Use of shallot (Allium ascalonicum) and leek (Allium tuberosum) to improve the in vitro available iron and zinc from cereals and legumes

Abstract

In this work, the effects of shallot and leek added to food grains on the available iron and zinc (in vitro) were examined. Two representative cereals and pulses were studied in both raw and cooked condition employing two levels of shallot (25 and 50 g/kg of grain) and leek (150 and 300 g/kg of grain). The enhancing effect of these two spices on in vitro available iron was generally evidenced in the cereals (10.6–67.2% increase) and pulses (11.0–37.8% increase) in both raw and cooked conditions. The two spices similarly enhanced the in vitro available zinc from the food grains, the extent of increase in cereals ranging from 12.4% to 86.1% and in pulses from 9.0% to 45.5%. Thus, both shallot and leek were evidenced here to have a promoting influence on the in vitro available iron and zinc from food grains.

En la presente investigación, se estudió el efecto que produce el agregado de chalotas y de puerro a granos comestibles en el hierro y el cinc disponibles (in vitro). Para ello se analizaron tanto en forma cruda como cocida dos cereales y legumbres representativos, utilizándose dos niveles de chalotas (25 y 50 g/kg de grano) y dos niveles de puerro (150 y 300 g/kg de grano). Se hizo evidente el efecto de mejoría que estas dos especias produjeron en el hierro disponible in vitro en los cereales (10,6–67,2% de aumento) y en las legumbres (11,0–37.8% de aumento), tanto en su condición cruda como cocida. De manera similar, las dos especias mejoraron el cinc disponible in vitro de los granos comestibles, registrándose incrementos de 12,4% a 86,1% en los cereales y de 9,0% a 45,5% en las legumbres. Por lo tanto, las chalotas y los puerros demostraron tener una influencia profunda y favorable en el hierro y el cinc disponibles in vitro en granos comestibles.

Keywords:

• shallot
• leek
• in vitro available iron and zinc
• cereals
• legumes

Palabras claves:

• chalotas
• puerro
• hierro y cinc disponibles in vitro
• cereales
• legumbres

Introduction

Cereals and legumes are the major sources of minerals such as iron and zinc for the population dependent on plant foods. However, the bioavailability of iron and zinc from these food grains is reported to be poor (Gibson, 1994; Sandberg, 2002). Bioavailability of dietary iron and zinc is greatly influenced by both inhibitors (phytate, polyphenols, dietary fibre, oxalate, and calcium) and enhancers (organic acids) present in the diet (Lopez, Leenhardt, Coudray, & Remesy, 2002; Luo & Xie, 2012; Luo, Xie, & Cui, 2010a; Matuschek & Svanberg, 2002; Matuschek & Svanberg, 2005).

We have reported effects of phytases and dehulling treatments on iron and zinc bioavailability and relative contribution of phytates, fibres, and tannins to low iron and zinc in vitro solubility in faba bean (Vicia faba L.) flour and legume fractions (Luo & Xie, 2013; Luo et al., 2010a; Luo, Xie, & Cui, 2010b; Luo, Xie, Xu, & Luo, 2012). There is growing evidence that dietary sulphur amino acids influence the nutritional status of zinc. Renal metallothionine content has been reported to be higher in rats fed diets supplemented with cystine, suggesting improved zinc status (Yang, Ishii, Satoh, & Kato, 1995). Higher tissue zinc and zinc excretion was evidenced in rats fed diets supplemented with excessive cysteine (15 g/kg) (Greger & Mulvaney, 1985; Hsu & Smith, 1983). Increased absorption of zinc was 25% when rats were fed a cysteine-supplemented lactalbumin diet compared with an unsupplemented diet (Snedeker & Greger, 1983). Shallots (Allium ascalonicum) and leeks (Allium tuberosum), which are extensively consumed in Chinese diets, are rich sources of sulphur compounds (Yang et al., 1995). Previous reports on the influence of dietary sulphur-containing amino acids on iron and zinc status in animals concluded that it would be highly relevant to evaluate these two Allium spices for any influence on the mineral bioavailability. The objective of this investigation was therefore to examine the influence of exogenously added shallot and leek on the in vitro available iron and zinc from food grains consumed in China. For this purpose, two representative cereals and pulses were studied in both raw and cooked condition employing two levels of shallot (25 and 50 g/kg of grain) and leek (150 and 300 g/kg of grain). The information thus generated would make advances in human nutrition and is likely to help to evolve a food-based strategy to combat micronutrient malnutrition in a population dependent on plant foods.

Materials and methods

Materials

Two cereals, rice (Oryza sativa) and wheat (Triticum aestivum), and two legumes, faba bean (Vicia faba) and soybean (Glycine hispida), were procured locally, cleaned, and used. Fresh shallot and leek were procured from the local market and cleaned and were used for the experiment. All chemicals used were of analytical grade. Triply distilled water and acid washed glassware were used throughout the study.

In vitro available iron and zinc

In vitro available iron and zinc was determined using 10 g of the grain sample in the absence or presence of shallot/leek added at the specific proportion indicated below. Thus, the various food samples evaluated in vitro available zinc and iron were (1) food grain alone (10 g), (2) food grain (10 g) + shallot (0.25 g), (3) food grain (10 g) + shallot (0.50 g), (4) food grain (10 g) + leek (1.5 g), and (5) food grain (10 g) + leek (3.0 g). Whole grain samples were finely powdered in a hammer-mill type grinder (HY-04B, Beijing Xinhuanya, China) and sieved through a 1 mm screen; shallot or leek was finely chopped and ground into a smooth paste before addition to the powdered grain sample. The two levels of spices examined here correspond to the range of their normal intake by the China population (Kong, 2009).

In vitro available iron and zinc were defined as the relative amount of iron and zinc that becomes soluble after enzymatic treatment. Grain samples were sequentially digested with enzymes, including amylase, pepsin, pancreatin, and bile, under certain conditions following the enzymatic degradation procedure described by Kiers, Nout, and Rombouts (2000). Samples were suspended in distilled water (30 ml). The samples were incubated while stirring with an amylase solution (2 ml) consisting of 12,500 units/L amylase (Sigma A-1031, Sigma-Aldrich Co., St Louis, MO, USA), 1.5 g/L NaCl, 1.5 g/L K₂HPO₄, and 0.5 g/L Na₂CO₃ (pH 7.0) for 30 min at 37°C. Next the pH was adjusted to 4.0 using 5 M HCl and the suspensions were incubated with 8 ml of stomach medium (3.1 g/L NaCl, 1.1 g/L KCl, 0.6 g/L Na₂CO₃, 0.11 g/L CaCl₂, pH 4.0) for 1 h at 37°C. The pH was then adjusted to 6.0 using solid NaHCO₃. Finally, 10 ml of a 2% pancreatic solution (20.0 g/L pancreatin (Sigma P-1750, Sigma-Aldrich Co.), 5.0 g/L bile (Sigma B-3883, Sigma-Aldrich Co.), 5.0 g/L NaCl, 0.68 g/L KH₂PO₄, 0.3 g/L Na₂HPO₄, 0.84 g/L NaHCO₃) was added and the suspensions were incubated for 30 min at 37°C. Subsequently, mixtures were centrifuged at 5000 g for 15 min at 4 °C. The resulting supernatant was filtered (0.45 μm membrane, FP 030/3, Kaijie, Hangzhou, Zhejiang) and frozen until further analysis. Iron and zinc levels, including soluble free ionizable iron and zinc and soluble complexes of iron and zinc, were analysed by atomic absorption spectrophotometry. Each sample was enzymatically extracted in duplicate. In vitro available iron and zinc contents were determinated on three independent digests.

Determination of total iron and zinc content

Iron and zinc in grain samples were analysed by atomic absorption spectrophotometry (Varian SpectrAA 200, Victoria, Australia) after dry ashing for 2 h at 530°C. Depending on the different treatments, 2–4 g of ash were weighed in a silicon evaporating dish. Next, the ashes were wet-acid digested with nitric acid on a hot plate and solubilized with 25 ml of 0.5 N HCl.

Heat processing of cereals and legumes

All food samples were pressure cooked (15 psi) in 40 ml of triply distilled water for 15 min. The cooked samples were homogenized in a stainless steel blender and used for the determination of in vitro available mineral as described above.

Statistical analysis

Data were analysed with SPSS 15.0 for Windows (Shanghai Precision & Scientific Co. Ltd., Shanghai, China). The mean and standard deviation of means were calculated. The data were analysed by one-way analysis of variance (ANOVA). Duncan’s multiple range test was used to separate means. Significance was accepted at probability P < 0.05.

Results and discussion

Effect of shallot on the in vitro available iron

Table 1 presents the effect of shallot on the in vitro available iron from rice, wheat, faba bean, and soybean. Shallot exerted a promoting effect on from all of the four food grains examined. The increase in raw rice was 18.9% at the higher level of shallot (P < 0.05), while the same in cooked rice was 21.9% and 23.3% at the two levels of shallot, respectively (P < 0.05). In case of wheat, the positive effect of shallot was observed only in raw grains, the increase being 10.6% at the lower level, while it was 48.2% at the higher level (P < 0.05).

Table 1. Effect of shallot on the in vitro available iron (%) from raw and cooked cereals and legumes.

Efecto de la chalota en el hierro (%) disponible in vitro de cereales y legumbres crudos y cocidos.

	Rice (raw)	Rice (cooked)	Wheat (raw)	Wheat (cooked)	Faba bean (raw)	Faba bean (cooked)	Soybean (raw)	Soybean (cooked)
Grain	6.34 ± 0.11	11.38 ± 0.16	7.43 ± 0.10	13.31 ± 0.31	6.40 ± 0.09	7.72 ± 0.12	9.55 ± 0.06	14.19 ± 0.11
Grain + 0.25 g shallot	6.49 ± 0.04	13.87 ± 0.23*	8.22 ± 0.11*	13.33 ± 0.08	7.77 ± 0.12*	9.93 ± 0.28*	9.75 ± 0.08	15.75 ± 0.12*
Grain + 0.5 g shallot	7.54 ± 0.11*	14.03 ± 0.08*	11.01 ± 0.19*	14.23 ± 0.10	8.00 ± 0.11*	10.42 ± 0.09*	10.25 ± 0.15*	16.18 ± 0.18*

Notes: Values represent means ± SD of three independent determinations.The content of grain is 10 g.

*Significant (P < 0.05) increase compared to grain alone.

A similar positive influence of shallot on in vitro available iron was also evident in the legumes examined. Shallot enhanced raw faba bean by 21.4% and 25% at the two levels, respectively (P < 0.05). This positive effect of shallot was higher in the case of cooked faba bean, the increase being 28.6% and 34.8% at the two levels, respectively (P < 0.05). In the case of soybean, shallot enhanced by about 7.3% (higher level) in the raw grain and 11% and 14% at the two levels, respectively, in the cooked grains (P < 0.05).

Effect of shallot on the in vitro available zinc

The effect of shallot on the in vitro available zinc from the four food grains is presented in Table 2. Shallot significantly enhanced the in vitro available zinc only in cooked rice, the increase being 27.9% and 31.3% at 0.25 and 0.5 g levels, respectively (P < 0.05). Similar increases were evidenced in raw and cooked wheat, where shallot brought about 24.2% and 32.8% increase in the raw grain (P < 0.05), and in cooked grain, the increase was 78.1% and 86.1% at the two levels of shallot (P < 0.05). In the case of raw faba bean, shallot enhanced to an extent of 17.6% at the higher level, while in cooked faba bean the increase was 9.0% and 14.6% at the two levels, respectively (P < 0.05). No effects have been found in raw and cooked soybean.

Table 2. Effect of shallot on the in vitro available zinc (%) from raw and cooked cereals and legumes.

Efecto de la chalota en el cinc (%) disponible in vitro de cereales y legumbres crudos y cocidos.

	Rice (raw)	Rice (cooked)	Wheat (raw)	Wheat (cooked)	Faba bean (raw)	Faba bean (cooked)	Soybean (raw)	Soybean (cooked)
Grain	15.73 ± 0.41	14.70 ± 0.37	8.83 ± 0.26	6.27 ± 0.17	37.37 ± 1.91	26.70 ± 1.25	27.33 ± 2.00	22.40 ± 1.05
Grain + 0.25 g shallot	16.4 ± 0.26	18.80 ± 0.36*	10.97 ± 0.21*	11.17 ± 0.25*	38.73 ± 0.47	29.17 ± 0.31*	28.33 ± 0.21	23.80 ± 0.36
Grain + 0.5 g shallot	16.53 ± 0.06	19.30 ± 0.10*	17.73 ± 0.21*	11.67 ± 0.21*	43.93 ± 0.38*	30.60 ± 0.53*	28.80 ± 0.26	23.90 ± 0.35

Notes: Values represent means ± SD of three independent determinations.The content of grain is 10 g.

*Significant (P < 0.05) increase compared to grain alone.

Effect of leek on the in vitro available iron

Table 3 presents the effect of leek on the in vitro available iron from food grains. Leek had a significant positive effect on all of the four food grains examined. The positive effect of leek on in vitro available iron was higher in raw rice, the increase in the same being 35.4% and 37.3% at the two levels of leek (P < 0.05), while in cooked rice the increase was 19.5% and 32.9% at the two levels of leek, respectively (P < 0.05). Leek exerted a similar enhancing influence on the in vitro available iron from wheat. The increase was 19.4% and 32.9% in the raw, and 28.3% and 67.2% in the cooked, grain at the two levels of leek, respectively (P < 0.05). In the case of raw faba bean, leek enhanced to an extent of about 23% at both of the levels, while in cooked faba bean, the same was 25.6% and 31.5% at the two levels of leek, respectively (P < 0.05). In the case of soybean, the positive effect of leek on was higher in the raw grains (P < 0.05), the increase being 28.2% and 37.8% at the lower and higher levels, respectively. Leek increased cooked soybean to an extent of 19.2% and 20.5% at the two levels, respectively (P < 0.05).

Table 3. Effect of leek on the in vitro available iron (%) from raw and cooked cereals and legumes.

Efecto del puerro en el hierro (%) disponible in vitro de cereales y legumbres crudos y cocidos.

	Rice (raw)	Rice (cooked)	Wheat (raw)	Wheat (cooked)	Faba bean (raw)	Faba bean (cooked)	Soybean (raw)	Soybean (cooked)
Grain	8.47 ± 0.26	15.57 ± 0.32	7.65 ± 0.15	12.10 ± 0.30	3.53 ± 0.15	3.40 ± 0.20	12.50 ± 0.40	15.63 ± 0.55
Grain + 1.5 g leek	11.47 ± 0.25*	18.60 ± 0.20*	10.50 ± 0.30*	15.53 ± 0.21*	4.34 ± 0.18*	4.27 ± 0.21*	16.03 ± 0.21*	18.63 ± 0.45*
Grain + 3 g leek	11.63 ± 0.15*	20.70 ± 0.36*	12.10 ± 0.20*	20.23 ± 1.02*	4.44 ± 0.07*	4.47 ± 0.09*	17.23 ± 0.35*	18.83 ± 0.40*

Notes: Values represent means ± SD of three independent determinations.The content of grain is 10 g.

*Significant (P < 0.05) increase compared to grain alone.

Effect of leek on the in vitro available zinc

The effect of leek on the in vitro available zinc from food grains is presented in Table 4. The effect of leek on (12.4% increase) raw rice was evident only at the higher level of leek (P < 0.05). This positive effect of leek was more pronounced in the case of cooked rice, the increase being 41.6% and 52.5% at the two levels of leek (P < 0.05). In the case of raw wheat, leek increased zinc by 27.6% and 36.0%, while an increase of 25.3% was seen with a higher level of leek in the case of the cooked grain (P < 0.05). Leek marginally increased cooked faba bean, the increase being 15.9% and 18.0% at the two levels, respectively. The in vitro available zinc from raw faba bean in the presence of leek was comparable to that from the grain alone. About 45% increase in in vitro available zinc was seen from raw soybean in the presence of either of the levels of leek, while the increase was around 10.1% in the case of cooked soybean. Thus, the promotive effect of leek on the in vitro available zinc was generally higher in the raw grains as compared to the cooked grains.

Table 4. Effect of leek on the in vitro available zinc (%) from raw and cooked cereals and legumes.

Efecto de puerro en el cinc (%) disponible in vitro de cereales y legumbres crudos y cocidos.

	Rice (raw)	Rice (cooked)	Wheat (raw)	Wheat (cooked)	Faba bean (raw)	Faba bean (cooked)	Soybean (raw)	Soybean (cooked)
Grain	21.80 ± 0.30	10.60 ± 0.36	8.41 ± 0.25	5.42 ± 0.26	36.93 ± 1.31	17.80 ± 0.30	41.73 ± 0.47	38.80 ± 0.36
Grain + 1.5 g leek	22.80 ± 0.36	15.01 ± 0.18*	10.72 ± 0.17*	5.53 ± 0.07	37.07 ± 0.25	20.63 ± 0.45*	60.47 ± 0.70*	42.73 ± 0.32
Grain + 3 g leek	24.50 ± 0.87*	16.17 ± 0.25*	11.44 ± 0.12*	6.79 ± 0.10*	39.67 ± 0.38*	21.00 ± 0.17*	59.40 ± 0.75*	43.07 ± 0.15

Notes: Values represent means ± SD of three independent determinations.The content of grain is 10 g.

*Significant (P <  0.05) increase compared to grain alone.

The Allium spices shallot and leek, common ingredients of the Chinese diet, are characterized by a rich content of thiosulphinates, sulphides, polysulphides, mercaptans, and other odoriferous sulphur compounds. The sulphur content of shallot is 2.6 mg/g fresh weight, four times the level in leek (Fenwick & Hanley, 1985). Leek is used both as a spice and as a vegetable. As much as 500 g or more of fresh leek may be consumed per adult per day when used as a vegetable. Iron and zinc content of shallot and leek has been measured and taken into consideration while calculating the percentage bioavailability of these minerals from grain samples to which these were added. Additional amounts of iron contributed by these exogenous Allium spices at the higher level ranged from 1.6% to 4% of the amount intrinsically present in the grains tested. Similarly, these Allium spices contributed zinc to the extent of 1.8–6.1% when added at the higher level.

Supplemental sulphur amino acids, particularly cysteine, have been shown by several workers to enhance zinc status in experimental animals (Hsu & Smith, 1983; Snedeker & Greger, 1983; Yang et al., 1995). This has been attributed to a possible promotion of zinc absorption from the diet by the sulphur amino acids, which, however, has not been quantitated and merits further investigation. Such a possible beneficial effect of sulphur amino acids on zinc absorption may also be exerted by other natural sulphur compounds such as those present in plant foods. Shallot and leek, being abundant in sulphur compounds, qualify for evaluation of their influence on mineral absorption.

The highest effect of added Allium spices on in vitro available mineral was that of shallot, which produced more than 80% increase in in vitro available zinc from cooked wheat. The promotive effect of shallot on the in vitro available zinc was generally higher in the cooked grains as compared to the raw grains, while that of leek on the same was generally higher in the raw grains. The increased in vitro available iron and zinc observed here as a result of addition of Allium spices is not attributable to increase in the content of these minerals alone, since the increase in percentage bioaccessibility far exceeded the increase in the mineral content. For example, the iron content of the four grains was increased by 2.4–3.8% as a result of exogenous shallot at the higher level, while the increase in bioaccessible iron ranged from 11% to 67% in these grains. In the case of zinc, the content of this mineral of only wheat was increased by 5.2% and 3.5% by the addition of leek and shallot, respectively, at the higher level; while the increase in bioaccessible zinc was 33% in the raw and 86% in the cooker grain, by the addition of shallot. Similarly, leek enhanced in vitro available zinc by 36% and 25% in the raw and cooked wheat, respectively.

Among the two levels of the spices examined for an effect on in vitro available mineral, the higher level, which was double the lower level, did not generally produce a proportionately higher quantum of the positive effect. This observation was true in the case of both shallot and leek, irrespective of the mineral in all of the food grains tested. This could be because the positive effect might have reached/approached saturation at the lower dose itself.

Conclusion

The present study has examined the influence of these two Allium spices on the in vitro available iron and zinc from representative cereals and pulses. The results of this study have evidenced a positive influence of both these spices on the in vitro available iron and zinc, when included at levels normally encountered in the Chinese diet. Such in vitro available values may provide an estimate of the amounts of the minerals likely to be absorbed.

Acknowledgements

The authors are grateful to Dr. Xueming Zai and Dr. Limei Zhu of Jinling Institute of Technology for their technical assistance. This work was supported by National Science Foundation of China (No. 31201318) and Qing Lan Project.