Production of single-cell protein: species selection and optimization of operational conditions
Abstract
O crescimento atual da população mundial está a levar a uma procura insustentável por proteína animal e vegetal. De modo a responder ao desafio de alimentar o mundo, este trabalho focou-se na obtenção de um produto rico em proteína e com valor nutricional, obtido a partir da fermentação em estado sólido (SSF) de cascas de fruta (laranja e banana) por fungos filamentosos. Inicialmente, foram estudados os efeitos de vários fatores (humidade - 60 % e 75 %; tempo de fermentação - 7 dias e 14 dias; tamanho do inóculo - 1×107 esporos/g e 5×107 esporos/g; concentração de sulfato de amónio - 0 g/g e 0.01 g/g; concentração de licor de maceração de milho (CSL) - 0 g/g e 0.01 g/g; e fungo filamentoso - Aspergillus ibericus e Rhizopus oryzae) na composição das cascas de fruta (nomeadamente proteína total, açúcares redutores, fenóis totais e atividade antioxidante) usando um desenho experimental de Plackett-Burman. Observou-se crescimento significativo das 2 espécies nos meios compostos por cascas de laranja e de banana. Para além disso, o conteúdo de proteína total, fenóis totais e atividade antioxidante das cascas de fruta aumentaram após a fermentação com os fungos filamentosos usados. A humidade (50 %, 60 %, 70 %), a concentração de sulfato de amónio (0 g/g, 0.005 g/g, 0.01 g/g) e a concentração de CSL (0 g/g, 0.005 g/g, 0.01 g/g) foram identificados como os fatores mais importantes para aumentar o valor nutricional das cascas de fruta (em particular o teor em proteína total) e foram posteriormente estudados através de um desenho experimental de Box-Behnken. Estes ensaios foram realizados com Aspergillus ibericus (concentração inicial de 5×107 esporos/g) durante 7 dias. O valor máximo de proteína total nas cascas de laranja fermentadas (16.29 %) foi obtido nas condições de 70 % de humidade, 0.005 g/g de sulfato de amónio e sem CSL. O valor máximo de proteína nas cascas de banana (17.67 %) foi obtido nas condições de 70 % de humidade, 0.005 g/g de CSL e sem sulfato de amónio. Por fim, as cascas fermentadas foram caracterizadas quanto ao seu teor em hidratos de carbono, açúcares redutores, fibra bruta, pectina, lípidos totais, minerais e atividade antioxidante. A SSF reduziu o conteúdo de hidratos de carbono, açúcares redutores e pectina das cascas, enquanto aumentou o conteúdo de proteína, fibra, lípidos, minerais, e atividade antioxidante, aumentando assim o seu valor nutricional.
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Helena Isabel Santos Vilela Production of single-cell protein: species selection and optimization of operational conditions outubro de 2023 Production of single-cell protein: species selection and optimization of operational conditions Helena Isabel Santos Vilela UMinho|2023 Universidade do Minho Escola de Engenharia
Helena Isabel Santos Vilela Production of single-cell protein: species selection and optimization of operational conditions outubro de 2023 Dissertação de Mestrado Mestrado Integrado em Engenharia Biológica Ramo de Tecnologia Química e Alimentar Trabalho efetuado sob a orientação da Doutora Marlene Alexandra da Silva Lopes e da Professora Rita Isabel Couto Pinheiro Universidade do Minho Escola de Engenharia
ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não revistas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
iii AGRADECIMENTOS Gostaria de agradecer a contribuição, o apoio e o esforço de várias pessoas envolvidas na elaboração desta Dissertação. Primeiramente, um grande obrigada à Doutora Marlene Lopes, pela minha orientação, pelo supervisionamento e ajuda no planeamento do trabalho experimental e pela revisão da escrita da Dissertação. Também à minha co-orientadora, a Professora Rita Pinheiro, gostava de agradecer pela contribuição com o seu conhecimento e por permitir a análise de algumas amostras no Instituto Politécnico de Viana do Castelo. A Professora Isabel Belo também prestou ajuda, na medida em que sugeriu melhorias para melhorar a apresentação de resultados e também contribuiu com o seu conhecimento na área de desenhos experimentais. Adicionalmente, um obrigada muito especial à minha colega e amiga Liliana Araújo, por me ajudar inúmeras vezes com ensaios e protocolos novos e por ter dado suporte emocional em dias de mais incerteza. Gostaria também de agradecer às outras meninas do Laboratório de Bioprocessos e Biossistemas, nomeadamente à Bruna Silva e à Marta Ferreira, por sempre me ajudarem com a localização de material de laboratório e com alguns protocolos. Um obrigada também aos alunos de licenciatura que acompanhei, o Luís Babo e a Raquel Figueiredo, a quem eu gosto de chamar “os meus pupilos”, por me terem ensinado a ensinar e por também terem acompanhado parte da minha jornada. Por fim, agradeço ao Professor Nelson Lima e ao Professor Armando Venâncio por me terem ajudado com a escolha do tema da Dissertação e por me terem redirecionado para o projeto da FCT da Doutora Marlene Lopes “Micro4Food - Upgrading fruit wastes for microbial protein production” (refª 2022.01705.PTDC), ao qual agradeço também.
iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho.
v PRODUÇÃO DE PROTEÍNA MICROBIANA: SELEÇÃO DE ESPÉCIES E OTIMIZAÇÃO DE CONDIÇÕES OPERACIONAIS RESUMO O crescimento atual da população mundial está a levar a uma procura insustentável por proteína animal e vegetal. De modo a responder ao desafio de alimentar o mundo, este trabalho focou-se na obtenção de um produto rico em proteína e com valor nutricional, obtido a partir da fermentação em estado sólido (SSF) de cascas de fruta (laranja e banana) por fungos filamentosos. Inicialmente, foram estudados os efeitos de vários fatores (humidade - 60 % e 75 %; tempo de fermentação - 7 dias e 14 dias; tamanho do inóculo - 1×107 esporos/g e 5×107 esporos/g; concentração de sulfato de amónio - 0 g/g e 0.01 g/g; concentração de licor de maceração de milho (CSL) - 0 g/g e 0.01 g/g; e fungo filamentoso - Aspergillus ibericus e Rhizopus oryzae ) na composição das cascas de fruta (nomeadamente proteína total, açúcares redutores, fenóis totais e atividade antioxidante) usando um desenho experimental de Plackett-Burman. Observou-se crescimento significativo das 2 espécies nos meios compostos por cascas de laranja e de banana. Para além disso, o conteúdo de proteína total, fenóis totais e atividade antioxidante das cascas de fruta aumentaram após a fermentação com os fungos filamentosos usados. A humidade (50 %, 60 %, 70 %), a concentração de sulfato de amónio (0 g/g, 0.005 g/g, 0.01 g/g) e a concentração de CSL (0 g/g, 0.005 g/g, 0.01 g/g) foram identificados como os fatores mais importantes para aumentar o valor nutricional das cascas de fruta (em particular o teor em proteína total) e foram posteriormente estudados através de um desenho experimental de Box-Behnken. Estes ensaios foram realizados com Aspergillus ibericus (concentração inicial de 5×107 esporos/g) durante 7 dias. O valor máximo de proteína total nas cascas de laranja fermentadas (16.29 %) foi obtido nas condições de 70 % de humidade, 0.005 g/g de sulfato de amónio e sem CSL. O valor máximo de proteína nas cascas de banana (17.67 %) foi obtido nas condições de 70 % de humidade, 0.005 g/g de CSL e sem sulfato de amónio. Por fim, as cascas fermentadas foram caracterizadas quanto ao seu teor em hidratos de carbono, açúcares redutores, fibra bruta, pectina, lípidos totais, minerais e atividade antioxidante. A SSF reduziu o conteúdo de hidratos de carbono, açúcares redutores e pectina das cascas, enquanto aumentou o conteúdo de proteína, fibra, lípidos, minerais, e atividade antioxidante, aumentando assim o seu valor nutricional. Palavras-chave: Cascas de banana; Cascas de laranja; Fermentação em estado sólido; Fungos filamentosos; Proteína microbiana.
vi PRODUCTION OF SINGLE-CELL PROTEIN: SPECIES SELECTION AND OPTIMIZATION OF OPERATIONAL CONDITIONS ABSTRACT The actual growth of global population is leading to an unsustainable rising demand for animal and plantbased protein. To address this challenge, this work focused on the production of a nutritious and proteinrich product, obtained by solid-state fermentation (SSF) of orange and banana peels with filamentous fungi. Firstly, the main effect of several factors (moisture content - 60 % and 75 %; fermentation time – 7 days and 14 days; inoculum size - 1×107 spores/g and 5×107 spores/g; concentration of ammonium sulfate - 0 g/g and 0.01 g/g; concentration of corn steep liquor (CSL) - 0 g/g and 0.01 g/g; and fungal species - Aspergillus ibericus and Rhizopus oryzae ) on fruit peels composition (specifically, total protein, reducing sugars, total phenols, and antioxidant activity), were studied in SSF by a variable screening PlackettBurman experimental design. A significant growth of both fungal species on orange and banana peels was observed. Additionally, the content of total protein, total phenols, and antioxidant activity increased in fruit peels after SSF with filamentous fungi. Moisture (50 %, 60 %, 70 %), ammonium sufate concentration (0 g/g, 0.005 g/g, 0.01 g/g), and CSL concentration (0 g/g, 0.005 g/g, 0.01 g/g) were considered important factors for upgrading the nutritional value of fruit peels (particularly protein content) and were studied with a Box-Behnken experimental design. These experiments were carried out with Aspergillus ibericus (initial concentration of 5×107 spores/g), for 7 incubation days. The highest protein content of fermented orange peels (16.23 %) was obtained with 70 % moisture content, 0.005 g/g ammonium sulfate, without CSL. For banana peels, the highest content of protein (17.49 %) was obtained with 70 % moisture content, 0.005 g/g CSL, without ammonium sulfate. Finally, the fermented fruit peels were characterized for carbohydrates, reducing sugars, crude fiber, pectin, total lipids, minerals, and antioxidant activity. Carbohydrates, reducing sugars, and pectin content of fruit peels decreased after SSF, while total protein, fiber, lipids, minerals, and antioxidant activities increased, enhancing the nutritional value of orange and banana peels. Key words: Banana peels; Filamentous fungi; Microbial protein; Orange peels; Solid-state fermentation.
vii TABLE OF CONTENTS DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS ........................ ii AGRADECIMENTOS ............................................................................................................................ iii STATEMENT OF INTEGRITY ................................................................................................................ iv RESUMO ............................................................................................................................................. v ABSTRACT ......................................................................................................................................... vi LIST OF FIGURES ............................................................................................................................... ix LIST OF TABLES .................................................................................................................................. x LIST OF NOMENCLATURES AND ABREVIATIONS ............................................................................... xii CHAPTER 1. STATE OF THE ART ........................................................................................................ 1 1.1. Current challenges and efforts for food production ............................................................... 1 1.2. Microbial protein as a potential protein source ..................................................................... 4 1.3. Microbial protein produced by fungi (mycoprotein) – high quality protein .............................. 7 1.4. Factors affecting microbial protein production .................................................................... 10 1.5. Microbial species: filamentous fungi ...................................................................................... 14 1.6. Fruit peels – an agro-industrial by-product with a valorization potential ................................... 15 1.7. Objectives ............................................................................................................................. 17 CHAPTER 2. MATERIALS AND METHODS ......................................................................................... 17 2.1. Raw material: fruit peels ........................................................................................................ 17 2.2. Microorganisms .................................................................................................................... 18 2.3. Preparation of spore suspension ............................................................................................ 18 2.4. Effect of medium composition and operational conditions on nutritional value of fruit peels..... 19 2.4.1. Plackett-Burman design ...................................................................................................... 19 2.4.2. Box-Behnken design ........................................................................................................... 21 2.5. Sample processing for analysis .............................................................................................. 22 2.6. Analytical methods ................................................................................................................ 23
2 for 70 % of total groundwater withdrawal, and groundwater accounts for 30 % of total water used for irrigation, increasing 2.2 % per year. This leads to an exhaustive exploitation of aquifers. Estimates indicate that 250 km3 of aquifer capacity is lost every year (FAO, 2022). In addition to water scarcity problems, agriculture contributes to climate change, as soils emit carbon dioxide, nitrous oxide and methane due to deforestation, use of fertilizers, and flooding of rice fields (Bailey et al. , 2014; UNICEF, 2020; FAO, 2022). In 2019, the agricultural sector alone emitted 31 % of global carbon dioxide equivalent (CO2-eq) emissions, corresponding to a 16 % increase in CO2-eq emissions since 1990 (FAO, 2022). The sustainable production of food to feed the growing population is limited due to the constrains of agriculture. The expansion of agricultural activities potential is reduced since everyday agriculture-grade land is lost to urbanization. In 2018, 55 % of people lived in urbanized areas, in which 80 % of total world food is consumed, and, by 2050, the population percentage might increase to 67 % (United Nations, 2018; FAO, 2022). Additionally, urbanization is associated with intensive agriculture and livestock exploitation due to the rising demand for fast food, particularly meat products. Meat consumption has increased almost 60 % over the last two decades and continues to increase every year at a 2 % rate. Near 80 % of agricultural land is used for livestock production, of which a third is destined for feed crop cultivation (FAO, 2022). Livestock productivity increased between 2000 and 2019 due to less use of land for this industry (a decrease of 0.2 billion ha), while techniques like zero-grazing allowed higher meat production yields. This productivity intensification, however, negatively impacts the environment due to overgrazing and poor livestock management, resulting in soil erosion and compaction and overuse of local water resources and their contamination with nutrients from animal waste and antibiotics. The production of meat requires large quantities of feed crops and water, having a higher impact on the environment than agriculture. For example, 15 415 L, 8 763 L, 5 988 L, and 4 325 L of water are needed to produce, respectively, 1 kg of bovine, sheep or goat, pig, and chicken meat. By contrast, 9 063 L, 1 644 L, 962 L, 387 L, and 322 L of water are needed to produce 1 kg of nuts, cereals, fruits, starchy roots and vegetables, respectively (Mekonnen and Hoekstra, 2012, 2010). A reduction in the consumption of meat, not only saves water, but also reduces land use and greenhouse gas emissions (GHG) (UNICEF, 2020). Due to their lower environmental footprint, high protein content and other health benefits (Thrane et al. , 2017), soy products are among the plant-based foods that are gaining popularity as an alternative protein source. Almost 14 % of the USA citizens avoid meat in their diet (Statista, 2022). The USA led soybean production between 2012 and 2017, but Brazil surpassed it in 2018. In 2022-2023, Brazil harvested
3 153 mega metric tons of soybeans, and the USA harvested 116.4 mega metric tons (Statista, 2023a). While these two American countries are the biggest producers of soy, Asian countries are the biggest consumers of soy products, with a total consumer sales market share of 72.2 % in 2020. China and Japan are the largest consumers of soy products, excluding natto, miso, and soy sauce (USSEC, 2021). The global market for plant-based foods had estimated worth of 44.2 billion U.S. dollars in 2022, and in 2025 it is estimated to be 77.8 billion U.S. dollars, and more than double of that in 2030 (Statista, 2023b). However, the soy and other plants production sectors are not currently capable of answering to the exponential rising demand of alternative protein-rich products to feed the future generations (Ahmad et al. , 2022). Besides, the high phytoestrogen content, allergenicity in some populations, low essential amino acids (EAAs) content, low bioavailability, and negative environmental and socioeconomic impacts are discouraging soy product consumption (Jargin, 2014; Taylor et al. , 2021; Mulalapele and Xi, 2021; Vallikkadan et al. , 2023). Moreover, the environmental and socioeconomic negative impacts also contribute to the reduction of soy market (da Silva et al. , 2021; Dreoni et al. , 2022; Gollnow et al. , 2018; Lopes et al. , 2021). Another common alternative to meat products is fish. Fish accounts for 10 % of protein consumption and less than 5 % of global food production. The most common means for fish production are inland fisheries and inland aquaculture, which, together, account for 75 % of total fish production. Inland fisheries and inland aquaculture rely on local freshwater resources, such as rivers or lakes, and currently more than 40 % of inland fish comes from poor food-scarce countries. However, these systems are vulnerable to human and climate change-induced changes on local freshwater bodies, such as reduced water availability due to competition with agriculture for irrigation water and damns for water storage, pollution, and eutrophication accompanied by fish mortality (FAO, 2022; Funge-Smith, 2018). From 1984 to 2015, 90 000 km2 of water bodies were lost and another 70 000 km2 transitioned from permanent water bodies to seasonal water bodies (Pekel et al. , 2016), thus reducing growth potential of inland fish capture/production. New protein sources are being explored, such as insect protein, cultured meat (created in vitro with animal cell cultures or from genetically modified animals), and microbial protein produced by microorganisms (Hashempour-Baltork et al. , 2020; Ahmad et al. , 2022; Vallikkadan et al. , 2023; Mateti et al. , 2022). Insect protein is a viable protein source due to its easily scalable production, high bioavailability, and high content of protein, essential amino acids, vitamins, minerals, and unsaturated fatty acids. There have been identified around 2000 edible insect species so far (Vallikkadan et al. , 2023).
4 Generally, protein content in insects ranges from 50 % to 82 %, containing high amounts of threonine and lysine (often not present in plants), and low amounts of methionine and cysteine, which can be supplemented with plant protein (Vallikkadan et al. , 2022; Agbidye et al. , 2009). Additionally, Fe, Na, Zn, P, Ca, Mg, Cu, Mn, pantothenic acid, riboflavin, and biotin can also be found at high concentrations in insect protein (Vallikkadan et al. , 2023). In contrast to meat and plant-based protein, insect protein has low requirements for water, land, and feed, however, its main downside is the low acceptability of the market in developed countries, due to a preconception that insect protein is unclean (Lee et al. , 2023). Insect protein is already consumed in African and Asian countries, but allergic reactions to insect protein, such as urticaria, angioedema, dyspnea, nausea, vomiting, and diarrhea have been reported (Vallikkadan et al. , 2023). Cultured meat refers to meat or fish analogs produced using cell engineering technology, where tissue cells from animals are collected and proliferated in vitro in order to culture animal tissues (Lee at al., 2023; Zhang et al. , 2020; Stephens et al. , 2018). This emerged as an alternative to obtain animal flesh products without requiring farming or slaughter, while also responding to the food and economic challenges that the population faces due to agriculture, livestock, and fishing’s constraints (Zhang et al. , 2020; Stephens et al. , 2018). In the cultured meat production process, animal stem cells are often used, specifically muscle satellite cells, a common type of adult stem cells, due to their capacity to differentiate into muscle and fat cells, commonly found in animal flesh, but already existing tissue can also be used. Already existing techniques are able to produce and structure cultured meat in a way to potentially confer it pleasant organoleptic properties, similar to those of natural meat, such as tissue engineering, the use of scaffolds ( e.g . chitosan, gelatin), microcarriers and hydrocolloids, freeze structuring, 3D printing, electrospinning, shear cell, biophotonics, nanotechnology, thermo-extrusion and cloning/genetically modifying animals (Vallikkadan et al. , 2023; Zhang et al. , 2020: Mateti et al. , 2022). However, the main disadvantages of this technology are that it is still expensive and stem cell maintenance is hard, which consequently limits its scale-up (Lee at al., 2023). 1.2. Microbial protein as a potential protein source Microbial protein is defined as a protein produced by microbial species such as bacteria, yeast, fungi, or microalgae (Upadhyaya et al. , 2016; Ahmad et al. , 2022). Microbial protein can be used as food additives or in industrial processes ( e.g. , as foam stabilizers and in leather and paper processing) (Junaid et al. ,
5 2020) but its most common application is for food and feed consumption. Microbial protein has the potential to substitute, partially or entirely, animal protein (Vallikkadan et al. , 2023). Microbial protein is commonly denominated single-cell protein (SCP) when it is obtained in the isolated microorganism form. Although it gained popularity after mycoprotein Quorn™ products entry into the market in 1985 (Ahmad et al. , 2022; Hashempour-Baltork et al. , 2020), microbial protein production goes way back in time, with the cultivation of microalgae Spirulina for food by African people in Lake Chad and the Aztecs in Mexico. Microbial protein produced by Saccharomyces cerevisiae , Candida utilis , Aspergillus oryzae, and Rhizopus arrhizus was used as a protein-rich food during the World Wars. Since 1960, some energy companies have been studying the production of microbial protein by yeasts from paraffin wax (Chama, 2019). Microbial protein has several advantages compared to other protein sources, including fast growth of microbial species (doubling time: microalgae: 2 h – 6 h; yeast: 1 h – 3 h; bacteria: 0.3 h – 2 h), high protein content, the possibility of using several by-products from agriculture and food processing activities, which are usually cheap and abundant, thus contributing to a circular economy, its production independency from climate conditions ( i.e. , production is viable in all seasons) and the fact that it requires less land and water comparatively to agriculture and livestock production (Chama, 2019; Hashempour-Baltork et al. , 2020; Bratosin et al. , 2021). Bacterial protein has a high protein concentration in the cellular wall and is more commonly used for feed rather than for human food. Its protein content ranges from 50 % to 80 %, however it is necessary to treat and refine it before its consumption (Junaid et al. , 2020; Raziq et al. , 2020; Pereira et al. , 2022). The main disadvantages of microbial protein produced from bacteria for human consumption are the low amount of sulfur-containing amino acids, the high nucleic acid content, and unattractive organoleptic properties without previous processing (Chama, 2019; Raziq et al. , 2020). The presence of nucleic acids in food for human consumption can have a negative impact on health, such as high levels of uric acid in the blood, which in turn can lead to kidney stones (Raziq et al. , 2020; Ritala et al. , 2017). Common bacterial species used for microbial protein production are Methylobacterium extorquens, Methylococcus capsulatus , Rhodobacter sphaeroides , Afifella marina, and Corynebacterium ammoniagenes (Pereira et al. , 2022). Imperial Chemical Industries used to produce a feed for pigs named Pruteen, composed of 70 % of bacterial protein but was discontinued due to the lack of competitivity in the market (Ahmad et al. , 2022). Another protein-producing group is microalgae, which are very popular in the vegetarian/vegan communities. Microalgae have been used for animal feed and human consumption for decades as protein
6 and vitamin supplements. Some popular microalgae belong to the genera Chlorella and Spirulina , but other commercially important microalgae are Dunaliella salina , Aphanizomenon flos-aquae , Euglena , Tetraselmis suecica , and Isochrysis galbana (Junaid et al. , 2020; Pereira et al. , 2022). An important aspect of microalgae is that they contain high concentrations of protein (40 % to 70 %) (Harun et al. , 2010; Draaisma et al. , 2013, Junaid et al. , 2020; Pereira et al. , 2022), and low content of nucleic acids (Ritala et al. , 2017). Other advantages of microalgae include the consumption of carbon dioxide for their metabolism, the production of metabolites under both fermentation and photosynthesis, and the production of omega-3 fatty acids (Ritala et al. , 2017; Pereira et al. , 2022). However, the main disadvantages of microalgae protein are the economically limited scale-up of production plants and the need to break the microalgae cell wall to increase digestibility (Pereira et al. , 2022). Filamentous fungi and yeasts are considered the most important microorganisms for protein production. Yeasts are the most studied microorganism group and the most accepted among consumers. In addition to S. cerevisiae and C. utilis , Yarrowia lipolytica is a non-pathogenic and food-grade yeast commonly studied to produce microbial protein (Sotiris et al. , 2020; Lopes et al. , 2022; Carranza-Méndez et al. , 2022). The main advantages of using yeasts include the high protein content (38 % - 65 %), the nutritional quality, the easy recovery, the excretion activity, the lower nucleic acid content compared with bacterial species, the high lysine and malic acid content, and the low risk for spoilage due to their capability to grow in acidic media (Sotiris et al. , 2020; Raziq et al. , 2020; Pereira et al. , 2022). Filamentous fungi have several advantages compared to yeasts and other microbial species regarding protein production. Firstly, fungal protein (also known as mycoprotein) has a lower content of nucleic acids and higher digestibility than bacteria or yeasts. Filamentous fungi can also penetrate solid substrates, which allows the fermentation of solid food-grade by-products. Protein from filamentous fungi can easily be transformed into foods with diverse pleasant textures, and its aroma and taste are similar to those of mushrooms, thereafter, having high acceptability by consumers (Chama, 2019). It is estimated that the market of mycoprotein will grow 20.3 % between 2022 and 2031 (PR Newswire, 2023), reaching more than 460 million EUR in 2027 (Derbyshire & Delange, 2021). Examples of filamentous fungi species used for mycoprotein production are Fusarium venenatum , Aspergillus niger , Aspergillus oryzae , Rhizopus oligosporus , Trichoderma reesei , and Neurospora intermedia . The protein content of filamentous fungi could reach up to 50 % (Pereira et al. , 2022; Chama, 2019). Table 1.1 summarizes the protein content and characteristics of microbial protein produced by microalgae, fungi, yeasts and bacteria.
7 Table 1.1. Average protein content and characteristics of protein produced by microbial species. Adapted from Matassa et al. (2016) Microorganism Protein content Characteristics References Microalgae 40 % - 70 % Similar to eggs, soy, and wheat proteins. Cell wall digestibility is an issue. Contains omega-3 fatty acids. Harun et al. (2010), Draaisma et al . (2013), Pereira et al. (2022) Filamentous fungi and yeasts 0.23 % to 50 % (filamentous fungi) 38 % - 65 % (yeasts) Amino acids profile and digestibility of mycoprotein are similar to those of eggs and milk. Low fat. High unsaturated/saturated fatty acids ratio. Raziq et al. (2020), Chama (2019), Pereira et al. (2022) Bacteria 50 % - 80 % Amino acid’s profile and digestibility are similar to those of fishmeal. Junaid et al. (2020), Raziq et al. (2020), Pereira et al. (2022) Microbial protein is a potential nutritious alternative to animal-based protein. The target markets of microbial protein include vegetarian/vegan populations and Muslims, Jews, and Buddhists, which have religious restrictions regarding the meat consumption of some animal species. Microbial protein not only mitigates the excessive use of antibiotics, pesticides, fertilizers, water, and land used for animal and vegetable protein production but is also a solution to food shortages (Hashempour-Baltork et al. , 2020; Ahmad et al. , 2022). 1.3. Microbial protein produced by fungi (mycoprotein) – high quality protein The protein produced by food-grade fungi (mycoprotein) is classified as Halal by the Halal Food Authority and as GRAS (Generally Recognized as Safe) by the U.S. Food and Drug Administration and is approved
8 for human consumption by the Ministry of Agriculture, Fisheries, and Food of the UK since 1984 (Hashempour-Baltork et al. , 2020; FDA, 2002; Thavamani et al. , 2020). The production of microbial protein by filamentous fungi requires 20 times less water and 23 times less land than animal protein, thus having a carbon footprint 4 to 10 times lower than meat products (Hashempour-Baltork et al. , 2020; Thavamani et al. , 2020). The commercial brands of mycoprotein products use a set of processes of fermentation, denaturation, separation, freezing, and mixing with food additives in order to produce mycoprotein products, such as chunks, sausages, burgers, nuggets, and minced “meat”, with pleasant textures and flavors similar to animal meat (Vallikkadan et al. , 2023; Thavamani et al. , 2020; Wiebe, 2002). Although there were some reports of sensitivity and intolerance to mycoprotein in humans, the data relative to these reactions is still scarce, and intolerance seems to be statistically lower than soy and egg intolerance (Finnigan et al. , 2017; Vallikkadan et al. , 2023). Mycoprotein contains all essential amino acids (EAAs) and in higher content than vegetable protein, is rich in fiber, specially β-glucans and chitin poly-N-acetyl glucosamines, and poor in fats (predominantly constituted by polyunsaturated fatty acids such as linoleic and linolenic acids). Mycoprotein is also a source of minerals, such as copper, zinc, selenium, manganese, phosphorous, iron, and sodium, and vitamins, such as vitamins B2 and D, which makes mycoprotein a perfect option for consumers who are concerned with their health (Ahmad et al. , 2022; Hashempour-Baltork et al. , 2020; Thavamani et al. , 2023; Vallikkadan et al. , 2023). However, the content of iron, vitamin B12, and sodium is lower in mycoprotein than in beef and it does not contain vitamins A, C, and E (Ahmad et al. , 2022). It was demonstrated that mycoprotein reduces blood cholesterol levels and energy intake, promotes healthy muscle growth, and increases satiety and blood sugar control (Cherta-Murillo et al. , 2020; Coelho et al. , 2021; Monteyne et al. , 2020). Mycoprotein is safe for children and babies, however, due to its high fiber content and low energetic value, they are not recommended for children under 3 years (Denny et al. , 2008). Table 1.2 summarizes the nutritional value of mycoprotein compared with other protein sources. The content of essential amino acids and nucleic acids are important quality factors in microbial protein. Generally, mycoprotein has high amount of lysine and threonine but low content of cysteine and methionine (though methionine levels still meet international recommendations) (Upadhyaya et al. , 2016; Ritala et al. , 2017). The amino acids profile of mycoprotein depends not only on the microorganism but also on the substrate used for the fermentation and on the processing techniques (Upadhyaya et al. , 2016). The protein quality of mycoprotein can also be verified by its high PDCAAS (Protein Digestibility
9 Corrected Amino Acid Score). Table 1.3 summarizes the PDCAAS values of mycoprotein and of other protein sources. Table 1.2. Nutritional value of mycoprotein, animal protein, and soy protein. Adapted from Ahmad et al. (2022) Food (100 g) Mycoprotein Milk Eggs Beef Chicken Soybean Energy (kcal) 85 46 151 172 106 141 Protein (g) 11.25 3.40 12.56 20.20 24.00 14.00 EAAs (g) 4.59 1.53 5.55 7.92 9.11 8.50 Carbohydrates (g) 3.0 4.7 Trace 0.06 Trace 5.10 Total fat (g) 2.90 1.70 9.51 4.30 1.10 7.30 Saturated fat (g) 0.60 1.10 3.13 1.70 0.30 0.90 Fiber (g) 6.0 Trace Trace Trace Trace 6.10 Sodium (mg) 5.00 43.00 142.00 43.00 60.00 1.00 Iron (mg) 0.50 0.02 1.75 3.50 0.40 3.00 Zinc (mg) 9.00 0.40 1.29 0.40 0.70 0.90 Vitamin B12 (mg) Trace 0.40 0.89 0.40 Trace Trace Selenium (mg) 20.00 1.00 11.00 1.00 12.00 5.00 Table 1.3. Protein Digestibility-Corrected Amino Acid Scores (PDCAAS) of some traditional sources of protein and microbial protein sources Protein source PDCAAS (%) Reference Egg 100 Hoffman and Falvo (2004) Milk/Casein 94 - 100 Yamada and Sgarbieri (2005) Hoffman and Falvo (2004) Whey protein 100 Hoffman and Falvo (2004) Beef 92 Hoffman and Falvo (2004) Soy 100 Hoffman and Falvo (2004) Mycoprotein 99.6 Edwards and Cummings (2010) Baker’s yeast 62 - 84 Yamada and Sgarbieri (2005) Other important factor for mycoprotein quality is its concentration in nucleic acids, specifically RNA. Microbial biomass generally contains high concentrations of RNA, depending on the microorganism species, which can be detrimental to health for humans and animals. While the safe limit for human
10 consumption of RNA in food is a concentration of 2 %, this nucleic acid is usually found at concentrations between 7 % to 10 % in dry basis in mycoprotein, thereafter it must be removed or inactivated prior to consumption (Nasseri et al. , 2011). The methods for RNA removal or inactivation include chemical, enzymatic, and physical treatments. Chemical treatments, such as nucleic acid removal by extraction with alcohol, salt, acids, and alkalis, are not the most suitable for animal and human consumption applications, since most solvents are not food-grade and toxic compounds may be formed, especially if high temperatures are also applied ( e.g. , lysinoalanine) (Nasseri et al. , 2011). Enzymatic treatment is another possible method for nucleic acids inactivation, for example with ribonucleases. Optimal conditions for the degradation of ribonucleic acids by ribonucleases are also optimal for protein degradation by endogenous proteases. Therefore, using exogenous ribonucleases or immobilized enzymes is preferable to not reduce protein yield in mycoprotein. Disadvantages of enzymatic treatments are: 1) the process is slow, and 2) is costly (Nasseri et al. , 2011). Physical treatments, such as high temperature, are the most used methods for the reduction of nucleic acid content. In mycoprotein production, the fungal biomass is generally heated to 72 °C – 74 °C for 30 min – 45 min (Ahmad et al. , 2022). In this case, the disadvantages are: (1) loss of protein and (2) loss of nutritional value (Nasseri et al. , 2011; Wiebe, 2002; Souza Filho et al. , 2018). 1.4. Factors affecting microbial protein production Generally, microbial species require carbon and nitrogen sources for cellular growth and metabolite production. Substrates for fermentation can either be pure components, for example as in Quorn™’s method of production (where glucose is used directly as a substrate, along with ammonium salts and biotin), or they can be by-products of the food processing industry, agricultural and forest cleaning activities, or the oil refining industry ( e.g., simple sugars, starches, cellulose, hydrocarbons). In mycoprotein production for human consumption, it is a requirement that the substrate used is food-grade. Thus, by-products like fruit peels, rice bran and straw, corn cobs, wheat straw, vegetable pulps, and fruit pomace are examples of appropriate and cheap substrates for mycoprotein production for food application (Junaid et al. , 2020; Barzee et al. , 2021; Bratosin et al. , 2021). Depending on the substrate and microorganism, three types of fermentation can be carried out to produce mycoprotein: submerged (SmF), semi-solid and solid-state (SSF). Filamentous fungi are known for their ability to use solid by-products as substrates for SSF. This has several advantages in substrate acquisition
11 (abundant and cheap) and preparation (fewer pre-treatments are needed) and energy costs (Barzee et al. , 2021). With solid-state fermentation, it is possible to achieve high yields, and the substrate can simultaneously be a physical support and a nutrient source for fungi. Using low-cost food by-products reduces the global costs of the process and is more environmentally friendly. The seasonality and distance to production centers should be considered in the industrial production of mycoprotein from food by-products (Hashempour-Baltork et al. , 2020; Koukoumaki et al. , 2023). The operational conditions that may affect mycoprotein production by SSF are nutrient availability, aeration, the particle size of the substrate, temperature, pH, moisture content, water activity, and inoculum’s size, age, and physiological state (Junaid et al. , 2020; López-Gómez et al. , 2020). SSF ideally must have an aeration system, as oxygen is an important factor for aerobic species. Forced humid air and the mixture of the solid matrix are commonly applied to overcome the diffusion limitations between the solid particles and the air and to prevent moisture gradients and heterogenicity (López-Gómez et al. , 2020). The particle size of the substrate also plays a role in oxygen diffusion. Fungi easily penetrate substrates with small particle size, however, the oxygen diffusion coefficient is also smaller due to smaller interparticle space. A compromise between substrate penetration and oxygen diffusion has to be evaluated (López-Gómez et al. , 2020). Solid-state fermentation with filamentous fungi enables to produce high quality mycoprotein or a proteinenriched food from GRAS by-products. For SSF, substrate is collected, homogenized and pre-treatments are applied, and nutritional supplements are added, if needed. After sterilization of the medium and equipment, the medium is inoculated with the fungal species. During SSF, aeration of the medium or other growth factors are regulated. For food application purposes, a reduction of nucleic acid concentration step is necessary. Table 1.4 summarizes some operational conditions used for mycoprotein production by filamentous fungi. Schmidt and Furlong (2012) studied the effect of particle size of rice bran and supplementation with ammonium sulfate on protein enrichment of rice bran and phenolic compounds production by Rhizopus oryzae under semi-solid-state fermentation. The authors found that an increase in particle size decreased protein production, whereas biomass production was improved. This factor was significative for protein, biomass, and phenolic compounds production. Additionally, the authors concluded that ammonium sulfate supplementation was significative for biomass and phenolic compounds production but not for protein production. They reported an increase of 53 % in total protein content and 56 % of total phenolic
18 Figure 1.1. Preparation of (A) orange and (B) banana peels before storage at – 20 °C. 2.2. Microorganisms Aspergillus ibericus MUM 01.294 and Rhizopus oryzae MUM 10.260, stored in cryostocks at – 80 °C, were obtained from the culture collection of Micoteca da Universidade do Minho (MUM - Braga, Portugal). The fungal strains were grown in potato dextrose agar (PDA) petri dishes (4 g/L potato extract, 20 g/L dextrose, 15 g/L agar) at 25 °C for 5 days and stored at 4 °C for a maximum of 2 weeks. 2.3. Preparation of spore suspension Spore suspensions of A. ibericus MUM 01.294 or Rhizopus oryzae MUM 10.260 were prepared by adding a sterile peptone solution (1 g/L peptone, 0.1 g/L Tween 80) to 5-day-grown agar plates. After appropriate dilution, 1×107 spores/g solid or 5×107 spores/g solid solutions were used to inoculate the fruit peels medium.
19 2.4. Effect of medium composition and operational conditions on nutritional value of fruit peels The dried orange (OP) and banana (BP) peels were weighted (5 g of dry basis) to 250-mL Erlenmeyer flasks and the nitrogen supplements (ammonium sulfate and corn steep liquor, CSL) were added, according to the experimental assay matrixes (Chapters 2.4.1 and 2.4.2). The flasks containing the medium for SSF were sterilized at 121 °C for 15 min. A spore suspension prepared according to Chapter 2.3 was added to each flask, according to the experimental assay matrixes. The initial moisture of the OP and BP was adjusted with sterilized distilled water. The fermentations were carried at 25 °C in a ventilated oven without agitation for 7 days or 14 days (Figure 1.2). Figure 1.2. Visible growth of filamentous fungi in orange and banana peels. (A) Rhizopus oryzae growing in banana peels. (B) Aspergillus ibericus growing in orange peels. 2.4.1. Plackett-Burman design The Plackett-Burman design (Plackett and Burman, 1946) is a statistical experimental design derived from the full factorial design that was created in order to study the main effects of several independent variables (n), called factors, in a dependent variable, called response, generally at 2 levels (L=2), with the least possible number of experiments, when the complete factorial number of experiments (Ln) is realistically unfeasible. For this design, a Hadamard matrix is created, where “1” and “-1” represent the 2 quantitative levels studied for each factor. This experimental design is commonly used for variable screening, since it assumes that factor interaction is negligible, thus only considering main effects. The
20 Plackett-Burman design adjusts the experimental data to a linear model, that establishes a relationship between response variables (Y) and factors (xi) and their main effect coefficients (ai): Y = A + ∑𝑎𝑖•𝑥𝑖𝑖 The main effect of 6 SSF variables (time, inoculum size, moisture, ammonium sulfate concentration, CSL concentration and fungus species) on protein production, reducing sugars, total phenols, and antioxidant activity responses in fermented OP and BP (Batch 1) were studied at 2 levels, in order to evaluate the potential to upgrade fruit peels’ nutritional value. Table 2.1 depicts the matrix of factors and levels studied with Plackett-Burman design in SSF of OP and BP, by filamentous fungi. Table 2.1. Plackett-Burman design matrix used to study the main effects of fermentation time, inoculum size, moisture content, ammonium sulfate (AS) concentration, corn steep liquor (CSL) concentration and fungal species, at 2 levels, on protein production, reducing sugars, total phenols, and antioxidant activity responses in fermented orange and banana peels Run/Level Time Inoculum size Moisture AS concentration CSL concentration Fungus species 1 -1 1 1 1 -1 1 2 -1 1 1 -1 1 -1 3 1 -1 1 -1 -1 -1 4 1 -1 1 1 -1 1 5 1 1 1 -1 1 1 6 -1 -1 -1 -1 -1 -1 7 -1 -1 1 1 1 -1 8 1 -1 -1 -1 1 1 9 1 1 -1 1 -1 -1 10 -1 -1 -1 1 1 1 11 1 1 -1 1 1 -1 12 -1 1 -1 -1 -1 1 -1 7 days 1×107 spores/g 60 % 0 g/g 0 g/g Aspergillus ibericus 1 14 days 5×107 spores/g 75 % 0.01 g/g 0.01 g/g Rhizopus oryzae
21 2.4.2. Box-Behnken design The Box-Behnken design (Box and Behnken, 1960) is a statistical experimental design which generates response surfaces that requires 3 equally spaced levels and 3 central points. This experimental design is used for optimization of quantitative response variables. The Box-Behnken design has the advantage of needing fewer experimental assays (runs) with 3 to 4 factors, relatively to other optimization experimental designs ( e.g ., Central Composite Circumscribed design and Central Composite Inscribed design). The Box-Behnken design adjusts experimental data to a quadratic model, that establishes a relationship between response variables (Y), factors (xi), main effect coefficients (ai), and interaction effects coefficients (aij): Y= A + ∑𝑎𝑖•𝑥𝑖𝑖 + ∑𝑎𝑖𝑖 •𝑥𝑖 2 𝑖 + ∑𝑎𝑖𝑗 •𝑥𝑖• 𝑥𝑗𝑖𝑗 After variable screening with the Plackett-Burman design, a Box-Behnken design was employed to maximize the production of microbial protein from OP and BP (Batch 2). Table 2.2 depicts the matrix of factors and levels studied with Box-Behnken design for the optimization of total protein from OP and BP. Table 2.2. Box-Behnken design matrix used to study the effect of different levels of moisture content, ammonium sulfate (AS) concentration, and corn steep liquor (CSL) concentration on total protein production from orange and banana peels Run/Level Moisture AS concentration CSL concentration 1 -1 -1 0 2 1 -1 0 3 -1 1 0 4 1 1 0 5 -1 0 -1 6 1 0 -1 7 -1 0 1 8 1 0 1 9 0 -1 -1 10 0 1 -1
22 Table 2.2. Box-Behnken design matrix used to study the effect of different levels of moisture content, ammonium sulfate (AS) concentration, and corn steep liquor (CSL) concentration on total protein production from orange and banana peels ( continued ) Run/Level Moisture AS concentration CSL concentration 11 0 -1 1 12 0 1 1 13 0 0 0 14 0 0 0 15 0 0 0 -1 50 % 0 g/g 0 g/g 0 60 % 0.005 g/g 0.005 g/g 1 70 % 0.01 g/g 0.01 g/g The fermentations were carried at 25 °C in a ventilated oven without agitation for 7 days. Each Erlenmeyer flask was inoculated with 1×107 spores/mL of A. ibericus . A control experiment without inoculation was also performed. 2.5. Sample processing for analysis After solid-state fermentation, the final solid (fruit peels and grown fungi) was mixed to reduce its heterogeneity and it was separated into 2 fractions: 2 g - 3 g of solid were saved for final moisture and total protein analysis and its remainder was utilized for a room temperature distilled water extraction (1:10 w/v, 200 rpm, 30 min) for analysis of soluble compounds (reducing sugars and total phenols) and antioxidant activity. In the final experiments (verification of conditions predicted by Box-Behnken design), fermented solid was also characterized for carbohydrates, crude fiber, pectin, and lipids, in addition to moisture and total protein.
23 2.6. Analytical methods 2.6.1. Moisture Moisture content of non-fermented and fermented fruit peels was measured according to a modified standard method from AOAC (2023). Briefly, aluminum cups were identified and placed in the drying oven at 105 °C for a minimum of 6 hours or until constant weight. Once cooled in a desiccator, the aluminum cups were weighed empty and 0.5 g to 1.0 g of sample were weighed into the aluminum cups, with an analytical balance. The samples were dried at 105 °C until constant weight. After reaching room temperature in the desiccator, the cups’ final weigh was registered. Moisture content was determined by: Moisture (%)= (Mass of cup and fresh sample)+(Mass of cup and dried sample) (Mass of cup and fresh sample)+(Mass of empty cup)× 100 2.6.2. Total protein The total protein content was quantified by the Kjeldahl method (Kjeldahl, 1883). The nitrogen content was converted into crude protein using a factor of 6.25. Briefly, 0.5 g of sample, 10 mL of concentrated sulfuric acid and a selenium tablet were added into each digestion tube. After digestion and cooling (1 h + 15 min, 420 °C) in the digestor (FOSS Kjeldahl Digestion Systems, Thermo Fisher Scientific, USA), the tubes were transferred into a distiller (FOSS Kjeltec 8400 system, Thermo Fisher Scientific, USA), where the samples were neutralized. 2.6.3. Reducing sugars Reducing sugars content was measured by the dinitro-salicylic (DNS) acid reagent (Miller, 1959), using glucose as standard. Briefly, 100 μL of extracts (or distilled water for the blank) and 100 μL of DNS reagent were added to the test tubes. The tubes were boiled in a water bath at 100 °C, for 5 min, placed
24 immediately in a cold-water bath, and 1 mL of distilled water was added. After vortex-mixing, the absorbance was read at 540 nm (Multiskan Sky, Thermo Fisher Scientific, USA), in 96-well plates. 2.6.4. Carbohydrates Carbohydrates were determined after acidic hydrolysis and the quantification of the resulting total sugar monomers by the DNS method. 0.1 g to 0.2 g of sample were weighed to hydrolysis tubes, and 10 mL of H2SO4 1.5 M were added. The tubes were boiled in a water bath for 20 min and 12 mL of NaOH 10 % were added to the tubes, once cooled. The mixture was vortex-mixed and filtered into a 100-mL volumetric flask and the remaining volume was made up with distilled water. For the quantification of sugar monomers, the DNS method protocol mentioned in Chapter 2.6.3. was employed. The carbohydrates content was determined by: Carbohydrates (%)= Concentration of reducing sugars Mass of sample used for hydrolysis × 0.1 L × 100 2.6.5. Crude fiber The crude fiber was quantified according to the AOAC Official Method 962.09, with some modifications. Briefly, 2 glass microfiber filters were calcined at 500 °C for 4 h in a muffle furnace and allowed to cool in a glass desiccator. 2 g of sample and 150 mL of H2SO4 0.13 M were added into a 250 mL reflux flask. The mixture was boiled for 30 min, vacuum filtered and washed, along with the reflux flask, with 90 mL of boiling water. The filter was then placed inside the reflux flask, and 150 mL of KNO 0.23 M were added. The mixture was boiled again for 30 min and repeated the washing step with boiling water. The mixture and the reflux flask were washed with 75 mL of acetone. Afterwards, the 2 filters were dried at 105 °C for 4 h in a drying oven and weighed after cooling in the desiccator. The filters were calcined for 30 min, at 500 °C, in the muffle furnace. Finally, the 2 filters were weighed after cooling in the desiccator. The crude fiber content corresponds to the fraction of residue left in the sample: Crude Fiber (%)= Mass of 2 filters after drying − Mass of 2 filters after calcination Mass of sample × 100
25 2.6.6. Pectin Pectin was quantified according to the method described by Rodsamran & Sothornvit (2018) with some modifications. Briefly, 5 g of sample powder was weighed into a bottle Schott, and 100 mL of HCL 0.05 M were added. The bottle Schott was placed in an agitated water bath at 95 °C for 1 h. The content was filtered through a nylon net and vacuum filtered, and the solid residues went through a second extraction, following the same previous steps. The solution volume was measured and absolute ethanol (1:2, v/v) was added. The bottle Schotts were agitated for 10 min in an incubator (100 rpm, room temperature) and left to rest for 2 h to precipitate the pectin. The pectin was vacuum filtered and washed with absolute ethanol (3 times) and acetone (1 time). The pectin was dried overnight at 40 °C and it was finally weighed. The pectin content was determined by: Pectin (%) = Mass of pectin Mass of sample × 100 2.6.7. Total lipids The total lipids content was determined by the Soxhlet method, with petroleum ether as extraction solvent, in a Soxtec 8000 system. Firstly, the Soxhlet aluminum cups were dried at 105 °C for 2 h with 5 glass spheres inside. Once cooled in the desiccator, the Soxhlet cups were weighed. 1 g of sample was weighed into the cellulose thimbles, which were covered with cotton. 50 mL of petroleum ether were added to the Soxhlet cups and these, along with the cellulose thimbles were inserted and adapted into the Soxtec system, and the extraction occurred overnight (70 °C). In the morning, the cups were removed and placed in the drying oven at 105 °C for 15 min to evaporate the residual petroleum ether. The weight of the Soxhlet cups was registered, after cooling in the desiccator. The total lipids content of the sample was determined as followed: Total lipids (%) = Mass of Soxhlet cups after extraction − Mass of Soxhlet cups before extraction Mass of sample × 100
26 2.6.8. Ashes Ashes content was determined according to the standard methods from AOAC (2023). Firstly, porcelain crucibles were calcined at 550 °C, for 4 h, cooled in the desiccator and weighed. 0.5 g to 1 g of sample were weighed into the crucibles, which were heated at 550 °C for 4 h, cooled in the desiccator, and weighed. The ashes content of the fruit peels was determined as: Ashes (%) = Mass of crucible and ash − Mass of crucible Mass of sample × 100 2.6.9. Free carbon and nitrogen in aqueous extracts Free carbon and nitrogen concentrations in fruit peels were determined in aqueous fruit peel extracts (Chapter 2.5). For free carbon, a Total Organic Carbon LCK387 (300 mg/L – 3000 mg/L, Hach-Lange GmbH, Berlin, Germany) kit was used and, for free nitrogen, a Total Nitrogen LCK 138 (1 mg/L – 16 mg/L, Hach-Lange GmbH, Berlin, Germany) kit was used. 2.6.10. Total phenols Total phenols in fruit peels extracts were quantified by the Folin-Ciocalteau method (Commission Regulation (EEC) No. 2676/90), using gallic acid as standard. Briefly, 20 μL of sample (or distilled water for blank), 400 μL of Na2CO3 15 %, 100 μL of Folin-Ciocalteau reagent, and 1480 μL of distilled water were added to a tube, in triplicate for each sample. The assay tubes were placed in a 50 °C water bath for 5 min and then cooled with water and vortex-mixed for 30 s. The absorbance was read at 740 nm (Multiskan Sky, Thermo Fisher Scientific, USA) and converted to total phenols concentration using the calibration curve.
27 2.6.11. Antioxidant activity Antioxidant activity was measured in fruit peels aqueous extracts by the modified DPPH (2,2-diphenyl-1picrylhydrazyl) antioxidant assay method (Blois, 1958). Briefly, 200 μL of diluted sample extracts (trolox solution for calibration curve) and 100 μL of a methanolic solution of DPPH 0.5 mM were added into microplate wells. The mixture was incubated for 30 min in the dark at room temperature. The absorbance was measured at 517 nm (Multiskan Sky, Thermo Fisher Scientific, USA). Two controls were also carried out: (1) 200 μL of diluted sample extracts and 100 μL of methanol (individual control); and (2) 200 μL of methanol and 100 μL of DPPH solution (microplate control). Antioxidant activity was expressed as micromoles of trolox per g of sample and obtained through the trolox concentration vs free radical scavenging activity calibration curve. The absorbance was converted to scavenging activity with the following equation: Scavenging activity (%) = (1 − 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑖𝑡ℎ 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑏𝑙𝑎𝑛𝑘 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑏𝑙𝑎𝑛𝑘 ) × 100 2.6.12. Minerals Mineral composition of fruit peels was analyzed by Inductively coupled plasma optical emission spectrometry (ICP-OES). Fruit peels were previously digested using a Berghof microwave digestion system (Speedwave four DAP-60+, Berghof Products + Instruments GmbH, Germany). Briefly, 0.25 g of dried peels or 0.5 g of humid sample were weighed into the digestion vessels, and 6 mL of HNO3 69 % and 1 mL of H2O2 30 % were added. The vessels were slowly shaken and, after 10 min, closed. The digestion program was chosen, based on the equipment user manual chapter “Microwave Digestion of Fruits”. Firstly, samples were subjected to temperature and pressure values of 170 °C and 30 bar, respectively, for 10 min, and then of 200 °C and 30 bar, for 15 min. The final step of digestion consisted of applying temperature and pressure of 50 °C and 25 bar, for 10 min. The digested liquid samples were filtrated with 0.22 μm pore syringe filters and analyzed in the ICP-OES equipment (Optima 8000, PerkinElmer, Waltham, MA, USA). The operating conditions were as follows: radio frequency power 1400 W; 12 L/min argon plasma flow; 0.2 L/min auxiliary gas flow; 0.75 L/min nebulizer gas flow. The plasma view was axial for all analyzed elements. The wavelengths (nm) used for
34 Table 3.5. Experimental results of total protein, soluble sugars, total phenols, and antioxidant activity responses obtained in Plackett-Burman variable screening analysis after fermentation of orange peels. Data are represented in w/w (dry basis) Run Total protein (%) Reducing sugars (%) Total phenols (mg/g) Antioxidant activity (μmol/g) 1 6.30 15.54 10.08 15.56 2 10.50 2.71 8.21 15.08 3 10.07 22.34 7.19 14.02 4 6.60 32.92 10.46 13.60 5 6.02 10.34 9.71 13.43 6 7.92 15.68 8.71 13.52 7 13.33 36.44 7.06 14.00 8 5.64 20.07 8.27 14.55 9 10.73 37.34 5.85 16.12 10 7.07 9.02 10.17 15.92 11 12.28 29.97 6.12 13.81 12 5.44 21.92 10.80 14.53 Table 3.6. Experimental results of total protein, soluble sugars, total phenols, and antioxidant activity responses obtained in Plackett-Burman variable screening analysis after fermentation of banana peels. Data are represented in w/w (dry basis) Run Total protein (%) Reducing sugars (%) Total phenols (mg/g) Antioxidant activity (μmol/g) 1 9.21 8.44 2.28 9.36 2 9.01 5.31 2.13 9.37 3 9.44 3.60 1.92 10.14 4 11.01 2.22 1.63 7.36 5 8.39 2.66 1.78 7.33 6 7.95 14.99 2.11 6.30 7 9.39 2.44 2.18 8.23 8 8.24 11.00 1.76 8.07 9 9.05 12.81 1.59 5.22 10 9.04 13.39 2.10 12.89 11 8.45 11.67 1.99 12.93 12 7.74 12.26 1.84 14.66
35 Tables 3.7 and 3.8 show coefficients of regression and correlation coefficients (R2) of Plackett-Burman design for orange and banana peels, respectively. Significant coefficients were identified by asterisk. Regression coefficients allow the determination of the effect of each constituent. A high value of regression coefficient indicates that the factor has a large impact on dependent variable. A R2 close to 100 indicates a good correlation between the predicted response value and the actual response value. However, in the range of values tested in this work, it was not possible to predict the entire variability of the results, particularly for protein content (orange and banana peels) and total phenols (banana peels). Furthermore, due to the inability of the Plackett-Burman test to account for interactions between factors, the results may miss some factors where significant interactions occurred. In the experiments carried out with orange peels, only time was found as a significant factor for the antioxidant activity (Table 3.7). In the SSF with banana peels, inoculum size had a significant impact on protein and reducing sugars content. Moisture and CSL had a significant effect on antioxidant activity (Table 3.8). Table 3.7. Regression coefficients and parameters for the linear model obtained by Plackett-Burman analysis for each response (total protein, reducing sugars, total phenols, and antioxidant activity) in orange peels solid-state fermentation Linear coefficients Total Protein Reducing sugars Total phenols Antioxidant activity Average 8.49167 21.1917 855.25 14.5 Time -0.546667 8.17 -142.833 -1.06667* Inoculum size 0.88 -4.45667 27.1667 -0.333333 Moisture -1.11333 -12.5467 128.5 0.433333 AS -0.493333 8.67667 13.1667 0.9 CSL -1.72 -2.74 -13.8333 0.433333 Fungus species -1.99333 -2.04 161.5 0.466667 R2 (%) 34.056 71.9304 59.1577 78.7789 Adjusted R2 (%) 0 38.247 10.147 53.3135
36 Table 3.8. Regression coefficients and parameters for the linear model obtained by Plackett-Burman analysis for each response (total protein, reducing sugars, total phenols, and antioxidant activity) in banana peels solid-state fermentation Linear coefficients Total Protein Reducing sugars Total phenols Antioxidant activity Average 8.91 8.39917 194.25 9.325 Time -0.08 -2.715 -15.8333 0.883333 Inoculum size 0.94* -6.925* 2.83333 -0.216667 Moisture -0.54 1.11167 15.5 3.55* AS 0.66 0.388333 -1.16667 0.616667 CSL 0.483333 -2.65167 -16.1667 -3.05* Fungus species -0.52 1.775 -22.1667 0.783333 R2 (%) 76.8629 77.3715 67.9501 77.5342 Adjusted R2 (%) 49.0984 50.2173 29.4901 50.5752 The main effects of each studied factor on total protein, reducing sugars, total phenols, and antioxidant activity after SSF of orange peels are depicted in Figure 3.1. A negative effect of time was observed for protein, total phenols, and antioxidant activity, while a positive effect was observed for reducing sugars. This indicates that, as time increases from 7 days to 14 days, reducing sugars concentration increases. By contrast, total protein, total phenols, and antioxidant activity were favored by a 7-day fermentation. In fact, the highest amount of protein and total phenols were obtained in runs 7 and 12, respectively, which were carried out for 7 days (Tables 2.1 and 3.5). The highest concentration of reducing sugars was obtained in a 14-day SSF (run 9). A positive effect of inoculum size was observed for protein and total phenols, while a negative effect was observed for reducing sugars and antioxidant activity. This indicates that, as the inoculum size increases from 1×107 spores/g to 5×107 spores/g, reducing sugars and antioxidant activity reduce. In fact, the highest concentration of total phenols was obtained in run 12, which was inoculated with 5×107 spores/g (Tables 2.1 and 3.5). By contrast, reducing sugars and antioxidant activity were favored by a SSF inoculated with 1×107 spores/g (Figure 3.1). A negative effect of moisture was observed for total protein and reducing sugars, while a positive effect was observed for total phenols and antioxidant activity (Figure 3.1). This indicates that, as moisture increases from 60 % to 75 %, total phenols and antioxidant activity increase.
37 Figure 3.1. Estimated effects of several parameters (fermentation time, inoculum size, humidity of peels, ammonium sulfate (AS) and corn steep liquor (CSL) supplementation, and fungal species) on (A) total protein, (B) reducing sugars, (C) total phenols, and (D) antioxidant activity, obtained for solid-state fermentation of orange peels. A positive effect of ammonium sulfate was observed for reducing sugars, total phenols, and antioxidant activity, while a negative effect was observed for total protein (Figure 3.1). This indicates that the supplementation of orange peels with ammonium sulfate did not increase protein content. By contrast, the supplementation of orange peels with 0.01 g/g ammonium sulfate during SSF, favored the content of reducing sugars, total phenols, and antioxidant activity. The highest antioxidant activity and reducing
38 sugars concentration were obtained in run 9, which was supplemented with 0.01 g/g ammonium sulfate (Tables 2.1 and 3.5). A negative effect of corn steep liquor (CSL) was observed for protein, reducing sugars, and total phenols, while a positive effect was observed for antioxidant activity (Figure 3.1). This indicates that the supplementation of orange peels with 0.01 g/g CSL favored the antioxidant activity of orange peels after SSF. The level (+1) of fungal species had a positive effect on total phenols and antioxidant activity, while the level (-1) favored total protein and reducing sugars (Figure 3.1). This indicates that SSF of orange peels with A. ibericus favored protein and reducing sugars. In fact, the highest protein content and the second highest reducing sugars concentration were obtained in the experiments inoculated with A. ibericus (run 7). By contrast, SSF of orange peels with R. oryzae favored total phenols and antioxidant activity. The highest content of total phenols was obtained in the experiments inoculated with R. oryzae (run 12) (Tables 2.1 and 3.5). The main effects of each studied factor on total protein, reducing sugars, total phenols, and antioxidant activity after SSF of banana peels are depicted in Figure 3.2. A negative effect of time was observed for protein, reducing sugars, and total phenols, while a positive effect was observed for antioxidant activity. This indicates that, as time increases from 7 days to 14 days, antioxidant activity increases. By contrast, total protein, reducing sugars, and total phenols were favored by a 7-day fermentation. In fact, the highest amount of reducing sugars and total phenols of fermented banana peels were obtained in runs 6 and 1, respectively, which were carried out for 7 days (Tables 2.1 and 3.6). A positive effect of inoculum size was observed for protein and total phenols, while a negative effect was observed for reducing sugars and antioxidant activity (Figure 3.2). This indicates that, as the inoculum size increases from 1×107 spores/g to 5×107 spores/g, reducing sugars and antioxidant activity reduce. In fact, the highest concentration of total phenols was obtained in run 1, which was inoculated with 5×107 spores/g. The highest concentration of reducing sugars was obtained in run 6, which was inoculated with 1×107 spores/g (Tables 2.1 and 3.6). A negative effect of moisture was observed for protein content, and a positive effect for all the other responses (Figure 3.2). This indicates that, as moisture increase from 60 % to 75 %, reducing sugars, total phenols, and antioxidant activity increase. In contrast, an increase moisture content did not increase
39 protein production. The highest total phenols content in fermented banana peels was obtained in run 1, which was carried out with 75 % of moisture (Tables 2.1 and 3.6). Figure 3.2. Estimated effects of several parameters (fermentation time, inoculum size, humidity of peels, ammonium sulfate and corn steep liquor supplementation and fungal species) on (A) total protein, (B) soluble sugars, (C) total phenols, and (D) antioxidant activity, obtained for solid-state fermentation of banana peels. A positive effect of ammonium sulfate was observed for total protein, reducing sugars, and antioxidant activity, while a negative effect was observed for total phenols (Figure 3.2). This indicates that the
40 supplementation of banana peels with ammonium sulfate did not increase total phenols content. By contrast, the supplementation of banana peels with 0.01 g/g ammonium sulfate during SSF, favored the content of total protein, reducing sugars, and antioxidant activity. The highest protein content was obtained in run 4, which was supplemented with 0.01 g/g ammonium sulfate (Tables 2.1 and 3.6). A negative effect of corn steep liquor (CSL) was observed for reducing sugars, total phenols, and antioxidant activity, while a positive effect was observed for total protein (Figure 3.2). This indicates that the supplementation of banana peels with 0.01 g/g CSL favored the protein content of banana peels after SSF. In fact, the highest content of reducing sugars, total phenols and antioxidant activity were obtained in runs 6, 1 and 12, respectively, which were not supplemented with CSL (Tables 2.1 and 3.6). The level (+1) of fungal species had a positive effect on reducing sugars and antioxidant activity, while the level (-1) favored total protein and total phenols (Figure 3.2). This indicates that SSF of banana peels with A. ibericus favored protein and total phenols. By contrast, SSF of banana peels with R. oryzae favored reducing sugars and antioxidant activity. In fact, the highest concentration of antioxidant activity was obtained in run 12, which was inoculated with R. oryzae (Tables 2.1 and 3.6). Schmidt and Furlong (2012) concluded that supplementation with ammonium sulfate was not a significative factor for protein production from rice bran under semi-SSF with Rhizopus oryzae , but it was significative for total phenols increase. Particle size was also significative for total protein and total phenols increase. Olorunnisola et al. (2018), however, reported a positive effect on protein production by ammonia supplementation (with ammonium phosphate), in sequential SSF of banana peels with Phanerochaete chrysosporium and Candida utilis . Kamal et al (2019) reported that fermentation time was a significative factor, with positive effect (in the range of 1 day – 5 days), for protein production from banana peels with Aspergillus niger , under SmF. 3.2.2. Variable optimization with Box-Behnken design Since the results of regression coefficients obtained in the Plackett-Burman design were not significant, and also considering information reported in the literature, moisture content, and ammonium sulfate and corn steep liquor concentration were considered as three substantial factors for further SSF experiments. A Box-Behnken experimental design was used to optimize the level of each factor in order to attain a maximum content of total protein.
41 Table 3.9 summarizes the total protein content obtained in fermented orange peels (OP) and fermented banana peels (BP) in the Box-Behnken experimental matrix. The total protein content varied from 7.32 % (run 5) to 16.29 % (run 6) in fermented OP and from 8.35 % (run 14) to 17.67 % (run 2) in fermented BP. In general, the total protein content increased in fruit peels after SSF, relatively to unfermented peels. In SSF of orange peels, the lowest protein content was obtained in the run 5, which had as conditions 50 % moisture content, 0.005 g/g ammonium sulfate and no CSL. By contrast, the highest protein content was achieved in run 6, which had as conditions 70 % moisture content, 0.005 g/g ammonium sulfate and no CSL. The second highest protein content (13.49 %) was obtained in run 8, which had the same level of moisture and ammonium sulfate of run 6, but 0.01 g/g CSL. These results suggest that high level of moisture content favored protein production in SSF of orange peels, while CSL supplementation may had a negative effect in protein production. This was confirmed in Table 3.10, with the estimated factor coefficients by the quadratic model obtained by response surface methodology. Moisture content and the quadratic term of CSL were considered significant factors, with positive effect, for protein production (p<0.05). The individual effect of CSL, even though not significant (p > 0.05), was negative for protein production in SSF from orange peels. Table 3.9. Experimental results of total protein content in fermented orange peels (OP) and in fermented banana peels (BP), obtained for Box-Behnken optimization analysis Run Total protein (%) in fermented OP Total protein (%) in fermented BP 1 7.89 10.67 2 9.78 17.67 3 8.18 11.92 4 12.74 10.75 5 7.32 10.39 6 16.29 11.63 7 10.41 10.19 8 13.49 10.96 9 13.31 10.65 10 11.96 13.65 11 12.53 9.94 12 11.78 12.92 13 10.32 8.72
42 Table 3.9. Experimental results of total protein content in fermented orange peels (OP) and in fermented banana peels (BP), obtained for Box-Behnken optimization analysis ( continued ) Run Total protein (%) in fermented OP Total protein (%) in fermented BP 14 10.64 8.35 15 10.68 9.09 Table 3.10. ANOVA results of the quadratic models obtained for protein production under solid-state fermentation (SSF) of orange and banana peels using a Box-Behnken experimental design. Significant factors are identified with an asterisk SSF of orange peels SSF of banana peels Factor Coefficient estimate Standard error p -value Coefficient estimate Standard error p -value Intercept 10.55 0.7198 - 8.72 1.13 - A (Moisture) 2.31 0.4408 0.0033* 0.9800 0.6911 0.2154 B (SA) 0.1437 0.4408 0.7575 0.0387 0.6911 0.9575 C (CSL) -0.0838 0.4408 0.8568 -0.2888 0.6911 0.6934 AB 0.6675 0.6233 0.3332 -2.04 0.9773 0.0909 AC -1.47 0.6233 0.0646 -0.1175 0.9773 0.9090 BC 0.1500 0.6233 0.8194 -0.0050 0.9773 0.9961 A² -0.7083 0.6488 0.3247 1.52 1.02 0.1960 B² -0.1908 0.6488 0.7805 2.52 1.02 0.0564 C² 2.04 0.6488 0.0256* 0.5550 1.02 0.6088 R2 90.26 % 74.30 % Adjusted R2 72.72 % 28.05 % Model p -value 0.0430* 0.3128 Residual p -value 0.0150* 0.0214* In SSF of banana peels, the lowest protein content was obtained in the run 14, which was a central point for the experimental design and had as conditions 60 % moisture content, 0.005 g/g ammonium sulfate and 0.005 g/g CSL. By contrast, the highest protein content was attained in run 2, which had as
43 conditions 70 % moisture content, no ammonium sulfate and 0.005 g/g CSL. The second highest total protein content obtained in fermented banana peels (13.65 %, run 10) was performed with 60 % moisture content, 0.01 g/g ammonium sulfate and no CSL. The total protein content results for fermented banana peels could not be adjusted with a significant model, since many results with different operational conditions resulted in very similar total protein values (around 10 % total protein), specifically runs 1, 4, 5, 7, 8, 9, and 11 (Table 3.9). Consequently, it was not possible to determine the significancy of any factors or interactions between them (Table 3.10), in the range of operational conditions studied. The quadratic models for the relation among protein production and moisture content (%), supplementation with ammonium sulfate (g/g), and supplementation with CSL (g/g), from SSF in orange (OP) and banana (BP) peels are the following equations: Total Protein (%) in fermented OP = - 31.72 + 1.16175×Moisture − 725.91667×SA + 904.58333×CSL + 13.35×Moisture×SA − 29.45×Moisture×CSL + 6000×SA×CSL − 0.007083×Moisture² − 7633.33333× SA² + 81566.66667×CSL² Total Protein (%) in fermented BP = 47.82500 − 1.50700×Moisture + 1453.75000×SA − 137.75×CSL − 40.85000×Moisture×SA − 2.35×Moisture×CSL − 200×SA×CSL + 0.015175×Moisture² + 100600× SA² + 22200×CSL² These models predicted a maximum total protein of 16.22 % (with 0.992 desirability) in fermented orange peels at the conditions 70 % moisture, 0.01 g/g ammonium sulfate and without CSL. For banana peels, the quadratic model predicted a maximum total protein of 16.69 % (with 0.895 desirability) at the conditions 70 % moisture content, and without ammonium sulfate and CSL (Figure 3.3). Additionally, the 95 % confidence level intervals for these total protein content points were wide (12.43 % - 20.00 %, OP; 10.76 % - 22.63 %, BP). The 3D surface plots for total protein content in fermented fruit peels are depicted in Figure 3.4. It can be observed that neither for orange peels nor for banana peels the plots are completely concave down surfaces. In fact, for SSF of orange peels, the plot for ammonium sulfate vs moisture (Fig. 3.4A1) is a concave down surface but has no maximum point; the plot for CSL vs moisture (Fig. 3.4A2) is a curved surface with inflexion after which the concave surface becomes a convex surface; and the plot for CSL vs ammonium sulfate (Fig. 3.4A3) is a concave up (convex) surface. For SSF of banana peels, all plots of moisture, ammonium sulfate and CSL are convex surfaces. This means that, although the quadratic
50 REFERENCES Abdel-Azeem, A.M., Yadav, A.N., Yadav, N., Usmani, Z. (Eds.). (2021). Industrially Important Fungi for Sustainable Development . Volume 1. Fungal Biology, Springer Cham. Abdullahi, N., Dandago, M.A., Yunusa, A.K. (2021). Review on Production of Single-Cell Protein from Food Wastes. Turkish Journal of Agriculture - Food Science and Technology 9, 968-974. Abera, W.G., Kasirajan, R., Majamo, S.L. (2023). Synthesis and characterization of bioplastic film from banana (Musa Cavendish species) peel starch blending with banana pseudo-stem cellulosic fiber. Biomass Conversion and Biorefinery. Aboul-Enein, A.M., Salama, Z.A., Gaafar, A.A., Aly, H.F., About-Elella, F., Ahmed, H.A. (2016). Identification of phenolic compounds from banana peel (Musa paradaisica L.) as antioxidant and antimicrobial agents . Journal of Chemical and Pharmaceutical Research, 8 (4), 46-55. Abrunhosa, L., Oliveira, F., Dantas, D., Gonçalves, C., Belo, I. (2013). Lipase production by Aspergillus ibericus using olive mill wastewater. Bioprocess and Biosystems Engineering, 36 (3), 285-291. Adoki, A. (2008). Factors affecting yeast growth and protein yield production from orange, plantain and banana wastes processing residues using Candida sp. African Journal of Biotechnology, 7 (3), 290-295. Agbidye, F.S., Ofuya, T.I., Akindele, S.O. (2009). Marketability and nutritional qualities of some edible forest insects in Benue State, Nigeria. Pakistan Journal of Nutrition 8, 917-922. Agourram, A., Ghirardello, D., Rantsiou, K., Zeppa, G., Belviso, S., Romane, A., Oufdou, K., Giordano, M. (2013). Phenolic Content, Antioxidant Potential, and Antimicrobial Activities of Fruit and Vegetable By-Product Extracts . International Journal of Food Properties, 16 (5), 1092-1104. Ahmad, M.I., Farooq, S., Alhamoud, Y., Li, C., Zhang, H. (2022). A review on mycoprotein: History, nutritional composition, production methods, and health benefits . Trends in Food Science & Technology 121, 14-29. Anhwange, B., Ugye, J.T., Nyiatagher, T.D. (2009). Chemical Composition of Musa sapientum (Banana) Peels . Electronic Journal of Environmental, Agricultural and Food Chemistry, 8 (6), 437-4442. AOAC, Dr. Latimer, George W, Jr. (ed.) (2023). Official Methods of Analysis of AOAC INTERNATIONAL, 22nd online edition. New York, Oxford Academic. Bailey, R., Froggatt, A., Wellesley, L. (2014). Livestock - Climate Change’s Forgotten Sector. Global Public Opinion on Meat and Dairy Consumption . Energy, Environment and Resources. The Royal Institute of International Affairs, Chatham House. Bakir, U., Yavascaoglub, S., Guvencb, F., Ersayinb, A. (2001). An endo-beta-1,4-xylanase from Rhizopus oryzae: production, partial purification and biochemical characterization . Enzyme and Microbial Technology. Bampidis, V.A., Robinson, P.H. (2006). Citrus by-products as ruminant feeds: A review . Animal Feed Science and Technology 128, (3-4), 175-217. Banerjee, R., Mukherjee, G., Patra, K.C. (2005). Microbial transformation of tannin-rich substrate to gallic acid through co-culture method. Bioresource Technology 96, 949-953.
51 Barzee, T.J., Cao, L., Pan, Z., Zhang, R. (2021). Fungi for future foods . Journal of Future Foods, 1 (1), 25-37. Battaglia, E., Benoit, I., van den Brink, J., Wiebenga, A., Coutinho, P.M., Henrissat, B., de Vries, R.P. (2011). Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level . BMC Genomics 12, 38. Bhanja Dey, T., Kuhad, R.C. (2014). Upgrading the antioxidant potential of cereals by their fungal fermentation under solid-state cultivation conditions . Letters in Applied Microbiology 59, 493-499. Bishnoi, S., Sharma, S., Agrawal, H. (2023). Exploration of the Potential Application of Banana Peel for Its Effective Valorization: A Review. Indian Journal of Microbiology. Biz, A., Finkler, A.T.J., Pitol, L.O., Medina, B.S., Krieger, N., Mitchell, D.A. (2016). Production of pectinases by solid-state fermentation of a mixture of citrus waste and sugarcane bagasse in a pilot-scale packed-bed bioreactor . Biochemical Engineering Journal 111, 54-62. Blois, M.S. (1958). Antioxidant determinations by the use of a stable free radical . Nature 181, 1199-1200. Box, G.E.P., Behnken, D.W. (1960). Some new three level designs for the study of quantitative variables . Technometrics 2, 455-475. Bratosin, B.C., Darjan, S., Vodnar, D.C. (2021). Single Cell Protein: A Potential Substitute in Human and Animal Nutrition. Sustainability 13, 9284. Bulut, S., Elibol, M., Ozer, D. (2004). Effect of different carbon sources on L(+)-lactic acid production by Rhizopus oryzae . Biochemical Engineering Journal 21, 33-37. Cai, S., Gao, F., Zhang, X., Wang, O., Wu, W., Zhu, S., Zhang, D., Zhou, F., Ji, B. (2012). Evaluation of γaminobutyric acid, phytate and antioxidant activity of tempeh-like fermented oats (Avena sativa L.) prepared with different filamentous fungi . Journal of Food Science and Technology 51, 2544-2551. Carranza-Méndez, R.C., Chavez-González, M.L., Sepúlveda-Torre, L., Aguilar, C.N., Govea-Salas, M., Ramos-Gonzalez, R. (2022). Production of single cell protein from orange peel residues by Candida utilis. Biocatalysis and Agricultural Biotechnology 40, 102298. Chama, N.T. (2019). Production of SCP from different substrates . Australian Journal of Science and Technology 3, 4. Chau, C.-F., Huang, Y.-L. (2003). Comparison of the Chemical Composition and Physicochemical Properties of Different Fibers Prepared from the Peel of Citrus sinensis L. Cv. Liucheng . Journal of Agricultural and Food Chemistry, 51 (9), 2615–2618. Cherta-Murillo, A., Lett, A.M., Frampton, J., Chambers, E.S., Finnigan, T.J.A., Frost, G.S. (2020). Effects of mycoprotein on glycaemic control and energy intake in humans: A systematic review . British Journal of Nutrition, 123 (12), 1321-1332. Coelho, M.O.C., Monteyne, A.J., Dirks, M.L., Finnigan, T.J.A., Stephens, F.B., Wall, B.T. (2021). Daily mycoprotein consumption for 1 week does not affect insulin sensitivity or glycaemic control but modulates the plasma lipidome in healthy adults: A randomised controlled trial . British Journal of Nutrition, 125 (2), 147-160. Commission Regulation (EEC) No 2676/90 of 17 September 1990 determining Community methods for the analysis of wines.
52 Czech, A., Zarycka, E., Yanovych, D., Zasadna, Z., Grzegorczyk, I., Kłys, S. (2020). Mineral content of the pulp and peel of various citrus fruit cultivars. Biological Trace Element Research 193, 555-563. da Silva, R.F.B., Viña, A., Moran, E.F., Dou, Y., Batistella, M., Liu, J. (2021). Socioeconomic and environmental effects of soybean production in metacoupled systems . Scientific Reports 11, 18662. Denny, A., Aisbitt, B., Lunn, J. (2008). Mycoprotein and health . Nutrition Bulletin 33, 298-310. Derbyshire, E.J., Delange, J. (2021). Fungal protein - what is it and what is the health evidence? A systematic review focusing on mycoprotein . Frontiers in Sustainable Food Systems 5, 581682. Draaisma, R.B., Wijffels, R.H., Slegers, P.M.E., Brentner, L.B., Roy, A., Barbosa, M.J. (2013). Food commodities from microalgae. Current Opinion in Biotechnology 24, 169-177. Dreoni, I., Matthews, Z., Schaafsma, M. (2022). The impacts of soy production on multi-dimensional well-being and ecosystem services: A systematic review . Journal of Cleaner Production 335, 130182. Dulf, F.V., Vodnar, D.C., Socaciu, C. (2016). Effects of solid-state fermentation with two filamentous fungi on the total phenolic contents, flavonoids, antioxidant activities and lipid fractions of plum fruit (Prunus domestica L.) by-products. Food chemistry 209, 27-36. Edwards, D., Cummings, J. (2010). The protein quality of mycoprotein . Proceedings of the Nutrition Society, 69 (OCE4), E331. Erukainure, O.L., Ebuehi, O.A.T., Choudhary, M.I., Mesaik, M.A., Shukralla, A., Muhammad, A., Zaruwa, M.Z., Elemo, G.N. (2016). Orange Peel Extracts: Chemical Characterization, Antioxidant, Antioxidative Burst, and Phytotoxic Activities . Journal of Dietary Supplements, 13 (5), 585-594. Essien, J.P., Akpan, E.J., Essien, E.P. (2005). Studies on mould growth and biomass production using waste banana peel . Bioresource Technology, 96 (13), 1451-1456. FAO (2019). Food Outlook - Biannual Report on Global Food Markets – November 2019 . Rome. FAO (2021). Citrus Fruit Statistical Compendium 2020 . Rome. FAO (2022). The State of the World’s Land and Water Resources for Food and Agriculture – Systems at breaking point . Main report. Rome. FAO (2023a). Ukraine: Impact of the war on agricultural enterprises – Findings of a nationwide survey of agricultural enterprises with land up to 250 hectares, January–February 2023. Rome. FAO (2023b). Banana Market Review 2022 . Rome. FDA, 2002. Marlow Foods. GRAS Notification for Mycoprotein (submitted as: US FDA. Marlow Foods Ltd, Stokesley (UK). GRN 091). Finnigan, T., Needham, L., Abbott, C. (2017). Mycoprotein: A Healthy New Protein with a Low Environmental Impact . Nadathur, S.R., Wanasundara, J.P.D., Scanlin, L. (Eds.). Chapter 19 in Sustainable Protein Sources . Academic Press. Funge-Smith, S.J. (2018). Review of the state of world fishery resources: Inland fisheries. FAO Fisheries and Aquaculture Circular No. 942, Rev. 3. Rome. Ghosh, B., Ray, R.R. (2011). Extra-cellular isoamylase production by Rhizopus oryzae in solid-state fermentation of agro wastes. Brazillian Archives of Biology and Technology 54, 867-876.
53 Gmoser, R., Fristedt, R., Larsson, K., Undeland, I., Taherzadeh M.J., Lennartsson, P. R. (2020). From stale bread and brewers spent grain to a new food source using edible filamentous fungi . Bioengineered, 11 (1), 582-598. Gollnow, F., Hissa, L.B.V., Rufin, P., Lakes, T. (2018). Property-level direct and indirect deforestation for soybean production in the Amazon region of Mato Grosso, Brazil . Land Use Policy 78, 377-385. Harun, R., Singh, M., Forde, G.M., Danquah, M.K. (2010). Bioprocess engineering of microalgae to produce a variety of consumer products. Renewable and Sustainable Energy Reviews 14, 1037-1047. Hashempour-Baltork, F., Khosravi-Darani, K., Hosseini, H., Farshi, P., Reihani, S. (2020). Mycoproteins as safe meat substitutes. Journal of Cleaner Production 253. Hoekstra, A.Y. (2014). Water for animal products: a blind spot in water policy . Environmental Research Letters, 9 (9), 091003. Hoffman, J.R., Falvo, M.J. (2004). Protein - Which is Best? Journal of Sports Science and Medicine, 3 (3),118-130. Ibarruri, J., Cebrián, M., Hernández, I. (2021b). Valorisation of fruit and vegetable discards by fungal submerged and solid-state fermentation for alternative feed ingredients production . Journal of environmental management 281, 111901. Ibarruri, J., Goiri, I., Cebrián, M., García-Rodríguez, A. (2021a). Solid State Fermentation as a Tool to Stabilize and Improve Nutritive Value of Fruit and Vegetable Discards: Effect on Nutritional Composition, In Vitro Ruminal Fermentation and Organic Matter Digestibility . Animals, 11 (6), 1653. Jacobo-Velázquez, D.A., Cisneros-Zevallos, L. (2009). Correlations of Antioxidant Activity against Phenolic Content Revisited: A New Approach in Data Analysis for Food and Medicinal Plants . Journal of Food Science 74, 107-113. Jargin, S. V. (2014). Soy and phytoestrogens: possible side effects . German medical science: GMS e-journal, 12, Doc18. Junaid, F., Khawaja, L.A., Ali, S. (2020). Single-cell protein as a potential meat substitute: a critical review . World Journal of Pharmaceutical Research, 9 (2), 141-161. Kamal, M.M., Ali, M.R., Shishir, M.R.I., Saifullah, M., Haque, M.R., Mondal, S.C. (2019). Optimization of process parameters for improved production of biomass protein from Aspergillus niger using banana peel as a substrate . Food Science and Biotechnology 28, 1693-1702. Kjeldahl, J. (1883). Neue Methode zur Bestimmung des Stickstoffs in organischen Körpern (New method for the determination of nitrogen in organic substances) . Zeitschrift für analytische Chemie, 22 (1), 366-383. Koukoumaki, D.I., Tsouko, E., Papanikolaou, S., Ioannou, Z., Diamantopoulou, P., Sarris, D. (2023). Recent advances in the production of single cell protein from renewable resources and applications . Carbon Resources Conversion, Pre-Journal Proof. Kupski, L., de Carvalho Silvello, M.A., Fontes, M.R.V., Lima, T.S., Treichel, H., Badiale, Furlong, E. (2015). R. oryzae cellulases: a new approach to degrading lignocellulosic material . Journal of Food Biochemistry 39, 129-138.
54 Lakner, C., Yonzan, N., Mahler, D.G, Aguilar, R.A.C., Wu, H. 2021. Updated estimates of the impact of COVID-19 on global poverty: Looking back at 2020 and the outlook for 2021 . In World Bank . Washington, DC. Lee, S.Y., Lee, D.Y., Jeong, J.W., Kim, J.H., Yun, S.H., Joo, S., Choi, I., Choi, J.S., Kim, G., Hur S.J. (2023). Studies on Meat Alternatives with a Focus on Structuring Technologies . Food and Bioprocess Technology 16, 1389-1412. Liao, W., Liu, Y., Frear, C., Chen, S. (2007). A new approach of pellet formation of a filamentous fungus - Rhizopus oryzae . Bioresourse Technology 98, 3415-3423. Lo Curto, R., Tripodo, M.M., Leuzzi, U., Giuffrè, D., Vaccarino, C. (1992). Flavonoids recovery and SCP production from orange peel . Bioresource Technology, 42 (2), 83-87. Londoño-Hernández, L., Ramírez-Toro, C., Ruiz, H. A., Ascacio-Valdés, J.A., Aguilar-Gonzalez, M.A., Rodríguez-Herrera, R., Aguilar, C. N. (2017). Rhizopus oryzae - Ancient microbial resource with importance in modern food industry . International journal of food microbiology 257, 110-127. Lopes, G.R., Lima, M.G.B., dos Reis, T.N.P. (2021). Maldevelopment revisited: Inclusiveness and social impacts of soy expansion over Brazil’s Cerrado in Matopiba. World Development 139, 105316. Lopes, M., Miranda, S.M., Costa, A.R., Pereira, A.S, Belo I. (2022). Yarrowia lipolytica as a biorefinery platform for effluents and solid wastes valorization - challenges and opportunities . Critical Reviews in Biotechnology, 42 (2), 163-183. López-Gómez, J.P., Manan, M.A., Webb, C. (2020). Solid-state fermentation of food industry wastes . Food Industry Wastes, 135-161. Lv, X.C., Cai, Q.Q., Ke, X.X., Chen, F., Rao, P.F., Ni, L. (2015). Characterization of fungal community and dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine by means of multiple cultureindependent methods. Food Control 54, 231-239. Lv, X.C., Weng, X., Zhang, W., Rao, P.F., Ni, L. (2012). Microbial diversity of traditional fermentation starters for Hong Qu glutinous rice wine as determined by PCR-mediated DGGE . Food Control 28, 426-434. Maas, R.H.W., Bakker, R.R., Eggink, G., Weusthuis, R.A. (2006). Lactic acid production from xylose by the fungus Rhizopus oryzae . Applied Microbiology and Biotechnology 72, 861-868. Mahesh, M., Arivizhivendhan, K.V., Maharaja, P., Boopathy, R., Hamsavathani, V., Sekaran, G. (2016). Production, purification and immobilization of pectinase from Aspergillus ibericus onto functionalized nanoporous activated carbon (FNAC) and its application on treatment of pectin containing wastewater . Journal of Molecular Catalysis B: Enzymatic 133, 43-54. Mahmoud, M.H., Abou-Arab, A.A., Abu-Salem, F.M. (2015). Effect of some different drying methods on the chemical analysis of citrus by-products . Research Journal of Pharmaceutical, Biological and Chemical Sciences 6, 105-116. Matassa, S., Boon, N., Pikaar, I., Verstraete, W. (2016). Microbial protein: future sustainable food supply route with low environmental footprint . Microbial Biotechnology 9, 568-575. Mateti, T., Laha, A., Shenoy, P. (2022). Artificial Meat Industry: Production Methodology, Challenges, and Future . JOM (The Journal of The Minerals, Metals & Materials Society) 74, 3428-3444.
55 Mekonnen, M.M., Hoekstra, A.Y. (2010). The green, blue and grey water footprint of farm animals and animal products. Value of Water Research Report Series No. 48, UNESCO-IHE, Delft, the Netherlands. Mekonnen, M.M., Hoekstra, A.Y. (2012). A global assessment of the water footprint of farm animal products . Ecosystems, 15(3), 401-415. Mertens, J.A., Burdick, R.C., Rooney, A.P. (2008). Identification, biochemical characterization, and evolution of the Rhizopus oryzae 99–880 polygalacturonase gene family. Fungal Genetics and Biology, 45 (12), 1616-1624. Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar . Analytical Chemistry, 31 (3), 426-428. Monteyne, A.J., Coelho, M.O., Porter, C., Abdelrahman, D.R., Jameson, T.S., Jackman, S.R., Blackwell, J.R., Finnigan, T.J.A., Stephens, F.B., Dirks, M.L., Wall, B.T. (2020). Mycoprotein ingestion stimulates protein synthesis rates to a greater extent than milk protein in rested and exercised skeletal muscle of healthy young men: A randomized controlled trial. The American Journal of Clinical Nutrition, 112 2, 318-333. Mulalapele, L.T., Xi, J. (2021). Detection and inactivation of allergens in soybeans: A brief review of recent research advances . Grain & Oil Science and Technology, 4 (4), 191-200. Nasseri, A., Rasoul-Amini, S., Morowvat, M., Ghasemi, Y. (2011). Single-cell protein: production and process . American Journal of Food Technology 6, 103-116. Nobre, C., Alves Filho, E.G., Fernandes, F.A.N., Brito, E.S., Rodrigues, S., Teixeira, J.A., Rodrigues, L.R. (2018). Production of fructo-oligosaccharides by Aspergillus ibericus and their chemical characterization . LWT - Food Science and Technology 89, 58-64. Nobre, C., Sousa, S.C., Silva, S.P., Pinheiro, A.C., Coelho, E., Vicente, A.A., Gomes, A.M.P., Coimbra, M.A., Teixeira, J.A., Rodrigues, L.R. (2018). In vitro digestibility and fermentability of fructooligosaccharides produced by Aspergillus ibericus . Journal of Functional Foods, 46, 278-287. O’Shea, N., Arendt, E.K., Gallagher, E. (2012). Dietary fibre and phytochemical characteristics of fruit and vegetable by-products and their recent applications as novel ingredients in food products . Innovative Food Science & Emerging Technologies 16, 1-10. Oliveira, F., Souza, C.E., Peclat, V.R.O.L., Salgado, J.M., Ribeiro, B.D., Coelho, M.A.Z., Venâncio, A., Belo, I. (2017). Optimization of lipase production by Aspergillus ibericus from oil cakes and its application in esterification reactions. Food and Bioproducts Processing 102, 268-277. Olorunnisola, K., Jamal, P., Alam, M. (2017). Optimization of protein enrichment of fruit peels by mixed culture of Phanerochaete chrysoporium and Schizophyllum commune as animal feed supplement. International Food Research Journal 24, 2632-2639. Olorunnisola, K.S., Jamal, P., Alam, M. Z. (2018). Protein improvement of banana peel through sequential solid-state fermentation using mixed-culture of Phanerochaete chrysosporium and Candida utilis . 3 Biotech, 8 (10), 416. Ottaviani, J.I., Ensunsa, J.L., Fong, R.Y., Kimball, J., Medici, V., Kuhnle, G.G.C., Crozier, A., Schroeter, H., Kwik-Uribe, C. (2023). Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study . Food & Function 14, 8217-8228.
56 Park, E.Y., Anh, P.N., Okuda, N. (2004). Bioconversion of waste office paper to L(+)-lactic acid by the filamentous fungus Rhizopus oryzae . Bioresource Technology 93, 77-83. Pekel, J.F., Cottam, A., Gorelick, N., Belward, A.S. (2016). High-resolution mapping of global surface water and its long-term changes. Nature, 540 (7633), 418-422. Pereira, A.G., Fraga-Corral, M., Garcia-Oliveira, P., Otero, P., Soria-Lopez, A., Cassani, L., Cao, H., Xiao, J., Prieto, M.A., Simal-Gandara, J. (2022). Single-Cell Proteins Obtained by Circular Economy Intended as a Feed Ingredient in Aquaculture. Foods 11, 2831. Plackett, R.L., Burman, J.P. (1946). The design of optimum multifactorial experiments . Biometrika, 33 (4), 305-325. PR Newswire. (2023). Protein Alternatives Market to Reach $73.9 Billion, Globally, by 2031 at 16.2% CAGR: Allied Market Research . https://www.prnewswire.com/news-releases/proteinalternatives-marketto-reach-73-9-billion-globally-by-2031-at-16-2-cagr-allied-market-research301724499.html assessed on 28th January 2023. Qureshi, A.S., Khushk, I., Naqvi, S.R., Similar, A.A., Ali, C.H., Naqvi, M., Danish, M., Ahmed, A., Majeed, H., Jatt, A.N.M., Rehan, M., Nizami, A. (2017). Fruit Waste to Energy through Open Fermentation . Energy Procedia 142, 904-909. Ramachandran, S., Roopesh, K., Nampoothiri, K.M., Szakacs, G., Pandey, A. (2005). Mixed substrate fermentation for the production of phytase by Rhizopus spp. using oilcakes as substrates . Process Biochemistry 40, 1749-1754. Raziq, A., Lateef, M., Khan, A., Ullah, H., Khan, M. (2020). Single cell protein (SCP) production and potential substrates: A comprehensive review . Pure and Applied Biology, 9 (3), 1743-1754. Rebello, L., Ramos, A., Pertuzatti, P., Barcia, M., Castillo-Muñoz, N., Hermosín-Gutiérrez, I. (2014). Flour of banana (Musa AAA) peel as a source of antioxidant phenolic compounds . Food Research International 55, 397-403. Rella, A., Farnoud, A.M., Poeta, M.D. (2016). Plasma membrane lipids and their role in fungal virulence . Progress in Lipid Research 61, 63-72. Ritala, A., Häkkinen, S.T., Toivari, M., Wiebe, M.G. (2017). Single-Cell Protein-State-of-the-Art, Industrial Landscape and Patents 2001-2016 . Frontiers in microbiology 8, 2009. Rivas, B., Torrado, A., Torre, P., Converti, A., Domínguez, J.M. (2008). Submerged Citric Acid Fermentation on Orange Peel Autohydrolysate . Journal of Agricultural and Food Chemistry, 56 (7), 2380-2387. Roa Engel, C.A., Van Gulik, W.M., Marang, L., Van der Wielen, L.A.M., Straathof, A.J.J. (2011). Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae . Enzyme and Microbial Technology 48, 39-47. Rodsamran, P., Sothornvit, R. (2018). Microwave heating extraction of pectin from lime peel: Characterization and properties compared with the conventional heating method . Food Chemistry 278, 364-372. Said, S.D., Zaki, M, Novita, E., Asnawi, T.M. (2019). Production of single-cell protein by a local Trichoderma reesei in solid state fermentation: effects of process variables . Journal of Physics: Conference Series (1376), 012043.
57 Salgado, J.M., Abrunhosa, L., Venâncio, A., Domínguez, J.M., Belo, I. (2016). Combined bioremediation and enzyme production by Aspergillus sp. in olive mill and winery wastewaters . International Biodeterioration & Biodegradation 110, 16-23. Salgado, J.M., Abrunhosa, L., Venâncio, A., Domínguez, J.M., Belo, I. (2014). Integrated Use of Residues from Olive Mill and Winery for Lipase Production by Solid State Fermentation with Aspergillus sp.. Applied Biochemistry and Biotechnology, 172 (4), 1832-1845. Schmidt, C.G., Furlong, E.B. (2012). Effect of particle size and ammonium sulfate concentration on rice bran fermentation with the fungus Rhizopus oryzae . Bioresource Technology 123, 36-41. Serra, R., Cabañes, F., Perrone, G., Castellá, G., Venâncio, A., Mule, G., Kozakiewicz, Z. (2006). Aspergillus ibericus: A new species of section Nigri isolated from grapes . Mycologia 98, 295-306. Sharif, M., Zafar, M.H., Aqib, A.I., Saeed, M., Farag, M.R., Alagawany, M. (2021). Single cell protein: Sources, mechanism of production, nutritional value and its uses in aquaculture nutrition . Aquaculture 531, 735885. Siyal, F., Wagan, R., Bhutto, Z., Tareen, M., Arain, M., Saeed, M., Brohi, S., Soomro, R. (2016). Effect of Orange and Banana Peels on the Growth Performance of Broilers . Advances in Animal and Veterinary Sciences 4, 376-380. Sotiris, I.P., Dedousi, A., Sossidou, E.N., Zdragas, A. (2020). Sustainable animal feed protein through the cultivation of Yarrowia lipolytica on agro-industrial wastes and by-products . Sustainability 12, 1398. Souza Filho, P.F., Nair, R.B., Andersson, D., Lennartsson, P.R., Taherzadeh, M.J. (2018). Vegan-mycoprotein concentrate from pea-processing industry byproduct using edible filamentous fungi . Fungal Biology and Biotechnology 5, 5. Statista (2022). Soy food products in the U.S. - statistics & facts . Statista Research Department. https://www.statista.com/topics/2218/soy-food-products-industry-statistics-and-facts/#topicOverview assessed on 26th September 2023. Statista (2023a). Leading soybean producing countries worldwide from 2012/13 to 2022/23 (in million metric tons) . https://www.statista.com/statistics/263926/soybean-production-in-selected-countriessince-1980/ assessed on 26th September 2023. Statista (2023b). Value of the plant-based food market worldwide from 2020 to 2030 (in billion U.S. dollars). https://www.statista.com/statistics/1280394/global-plant-based-food-market-value/ assessed on 26th September 2023. Stephens, N., Silvio, L.D., Dunsford, I., Ellis, M., Glencross, A., Sexton, A. (2018). Bringing cultured meat to market: Technical, socio-political, and regulatory challenges in cellular agriculture. Trends in Food Science & Technology 78, 155-166. Sui, X., Meng, Z., Dong, T., Fan, X., Wang, Q. (2023). Enzymatic browning and polyphenol oxidase control strategies . Current Opinion in Biotechnology 81, 102921. Suri, S., Singh, A., Nema, P.K. (2022). Current applications of citrus fruit processing waste: A scientific outlook . Applied Food Research, 2 (1), 100050. Taylor, S.L., Houben, G.F., Blom, W.M., Westerhout, J., Remington, B.C., Crevel, R.W.R., Brooke-Taylor, S., Baumert, J.L. (2021). The population threshold for soy as an allergenic food – Why did the Reference Dose decrease in VITAL 3.0? Trends in Food Science & Technology 112, 99-108.
58 Thavamani, A., Sferra, T.J., Sankararaman, S. (2020). Meet the Meat Alternatives: The Value of Alternative Protein Sources . Current Nutrition Reports 9, 346-355. Thrane, M., Paulsen, P.V., Orcutt, M.W., Krieger, T.M. (2017). Soy Protein: Impacts, Production, and Applications , Nadathur, S.R., Wanasundara, J.P.D., Scanlin L. (Eds.). Chapter 2 in Sustainable Protein Sources . Academic Press, 23-45. Tsado, A.N., Okoli, N.R., Jiya, A.G., Gana, D., Saidu, B., Zubairu, R., Salihu, I.Z. (2021). Proximate, Minerals, and Amino Acid Compositions of Banana and Plantain Peels . BIOMED Natural and Applied Science, 1 (1), 32-42. UNCTAD (United Nations Conference on Trade and Development) (2022). The impact on trade and development of the war in Ukraine – UNCTAD Rapid Assessment. Geneva. UNICEF (2020). The state of food security and nutrition in the world 2020. Full report. United Nations (2018). World urbanization prospects: The 2018 revision . Department of Economic and Social Affairs, Population Division. New York. United Nations (2019). World population prospects 2019: Highlights . Department of Economic and Social Affairs, Population Division. New York. United Nations (2022). Global Impact of war in Ukraine on food, energy and finance systems . Brief report. Upadhyaya, S., Tiwari, S., Arora, N., Singh, D. (2016). Microbial Protein: a Valuable Component for Future Food Security . Singh, J.S., Singh, D.P. (Eds.). Chapter 12 in Microbes and Environmental Management . Studium Press. USSEC (United States Soybean Export Council) (2021). The Global Soy Foods Market: An Overview . Prepared by Agromeris LLC. Vallikkadan, M.S., Dhanapal, L., Dutta, S., Sivakamasundari, S.K., Moses, J.A., Anandharamakrishnan, C. (2023). Meat Alternatives: Evolution, Structuring Techniques, Trends, and Challenges . Food Engineering Reviews 15, 329-359. Wiebe, M.G. (2002). Myco-protein from Fusarium venenatum: a well-established product for human consumption. Applied Microbiology and Biotechnology 58, 421-427. Yadav, G., Singh, A., Bhattacharya, P., Yuvraj, J., Banerjee, R. (2013). Comparative analysis of solid-state bioprocessing and enzymatic treatment of finger millet for mobilization of bound phenolics . Bioprocess and Biosystems Engineering 36, 1563-1569. Yamada, E.A., Sgarbieri, V.C. (2005). Yeast (Saccharomyces cerevisiae) protein concentrate: preparation, chemical composition, and nutritional and functional properties . Journal of Agricultural and Food Chemistry, 53 (10), 3931-3936. Zaini, H.M., Roslan, J., Saallah, S., Munsu, E., Shaeera, N., Sulaiman, Pindi, W. (2022). Banana peels as a bioactive ingredient and its potential application in the food industry . Journal of Functional Foods 92, 105054. Zhang, G., Zhao, X., Li, X., Du, G., Zhou, J., Chen, J. (2020). Challenges and possibilities for biomanufacturing cultured meat. Trends in Food Science & Technology 97, 443-450.