In-vitro bioavailability of selected minerals in dry and green shelled beans

1 Department of Food Science and Technology, Sokoine University of Agriculture, P. O. Box 3006, Morogoro. 2 Department of Crop Science and Production, Sokoine University of Agriculture, P. O. Box 3005, Morogoro, Tanzania. 3 The Children's Investment Fund Foundation (UK), 3rd floor, ABC Place, Waiyaki Way, PO Box 1694-00606, Nairobi, Kenya. 4 School of Nursing and Public Health, Department of Public Health, College of Health Sciences University of Dodoma, P. O. Box 259 Dodoma, Tanzania.


INTRODUCTION
In-vitro bioavailability of a mineral is generally defined as a measure of proportion of the total minerals in food or a meal that is utilized for normal body functions.For most minerals the amount that is absorbed from the gastrointestinal tract, is the major determinant of bioavailability but this varies greatly between minerals (Sandberg, 2002).Absorption is affected by a range of factors including interactions with other dietary components in the gastrointestinal tract, for example vitamin C enhances iron absorption while tannins and phytates have an inhibitory effect.Several antinutritional factors have been implicated as the main cause of reduced micronutrient availability such as phytic acid and tannins although phytic acid has a more pronounced  Corresponding author.E-mail: petermamiro@suanet.ac.tz, petermamiro@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License effect (Zhou and Erdman, 1995;Zdunczyk et al., 1996;Antony and Chandra, 1998;Manary et al., 2000).
The quantification of bioavailability has been done by complex processes comprising isotopic elemental digestion.However, various techniques have been established by nutritionists to determine bioavailability.For instance, In-vitro studies range from measurements of solubility, extractability, and fractional dialysability to studies of the nutrient uptake in experimental animals (Miller et al., 1981;Sripriya et al., 1997;Mamiro et al., 2001).The nutrients are incubated in intestinal preparations to simulate the processes that take place in the gastrointestinal tract (Blenford, 1995).Contemporary developments have used cell culture called the Caco-2 as an in-vitro method to assess iron bioavailability in human beings (Glahn et al., 2000;Kim et al., 2011).When compared with other In-vitro methods used previously by other scientists a significant correlation (r=0.97 and P=0.0001) was found (Yun et al., 2004).The results obtained from the above methods are used as proxy to estimate bioavailability (Watzke, 1998).
Beans are naturally low in fat, with little saturated fats.The average lipid content is only 1.5% of the dry bean and unsaturated fatty acids make up 75% of the lipid material.Beans consumption does not improve only micro and macronutrient status but also they play a role in other health parameters such as improving glycemic control (Kabagambe et al., 2005), decrease the risk of coronary heart disease and lowers cholesterol levels (Bazzano et al., 2011).
Minerals and trace elements play essential roles in numerous biochemical and physiological processes in animals and man.A deficiency, an overdose or imbalances between minerals or trace elements will exert a negative effect on health or nutritional status of an individual.Generally, it is not the ingested dose of minerals and trace elements that is important to maintain balance, but rather the amount that is bioavailable (available for biological and biochemical processes in the organism).Several food components are able to form soluble or insoluble complexes with minerals and trace elements under gastrointestinal conditions.These food components thereby increase or decrease the availability for absorption in the small intestine, and thus the bioavailability of minerals and trace elements.Due to the complexity of food products, however, the relative contributions of food components to the bioavailability of minerals and trace elements are often not clear.
Although in-vivo experiments (using actual animals or human beings) are the best way to study the bioavailability of minerals and trace elements, In-vitro methods offer an appealing alternative because they are relatively simple, rapid and inexpensive (Mamiro et al., 2001).Therefore, there is a great need in human nutrition and animal nutrition for an In-vitro method, which predicts the bioavailability of minerals and trace elements in vivo.The aim of this study therefore was to compare in-vitro bioavailability of selected minerals in dry and shelled green beans for raw and cooked samples.

METHODOLOGY Bean samples
Bean seeds of 38 varieties were brought from University of Nairobi and multiplied on experimental plots at Sokoine University of Agriculture Morogoro.The objective was to harvest enough beans to perform a number of laboratory experiments on each variety.A one acre plot was cleared and seed-bed prepared.Each variety was planted on four lines 50 cm apart and 20 cm from seedling to seedling (one stand per hill).This gave on average about 4 kg per variety.
All analyses were done in the Food Science and Technology Laboratory in collaboration with the Department of Soil Science.In-vitro bioavailability of minerals was determined on raw and cooked dry and shelled bean samples.

Total In-vitro bioavailable minerals
Total in-vitro bioavailable Iron and Zinc of the raw and cooked dry and green shelled bean samples was carried out by AOAC method No 968.08 and determined by atomic absorption spectrophotometry method No AOAC method 970.12, (AOAC, 1995).

HCl-pepsin and pepsin-pancreatin mineral bioavailability
In-vitro bioavailability of iron and zinc by HCl-pepsin (HCl-P) was carried out by a method described by Kumar and Chauhan (1993).Two grams of the dried bean samples were weighed and 25 ml of HCl-Pepsin solution was added in a 100 ml conical flask.The pH was adjusted to 1.35 and the mixture was incubated at 37ºC in a metabolic shaker water bath for 90 min.At the end of 90 min, the digest was centrifuged at 3000 rpm for 45 min and the supernatant filtered through whatman filter paper no 41.The filtrated was subjected to Pepsin-Pancreatin (P-P) method as described by Miller et al. (1981).Fifteen milliliters of the filtrate was mixed in 10 ml of pancreatin bile in tyrode buffer and pH adjusted to 7.5 and incubation continued for 2 h.The digest was centrifuged at 10,000 g for 20 min and then filtered through Whatman filter paper No 41.Analysis of the filtrate for soluble iron and zinc was performed using the Atomic Absorption Spectrophotometer (Shimadzu UNICAM 919, England).

Statistical analysis
Data for the In-vitro solubility were entered in excel office 2010 computer software.Descriptive statistics was used to compare the solubility's in raw and cooked samples and Students t-test was used to test for significant differences among the treatments at 95% confidence interval.

RESULTS
In-vitro bioavailability of iron and zinc in raw and cooked dry beans are presented in Figures 1 and 2. In both minerals there were a small but significant (P=0.009) and (P=0.0003)increase in mineral bioavailability after  cooking.The average increase for all the varieties was 3.2 to 3.4% for iron and 1.3 to 1.6% for zinc.
A similar trend was observed in the In-vitro bioavailability of iron and zinc in raw and cooked shelled beans (Figures 3 and 4).A relative wider gap is observed for iron and zinc bioavailability in shelled beans.In both minerals, there was a significant (P=0.000)increase in bioavailability after cooking the shelled beans.The average increase for all the varieties was 4.9 to 7.4% for iron and 2.4 to 3.6% for zinc.The highest difference for iron bioavailability was observed in Maharagi soja (12.9%) while lowest was shown by TY 3396-12 (-1.4%).The highest observed for zinc was 3% in G59/1-2.

DISCUSSION
Variety of beans displays a diverse variation in mineral bioavailability.This might probably be brought up by the variation in the content of antinutritional factors such as oxalates, lectins, hemaglutins, tannins and phytates (Sandberg, 2002).The most important antinutritional factors in the dietary minerals bioavailability are the phytates and tannins.The bioavailability of iron, which is the amount absorbed from food can be less than one percent to over 50%.Iron is found in food in two different forms, either as heme iron, found in the haemoglobin and myoglobin of meat, poultry and fish, or as non-heme iron found in plant products.Heme iron is better absorbed (about 15 to 40%) than non-heme iron (1 to 15%) (Roughhead and Hunt, 2000).Thus, although heme iron contributes  only about 10 to 15% of total iron intake but may provide substantial amount of total iron absorbed (Allen andAhluwalia 1997, EFSA, 2010).Nonheme iron is relatively consumed in much greater quantities, but is greatly affected by both inhibitors and enhancers in the diet such that there can be up to ten fold variations in absorption rates (Gropper et al., 2005).Non-heme iron absorption from a meal containing enhancers of absorption, such as meat, fish, or chicken, is about four times greater than it would be if the major protein sources were eggs or pulses.The addition of even small amounts of meat or a source of vitamin C (ascorbic acid) substantially increases the non-heme iron absorption from the entire meal (Lynch and Cook, 1984;Baech et al., 2003;Nielsen et al., 2013).In contrast, tea, coffee or eggs decreases the absorption of non-heme iron from a meal.
Low bioavailability diets (5% of the total iron absorbed) are based mainly on cereals and root vegetables with only very small quantities of meat, fish or vitamin C-containing foods (Baech et al., 2003).Such diets often contain foods that inhibit iron absorption (maize, beans, whole-grain flour) and are dominant in many developing countries.Intermediate bioavailability diets (10% of the iron absorbed) consist mainly of cereals and root vegetables but contain some meat and some foods containing vitamin C. High bioavailability  diets (15% of the iron absorbed) contain regular intakes of meat, poultry and fish (Baech et al., 2003).They also contain vitamin C-rich foods such as citrus fruits and some vegetables.A high bioavailability diet containing inhibitors of iron absorption, such as tea, coffee, cereal fibre, and dairy foods with main meals, would become an intermediate bioavailability diet.
In legume seeds, phytate is located in the protein bodies in the endosperm (Sandberg, 2002).
Phytate occurs as a mineral complex, which is insoluble at the physiological pH of the intestine.It is considered antinutritional, causing reduced uptake in the human intestine of essential dietary minerals such as Fe, Zn and Ca.A dosedependent inhibition of Fe, Zn and Ca absorption by phytate has been demonstrated in humans (Hallberg et al., 1989;Brune et al., 1992;Hurrell et al., 1992;Fredlund et al., 2002;Petry et al., 2010;La Frano et al., 2014).Inositol pentaphosphate has also been identified as an inhibitor of Fe and Zn absorption (Sandstrom and Sandberg, 1992;Sandberg et al., 1999).Furthermore, it was found that inositol tri and tetra-phosphate contribute to the negative effect on Fe absorption of processed foods containing a mixture of inositol phosphates (Sandberg et al., 1999), probably by interactions with the higher phosphorylated inositol phosphates.Fe absorption from soya beans and soya protein  products studied in single meals, using extrinsic labeling of meals with radioactive isotopes or studied by a stable isotope technique, was found to be low (Cook et al., 1981;Hurrell et al., 1992;Davidsson et al., 1994).Fe absorption from single meals based on black beans, lentils, mung beans, split beans and from two bean phenotype varieties was found to be very low, ranging from 0.8 to 1.9% (Lynch et al., 1984;Donangelo et al., 2003).
Legumes contain varying amounts of polyphenols and generally the amounts are considered higher in the coloured seeds.Beans of the species Phaseolus vulgaris were found to contain high amounts of polyphenols (Paredes-Lopez and Harry, 1989), whereas the content of polyphenols in peas (Pisum sativum) was very low.Another study which used In-vitro digestion/human Caco-2 model reported high bioavailability of iron in white compared to colored beans which was explained by presence of flavonoids in colored beans (Hu et al., 2006).
For shelled beans the percentage In-vitro bioavailability was comparatively higher than that observed in the dry beans.This is probably because the seeds were still maturing and therefore the antinutritional factors were not fully formed with regard to quantities and so the amount that was required to bind the minerals was not sufficient.Consuming green shelled beans might be rather beneficial with regard to mineral uptake than the dry beans.

Conclusion
Minerals from green shelled beans have been observed to be more bioavailable compared to dry beans.Vulnerable groups who suffer from micronutrient deficiencies especially iron and zinc, such as children below five years of age, pregnant and lactating mothers and the sick, should be encouraged to consume green shelled beans more often to improve their mineral uptake.

Figure 1 .
Figure 1.In-vitro bioavailability of iron from raw and cooked beans.

Figure 1 .
Figure 1.In-vitro bioavailability of iron from raw and cooked beans.

Figure 2 .
Figure 2. In-vitro bioavailability of zinc from raw and cooked beans.

Figure 2 .
Figure 2. In-vitro bioavailability of zinc from raw and cooked beans.

Figure 3 .
Figure 3. In-vitro bioavailability of iron from raw and cooked green shelled beans.

Figure 3 .
Figure 3. In-vitro bioavailability of iron from raw and cooked green shelled beans.

Figure 4 .
Figure 4. In-vitro bioavailability of zinc from raw and cooked green shelled beans.

Figure 4 .
Figure 4. In-vitro bioavailability of zinc from raw and cooked green shelled beans.