*2.1. Goal and Scope Definition*

The goal of this study is to quantify the potential environmental impact of the use of phenolic extracts (PE) from olive mill wastewater (OMWW) and from olive leaves (OL) in particular case studies of food processes to improve nutritional and technological parameters.

The functional unit (FU) defines the reference unit of the system under analysis (ISO 14040 and 14044) [12]. To evaluate the environmental impact of the polyphenol's extraction processes and encapsulating process, 1 g of TPC was defined as FU.

Differently, a commercial unit was used to evaluate the weight of the PE as ingredients in four food formulations: (i) 350 g for vegan mayonnaise, (ii) 135 mL for salad dressing, (iii) 160 g for biscuits, and (iv) 300 g for gluten free breadsticks.

Moreover, the commercial FU for each product was normalized considering technological and nutritional parameters.

The system under study follows an approach called "from cradle to grave" where all the factors were considered from the olive oil extraction process to the formulation of the four food preparations. In detail, as reported in Figure 1 (detailed system boundaries are reported in supplementary data Figure S1), every input (extraction of raw materials, energy and water consumption, chemicals) and output (hazardous waste and food production waste) were considered. Regarding the food preparations, the consumption and the packaging were neglected.

**Figure 1.** Simplified system boundaries.

#### 2.1.1. Normalization Factors

Since the PE addiction implies advantages in term of technological and nutritional aspects, the environmental impact calculated considering commercial FU was normalized based on Total Phenolic Content (TPC) and oxidative stability. In order to quantify (i) the TPC, Singleton and Rossi's method [13] was performed and (ii) the oxidative stability method used by Paciulli et al. [14] was carried out. Gallic acid equivalent expressed as TPC

(mg/g) and oxidative stability expressed in oxidation induction days represent the units describing nutritional and technological parameters respectively.

In this work different combinations of extracts (OMWW PE, OLE, and eOLE) and food preparations were considered and only the best performing enriched food preparations were studied (higher values of induction days and mg of TPC/g of product) and reported in Table 1. To evaluate the technological and nutritional performance of enriched food preparations, a conventional production process (control) was also analyzed.


**Table 1.** Technological and nutritional characterization of food preparations.

#### 2.1.2. Allocation Criteria

Considering the olive oil mill process and according to Parascanu et al. [20], olive oil has a much higher economic value compared to the other by-products, not only representing the higher output in terms of product amount (mass allocation in Table 2). To better evaluate the environmental impact of every olive oil mill output, an economic allocation was used. In particular, according to Tsagaraki et al. [4], in addition to the conventional subdivision of the by-products, it was possible to identify in a more specific way the average composition of the different outputs: (i) pomace, (ii) wastewater, (iii) stone, and (iv) leaves and dust (Table 2).

**Table 2.** Economic values and average percentage of mass and economic allocation for the olive oil and by-products of the milling process.


Details about the transition from the mass allocation to the economic one was reported in Table 2. Overall, OMWW and OL values represent a cost for the milling process instead of a revenue, not having economic allocation (waste products). In this work, in order to calculate the relative environmental impact, a minimum economic value attribution was chosen also for these products (by-products). Considering the four food preparations, a mass allocation was used.

#### *2.2. Life Cycle Inventory (LCI)*

Regarding the olive oil milling process, secondary data from the WFLD (World Food LCA Database) were used and modeled in to obtain the five different outputs as argued in the allocation criteria paragraph. OMWW and OL were used as inputs for the following extraction processes. The inventory of OMWW PE and OLE extraction phases, encapsulation phase (eOLE), and the related applications on food preparations, on the contrary, were performed separately and reported below using primary data.

#### 2.2.1. OMWW PE Extraction Phase LCI

Following the method used by Romeo et al. [24], 2 L of olive mill wastewater were acidified with 1 mL of HCl to obtain a pH 2 mixture. The mixture passed three cycles which required 2 L of hexane and 3 min of centrifuge (0.5 kW) for each cycle. Then, 0.625 L of ethyl acetate were added, and using (i) the centrifuge (3000 rpm 0.8 kW, 18 min) and (ii) the evaporator for 2 h, 3 g of dry residue were obtained. Then, 100 mL of water were added to the dry residue to obtain a solution, which was filtered to obtain 103 mL of OMWW PE. Hexane and ethyl acetate were recovered after use for 75%, and the remaining 25% were treated as hazardous waste. Input and output data related to this analysis and allocated to the FU (equivalent to 34.33 mL) are reported in Table 3.

**Table 3.** Input, output data and allocated quantity per FU, related to the OMWW PE process.


2.2.2. OLE Extraction Phase LCI

According to Difonzo et al. [25], 200 g of olive leaves were washed using 1 L of water, dried firstly with paper (2–3 pieces) and then using an oven (0.53 kW, 8 min, 120 ◦C). Then, 100 g of hot air-dried leaves (3–4% water content) were milled for 30 s using a mill (0.175 kW). The powder obtained passed three cycles, which required (i) 2L of water, (ii) the use of ultrasound (0.2 kW, 30 min), and (iii) filtering to obtain 6 L of filtered aqueous extract. Using a freeze dryer (1.4 kW, 24 h), 10 g of OLE were obtained. Then, 100 g of exhausted leaves were treated as biowaste. Input and output data related to this analysis and allocated to the FU (equivalent to 6.67 g) are reported in Table 4.


**Table 4.** Input, output data, and allocated quantity per FU, related to the OLE process.

2.2.3. eOLE Encapsulation Phase LCI

According to Flamminii et al. [26], 0.4 g of OLE, 0.4 g of pectin, 1.64 g of calcium citrate, 0.4 g of alginate, and 17.16 mL of water were mixed to 98 g of sunflower oil and 2 g of Span 80 (emulsifier) and agitated using a stirring plate (0.4 kW, 15 min). Then, 20 g of sunflower oil and 0.5 g of glacial acetic acid were added and agitated using a stirring plate (0.4 kW, 30 min). Then, 3 g of OLE, 145.42 mL of water, 0.83 g of calcium chloride, and 0.75 g of Tween20 (surfactant) were added to the mixture and agitated again using a stirring plate (0.4 kW, 30 min). To obtain the beads (92% water content and 8% dried matter), a centrifuge was used for 5 min (0.800 kW). Then, 30 mL of ethanol solution (21 mL ethanol, 8.94 mL water, and 0.6 g OLE) was added to obtain 20 g of cleaned beads. Lastly, the beads were lyophilized using a freeze drier for 24 h (1.4 kW) to obtain 1.60 g of eOLE. Then, 300.5 g of exhausted oil were treated as waste oil. Input and output data related to this analysis and allocated to the FU (1 g of TPC equivalent to 5 g of eOLE) are reported in Table 5.


**Table 5.** Input, output data, and allocated quantity per FU, related to the eOLE process.
