**4. Conclusions**

The general purpose of this study was to produce Zn-biofortified rocket and purslane and to propose a workflow for studying their nutritional qualities based on the analysis of the bioaccessible fraction of the overall mineral elements.

The agronomic biofortification protocol used in this study was based on increasing the concentration of Zn in the NS used for the cultivation of rocket and purslane in soilless conditions. This protocol allowed Zn-biofortified plants with a higher nutritional quality to be obtained. The amount of bioaccessible Zn released by the plants during the digestion process was influenced by the species (rocket and purslane) and by the initial Zn content accumulated in the edible parts of the plants in soilless cultivation using Zn-enriched NS.

The use of the in vitro gastrointestinal digestion protocol allowed the evaluation of the bioaccessible fraction of Zn and other mineral elements. Antinutritional factors (carbonate, phytic and oxalic acids) and some healthy food components (proteins, fibers, and polyphenols) can modify the release of nutrients from the food matrix, generating insoluble salts and determining the reduction of bioaccessibility and absorption of the mineral elements. Hence, it is important to quantify the bioaccessible fraction of the target mineral and also of the other mineral elements.

Our results confirmed that in vitro digestion is a valuable method for assessing the nutritional efficiency of the biofortification process. This approach can be efficiently used to improve the design process for biofortified products. Furthermore, the calculated hazard quotient demonstrates the safety of biofortified rocket and purslane.

Overall, the consumption of biofortified rocket and purslane would provide greater intake of Zn in the human diet without causing harm to the consumer, thus, providing benefits for different classes of consumers, such as the elderly, vegetarians, vegans, and people with gastrointestinal and other diseases. However, more research is needed to further explore and validate the applicability of the proposed workflow to biofortification processes for other mineral elements and in other plant species.

**Author Contributions:** Conceptualization, M.D. and A.P.; methodology, M.D. and A.P.; crop performance measurements, F.F.M. and F.S.; chemical analysis, M.D. and A.P.; in vitro digestion process, M.D.; statistical analysis, A.P.; original draft preparation, M.D. and A.P.; writing—review and editing, M.D., F.F.M., F.S., E.S., and A.P.; supervision of the study, M.D., F.F.M., F.S., and A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was founded by the National Research Council (CNR) project NUTR-AGE (Ordinary fund for research organizations and institutions FOE-2019), DSB.AD004.271, and SOILLESS GO project (project code (CUP) B97H20000990009), funded by the Rural Development Program of the Apulia Region (Italy) 2014–2020, sub-measure 16.2 (Support for pilot projects and development of new products, practices, processes, and technologies and transfer and dissemination of results obtained by operational groups) (Paper n. 14).

**Acknowledgments:** The authors thank Nicola Gentile for the technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.
