Iron (Fe) and zinc (Zn) are essential micronutrients crucial for various metabolic activities, including oxygen transport (Fe) and enzymatic regulation (Zn). Deficiencies in these nutrients lead to significant health problems such as iron-deficiency anemia and impaired immunity, affecting billions worldwide, particularly preschool children and pregnant women in low-income regions. Strategies to address these deficiencies include direct supplementation, food biofortification, increasing bioavailability by reducing anti-nutrients, and dietary diversification. Bamboo seeds, a rare and valuable food source in some Asian regions, have shown promise due to their high nutritional value. Previous research indicated high Fe and Zn concentrations in Moso bamboo seeds, prompting this study to further investigate their bioaccessibility, bioavailability, and micronutrient localization to assess their potential for natural biofortification.

Extensive literature highlights the global prevalence of iron and zinc deficiencies and their adverse health consequences. Various approaches to combat these deficiencies, including biofortification strategies targeting staple crops like rice, wheat, and maize, have been explored. Studies emphasize the challenges of ensuring bioavailability due to the presence of anti-nutrients like phytate and tannins. While the nutritional value of bamboo seeds has been investigated, detailed analysis of iron and zinc bioaccessibility and bioavailability in Moso bamboo seeds was lacking, leading to the current study's focus.

Moso bamboo (*Phyllostachys edulis*) seeds were collected from three geographically diverse sites in Guangxi Province, China, with varying precipitation and temperature but similar subtropical monsoon climates and krasnozem soil types. Rice (*Oryza sativa*, cv Nipponbare) served as a control. Fe and Zn concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS) after microwave digestion. An in vitro gastrointestinal digestion protocol simulated oral, gastric, and intestinal digestion to assess Fe and Zn bioaccessibility and bioavailability in both raw and boiled seeds. Phytic acid (PA) and tannin concentrations were measured using spectrophotometric methods. Cellular localization of Fe and Zn was investigated using Prussian blue and dithizone staining, respectively, followed by laser ablation ICP-MS (LA-ICP-MS) for elemental mapping in longitudinal and transverse seed sections. Statistical analysis employed one-way ANOVA with Tukey's multiple comparison test (P<0.05).

Moso bamboo seeds exhibited significantly higher Fe and Zn concentrations than rice (P<0.05). Fe concentrations in bamboo seeds ranged from 45.6 to 56.2 mg kg⁻¹, while Zn concentrations ranged from 71.6 to 84.9 mg kg⁻¹, both significantly exceeding those in rice. Bioaccessibility and bioavailability of Fe and Zn were also substantially higher in bamboo seeds compared to rice. For example, Fe bioaccessibility in bamboo seeds averaged around 25 mg kg⁻¹, approximately two-fold higher than in rice. Similar trends were observed for Zn. Boiling decreased bioaccessibility and bioavailability of both minerals in both bamboo seeds and rice but bamboo seeds maintained higher values even after boiling. Phytic acid concentrations were significantly lower in bamboo seeds than in rice, suggesting it's not a major inhibitor. However, tannin concentrations were considerably higher in bamboo seeds, potentially influencing bioavailability. Histochemical staining and LA-ICP-MS revealed that Fe and Zn were primarily concentrated in the embryo and aleurone layer of the bamboo seeds, while the starchy endosperm contained much lower amounts. The molar ratios of phytate to Fe and Zn were much lower in bamboo seeds than in rice (e.g., 11:1 to 12:1 for PA:Fe in bamboo vs. 68:1 in rice).

The high Fe and Zn concentrations and their enhanced bioaccessibility and bioavailability in Moso bamboo seeds, despite higher tannin content, demonstrate their potential as a valuable dietary source for natural biofortification. The significant difference in mineral content between bamboo seeds and rice, along with the observation that boiling negatively impacts bioavailability for both, highlights the need for consumption strategies that maintain the integrity of the embryo and aleurone layer, similar to the preference for brown rice over white rice. The lower phytic acid levels and the predominantly embryo and aleurone layer localization of iron and zinc suggest that intrinsic factors within the bamboo plant may play a greater role than external factors in the high accumulation of Fe and Zn, although further investigation is needed.

This study provides strong evidence that Moso bamboo seeds are a naturally rich source of bioaccessible and bioavailable iron and zinc. The findings suggest that incorporating bamboo seeds into diets could contribute significantly to combating micronutrient deficiencies. Future research should focus on understanding the specific mechanisms underlying the high Fe and Zn accumulation in bamboo seeds and exploring optimal processing methods to maximize nutrient retention and bioavailability.

The study employed an in vitro digestion model, which may not perfectly replicate the complex in vivo digestion processes. Further research using in vivo studies is needed to confirm the findings. The analysis focused solely on Moso bamboo, and investigations involving other bamboo species would broaden the understanding of their potential for biofortification. The influence of soil composition and microbial communities on mineral uptake in bamboo requires further exploration.