹ with sequential combination of vacuum impregnation (VI), 24-h immersion (IM), incubation in a water bath at 30 °C for 30 min (WB), MEF (15 V/cm at 60 Hz for 30 min) in an iron and AA (molar ratio = 1) solution. The combination-MEF treated kale was found to contain 514 mg Fe and 518 mg AA per 100 g treated kale leaf. The Fe was ~ 160 × higher,⁹ and AA was 2.9 × higher as compared to untreated kale. Iron or AA in the positive controls, i.e. FeSO₄ or FeSO₄+AA or hemoglobin, was dose-matched with the level of iron and AA measured in the kale exposed to combination-MEF treatment (see Methods 2.4).

Digesta containing iron-enriched kale, FeSO₄ or FeSO₄+AA produced iron which was ~ 50–60% bioaccessible, and ~ 5 × more bioaccessible as compared to iron from digesta containing hemoglobin (Fig. 1). No significant differences in iron bioaccessibility were observed between the digesta containing iron-enriched kale, FeSO₄ or FeSO₄+AA. Caco-2 cells incubated with digesta containing the iron-enriched kale had a 4.4 × and 1.8 × increase in cellular ferritin, as compared to cells treated with digesta containing FeSO₄ alone or digesta containing hemoglobin, respectively (Fig. 2).

Fig. 1.

Iron bioaccessibility after in vitro digestion. Iron-enriched kale was treated by vacuum (VI) +24-h immersion (IM) +incubation in a water bath at 30 °C for 30 min (WB) + MEF (15 V/cm at 60 Hz for 30 min). The bioaccessibility of iron after in vitro digestion was calculated as the percentage of iron in the filtered digesta (i.e., the chyme) relative to the amount of iron added to the initial digestion (i.e., 50 µg/mL). Bars represent average ± SEM, with n = 6 per group. Letters indicate a statistically significant difference between the groups, as assessed via ANOVA, followed by Tukey’s post-hoc test (P < 0.05 considered statistically significant).

Fig. 2.

Cell ferritin response after incubation with 16 × diluted filtered digesta. Iron-enriched kale was treated by vacuum (VI) + 24-h immersion (IM) + incubation in a water bath at 30 °C for 30 min (WB) +MEF (15 V/cm at 60 Hz for 30 min. Bars represent average ± SEM, with n = 6 per group. Letters indicate a statistically significant difference between the groups, as assessed via ANOVA, followed by Tukey’s post-hoc test (P < 0.05 considered statistically significant).

The effect of a variety of combination treatments on β-carotene, lutein, and α-tocopherol concentrations is shown in Fig. 3. Compared to raw untreated kale (control), all combined treatments, besides VI+WB+IM, resulted in a significant decrease in β-carotene content in kale leaves. β-Carotene in kale treated with VI+WB+MEF decreased the most, showing a 17.7 × reduction compared to the β-carotene content in the control. β-Carotene contents in kale treated with VI+WB and VI+WB+IM+MEF were 1.9 × and 4.3 × lower, respectively, than in the control. Under MEF-based combination treatments, lutein content in kale leaves was 3.2 × to 9.7 × lower as compared to lutein content in the control. VI+WB and VI+WB+IM did not significantly affect lutein contents in kale leaf. α-Tocopherol (i.e., vitamin E) contents in all treated kale samples were comparable to that of untreated control kale leaves, except for the VI+WB+MEF, where α-tocopherol degraded below the limit of detection.

Fig. 3.

β-Carotene, lutein and α-tocopherol contents in treated kale leaves. Vacuum (VI), 24-h immersion (IM), incubation in a water bath at 30 °C for 30 min (WB), MEF (15 V/cm at 60 Hz for 30 min). Bars represent average ± SEM, with n = 3 per group. Letters indicate a statistically significant difference between different treatments, within the same nutrient, as assessed via ANOVA, followed by Tukey’s post-hoc test (P < 0.05 considered statistically significant).

Discussion

The MEF-based combination treatments produced both (1) notable increases in leaf iron and AA concentrations and (2) demonstrated meaningful improvements in intestinal cell uptake of iron, enriching cells by 4.4 × relative to a common supplemental form of iron, FeSO₄ (Fig. 2). The AA present in the iron-enriched kale is believed to contribute, at least partially, to the increased cellular absorption of non-heme iron as measured by ferritin levels, consistent with previous studies.¹⁴.

The iron delivery observed from iron-enriched kale surpasses that reported for other iron-fortified chlorophyll-rich plant foods. Nori (Porphyra spp.) and sea lettuce (Ulva lactuca) were digested alone or together with FeSO₄ (5.5 μg) and AA (0.28 mM).¹⁵ Ferritin formation in Caco-2 incubated with the fortified digesta was ~ 2.5 × and 3.5 × higher for nori and sea lettuce, respectively, as compared to FeSO₄ digested alone.¹⁵ Others have reported 2 × higher ferritin levels in Caco-2 cells following incubation with digesta containing iron-fortified spirulina, as compared to digesta containing FeSO₄.¹⁶ Likewise, ferritin formation in Caco-2 cells following incubation with digesta containing iron-fortified cereals has also been more attenuated, with 1.1–1.6 × more ferritin synthesized, as compared to digesta containing FeSO₄ alone.¹⁷.

The concentration of bioaccessible iron in digesta of iron-enriched kale was comparable to the concentration of iron in digesta containing FeSO₄ or FeSO₄+AA (Fig. 1). A previous study reported that iron bioaccessibility from digesta containing raw kale was ~ 3 × lower as compared to iron bioaccessibility from digesta containing FeSO₄.¹⁸ In contrast, the bioaccessible iron released from combination-MEF treated iron-enriched kale was improved as compared to raw “control” kale. The MEF-combination treatments may have increased the permeability of cell membranes thus increasing tissue breakdown, permitting better iron release from vegetable tissue.¹⁹.

MEF-based combination treatments, which involve applying iron and AA solution together to kale leaves, carry a risk of increasing oxidation of the endogenous β-carotene, a provitamin A carotenoid. Indeed, β-carotene was significantly reduced in VI+WB+MEF and VI+WB+IM+MEF treated kale, as compared to the VI+WB and VI+WB+IM treated kale (Fig. 3). Similarly, under pulsed electric field treatment, β-carotene concentration in kale-based juice decreased 2.4x, as compared to concentrations in raw kale juice.²⁰ Ferric iron can directly oxidize β-carotene and form β-carotene cation radicals, which can further react with the ferric iron and produce dications.²¹ Despite the fact that β-carotene concentrations in iron-enriched kale decreased, a half cup serving of the VI+WB+IM+MEF treated kale (i.e., the enhanced kale providing the highest amount of iron/100 g kale leaf) would still provide ~ 10% of the recommended dietary allowance of vitamin A as the provitamin A form of β-carotene.²².

MEF-based combination treatments and iron may also cause the degradation of the carotenoid lutein, a xanthophyll that is associated with ocular and cognitive health.^23,24 In kale leaves treated with VI+WB+MEF and VI+WB+IM+MEF, lutein contents decreased 9.7 × and 3.2x, respectively, in relation to raw kale control (Fig. 3). This loss was expected, since kale-based juice treated with pulsed electric field showed a 1.3 × reduction in lutein concentration as compared to raw kale juice.²⁰ The significant differences in lutein content between VI+WB vs. VI+WB+MEF treated kale, as well as between VI+WB+IM vs. VI+WB+IM+MEF treated kale, strongly support MEF treatment specifically in facilitating lutein degradation. We purport that iron-catalyzed lipid peroxidation in kale leaves is at least partially responsible for degrading lutein.^25,26.

As compared to untreated kale, α-tocopherol concentrations did not decrease in VI+WB+IM+MEF treated kale leaves but decreased in kale leaves under VI+WB+MEF. This result suggests that the IM treatment of the kale leaf in the iron and AA-rich fluid best conserved α-tocopherol, possibly via AA regeneration of α-tocopherol, a well-documented phenomenon.²⁷.

Taken together, our results strongly suggest that the iron-enriched kale is an excellent dietary iron source (i.e., > 20% daily value per serving) with great potential for future applications, both in the quantity of iron per leaf, but also in the absorption potential of iron from each leaf. Sensory tests and clinical studies are expected to be conducted in the future in iron-deficient populations to provide clear insight of customer acceptance and in vivo iron bioavailability. Our results also make the case that combination-MEF processing studies are merited to evaluate their ability to “iron-enrich” other vegetables and fruits.

Materials and methods

Materials

The kale (Brassica oleracea L. var. acephala) was purchased from local grocery (Columbus, OH). Supplies purchased from Sigma-Aldrich (St. Louis, MO) included: bovine hemoglobin, ferrous sulfate (FeSO₄·7H₂O), ascorbic acid, α-tocopherol, retinyl acetate, pepsin (P6887, 3883 U/mg protein), pancreatin (P7545, 8 × USP/mg), and porcine bile extract (B8631), reagents suitable for cell culture: D-glucose, piperazine-N–N’-bis(2-ethanesulfonic acid) (PIPES), hydrocortisone, insulin, Na₂SeO₃, triiodothyronine, epidermal growth factor solution. ACS grade KCl, NaCl, NaHCO₃, NaOH, acetic acid, certified 1 N HCl, molecular biology grade 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), bicinchoninic acid (BCA) protein assay kits, HPLC-grade methanol, methyl tertiary-butyl ether (MTBE), hexane, acetone, Optima grade LC–MS formic acid, were purchased from Fisher Scientific (Pittsburgh, PA). Collagen was purchased from Corning Inc. (Corning, NY). Hank’s balanced salt solution (HBSS) powder was purchased from Cytiva Life Sciences (Marlborough, MA). β-Carotene and lutein standards were purchased from Cayman Chemical (Ann Arbor, MI). Dulbecco’s modified Eagle medium (DMEM,9,612,100–046), Minimum Essential Medium (MEM, 61,100–061), and fetal bovine serum (FBS, 10,082,147) were purchased from Gibco (Grand Island, NY). Zellshield was purchased from Thomas scientific (Chadds Ford Township, PA). Ferritin assay kits were purchased from Eagle Biosciences (Amherst, NH). Type Ⅰ water was produced by Milli-Q® reference water purification system (Millipore, Sigma, Burlington, MA). The Caco-2 cells (HTB37, American Type Cell Collection) were a generous gift from Dr. Mark Failla.

Novel processing treatments

Full details of novel processing treatments can be found in the previous publication.⁹ In brief, to perform the MEF treatments, a pair of curved platinized-titanium electrodes (5 cm apart) were installed in the treatment chamber and connected to a MEF unit. The unit was powered by a 60 Hz, 75 V sinusoidal AC, generating a field strength of 15 V/cm. Samples of Fe-AA solution in a laboratory-scale liquid-jacketed beaker (up to 50 mL in volume capacity), were treated at 50 °C for 15 min, with temperature controlled by an external refrigerated circulator (RTE10 Bath Circulator) to offset ohmic heating. Sample temperature was monitored with a T-type thermocouple (SCPSS-062G-6, Omega), and voltage, current, and temperatures (sample and bath) were recorded at 1 s intervals using a DAQ system (Agilent 39704A).

Ascorbic acid quantitation

Lyophilized kale samples (fresh weight ~ 1 g, VI+WB+IM+MEF treated) were powdered, followed by the addition of methanol (10 mL). Meta-phosphoric acid (4.5% aq. concentration, 1 mL) was added to the methanol-kale mixture (0.5 mL).²⁸ Polar extracts were separated using a high-performance liquid chromatography-photo diode array (HPLC–PDA) system equipped with an Atlantis HILIC column (150 mm × 2.1 mm diameter, 3 µm particle size). An isocratic mobile phase of KH₂PO₄ (5%) at 0.25 mL/min was used for separation, with the column held at 25 ℃. An external standard curve was used for identification and quantitation of AA at 254 nm.

In vitro digestion of iron-enriched kale

All glassware used during the in vitro digestion procedure was soaked in 20% HNO₃ for at least 24 h before using to remove any residual iron.

Iron-enriched kale (1 g) was blended with 10 mL methanol. The combination-MEF treated kale mixture, providing 0.25 mg iron (~ 500 µL), was added to centrifuge tubes (15 mL) and the methanol removed under argon gas. The dose of hemoglobin (78 mg providing 0.25 mg iron) was weighed into a separate tube. To both the MEF-treated kale and hemoglobin digests, acidified distilled water (pH = 2, 1 mL) was added. For the FeSO₄ group, FeSO₄·7H₂O (25 mg) was dissolved in acidified distilled water (pH = 2, 20 mL), and then the FeSO₄•7H₂O solution (1.25 mg/mL, 1 mL) was aliquoted into a digestion tube. AA stock solution (100 µg/µL) was prepared using distilled water acidified with 1N HCl to pH = 2. A dose of AA matching that measured in iron-enriched kale (252 µg) was added to the FeSO₄ digestion group (i.e. FeSO₄+AA).

Samples were digested following our previous publication,²⁹ except the volume was scaled down 10x. Additionally, following intestinal digestion, the samples were centrifuged at 3200 × g at 25 °C and the supernatant was aliquoted for cell incubation.

Assessment of iron bioaccessibility

Quantitative measurements of total iron in the filtered supernatant fraction following in vitro digestion was performed using a Perkin-Elmer Optima 8300DV inductively coupled plasma optical emission spectrometer (ICP-OES, Waltham, MA) with an external calibration curve. Bioaccessibility was calculated as the percentage of iron recovered in the filtered digesta relative to the initial iron concentration digesta (normalized to 50 µg/mL).

Caco-2 cell culture, incubation, and analysis

Caco-2 cells (HTB 37, passages 31–32) were seeded at 5 × 10⁴ cells per cm² and were grown in collagen-coated 6-well plates.³⁰ Caco-2 cell treatment procedures were conducted as previously described.²⁹ After adding distilled water (2 mL), Caco-2 cells were scraped, harvested, and stored at -80 °C prior to analysis.

Cell pellets were probe sonicated on ice twice for 10 s each. Ferritin assay kits were used to assess Caco-2 cell iron uptake, previously validated by Glahn et al.³⁰ A cell suspension (20 µL) was used to quantify total cell protein, following the protocol of BCA protein assay kit.

Carotenoid and α-tocopherol quantitation

The lipophilic analytes (i.e., α-tocopherol, β-carotene, and lutein) were extracted following a previously developed method.⁹ An internal standard (IS) of retinyl acetate (10 µL of a 1 nmol/µL in hexane solution) was added before extraction. Extracts were separated using the C30 method previously published on an HPLC equipped with diode-array detection (DAD),²⁰ with slight modifications. The gradient was as follows: 20% B at 0 min, linearly increasing to 95% B over 13 min, and returning and holding at 20% B for 2 min, with a flow rate of 1 mL/min. External standard curves normalized to the IS were used for quantitation.

Statistics

All statistics were performed in R Studio version 4.4.0 (www.r-project.org) by using the package “stats”. An analysis of variance (ANOVA) test, followed by Tukey’s post-hoc test, was performed to evaluate significant differences to compare iron bioaccessibility, Caco-2 cell ferritin formation, and lipophilic bioactive compounds among different groups. T-test was performed to compare AA content of control kale with AA content of VI+WB+IM+MEF-treated kale. A P-value < 0.05 was considered statistically significant.

Author contributions

Ziqi Li: methodology, formal analysis, investigation, writing—original draft, visualization. Jin Hong Mok: investigation, writing—review & editing Sudhir K. Sastry: conceptualization, funding acquisition, supervision, project administration, investigation, writing—review & editing, Amanda J. Bird: investigation, writing—review & editing Rachel E. Kopec: conceptualization, funding acquisition, validation, supervision, project administration, writing—review & editing.

Funding

This research was supported by the USDA NIFA Multistate #W5122 and USDA 2019–68015-29229.

Data availability

The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.