**1. Introduction**

Zinc (Zn) is an essential key nutrient for several biochemical activities, such as human growth and development, immune system functions, and gene regulation. After iron, Zn is the second most abundant metal ion in organisms [1,2].

The Zn content in vegetables is related to various factors, such as species, genotype, type of edible portion (seed, leaf, fruit, or roots), phenological stage (microgreens, baby leaf, or mature vegetables), production method, and type of soil [3–6]. The recommended dietary allowance (RDA) of Zn for adults is 11 mg/day for men and 8 mg/day for women [7]. However, in some physiological conditions (such as pregnancy and lactation), chronic diseases (such as liver cirrhosis), diet (vegans/vegetarians), and in the elderly, it is necessary to increase the Zn intake with nutrition [7]. In humans, Zn deficiency is mostly associated with poor nutrition and poor dietary variegation and is aggravated by its poor availability in soils [5].

Zn deficiency is estimated to affect more than 3 billion of the world's population, with the vast majority occurring in underdeveloped countries [8,9].

The human and economic cost of Zn malnutrition is noteworthy, considering that about 17% of the global population suffers from this condition in developed and underdeveloped countries. More than 100,000 deaths per year in children under the age of 5 with various pathologies are attributable to the Zn deficiency [1–9]. Consequently, a series of international actions have been undertaken to improve the nutritional status of the population exposed to Zn malnutrition through the use of different approaches [10–13]. Among these, biofortification is the practice of deliberately increasing nutrients and healthy

**Citation:** D'Imperio, M.; Montesano, F.F.; Serio, F.; Santovito, E.; Parente, A. Mineral Composition and Bioaccessibility in Rocket and Purslane after Zn Biofortification Process. *Foods* **2022**, *11*, 484. https://doi.org/10.3390/ foods11030484

Academic Editors: Arun K. Bhunia and Antonello Santini

Received: 19 November 2021 Accepted: 4 February 2022 Published: 7 February 2022

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compounds and/or decreasing antinutritional factors (such as phytic and oxalate acids) in plant-based foods (cereal, vegetables, and fruit) [14,15]. Biofortified crops can be obtained through various strategies, such as genetic engineering, plant breeding, and agronomic practices [14,15].

Agronomic biofortification is generally used to increase the content of mineral nutrients (iodine, silicon, calcium, iron, zinc, magnesium, selenium, and copper) in the edible parts of various leafy vegetables and fruits, such as mizuna, tatsoi, chicory, basil, purslane, lettuce, tomato, Swiss chard, rocket, potatoes, green beans, and others [16]. This approach can be applied in different cultivation conditions, such as open field, greenhouse, and indoors; in the latter cases, also using soilless cultivation systems. Indeed, several studies have reported that the efficiency of biofortification, especially in greenhouses and indoor cultivation, can be maximized by specific managemen<sup>t</sup> of the growing conditions [17–19]. The concentration of the nutrient solutions (NS) is an important characteristic for the quality of vegetables production [18]; therefore, changes in the composition of the NS can have a considerable impact on the nutritional quality of products, in particular, on the content of mineral elements [17,19] and bioactive organic compounds [20]. Furthermore, the choice of the plant species for biofortification represents an important aspect of the mineral biofortification process due to the effect of the phylogenetic heritage that inevitably affects plants' ability to accumulate essential mineral elements [21]. As an example, among leafy vegetables, purslane is considered a "new crop" for ready-to-eat products [22] and is characterized by a high oxalate content (2000 mg/kg of fresh weight). Rocket, on the other hand, is one of the most popular species grown in Mediterranean areas as a "ready-to-eat fresh-cut salads" product and is generally considered oxalate-free [23].

A crucial step after the biofortification process is the assessment of the bioaccessibility of the target nutrient. Ideally, in a successful biofortification protocol, the increase of a target nutrient in the edible parts parallels an increase in its bioaccessibility. The amount of nutrient that is released from the plant matrix during the gastrointestinal digestion process and its evaluation are independent of the approach and the method used to produce the biofortified crop. Furthermore, not all parts of a nutrient in the edible parts of biofortified vegetables can perform a biological activity. The release of nutrients in the intestinal tract (during the gastrointestinal digestion process) depends on different factors, such as species and type, and is subject to various influences, for example the concentration of nutrients, the activity of antinutritional compounds, texture, food processing, and the interaction of some nutrients with others [24–26]. During the gastrointestinal digestion process, the interaction of different mineral elements with similar electronic configurations (Zn2+, Ca2+, Fe2+, Mg2+, Mn2+, and Sr2+) can often lead to changes in the bioaccessibility and bioavailability of mineral nutrients [27,28]. Several methods are available to assess bioaccessibility using the in vitro digestion protocol. In these methods, the chemical, physical, and dynamic conditions of the gastrointestinal tract (mouth, stomach, and gut) are artificially reproduced in vitro [29].

Overall, the assessment of bioaccessibility provides information on the number of nutrients released from the food matrix, on nutrient–nutrient and nutrient–antinutrient interactions, on biochemical transformations, on chemical degradations, and on the effect of the matrix [30,31]. Furthermore, the assessment of bioaccessibility represents the starting point for the estimation of the beneficial effects of biofortified products on human health and can be used as a method to improve the food design process.

With all the above taken into account, the objectives of this study were:(i) to evaluate the overall mineral profile of rocket and purslane subjected to a process of Zn biofortification; (ii) to assess the quantity of mineral elements released by biofortified vegetables during the digestion process (bioaccessible fraction); and (iii) to calculate the RDA coverage and the hazard quotient (HQ) in relation to Zn bioaccessibility.

Two baby leaf vegetables (rocket and purslane) were produced and biofortified with Zn, the consumption of which allows an increase of zinc intake in the human diet without causing harm to the consumer. A workflow was proposed that was based on the evaluation of the efficiency of the biofortification process from a nutritional point of view, taking into account the overall bioaccessibility of the mineral nutrients.

#### **2. Materials and Methods**

#### *2.1. Production of Zn-Biofortified Purslane and Rocket*

Zn-biofortified rocket and purslane were produced in the experimental greenhouse "La Noria" located in Mola di Bari (BA), southern Italy (41◦03 N, 17◦04 E; 24 m a.s.l.) by using the floating hydroponic system. Rocket and purslane were grown in a complete NS with macro- and micro-nutrients [32]. Zn levels in the NS were 0.13 and 5.2 mg/L for growing non-biofortified and biofortified plants, respectively. The plants were harvested at the commercial stage of "baby leaf" (24 January 2020 and 30 July 2020, respectively, for rocket and purslane), as defined by Di Gioia et al. [33].

#### *2.2. Mineral Profile of Rocket and Purslane*

Al, B, Ca, Fe, K, Mg, Mn, Na, Sr, and Zn content was measured in dry samples by inductively coupled plasma optical emission spectrometry (ICP-OES) after mineralization of the dry samples with an acid microwave-assisted digestion system (MARS 6, CEM Corporation, Matthews, North Carolina) performed as reported by D'Imperio et al. [34]. To confirm the accuracy of the measurements, certified reference vegetable material (CRM, NIST tomato leaf 1535a) was analyzed using the same procedure as the rocket and purslane samples.

#### *2.3. In Vitro Gastrointestinal Digestion Process*

The assessment of mineral bioaccessibility (Al, B, Ca, Fe, K, Mg, Mn, Sr, and Zn) from plant samples (biofortified and not) during the digestion process was performed as reported by Ferruzzi et al. [35]. After the digestion process, samples were centrifuged at 10,000× *g* for 1 h at 4 ◦C to separate the aqueous intestinal digesta, called 'bioaccessible fraction' (BF), from the residual solids. The BFs were collected, filtered (0.2 μm PTFE filter), and dried at 50 ◦C for 48 h before the minerals content was measured. For the CRM sample only, the residual solids were washed with Milli-Q H2O (18 MΩ/cm) and dried (50 ◦C for 48 h) until use. To evaluate the accuracy of the measurement, CRM (NIST tomato leaf 1535a) was analyzed using the same procedure adopted for the rocket and purslane samples.

#### *2.4. Analysis of Mineral Content in Digested Sample*

After the digestion process, the BF and the residual solid were mineralized with HNO3 65% using the same protocol used for rocket and purslane (see Section 2.2). Blank correction was performed in all analyses. The protocol applied did not allow the estimation of Na bioaccessibility, because the blank correction was not performed for this mineral element. The amount of Na released from the food matrix during the digestion process was lower than the amount of Na in the blank sample (3.81 g/L). This is related to the reagents used, as also reported by another study [36]. The bioaccessibility fraction percentage (BF%), defined as the percentage of nutrient(s) released from the digested matrix in the gastrointestinal digestion process, was calculated as BF% = (total nutrient released during digestion/total nutrient in food) × 100.

#### *2.5. Percentage of Recommended Daily Allowance and Hazard Quotient for Zn Intake*

The recommended daily allowance of Zn (RDA-Zn) is equal to 11 and 8 mg, respectively, for male and female adults [7]. The daily intake of Zn and the percentage of coverage of RDA for Zn (% RDA-Zn) were calculated in relation to the quantity of Zn released from the vegetables during the gastrointestinal digestion process. Risk assessment was also performed by using HQ, considered as the risk to consumer health resulting from the consumption of Zn-biofortified, fresh baby leaf vegetables, based on a 70 kg adult. The HQ is the ratio of the potential exposure to an organic and/or inorganic substance and the level at which no negative effects are expected. HQ allows the estimation of the potential negative effects on health related to chronic consumption of food (in our case, biofortified rocket and

purslane). A HQ lower or equal to 1 indicates that adverse effects are unlikely to occur, and, thus, the product can be considered to have negligible hazard. For a HQ greater than 1, the potential for adverse effects increases [37]. The contribution of Zn from other nutritional sources was not examined. The HQ was calculated according to the protocol described by the Environmental Protection Agency [37], using the following equation: HQ = ADD/RFD, where ADD is the average daily dose of Zn (mg of Zn/kg body weight/day), and RFD is the recommended dietary tolerable upper intake level of Zn (mg of Zn/kg body weight/day). The I RFD value for a 70 kg adult is 3 × 10−<sup>1</sup> mg Zn/kg/day [38]. The ADD for 100 g portions of rocket or purslane was computed as follows: ADD = (MI × CF × DI)/BW. MI is the Zn concentration released during the gastrointestinal digestion process after the consumption of the two vegetables (mg/kg DW); CF is the fresh-to-DW conversion factor for vegetable samples (calculated as the ratio of FW to DW; rocket: 0.093 on average; purslane: 0.054 on average); DI is the daily intake of baby leaf vegetables (kg, taken as 100 g); BW is the body weight (kg) of humans, assumed as 70 kg.
