**1. Introduction**

In food production, extensive resources such as water, land, energy and nutrients are used with significant impact to the environment. It is forecasted that by 2050, the demand for agricultural products will increase by 35–50% due to a growing population and rising incomes, leading to higher pressure on the environment [1]. The problem is aggravated by the significant wastage of food throughout the supply chain. The wastage of food represents not only the loss of the product itself, but also all the resources used in food production, transportation and packaging.

The provision of animal-based protein generally has a higher environmental impact compared with vegetable or fungi-based proteins [2]. Concerns regarding the ethical and environmental implications of meat consumption have increased the demand for meat substitutes, such as those based on legumes and fungi [3]. Recently, the use of filamentous fungi as a commercial food product has gained considerable attention, due to its high protein content, the presence of essential amino acids and easy digestibility [4,5]. Filamentous fungi have traditionally been used by different societies as food, e.g., tempeh and oncom are indigenous staple foods in Indonesia [6,7]. One example of a product currently sold in the European market is Quorn™, which is made from the filamentous fungus *Fusarium venenatum* cultivated in synthetic medium with glucose, ammonium and mixed with egg albumen [8]. One problem with this product is the high cost of the cultivation medium [4]. Moreover, Smetana et al. [9] performed an environmental assessment of a similar product based on mycoprotein cultivated in molasses and concluded that the substrate is an environmental hotspot in the system. Therefore, it is relevant to consider other types of substrates to decrease production costs and environmental impacts.

**Citation:** Brancoli, P.; Gmoser, R.; Taherzadeh, M.J.; Bolton, K. The Use of Life Cycle Assessment in the Support of the Development of Fungal Food Products from Surplus Bread. *Fermentation* **2021**, *7*, 173. https://doi.org/10.3390/ fermentation7030173

Academic Editors: Alessia Tropea and Giuseppa Di Bella

Received: 19 July 2021 Accepted: 27 August 2021 Published: 30 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

One alternative substrate for fungal cultivation is surplus bread. The valorisation of food surplus into high-value products has gained interest as part of the strategies of transition to a bio-based economy grounded on a sustainable use of resources. Bread is rich in carbohydrates and its porosity makes it an ideal substrate for fungal cultivation [6,10]. Moreover, bread is a product with high waste generation, particularly at the level of the consumer and at the supplier-retailer interface. In the UK, it was estimated that 10% of all food waste are bakery products [11]. Brancoli et al. [12] studied food waste from retailers in Sweden and concluded that bread is a product category with large environmental impacts and economic costs [12]. In Sweden, the quantity of bread waste generated was estimated to be 80,410 tons/year, of which 28,200 tons/year are generated at the retail level [13]. The large waste volumes of uniform composition and quality, and the distribution model in which bread is sold makes it a good substrate for manufacturers who strive for stable delivery of raw materials.

There are mainly two different categories of bread, bake-off and pre-packaged. The technology proposed in this article can be used for both categories. Bake-off are products baked from pre-made dough in supermarkets or by a bakery. Pre-packaged bread is produced by the bakeries, and in some countries, it is often sold to retailers under takeback agreements (TBAs) [13–15]. In such agreements, the bakeries collect the bread that is not sold in the supermarkets, and here, bread waste is segregated from other food waste fractions from the retailers. This enables alternative pathways for the waste management and valorisation of the unsold bread. These pathways should preferably be located at higher levels in the food waste hierarchy, such as the production of fungal biomass for food, ethanol or animal feed [16,17]. Nevertheless, it is necessary to ensure that such technologies are sustainable and that they minimize the use of natural resources and the generation of waste as well as emissions of pollutants over their life cycles.

The life cycle assessment (LCA) methodology can be used to assess the environmental performance of a product to ensure its sustainability. The methodology can also be applied to support the early design stages in the development of the product by assessing the implication of design choices on the environmental performance of the technology [18]. Such studies are crucial since they can identify and prevent environmental impacts before the technology has been embedded by path dependency and lock-in [19]. Decisions made in the early stages of product development can have a significant influence on its subsequent environmental impacts. McAloone and Bey [20] estimated that 80% of the environmental performance of a product is determined by decisions made in the early stages of the technology development. For this reason, the European Union [21] recommends the use of LCA in product development to guarantee its sustainability.

Research on life cycle assessment of valorisation of bread waste is primarily focused on the lower stages of the food waste hierarchy. Few studies address bread waste valorisation into high-value products. Moreover, most of the research focuses on technologies with high technology readiness levels. Previous studies have investigated the environmental performance of bread waste management and valorisation alternatives, such as anaerobic digestion and incineration [22], conversion into biofuels [17], animal feed [17,23], and value-added chemicals [24]. There is a large body of research on the development of new technologies to valorise bread to high value products [6,25,26], but most of this research does not integrate lifecycle thinking into the early stages of the process development, increasing the risk for sub optimization of the technology.

The aim of this study was to integrate environmental considerations in the early stages of the development of a solid-state fermentation (SSF) process using bread as feedstock for the production of a protein-rich food product. The SSF process is proposed to be implemented as a stand-alone business, or on-site in small-scale bakeries to recover their otherwise discarded surplus bread. The technology can also be implemented in supermarkets that have a fresh bakery department in house, i.e., supermarkets that bake their own bread. The food product can be sold as a ready-to-eat meal directly to customers to reduce waste while generating additional income from the fungal product and provide a new sustainable food alternative that can replace other less sustainable options. new sustainable food alternative that can replace other less sustainable options. The goal of this study is to support the technology developers during the design and

otherwise discarded surplus bread. The technology can also be implemented in supermarkets that have a fresh bakery department in house, i.e., supermarkets that bake their own bread. The food product can be sold as a ready-to-eat meal directly to customers to reduce waste while generating additional income from the fungal product and provide a

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 3 of 13

The goal of this study is to support the technology developers during the design and development phases by assessing the environmental sustainability of a range of possible scenarios in which the technology can operate. This will enable a better understanding of the relation between design choices and the environmental performance of the technology, as well as allow the steering of the technology towards solutions with a lower environmental impact. Moreover, research on fungal growth patterns in solid-state cultures are limited and this study investigates whether morphological differences of the inoculation culture influence the performance in the subsequent SSF step. development phases by assessing the environmental sustainability of a range of possible scenarios in which the technology can operate. This will enable a better understanding of the relation between design choices and the environmental performance of the technology, as well as allow the steering of the technology towards solutions with a lower environmental impact. Moreover, research on fungal growth patterns in solid-state cultures are limited and this study investigates whether morphological differences of the inoculation culture influence the performance in the subsequent SSF step.

#### **2. Materials and Methods 2. Materials and Methods** The process development was iterative and used the concept of proxy technology

The process development was iterative and used the concept of proxy technology transfer-process [27], which considers that emerging technologies are not often based on completely new processes. Instead, it relies on a new application of existing technologies. The development of the process started with the assessment of an incumbent technology, namely the production of mycoprotein cultivated in molasses to identify hotspots in the system. The first results were then communicated to the researchers involved in the process development, starting an iterative approach wherein the hotspots in the system are identified and alternatives were proposed and assessed. In total, four different scenarios for the inoculation were proposed and compared in relation to the environmental performance and characteristics of the final product, such as protein content and morphology. A detailed description of the scenarios is available in Section 2.1. transfer-process [27], which considers that emerging technologies are not often based on completely new processes. Instead, it relies on a new application of existing technologies. The development of the process started with the assessment of an incumbent technology, namely the production of mycoprotein cultivated in molasses to identify hotspots in the system. The first results were then communicated to the researchers involved in the process development, starting an iterative approach wherein the hotspots in the system are identified and alternatives were proposed and assessed. In total, four different scenarios for the inoculation were proposed and compared in relation to the environmental performance and characteristics of the final product, such as protein content and morphology. A detailed description of the scenarios is available in Section 2.1.

#### *2.1. Process Description 2.1. Process Description*

The basic steps in the process are the drying and grinding of the bread, mixing with water and the starter spores (inoculum), and fermentation. The later processes after fermentation, such as frying, and freezing for later consumption are excluded in the study (Figure 1). The basic steps in the process are the drying and grinding of the bread, mixing with water and the starter spores (inoculum), and fermentation. The later processes after fermentation, such as frying, and freezing for later consumption are excluded in the study (Figure 1).

**Figure 1.** Basic process flow chart. The conditions of each process are described in brackets. The dotted box represents the system boundaries of the study. The further processing of the fungal biomass after fermentation is not included in the system boundaries. **Figure 1.** Basic process flow chart. The conditions of each process are described in brackets. The dotted box represents the system boundaries of the study. The further processing of the fungal biomass after fermentation is not included in the system boundaries.

#### 2.1.1. Substrate and Fungus 2.1.1. Substrate and Fungus

Steinbrenner & Nyberg (Mölndal, Sweden) kindly provided unsold fresh sourdough bread that showed no signs of mould contamination. The bread was prepared according to the process described in Gmoser et al [25]. Samples were autoclaved and stored in air-Steinbrenner & Nyberg (Mölndal, Sweden) kindly provided unsold fresh sourdough bread that showed no signs of mould contamination. The bread was prepared according to the process described in Gmoser et al [25]. Samples were autoclaved and stored in airtight containers at room temperature prior to use. The edible food grade filamentous fungus *Neurospora intermedia* CBS 131.92 (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used. The fungus was maintained on potato dextrose agar (PDA) plates containing 20 g/L glucose 20, 15 g/L agar and 4 g/L potato extract. The PDA plates were prepared by incubation for 5 days at 30 ◦C followed by storage at 4 ◦C. The spore solution was prepared by flooding each plate with 20 mL of distilled water, using a

disposable plastic spreader to gently release the spores and transferring them to a sterile slant tube. The spore solution (2.5 <sup>×</sup> <sup>10</sup><sup>6</sup> spores/mL) was used to prepare the four different inoculations (one for each of the scenarios). A detailed description of the conditions of fermentation is given in Section 2.1.3.
