Collection of Samples We conducted studies using Bangladeshi rice varieties to quantify the effect of reduced degree of milling and of modified parboiling processes on rice zinc content. The conditions tested for parboiling and pre-treatment of rice were simulated to reflect the range of conditions used in local small- and large-scale rice mills and that could potentially be optimized without the need for additional equipment. All studies were conducted in the Grain Quality & Nutrition Laboratories at the Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh. Parboiling studies For these studies, open (non-pressurized) and closed (pressurized) steaming systems were used after soaking. The open-steam method Autoclave was used at 100°C temperature but no pressure, and the pressure steam was obtained by placing the soaked paddy in a pressurized autoclave (Pressure 1kg cm-2) with a water vessel to produce steam. Soaking raw rice paddy in ambient temperature water without pre-steaming, following by open steaming represents conditions commonly used in traditional and semi-automatic rice mills, where batches of paddy are soaked in large tanks using tube well water to achieve adequate hydration, indicated when paddy husks begin to split, typically after 24-72 hours. During this time, soaking water may be changed every 12 hours to avoid microbial growth and fermentation. Soaked paddy is transferred to open tubular vessels, and steam is applied through a perforated pipe inserted in the center. Pre-steaming paddy prior to soaking has accelerates the time to achieve adequate hydration by raising the temperature of the paddy and soaking water. Pre-steaming followed by soaking and open-system steaming is used in small and large commercial mills using manual and semi-automatic operations. Pre-steaming followed by soaking and closed-system steaming is used in the larger commercial automatic mills. In this case, all steps are performed in a closed (pressurized) tubular tank with steam injectors. Our processing methods were designed to replicate these processes, as described below. The parboiling studies were conducted in two sets. One set using ambient soaking water conditions and the open-steaming system, which considered soaking time and the intermittent changing of soaking water as variables. The second set used pre-steaming followed by soaking and either open- or closed-system steaming, where pre-steaming time and soaking time were considered as variables. All studies used paddy of a common rice variety, BRRI dhan29, derived from one homogenized batch. Pre-steaming Pre-steaming was determined by steaming paddy for two or five minutes in an autoclave at 100 C, 0 g cm-2, with a vessel of distilled water placed inside as a source of steam. After two and five minutes pre-steaming water was added. The temperature of the soaking water increased to 37°C followed by 38°C but eventually water temperature became 30°C. Soaking Paddy samples (1 kg) were immersed in 2.5 litre distilled, deionized water (Barnstead Fistreem III Glass Still, Model A56220-857, Fistreem International Ltd., UK, and Barnstead E-Pure Ultrapure Water Purification Systems, Model: D4642-33, Thermo Fisher Scientific, USA). For the first studies, paddy was soaked in ambient temperature water (~25 C) for up to 48 hours, with samples drawn at 0, 12, 24, 36 and 48 hours. In a subset of samples, excess water was decanted at 12 hour intervals starting at 12 hours and replaced with fresh water. For the second studies, paddy was pre-steamed prior to soaking in distilled deionized water for 12 hours, with samples drawn at 0, 3, 6, 9 and 12 hours. Steaming Open steam parboiling was replicated by putting the soaked paddy samples in a mesh bag and autoclaving (JSAC-40, JS Research Inc, South Korea) at 100 C, 0 kg cm-2 for 30 minutes. A vessel of distilled water served as a source of steam. The closed steam parboiling system was replicated similarly but using the autoclave under pressure (10 minutes, reaching 121 C, 1 g cm-2). Parboiled paddy samples were left to cool and then laid out on individual polyethylene sheets; these were partially dried in the laboratory under a fan and drying was completed under the sun until a moisture content of 13-14% was reached. Dried samples were dehusked and milled to 10% degree of milling following procedures described below. Milling studies Ten rice varieties were selected to represent popular varieties produced in the two main growing seasons, Boro (irrigated) and Aman (rainfed). Ten rice varieties name are presented in table 1. Nine of the selected varieties were developed and released by BRRI and one is of Indian origin but popular in Bangladesh. For these studies, one standard soaking and parboiling method was used. Ambient temperature soaking water (25 C for 24 hours) followed by parboiling with the open steaming system described above was used. Dehusking and milling Outer husks were removed from dried paddy using a Satake Testing Husker (Model THU-35B, Satake Corporation, Hiroshima, Japan) with rubber rollers coated with polyvinyl chloride compound to avoid mineral contamination. The dehusked brown rice was milled using a Grainman tester mill (Model 60-220-50-DT, Grain Machinery Manufacturing Corporation, Miami, FL, USA). Six different degrees of milling were tested: 0%, 2%, 4%, 6%, 8%, and 10%, where 10% represents well-milled, polished rice, and the lower levels represent under milled rice. The degree of milling was calculated as the percent of outer milling fraction removed by weight using equation (1): % milling degree = 100 – [weight of milled rice (grams) / weight of brown rice (grams)] × 100. Analysis of zinc and phytate content Primary analysis for these samples was performed using atomic absorption spectrophotometry (AAS; Shimadzu Model AA-6800, Shimadzu Corporation, Tokyo, Japan). Samples were digested following an established method (IRRIASL, 2010). Briefly, 300-400 mg of oven-dried sample was weighed into 50 ml Erlenmeyer flasks, to which were added 12 ml each of 1:10 (v:v) 69-72% HClO4 and 65% HNO3. Duplicate samples were digested on a hotplate to completion (>7 hours), dissolved and made up to 25 ml using 1% HNO3, and transferred to polypropylene tubes for AAS analysis. Blanks and quality control samples were subjected to the same digestion procedure. A certified standard reference material (SRM1568a, rice flour, National Institute of Standards and Technology, Gaithersburg, MD, USA) and pooled internal control sample of rice grains were included in each run. Intra-run and inter-run CVs were calculated for the standard reference material. In addition, a subset of samples was submitted to a reference laboratory (Waite Analytical Services, Adelaide, Australia) for duplicate analysis by ICP-OES (Wheal et al., 2011). Phytate content was determined using Dionex liquid chromatography at the School of Biological Sciences, Flinders University, Adelaide, Australia. Phytate was extracted using 1.25% H2SO4 and 200 mmol /l NaOH in deonized water was used as an eluant. Data analysis For the degree of milling studies, data on zinc content (µg g-1) are presented on a dry weight basis as the mean ± SD of Boro and Aman rice varieties and all varieties combined. For the parboiling studies, data shown are the mean ± SD of duplicate analysis of the same sample. Differences in zinc content by degree of milling were determined by ANOVA with Tukey’s post-hoc analysis. For samples that were not pre-steamed, the effect of soaking time and changing of soaking water on the zinc content of 0% and 10% milled samples were determined independently by ANOVA. For samples that were pre-steamed, the independent effects of pre-steaming time and soaking time on zinc content of 0% and 10% milled rice were similarly determined for the open and closed parboiling systems.