Valorization of Agro-Industrial Wastes and Residues through the Production of Bioactive Compounds by Macrofungi in Liquid State Cultures: Growing Circular Economy

Abstract

Vast quantities of side streams produced worldwide by the agricultural and food industry present an environmental challenge and an opportunity for waste upcycling in the frame of the circular bioeconomy. Fungi are capable of transforming lignocellulosic residues and wastes into a variety of added-value compounds with applications in functional food products, pharmaceuticals, chemicals, enzymes, proteins, and the emerging sector of nutraceuticals. The liquid state culture of fungi is an efficient and potentially scalable and reproducible biotechnological tool that allows the optimized production of fungal metabolites. Particularly, the utilization of agro-industrial by-products, residues, and wastes as a substrate for the liquid culture of macrofungi is suggested as an attainable solution in the management of these streams, contributing to climate change mitigation. This review presents recently published literature in the field of liquid state cultures of macrofungi using agro-industrial side streams, the different substrates, methods, and factors affecting their growth and metabolite production, as well as their applications, focusing on the variety of natural valuable compounds produced.

1. Introduction

It is well understood that human activity on the planet is causing rising global environmental risks and for the first time constitutes the largest driver of planetary change [1]. Increasingly tightening legislation governing waste disposal makes conventional treatment of waste streams more expensive and energy demanding, creating the need for alternative solutions [2]. Circular economy transition is ongoing and increasingly supported and urged by policy makers, as a response to the global environmental problems earth is facing [3,4]. Circular bioeconomy is the term currently used to describe the bioconversion of waste streams into added-value products [5]. The European Union has published detailed reports quantifying the agro-industrial residual biomass production in the country members to promote their utilization as feedstock in the future bioeconomy [6]. Microbial biotechnology is indispensable for the creation of processes that will pave the transition to closed-loop circular production by exploiting residual biomass and re-introducing them into the food chain as novel products [7]. Various pathways to valorize biowaste into value-added products such as biofuel, biochar, and biomaterials that have emerged from research labs are currently being studied for their upscaling feasibility [8]. Bioconversion of lignocellulosic streams with the use of macrofungi can be a sustainable alternative to the energy-consuming production of biofuel and bioenergy. Especially, the concept of biorefineries that integrate various conversion and separation steps to utilize all process intermediates and by-products has been proposed as a feasible strategy for the valorization of lignocellulosic residues [9,10,11,12]. Although many side-streams are being utilized as animal feed, it is considered that they can potentially be used to produce higher-value products [7]. The high degree of competition in national and international markets provides another motive for the agri-food industry to seek economically sustainable solutions that will produce added-value products [13].

It is widely considered that fungi play an important role in tackling food security challenges [14]. Fungi are well-known for their ability to degrade lignocellulosic material in nature [15] and can be protagonists in this circle of transforming low-value substrates into a variety of valuable bio-compounds [14,15,16,17,18,19,20]. The metabolism of many macrofungi has been studied in synthetic media with various carbon and nitrogen sources to determine growth patterns and their productivity [21,22]. Current research indicates that satisfying quantities of macrofungi mycelial biomass can be produced in liquid cultures using agro-industrial side-streams as the sole carbon source [23,24,25,26,27]. Submerged cultivation of macrofungi is an efficient, reproducible, and potentially scalable method to produce fungal biomass and metabolites (Figure 1). Macrofungi can biosynthesize highly nutritional biomass containing a great variety of valuable bioactive compounds. Using fungal metabolites or biomass in industrial applications, known as fungal white biotechnology, is a field that has been gaining increased attention lately due to its eco-friendly nature. Applied fungal biotechnology is used for food, feed, biocontrol, bioenergy, chemicals, pharmaceuticals, detergents, enzymes, proteins, paper and pulp, textiles, materials for construction, and the automotive and transportation industries [14,28].

This article aims to provide a critical review of what has been achieved in the field of liquid state fermentation of macrofungi up to now, using agri-food residues as the substrate to produce several bioactive metabolites from a wide range of higher edible or/and medicinal fungi. In particular, Section 2 provides an overview of the volumes of agri-food side streams produced, as well as the key physicochemical characteristics that characterize them. Section 3 provides a succinct overview of macrofungi, while Section 4 is a detailed presentation of the fungal bioactive compounds with a focus on their diversity and activities. Section 5 presents liquid state cultures as a strategy for the biotechnological production of value-added fungal compounds. An overview of recent studies focused on producing macrofungal metabolites and biomass utilizing agri-food residues is provided in the form of a table in Section 6. Finally, Section 7 describes the challenges and prospects of the biotechnological exploitation of agri-food residues through submerged cultures using macrofungi. The present review, thus, could serve as a base for the design of new, more specified research targeting the optimum exploitation of selected liquid waste streams to produce biomass and metabolites with potential use in food or remedy applications.

2. Agri-Food Side Streams: Residues, By-Products, and Wastes

The agri-food production required to sustain the estimated population of nine billion people by 2050 will consequently increase the amounts of unavoidable agri-food wastes [29]. The agri-food system is currently producing in an inefficient manner [30,31] and polluting the planet while depleting natural resources [1,32,33]. The United Nations (UN) estimated that 7 to 10 billion tons of waste were generated globally in 2010 [34]. Gustavsson et al. [35] calculated that about one-third of the food produced globally is lost or wasted along the whole food chain. These losses equal 1.3 billion tons of wastes and by-products every year [36], and they are valued at about USD 936 billion dollars annually [37]. The European Union (EU) reported that 129Mt of food wastes were generated in the EU in 2011 [38]. These vast amounts of waste include agricultural lignocellulosic residues, agri-food by-products, food wastes, and industrial sludge and wastewater, all of them streams of organic waste suitable for exploitation [7]. Ensuring a sustainable food future for the world requires that the industry should be making the most out of the resources used [32].

Agricultural residues are produced during crop harvesting and consist of leaves, roots, stalks, stems, straw, seed pods, and husks. Asia is responsible for 47% of all the agricultural residues produced worldwide, while the United States produces 29%, Europe 16%, Africa 6%, and Oceania 2% [39]. Agricultural residues are inedible and unavoidable and have low bulk density, low ash melting point, fibrous nature, high volatile matter content, high calorific value, and low moisture [29]. Lignocellulosic residues are also being used for the production of bioenergy (heat and electricity generated by combustion) or biofuel production, but these are procedures that require considerable amounts of fossil fuel and other resources such as land and water [40]. Industrial residues derive from the food industries that use various methods and techniques to transform raw materials into food. Industries processing fruits and vegetables, grains, and dairy, meat, poultry, marine, winery, and brewery products are the main sources of huge quantities of solid and liquid effluents, which have to be sustainably managed without being an environmental burden [16]. Process residues include husks, peels, pulp and shells, brans, various pomaces, bagasse, and wastewaters. Agri-food side-streams are generally considered to have little value and are incorporated in animal feed [41], burned, end up in a landfill, or even worse, disposed of uncontrollably [42], causing severe environmental problems and health issues [43,44].

Agri-food residues are often called lignocellulosic biomass because they are mainly composed of cellulose, hemicelluloses, and lignin, which form composite, recalcitrant matrices that create structural complexity and deem them indigestible and hard to biodegrade. All these side streams and co-products exhibit a wide variety of physicochemical characteristics that determine their technical and economic potential for valorization [7]. The carbon/nitrogen ratio is a critical factor for macrofungi (mushroom) cultivation, as it can impact their growth, yield, and polysaccharide and protein content [26,45,46,47,48].

The properties of some of the most widely produced side streams and wastes are presented in

Table 1. Composition of various agri-food side streams.

Agri-Food Waste	Composition (% w/w)
	Cellulose	Hemicellulose	Lignin	Ash	Total Solids	Moisture	C/N Ratio	Reference
Barley straw	33.8	21.9	13.8	11	-	-	-	[18]
Wheat straw	32.9	24	8.9	6.7	95.6	7	50–80	[18,26,48]
Wheat bran	30	50	15	-	-	-	19	[18,26]
Rice straw	39.2	23.5	36.1	12.4	98.6	6.5	8–18	[18,49]
Rice bran	35	25	17		-	-	12–48	[28]
Oat straw	39.4	27.1	17.5	8	-	-	-	[18]
Sawdust	45.1	28.1	24.2	1.2	98.5	1.12	-	[18]
Sugar beet waste	26.3	18.5	2.5	4.8	87.5	12.4	-	[18]
Sugarcane bagasse	30.2	56.7	13.4	1.9	91.6	4.8	70–120	[18]
Corn stalks	61.2	19.3	6.9	10.8	97.7	6.4	-	[18,48]
Cotton stalks	58.5	14.4	21.5	9.9	-	7.4	15	[18,49]
Soya stalks	34.5	24.8	19.8	10.3	-	11.8	20–40	[18,45]
Sunflower stalks	24.1	29.7	13.4	11.1	-	-	97	[18,45]
Walnut shells	3	628	43	-	-	-	175	[28]
Almond shells	38	29	30	-	-	-	61	[28]
Chestnut shells	21	16	36	-	-	-	8	[28]
Pistachio shell	43	25	16	-	-	-	43	[28]
Hazelnut shell	55	34	35	-	-	-	50–58	[28]
Olive oil cake	31	21	26	-	-	-	14–17	[28]
Olive mill wastewater	-	-	-	2.61	0.41	-	35–55	[50]
Oil palm cake	64	15	5	-	-	-	-	[45]
Sunflower oil cake	25	12	8	-	-	-	-	[45]
Cotton seed hull	31	20	18	-	-	-	59–67	[45]
Potato peel waste	2.2	-	-	7.7	-	9.89	-	[45]
Orange peel	9.2	10.5	0.84	3.5	-	11.86	-	[45]
Lemon peel	12	5	2					[45]
Pineapple peel	18.11	-	1.37	-	93.6	91	41	[45]
Apple pomace	43	24	20	-	-	-	48	[45]
Tomato pomace	9	5	5	-	-	-	-	[45]
Coffee skin	23.7	16.6	28.5	5.3	-	-	14.4	[51]
Spent coffee grounds	12.4	39.1	23.9	1.3	-	-	16.9	[26]
Cheese Whey	-	-	-	0.5	0.1–22	-	57	[52]
Brewer’s spent grain	19	21	19	4.2	4.9	-	-	[53]
Distillery wastes	-	-	-	-	36	-	32	[54]
Grape marc	20.9	13.3	34.7		-	74	24	[55,56]
Wine lees	-	-	-	10.5	-	-	6–13	[57]

. An important characteristic that agri-food side streams share is wide variability in their physicochemical properties. Moisture, carbon, and nitrogen content are not consistent throughout the year, and the production cycles vary between production facilities and manufacturers. That is probably because the properties of the produced side streams and residues are affected by the location, the climate produced, and the properties of the original raw material [7]. Therefore, physicochemical analyses are needed as well as the mixtures’ formation before their use as a substate for mushroom cultivation [46,47,48].

Additionally, the use of microorganisms that produce valuable chemical compounds can be a sustainable alternative to the energy-consuming production of biofuel and bioenergy. The circular bioeconomy and the “zero-waste” policies set by the EU propose the bioconversion of agri-food residues to useful, higher-value products [58,59]. That is usually a multi-step process that includes the pretreatment of raw materials, hydrolysis of the polymers to readily metabolizable sugars, cultivation of microorganisms that will produce the wanted chemical compounds, and lastly, separation and isolation of the compounds [60]. Since fungi are well-known for their ability to degrade lignocellulosic material in nature [15,61], they can be selected as bioconversion agents to avoid the hydrolysis step of the process. Fungal mycelia are able to utilize the complex organic compounds of agro-industrial residues and produce useful bio-molecules that exhibit an amazing variety of functions, ranging from antibacterial antibiotics and anti-cancer agents to nutraceuticals, cosmetics, and industrial enzymes [62,63]. Particularly, macrofungi are not only beneficial for human health but also very nutritious and tasty, and this is the reason for being examined to such a great extent [14,17,61].

3. Macrofungi: Ascomycota and Basidiomycota

Fungi are eukaryotic, heterotrophic microorganisms with chitin-based cell walls and represent one of the seven kingdoms of life. They are the second most abundant group of organisms after insects. Fungi are subdivided into seven Phyla, 35 Classes, and 129 Orders. They are characterized by a wide range of diversity in their morphology, cycles, length of life, and the ecosystems they can be found in. According to the latest estimations, there are 2.2 to 3.8 million species of fungi, of which only 120,000 have been classified [64]. Out of those 120,000 fungi, more than 2000 macrofungi have been characterized as edible and/or medicinal [65]. Fungi exhibit a vast variety of life cycles, reproduction strategies, and ecological preferences, but most macrofungi are saprotrophic, which means that they obtain nutrients by decomposing animal and plant residues. They achieve that by secreting oxidative exoenzymes that degrade their growth substrate and absorbing the solubilized nutrients with their hyphae [49]. Thus, macrofungi are essential for the decomposition and recycling of dead organic matter in nature, converting complex lignocellulosic substrates into simpler organic matter, an ability that has been extensively exploited in a variety of biotechnological applications [66].

Macrofungi or “higher fungi” are the filamentous fungi that create spore-bearing fleshy fruiting bodies referred to as mushrooms and belong to Ascomycota and Basidiomycota [67]. Ascomycota have mycelia with simple tapered septa and their cells exist mainly as diploids with a transient dikaryotic cell persistence, whereas Basidiomycota mycelia have a complex dolipore septum with clamp connections and are dikaryotic throughout their life cycle. This difference in the mycelium of the two divisions of macrofungi creates implications for their successful submerged culture. The sex life of Ascomycetes is characterized by plasmogamy where gametangiums contact and produce asci, leading to karyogamy and meiosis which results in haploid spores. In Basidiomycetes, haploid basidiospores germinate into monokaryotic mycelia, which then meet to perform somatogamy and produce dikaryotic mycelium [68].

Many economically important mushrooms such as truffles and morels belong to the Ascomycota, while the fruiting bodies of many Basidiomycota are traditionally used as food and medicine. More than 35 species of macrofungi are being commercially cultivated, with Lentinula edodes being the most cultivated mushroom worldwide, followed by Pleurotus spp., Auricularia auricula, and Agaricus bisporus [69]. Other commonly cultivated mushrooms are Flammulina velutipes, Volvariella volvacea, Ganoderma lucidum [70], Grifola frondosa, Hericium erinaceus, and Trametes versicolor [49].

Traditionally, the fruiting bodies of edible macrofungi are collected in nature or cultivated in various substrates to be cooked or dried for consumption. Collecting fruiting bodies in nature is seasonal, weather dependent, and requires a considerable amount of time on the part of highly experienced mushroom hunters. Solid-state cultivation of mushrooms is a lengthy process that can take up to 6 months and is affected by numerous factors leading to variable final products with often no guaranteed quality. Modern biotechnology allows for fast and consistent production of fungal biomass rich in nutrients and high-value biological compounds, opening up new possibilities to include mushrooms in the everyday diet of humans as food, nutraceuticals, and medicines [71]. Information on these compounds, their composition, and their properties are analytically given in the next section.

4. Bioactive Metabolites

Humans have appreciated mushrooms for their edible and medicinal value since the Neolithic age [72]. Increased interest in the pharmaceutical potential of mushrooms has led to numerous publications that explore more than 100 reported medicinal functions [73]. These are functions such as antifungal, antibacterial, antiviral, antioxidant, anti-inflammatory, anti-tumor, antiviral, anti-diabetic, anti-thrombotic, anti-allergic, antidepressive, antihyperlipidemic, digestive, hypotensive, cytotoxic, hepatoprotective, neuroprotective, nephroprotective, osteoprotective, and immunomodulating activities, among others [72,73,74]. This therapeutic action is due to a wide array of bioactive metabolites isolated from the fruiting body, mycelium, and culture broth of macrofungi. Both complex high-molecular-weight compounds such as polysaccharides, proteins, and lipids and low-molecular-weight compounds such as terpenoids, polyketides, and alkaloids have been isolated and are under study [75]. In addition to their pharmaceutical potential, mushrooms are recognized as a rich source of nutraceuticals [70]. Nutraceuticals are substances that have positive effects, proven by clinical testing, on normal physiological functions that maintain health in humans, thus preventing and treating diseases and enhancing longevity [76,77]. There is a globally growing market for nutraceuticals sold as nutrients and supplements or in the form of enhanced “functional foods” [78,79]. Natural bioactive compounds found in macrofungi are ideal for this market and are already being used for their properties [80].

4.1. Carbohydrates: Polysaccharides (EPS and IPS) and Oligosaccharides

Polysaccharides are the most abundant carbohydrates found in nature; they are crucial for life maintenance in a variety of organisms such as plants, animals, microorganisms, and seaweeds. Polysaccharides are long-chain biopolymers composed of more than 10 monosaccharide units connected by glycosidic bonds, whereas oligosaccharides contain 3 to 10 monosaccharides [81]. Polysaccharides exhibit a wide variety of structures composed of different monosaccharides, glycosidic bonds, and functional groups. The precise chemical structure of polysaccharides can be investigated with a combination of analytical methods such as FTIR, NMR < Raman spectroscopy, GC, GC-MS, and HPLC. Their most common components are pyranoses such as glycose, galactose, mannose, xylose, and arabinose [82].

The diversity of their constituent monosaccharide composition and structure is responsible for their different biological activities. There is a close relationship between the polysaccharide’s structure and biological activity [83]. The different chemical structures and chain conformations are also responsible for the different activities of polysaccharides [84]. They play an important role in functions such as cell recognition, growth and differentiation, metabolism, embryonic development, immune response, energy storage, structural support, and antigenicity [85].

Polysaccharides found in fungi are made up of glucans, which are made of D-glycose monomers, two glucose units linked by α-glycosidic or β-glycosidic bonds. They all contain a β-linked glycose backbone, but the pattern and degree of branching vary from species to species. The α or β linkages can form heteroglycans such as mannose, galactose, arabinose, fucose, and xylose. Polysaccharides can also bind to proteins, protein residues, and peptides to create polysaccharide-protein complexes or peptide complex groups [86]. Microbial polysaccharides are categorized into intra-cellular polysaccharides (IPS) and extra-cellular polysaccharides (EPS), depending on their location. Due to the polysaccharides not being encoded in the genome and their structural complexity, there is little information available regarding their biosynthesis [87]. There are data on polysaccharides produced from 651 species of fungi [88] to which there are multiple confirmed biological functions attributed, including antidiabetic, antioxidant, immunomodulatory, hepatoprotective [89], antitumor, antiviral, anti-inflammatory, renal protective, and radioprotective actions [74], regulation of glycolipid metabolism and gut microbiota, anti-nephritic and antiangiongenic activity, and improvement of memory impairment [83].

Examples of mushroom polysaccharides isolated and characterized are pleuran from Pleurotus species, lentinan from L. edodes, ganoderan from G. lucidum, agaritine from Agaricus sp., grifolans from G. frondose, and calocyban from Calocybe indica [61]. Polysaccharides are major structural cell wall components of mushrooms, but they can be produced exo-cellularly in large amounts by the mycelium of macrofungi in liquid/submerged cultivation. This is not always the case, because the conditions required for their exo-cellular production are still under study [68]. Nevertheless, liquid cultivation of macrofungi mycelia results in much faster production of polysaccharides in comparison to fruitbody production and is a promising tool for mass-scale industrial production [17]. It should be mentioned that optimum conditions for EPS production can vary greatly for fungi in the same genus [90] and even between strains of the same species [91]. Apart from species and strain, the most important growth conditions for optimal EPS production are: type of culture, oxygen supply, temperature, pH, carbon and nitrogen source, content and ratio, salt content, and cultivation length [25,28,92,93]. Dedousi et al. (2021) used molasses as a substrate in liquid cultures of Morchella sp. producing IPS (4.8 g/L) and EPS (3.94 g/L) [28]. Diamantopoulou et al. report that adding OMW to the liquid substrate of G. resinaceum resulted in increased quantities of IPS (5.2 g/L) [92]. Many studies seek to optimize EPS production in liquid cultures by deploying a multitude of carbon and nitrogen sources, different light intensities, and wavelengths [94]. Homogenization of the mycelium inoculant by sonoporation proved to be effective in increasing EPS production and reducing the required quantity of starter mycelium in submerged cultures of G. lucidum [95]. It is important to mention that the production of EPS by fungi in liquid cultures is negatively correlated with IPS, indicating that the amount synthesized significantly decreases after the 8th or 12th day of culture [96]. IPS in liquid cultures of V. volvaceae were also found to start decreasing after the 8th day and continued to decrease until the end of the culture. IPS were produced in greater quantities when the substrate C/N ratio was 40 [22].

Beta-glucans are the most common polysaccharides found in the cell walls of Ascomycota and Basidiomycota [97]. Beta-glucans are groups of polysaccharides or dietary fibers composed of D-glycose monomers, linked by β (1→3) and β (1→6) glycosidic bonds [98]. Beta-glucans are classified as dietary fibers and prebiotics because they cannot be hydrolyzed by the gastric enzymes of mammals and reach the large intestine undigested, where they are fermented by the gut microbiota with potential health benefits for the host [99]. Beta-glucans cannot be synthesized by the human body, which leads to their recognition by the immune system, inducing both adaptive and innate immune responses. Current research has established that beta-(1→3,1→6)-D-glucans are efficient immunostimulant agents that can trigger a variety of molecular interactions that are not yet clearly understood [100]. It has been established that β-glucans can play a role in the prevention of insulin resistance, dyslipidaemia, hypertension, and obesity [101]. A recent systematic literature review of 34 clinical trials performed using fungal β-glucans summarized the main physiological impacts on patients as follows: a strengthened immune defense that reduces the incidence and symptoms of respiratory infections, improvement of allergic symptoms, improvement of mood states, and improvement of overall wellbeing. No adverse effects related to the supplementation of β-glucans were noted [102]. The content of fungi in beta-glucans varies from 16% to 53% and is influenced by factors such as species, strain, cultivation conditions, and total dietary fiber content. Several studies have studied the in vitro antibacterial and antiviral activity of beta-glucans through the activation of the non-specific immune system [103]. Beta-(1,3–1,6) glucans also exhibit a radioprotective effect, which has been investigated in vivo with mice [104] and has also been successfully used as a radioprotectant to treat and prevent radiation and chemotherapy-related injuries [105]. P. ostreatus fruiting bodies cultivated in agricultural residues contained high quantities of β-glucans (28.28% to 48.42% dw) [106]. Another study reports that the use of olive mill wastes in the substrate of G. lucidum resulted in up to a 43% increase in the β-glucan content of the produced mushrooms [107].

An extensive review of the mechanisms by which polysaccharides help the treatment of type 2 diabetes enumerates multiple compounds isolated from macrofungi [65]. Namely, submerged cultivation of Laetiporus sulphureus and G. lucidum produced polysaccharides with antidiabetic action in rats. A glucan-rich polysaccharide isolated from Pleurotus sajor-caju alleviated hyperglycemia and hyperinsulinemia in diabetic mice. Multiple polysaccharides from Coriolus versicolor successfully suppressed hyperglycemia, and polysaccharides from G. frondosa enhanced glucose metabolism and increased the synthesis of intracellular glycogen [65]. Lentinan has also been found to help prevent diabetes by protecting pancreatic β-cells [108]. Submerged liquid cultivation of Inonotus obliquus produced two novel polysaccharide fractions with hypoglycemic activity [109]. Polysaccharides from T. versicolor have been shown to alleviate bone deterioration caused by diabetes mellitus, in addition to improving hyperglycemic control [110]. The potential and limitations of using fungal polysaccharides for diabetes therapy are discussed in this review article [111]. A total of more than 47 EPS have been isolated from G. frondosa with antioxidant, antitumor, anti-hyperglycemia, and immunomodulatory activity, and have been used in the development of several patented healthcare and pharmaceutical products for cancer and diabetes [112]. Pleurotus eryngii produced significant quantities of EPS (5.16 g/L) in optimized submerged cultures, which show high antioxidant and antitumor abilities [113].

Fungal polysaccharides inhibit tumor cell proliferation, improve body immunity, cause tumor cell apoptosis with direct contact, and prevent the spread and migration of tumor cells in the body [114]. The antitumor action of polysaccharides is mediated through a thymus-dependent immune mechanism, which involves the activation of cytotoxic macrophages, monocytes, neutrophiles, natural killer cells, dendritic cells, and chemical messengers, which triggers the complementary and acute phase responses [115]. More specifically, lentinan from L. edodes and schizophyllan from Schizophyllum commune have a similar structure and show effective antitumor and immune potentiating activity through the cytokine production from immunocytes. Schizophyllan is being commercially used in vaccines and cancer therapy [116]. Krestin from C. versicolor, maitake D-fraction from G. frondosa, and ganoderan from Ganoderma spp. are all polysaccharides that have shown antitumor activity in clinical trials [86]. Many mushroom polysaccharides are being utilized as a co-treatment along with chemotherapy because of their ability to prevent oncogenesis and tumor metastasis [97].

4.2. Proteins and Peptides

Proteins are high-molecular-weight polymers made of amino acids linked together by peptide bonds and are essential for human health. Increased population and current dietary trends have resulted in increased demand for protein and meat alternatives, and it has been proposed that mushroom proteins can efficiently substitute meat. Fungal proteins contain all nine essential amino acids required by humans, in addition to carbohydrates, vitamins, and carotenes. Mycoprotein is considered a high-quality protein comparable to animal protein since it provides a high protein-to-energy ratio. At the same time, it contributes to minimizing animal slaughter, reducing the carbon footprint of food production, and can be produced with low total costs independently of climate and landscape limitations [117]. Pleurotus spp. are well known for efficiently recycling agro-industrial wastes and producing biomass with a protein content of up to 49% of their dry weight [118]. Submerged cultivation of P. sajor-caju in 5-L stirred tank bioreactor using 10 g/L glycose resulted in fast production of mycelium biomass (8.18 g/L) with a high content of protein (32%) [119]. Similarly, Cordyceps militaris mycelium produced in submerged cultivation had higher protein content than that of the fruiting body (21.10% vs. 18.47%), and higher total essential amino acid concentration than that found in eggs [120]. A. bisporus cultivated in shake flasks with synthetic media produced 12.67 g/L biomass containing 36% w/w protein and noteworthy amounts of IPS (2.57 g/L) in only 26 days [121]. The screening of Basidiomycetes: A. aegerita, Pleurotus sapidus, L. edodes, Wolfiporia cocos, Stropharia rugosoannulata, P. sajor-caju, and P. salmoneostramineus grown in submerged cultures using industrial side streams as the sole carbon successfully converted the residues into biomass containing protein enhanced with lipids, carbohydrates, and vitamin D₂. P. sapidus excelled in the screening and produced biomass containing 21% true protein, 4% lipids, and 115 μg/g vitamin D₂ in 4 days [24]. Making use of statistical optimization, Morchella fluvialis mycelium with 38% w/w protein content was produced in liquid culture, deeming it a promising source of essential amino acids for the human diet [122]. Pleurotus species cultivated on agro-industrial by-products produced a total of 22 free amino acids in quantities up to 130.12 mg/g dw plus the neurotransmitter GABA and ornithine [123]. The connection between dietary protein, the gut microbiota, and overall human health is also being investigated [124], and a single band protein (HEP3) isolated from H. erinaceus was found to improve the immune functions of mice by regulating the gut microbiota [125].

Apart from dietary proteins, macrofungi produce various bioactive proteins and peptides classified as lectins, fungal immunomodulatory proteins (FIP), ribosome-inactivating proteins (RIP), antimicrobial proteins, and ribonucleases. Lectins are nonimmune proteins or glycoproteins that bind specifically to carbohydrates and result in cell agglutination. This property of lectins makes them useful in studying the structure of cell surfaces and the isolation and purification of polymers with carbohydrate units [126]. In the last years, many lectins with antiproliferative, antitumor, cytotoxic, and immunomodulatory activities have been isolated from edible mushrooms such as A. bisporus, F. velutipes, G. lucidum, G. frondose, and V. volvacea [127]. FIPs are a novel family of bioactive proteins isolated from macrofungi that exhibit significant activity in suppressing the invasion and metastasis of tumor cells and show promise as immunologic adjuvants for tumor treatment [128]. RIPs are enzymatically active proteins that can inactivate ribosomes by eliminating adenosine residues from rRNA. They have been studied for their ability to inhibit HIV replication and viral disease prevention and are promising substances for developing drugs [129]. Considerable antifungal activity at the RNA level is attributed to ribonucleases isolated from mushrooms [130]. A ribonuclease isolated from Tuber indicum inhibited the proliferation of breast cancer cells [131], and another ribonuclease from G. lucidum suppressed autophagy and triggered apoptosis in colorectal cancer cells [132]. Amanitins are cyclic peptides of eight amino acids that are well known for their toxicity, but they have shown very promising results in anti-cancer pre-clinical trials [133].

4.3. Fatty Acids and Lipids

All fatty acids have important roles in the human body, and thus human diet must include foods containing fatty acids [134]. Macrofungi contain a wide variety of fatty acids, but the biggest percentage are polyunsaturated fatty acids (PUFA) and specifically linoleic acid, which is an important essential ω-6 fatty acid that cannot be synthesized in the body [135]. The PUFA contents of mushrooms are greater than those found in cattle and pork while containing low amounts of saturated fatty acids (SFA) [135]. PUFAs have been shown to reduce serum cholesterol [73] and play an important role in the regulation of cell physiology and cardiovascular health [136]. The fatty acids found in P. ostreatus were 28 in total, but linoleic acid was in the highest concentration (57–81%), followed by oleic (6–20%), and palmitic acid (8–12%) [91]. Agitated submerged cultures of G. applanatum, M. esculenta, A. aegerita, and P. pulmonarius produced significant quantities of unsaturated fatty acids [96]. Agitation of the shake flasks has been reported to have a positive impact on lipid biosynthesis by macrofungi [137]. Tuber aestivum cultivated in liquid cultures containing rice cereal hydrolysates as the sole carbon source produced the highest amount of lipids compared to M. vulgaris, M. elata L. edodes, and A. bisporus in substrates of agro-industrial waste [25]. Volvariella volvacea biomass produced in synthetic media contained 0.7 g/L of total lipids with linoleic acid being predominant. The researchers report that lipid synthesis was independent of the C/N ratio and that the considerable amounts of lipids (32% w/w) synthesized early in the culture started decreasing after the 8th day until they reached 3% by the end of cultivation. Fractionation of the produced lipids revealed that most of the lipids produced were neutral (65% w/w), followed by glycolipids and sphingolipids (30% w/w), and phospholipids in negligible amounts (2–3% w/w). The composition of fatty acids was not significantly influenced by the different C/N ratios, contrary to that of neutral lipids which increased in the high C/N ratio substrate [23].

The major sterol found in macrofungi is ergosterol, which is a well-known steroid precursor of vitamin D2 and a fundamental molecule for many biological processes. When commonly consumed mushrooms such as P. ostreatus, A. bisporus, and L. edodes are exposed to UV radiation for a short time (1–3 s), ergosterol is transformed into vitamin D2 in nutritionally relevant amounts [138]. Ergosterol has also been linked with preventive action against cardiovascular diseases and antioxidant properties[70]. Cultivation of macrofungi on agri-food residues produced fruiting bodies with enhanced quantities of ergosterol (11–26 mg/g) [106]. P. citrinopileatus cultivated on grape marc plus wheat straw produced significant quantities of ergosterol (5.53 mg/g dw) [139]. Other polyunsaturated fatty acids found in mushrooms, tocopherols, are natural antioxidants with high biological activity against degenerative malfunctions, and microbial and cardiovascular diseases [124]. Ceramides are waxy lipids composed of sphingosine, a fatty acid with application in foods and cosmetics. Novel ceramides have been isolated from G. frondosa and Armillaria mellea [88]. The lipids produced by macrofungi could also be utilized in the development of healthier and more sustainable food products [140]. It should be added that the fatty acids and their metabolic products are partially responsible for the characteristic umami aroma and flavor compounds of mushrooms [141].

4.4. Phenols

A wide variety of mycochemicals are grouped under phenolic compounds and contain the characteristic aromatic ring bearing one or more hydroxyl groups. Phenolic acids are the main phenolics found in macrofungi and specifically ferulic, sinapic, caffeic, and p-lo-coumaric acids. These phenolic acids are mostly bound in complex structures such as esters. [142]. Dedousi et al. performed detoxification of molasses using Morchella species in liquid agitated and static cultures producing impressive amounts of biomass (up to 18.16 g/L) with a maximum content of fungal phenolics of 32.2 mg/g. Although different ratios of C/N in the substrate were used, the total phenolics content of the produced biomass fluctuated without any apparent trends [28]. The biomass containing fungal phenolic compounds also exhibits antioxidant activity that is strongly correlated with the phenolics content. Specifically, a novel G. resinaceum strain was cultured in a liquid medium containing olive mill wastewater (OMW), and the resulting biomass contained a total phenolic content of 508.5 μg GAE/g dw [92]. A study comparing the total phenolics and their composition in the fruiting bodies versus the submerged culture mycelium of Coprinus comatus and Coprinellus truncorum reports that submerged culture mycelium contained the highest amount of total phenolics. The phenolic profiles of the fruiting bodies and the cultured mycelium of the species exhibited high variation and diversity with vanillic, gallic, gentisic, and cinnamic acids being the most abundant [143]. Phenolics are well-known antioxidants and additionally exhibit antiallergenic, antiatherogenic, anti-inflammatory, antimicrobial, antithrombotic, cardioprotective, and vasodilator effects [70]. Although hundreds of studies report various flavonoid contents in macrofungi, Gil-Ramirez et al. categorically declare that mushrooms do not contain flavonoids because they lack the enzymes required to synthesize flavonoids [144]. The antioxidant action of phenolic compounds and their ability to be produced by microorganisms in liquid cultures has gained the attention of researchers as an environmentally friendly alternative to the chemically synthesized antioxidant phenolics widely used in the food industry [145]. The liquid culture of higher fungi using agricultural residues as a substrate results in the production of high amounts of extracellular [146] and intracellular [147] phenolics, making this production method attractive especially for industrial applications.

4.5. Terpenes and Terpenoids

Ganoderma species are considered a panacea for all kinds of diseases in Chinese traditional medicine. They have been used to treat diseases of the liver, nephritis, hypertension, and hyperlipemia. Most of the known bioactive compounds found in Ganoderma are lanostane and ergostane pentacyclic triterpenes. Ganoderic acids especially have been studied extensively in vitro for their ability to induce cancer cell apoptosis [75]. In the past years, several new lanostane triterpenes have been isolated from Ganoderma species exhibiting hepatoprotective activities [148], antimalarial [149], and antifibrotic activity [150]. Another interesting group of metabolites is the sesquiterpenoids as tremulane sequiterpenoids isolated from P. igniarius have shown vascular-relaxing activities [151]. Nine alliacane sesquiterpenes isolated from the submerged culture of Inonotus sp. BCC 22,670 exhibited antibacterial activity against Bacillus cereus and antiviral activity against Herpes Simplex Virus type 1 [152]. Sesquiterpenes isolated from a mycelial culture of F. velutipes were also found to have antimicrobial activity [153]. Novel terpenoids, erinacines, and corallocines, isolated from mushrooms of the genus Hericium, have been shown to induce nerve growth factor [154] and brain-derived neurotrophic factor [155], which makes them promising substances for treating neurodegenerative disorders such as Alzheimer’s disease [156]. Cyathane diterpenoids produced by Hericium spp. exhibited antibacterial and cytotoxic activities [157] and enhanced neurotrophin induction in cell-based bioassays [158].

Carotenoids or tetraterpenoids are colored pigments ranging from light yellow to orange to deep red and they are produced by plants and microorganisms. They are powerful antioxidants that can scavenge free radicals, and thus can be used in food, as dietary supplements, and as cosmetic additives. Carotenoids have been found in the fruiting bodies of Cantharellus cibarius and Cordyceps militaris [126]. The novel carotenoids isolated from C. militaris were named cordyxanthins and are highly water-soluble, unlike the traditional hydrophobic carotenoids, a property that can extend their applications [159].

4.6. Nutrients

Macrofungi are also a good source of vitamins which are essential for human metabolism, immunity, and digestion. They contain above-average contents of ascorbic acid and B-complex vitamins such as thiamine (vitamin B1), riboflavin (vitamin B2), niacin, pantothenic acid (vitamin B5), pyridoxine (vitamin B6), folic acid (vitamin B9), vitamin A, and vitamin E. The levels of cobalamine (vitamin B12) in mushrooms are comparable to those in beef and liver. They also contain ergocalciferol, tocopherol, ergosterol, biotin, ergotheionine, and the minerals potassium, calcium, selenium, copper, magnesium, and other trace elements [70,160,161,162]. Although the contribution of mushrooms in the proposed daily values of nutrients is small, consistent consumption of mushrooms contributes to a nutritious diet and is associated with a higher intake of many nutrients [161].

4.7. Other Compounds

The rapid spread of antibiotic-resistant bacteria worldwide has led researchers to turn to compounds isolated from macrofungi. Clitopilus passeckerianus produces a tricyclic diterpenoid called pleuromutilin, which is a naturally occurring antibiotic and has a unique and highly specific mode of action, which inhibits protein synthesis in bacteria [163]. Extracts from A. blazei, H. erinaceus, and G. frondosa with documented effects against different diseases and infections were recently reviewed and presented as potential efficient tools against the lung inflammation that occurs with COVID-19 infection [164]. Comprehensive papers about the entirety of bioactive metabolites produced by macrofungi have been published by De Silva et al. [165], Schüffler [166], and Chen and Liu [167].

The synthesis of the above-mentioned major and minor metabolites is achieved through solid and/or liquid-state fermentations of lignocellulosic waste and agricultural bioproducts by macrofungi with high dietary and/or medicinal properties, procedures that are presented in detail in the following section.

5. Liquid Mycelial Cultivation of Macrofungi: Methods and Strategies

Global production of mushrooms grew from 6.90 to 10.24 million metric tons from 2008 to 2017 [168]. There is a great range of nutraceutical, pharmaceutical, and novel food products containing macrofungi fruiting bodies or mycelium in Asia and the USA and a growing market in the rest of the world [118]. Solid-state cultivation of fruiting bodies requires several months, a large volume of substrate and space, and the quality of the final product can be difficult to control [169]. In contrast, the submerged culture of macrofungi promises a faster, more efficient, and more consistent alternative for the production of fungal bioactive metabolites [88]. Submerged cultivation is any strictly controlled and monitored biochemical process where a liquid nutrient substrate is enzymatically converted by microbial species [60]. Submerged cultivation in bioreactors is considered the best method for producing bioactive molecules with medicinal properties from macrofungi. Process monitoring, control, reproducibility, and the high product quality required for such production can only be achieved in bioreactors [75,170]. That is because submerged liquid cultivation allows for the use of analytical tools that can supply at-line and online data, and thus a better characterization and modeling of the bioprocesses has been achieved in the last decades [171,172]. Gregory et al. [173] performed the first reported submerged cultivations of macrofungi in 1966, where they tested hundreds of Basidiomycota in search of antitumor substances.

Table 2. Comparison of fruiting body and mycelium biomass content in bioactive compounds (%, w/w; mg/g; μg/g) in different mushroom species (adapted from Bakratsas et al. [118]).

Mushroom Species	Bioactive Compound	Fruiting Body	Mycelium
Pleurotus ostreatus	Crude protein	36%	40.1%
	Total lipids	2%	3%
	Non-polar lipids	1.50%	4%
	Fat	1.90%	2.6%
Pleurotus ostreatoroseus	Free sugars	26 mg/g	58 mg/g
	Total organic acids	212 mg/g	91 mg/g
Pleurotus spp.	Proteins	24.30%	23.3%
	Carbohydrates	27.70%	70.4%
	Fatty acids	1.60%	1.5%
Pleurotus sajor-caju	Soluble carbohydrates	19.55%	4.1%
	Proteins	36.36%	32.1%
	Total fat	2.30%	10.2%
Agaricus campestris	Proteins	46.30%	44.4%
	Fat	2.80%	2.8%
Agaricus bisporus	Proteins	45.90%	47.1%
	Fat	2.70%	5.8%
Lentinus edodes	Proteins	23–24%	17%
L. edodes	Lipids	4%	20%
L. edodes	Proteins	26.50%	52.8%
Cordyceps militaris	Proteins	18.47%	21.1%
	Total amino acids	18.57%	28.7%
Tuber sinense	Total umami amino acids	1.5 mg/g	9.0 mg/g
	Sterol content	1883 μg/g	5740.4 μg/g
Tuber indigum	Total umami amino acids	5.3 μg/g	12.6 μg/g
	Sterol content	2204.2 μg/g	6638.1 μg/g
Tuber aestivum	Total umami amino acids	7.1 mg/g	11.6 mg/g
	Sterol content	3239.9 μg/g	5792.5 μg/g

presents the content of different bioactive metabolites in the fruiting bodies of the mushroom compared to that of the mycelium of the same species. Several studies indicate that the cell wall composition of mycelium can be similar to that of the mushroom fruiting body [174], whereas Bakratsas et al. [118] show that in many cases, the mycelium contains more bioactive compounds than the carposome, e.g., carbohydrates in P. ostreatus (70.4 vs. 27.7%), lipids in L. edodes (20.0 vs. 4.0%), or sterols in Tuber sinense (5740.5 vs. 1883.0 μg/g). In addition, a study comparing the chemical composition of the fruiting bodies with the submerged cultured mycelium of C. militaris demonstrated that the mycelium had a higher content of polysaccharides, phosphorus, cordycepin, cordycepic acid, alanine, and vitamins B3 and B2 [175]. There are more studies that confirm that macrofungi can produce extraordinarily high amounts of EPS in a small period of time under submerged cultivation conditions [94]. The mycelium biomass of P. ostreatus produced in a batch stirred-tank bioreactor contained enhanced quantities of bioactive metabolites known to be found in fruiting bodies, thus proving that submerged cultivation can be a promising alternative [176].

Advantages of liquid mycelial cultivation of macrofungi [177,178,179].

A wide range of products from a wide range of fungi can be produced, while many products are produced best in submerged conditions. Additionally, there are the following advantages:

• Kinetic, heat, and mass transfer parameters can be estimated;
• Easy control and adjustment of the operating parameters;
• Very good reproducibility when the medium composition is characterized;
• Defined medium allows for easier purification of desired substances;
• High yield potential;
• Mixing and stirring allow for very good diffusion of nutrients;
• Temperature control is precise and easier because of the high-water content and stirring;
• Existing kinetic and transfer information of bioprocess can guide the design and operation of bioreactors;
• A Variety of online sensors are available, and more are being developed;
• Sensors allow for the automatic addition of the proper reagents to control the ongoing process;
• Easy manipulation of environmental conditions, growth factors, and nutritional requirements;
• Short overall time of cultivation compared to solid state cultivation of fruit bodies that takes several months, a large volume of substrate and space, and the quality is not always reliable.

Disadvantages of liquid mycelial cultivation of macrofungi [177,178,180].

The raw material must be sterilized because the high-water activity creates the risk of contamination. In addition,

• Crude medium ingredients may need processing and/or solubilization and characterization which increases the cost of the process.
• Downstream processing of the products requires removal of large water volumes and can be more expensive;
• Media dilution leads to lower volumetric productivity;
• High substrate concentrations may cause rheological problems;
• Gas transfer from the gas to liquid requires high air pressure and may be slow and limiting;
• Scaling up from flasks to bioreactors can create technical problems that need to be tackled, such as increased broth viscosity and sufficient oxygen supply;
• Submerged cultivation can be more demanding in energy, water, and labor compared to solid-state fermentation;
• Optimization is necessary.

Optimization of submerged cultivation to produce specific metabolites can be costly, difficult, and time-consuming, especially if the classical empirical process of “change one-factor-at-a-time” is used. It is highly advised to employ experimental design such as principal component analysis to efficiently optimize the process of submerged cultivation to achieve specific goals (increase EPS production) while studying several contributing factors at the same time. Experimental design approaches are valuable tools for analyzing complex and multivariate processes such as fungal growth in liquid mediums because the data are analyzed in a formulated model that enables confident predictions of how certain variables might affect the experiments while discriminating true effects from random variation. Fazenda et al. [68] claim that the biochemistry and physiology of the fungus involved should not be neglected, since they play an important role in metabolic mechanisms that might lead to better scientific understanding and faster optimization. Koutrotsios et al. [91] and Diamantopoulou et al. [23]’s studies pinpoint that the production of bioactive compounds by macrofungi exhibits high intraspecific variability in the same cultivation conditions.

Liquid cultures are divided into static cultures, agitated cultures, which are the most common, and two-stage cultures. Two-stage cultures involve a classic agitated culture as a first step, which then continues as a static culture. Researchers have reported hyperproduction of carotenoids, cordycepin, and EPS after optimization of this strategy [177,178,179,180,181,182,183]. There are three main strategies for executing submerged cultivation. Batch culture is the simplest and most traditional technique; all the medium components and fungus are added at the start of cultivation. Batch culture is a closed system and is characterized by a long lag phase, low productivity, and a continuous change in nutrient concentrations over time [68]. Fed-batch is a semi-closed system, where nutrients and substrate are added aseptically as the fungus is consuming the nutrients. Most of the large-scale industrial submerged cultures are performed with this method because it promotes and prolongs metabolite production and maximizes substrate utilization. Continuous culture is a constant process that follows a batch culture until the fungus grows sufficiently, after which nutrients are continually added to the bioreactor and material is removed so that the concentration of nutrients remains the same [184].

Basic research on the behavior of macrofungi in submerged cultivation is first performed in flasks in shaker incubators, followed by laboratory-scale bioreactors of various volumes and geometries, equipped with a multitude of sensors, functions, and automation. Small laboratory bioreactors serve as the first step towards the scaling up of a bioprocess that is tested for industrial application. The most common bioreactor designs used for liquid fungal cultures are stirred-tank bioreactors, which incorporate an agitator or impeller for heat and mass transfer, aeration, and mixing and airlift bioreactors which utilize inserted gas from an airlift pump for mixing and aeration [68]. Filamentous fungi are morphologically complex and versatile microorganisms exhibiting different growth morphology throughout their life cycle. Macrofungi in submerged cultures can form filamentous growth, pellet growth, or a mixture of both. The fungal morphology and metabolite production affects the rheological properties and is simultaneously affected by the type and force of mixing applied in the bioreactor, which is one of the main challenges in industrial bioprocesses [185]. A study comparing different mushroom fungi cultivated in static and agitated liquid cultures reports that most fungi form thick mycelial layers, of varying fibrosity, on the surface of the liquid substrate under static conditions. On the other hand, agitated cultures result in the production of spherical mycelial pellets, of differing sizes depending on the fungi species, with the exception of G. applanatum, which produced compact and very dense mycelial clumps in static flasks and loose hyphal masses in agitated culture [96].

The principal factors affecting the process and products of submerged cultures are the subject of many studies. In short, physical factors are temperature, aeration, agitation, foaming, and fluid rheology. The design of the vessel and stirring type can have a big impact on those factors, and this has led to the building of numerous different types of bioreactors for lab and industrial use. Chemical factors are the initial pH and the pH correction during the process, dissolved oxygen, substrate composition, and more specifically: carbon and nitrogen sources and the C/N ratio [177], vitamins, minerals, and other additives such as surfactants and by-product formation [68]. Biological factors are inoculum size, age and type, and the species and strain of the fungus [186].

Lighting is another important factor that may contribute to increased productivity in macrofungi cultivation. Controlling the lighting intensity in C. militaris cultures resulted in increased yield and production of cordycepin, mannitol, and polysaccharides [187]. Another study examined the effect of blue and red LED lights on submerged cultures of C. militaris and concluded that red light enhances biomass production while blue light gives the highest EPS production [94]. Another group of researchers achieved maximum production of cordycepin in submerged cultures when they combined red:blue LED lights and extended the illumination time to 20 h/day [188].

Stimulatory agents such as fatty acids, organic solvents, and surfactants have been employed in submerged cultures of macrofungi to improve the production of biomass and EPS. Tween 80 added at the late stage of the exponential growth phase contributed to a 51.3% increase in biomass and a 41.8% increase in EPS [189]. The addition of vegetable oils to submerged cultures can increase the production of desired compounds. Tang et al. report that the low solubility of vegetable oils in the substrate of C. militaris inhibits the carbon catabolite repression and allows for an increased rate of secondary metabolites biosynthesis [190]. Supplementation of oleic acid in submerged cultivation of I. obliquus doubled the growth of the mycelium, while the use of a fungal elicitor stimulated mycelia growth and increased the betulinic acid accumulation by 429.5% [191]. Elicitors are widely used to boost the production of secondary metabolites in plants grown in vitro [192], but they remain relatively unexplored in the submerged cultivation of macrofungi. Various elicitors have been used for achieving the overproduction of desired triterpenoids from G. lucidum submerged cultures [193], but not much literature exists on the use of elicitors with other species.

6. Bioconversion of Wastes by Submerged Fermentation of Macrofungi

Table 3. Submerged cultivation of macrofungi using agri-food residues to produce bioactive compounds.

Sector	Substrate	Mushroom Species	Culture Conditions	Bioactive Compounds	Publication Year	Reference
Fruits and vegetables	Mandarin peels and tree leaves	Pleurotus dryinus	Shake flasks	Cellulase (47.7 U/mL), xylanase (71.7 U/mL), laccase (6493 U/mL), MnP (83 U/mL)	2006	[194]
	Mandarin peels and tree leaves	P. dryinus, P. ostreatus, P. tuberregium	Shake flasks	Cellulase (62.3 U/mL), xylanase (84.1 U/mL), laccase (4103 U/mL)	2008	[195]
	Mandarin peels	Coculture of Irpex lacteus and Schizophyllum commune	Shake flasks	Cellulase (7 U/mL), endoglucanase (142 U/mL), xylanase (104 U/mL), β-glycosidase (5.2 U/mL)	2017	[196]
	Loquat kernel	Morchella esculenta	Shake flasks	EPS (5.4 g/L)	2011	[197]
	Apple pomace	P. sapidus	Shake flasks	Protein (25.4%)	2019	[24]
		Agrocybe aegerita		Protein (18.6%)
		Lentinula edodes		Protein (20.4%)
		P. sajor-caju		Protein (14.6%)
		P. salmoneostramineus		Protein (20.9%)
		Stropharia rugosoannulata		Protein (12.3 %)
	Apple pomace	Agaricus brasiliensis	Shake flasks	Sterols (1053 μg/g) Ergosterol (323 μg/g)	2015	[198]
	Plantain peels	P. pulmonarius	Shake flasks	EPS	2020	[199]
	Mango peels			EPS
	Pineapple peels			EPS
	Kiwi peels	Cerrena unicolor	Shake flasks	Lectins	2011	[200]
	Banana peels combined with groundnut cake	Calocybe indica	Shake flasks	Biomass (5.3 g/L), protein, glucans, minerals, flavonoids	2018	[201]
	Sea buckthorn press cake	Inonotus obliquus	Shake flasks	EPS (0.67 g/L)	2021	[202]
	Yam dextrose	Pleurotus spp.	Shake flasks	EPS (0.54 g/L), Protein (55%)		[203]
		P. flabellatus		EPS (0.38 g/L), Protein (31%)
		Ganoderma lucidum		Protein (34%)
		Laetiporous sulphureus		Protein (39%)
	Tomato pomace	C. versicolor	Shake flasks	Polygalacturonase (1427 U/L)	2008	[204]
	Pectin			Polygalacturonase (3207 U/L)
Olive oil	Olive mill wastewater	Cerrena unicolor	Shake flasks	Laccase (112.8 U/mL)	2018	[205]
		P. οstreatus		Laccase (23.4 U/mL)
		G. lucidum		Laccase (18 U/mL)
		Pycnoporus coccineus		Laccase (18 U/mL)
		Trametella trogii		Laccase (29.7 U/mL)
		Trametes versicolor		Laccase (10.3 U/mL)
	Olive mill wastewater	T. versicolor	Shake flasks	Lacasse	2011	[206]
	Olive mill wastewater	Trametes ochracea	3 L Bioreactors	Laccase (14,967 IU/L) MnP (16,856 IU/L)	2009	[207]
	Olive mill wastewater	G. resinaceum	Static flasks	EPS (0.79 g/L), IPS (5.2 g/L)	2020	[92]
	Olive mill wastewater	P. citrinopileatus	2.5 L Lab scale bioreactor	Glucans (18.8 g/L)	2016	[208]
		G. lucidum		Glucans (19.7 g/L)
		P. ostreatus		Glucans (21.02 g/L)
	Olive mill wastewater	L. edodes	Shake flasks	β-1,3-Glucan synthase	2005	[60]
		P. ostreatus		β-1,3-Glucan synthase
	Olive mill wastewater and glycose	P. pulmonarius	Shake flasks	Biomass (32.76 g/L), IPS (4.38 g/L), lipids (2.85 g/L)	2022	[27]
Winery, Brewery, Distillery	Grape marc	G. lucidum	5 L Lab scale bioreactor	Protein (19.71%)	2016	[209]
	Grape marc	L. edodes		Protein (19.96%)
	Grape marc	P. ostreatus		Protein (17.62%)
	Grape pomace hydrolysate	P. ostreatus	Shake flasks	Biomass (0.5 g/g of dry substrate), laccase, endoglucanase	2019	[210]
		P. pulmonarius		Biomass (0.54 g/g of dry substrate), laccase, endoglucanase
	Brewery wastewater	P. ostreatus	Shake flasks	Biomass (1.78 g/L)	2017	[211]
		T. versicolor		Biomass (1 g/L)
	Brewer’s spent grains	Hericium erinaceus	Shake flasks	Biomass, erinacine C (175 mg/g)	2016	[212]
	Brewer’s spent grains	T. versicolor	Shake flasks	Laccase (560 U/L), polyphenols	2018	[213]
	Vinasse and cotton gin waste	T. versicolor	Shake flasks, 3.3 bioreactor, static tray	Laccase (5005.55 U/L)	2019	[214]
	Malt	A. brasiliensis	Shake flasks	Sterols (2659 μg/g), Ergosterol (1267 μg/g)	2015	[198]
Coffee	Spent coffee grounds	Cordyceps sinensis	Shake flasks	Glucosamine (140.3 μg/mL), Antioxidant activity (ABTS IC50, 0.93 mg/mL)	2021	[215]
	Spent coffee grounds hydrolysate	Flammulina velutipes	Shake flasks	Biomass, improved antioxidant activity	2018	[216]
		P. ostreatus		Biomass, improved antioxidant activity
		C. militaris		Biomass, improved antioxidant activity
		P. linteus		Biomass, improved antioxidant activity
Dairy	Deproteinized whey	P. sajor-caju	Shake flasks	Protein	2005	[217]
	Whey permeate	L. edodes	Shake flasks	Biomass (2 g/L)	2006	[218]
	Yogurt whey	H. erinaceus	Shake flasks	β-glucans (1.7 g/L)	2012	[219]
	Whey powder	P. djamor	Shake flasks	β-glucans, ergosterol	2019	[220]
	Cheese whey	T. versicolor	Shake flasks	Biomass (26 g/L), protein 19.8%	2022	[221]
Sugar industry	Beet molasses	M. rotunda	Static and shake flasks	EPS (3.94 g/L), IPS (3.34 g/L)	2021	[28]
		M. vulgaris		EPS (3.93 g/L), IPS (4.24 g/L)
		M. conica		EPS (3.18 g/L), IPS (4.8 g/L)
		M. vulgaris	Static flasks	EPS (1.17 g/L), Lipids (2.32 g/L)	2020	[25]
		M. elata		EPS (1.61 g/L), Lipids (1.93 g/L)
		Tuber aestivum		EPS (2.06 g/L), Lipids (5.91 g/L)
	Sugarcane bagasse hydrolysate	G. lucidum	Static flasks	Ganoderic acid (1.1 mg/L)	2019	[222]
	Blackstrap molasses	T. versicolor	Shake flasks	Biomass, ergosterol	2019	[223]
		Polyporus brumalis		Biomass, ergosterol
Biodiesel	Bio-diesel derived glycerol	L. edodes	Shake flasks	Lipids (0.1 g/g of biomass)	2010	[224]
	Bio-diesel derived glycerol	Τ. aestivum	Static flasks	EPS (0.54 g/L)	2020	[25]
Cereals, legumes, nuts	Expired rice cereal hydrolysate	L. edodes	Static flasks	EPS (1.75 g/L), Lipids (0.28 g/L)	2020	[25]
		M. vulgaris		EPS (1.67 g/L), Lipids (0.87 g/L)
		M. elata		EPS (2.17 g/L), Lipids (0.46 g/L)
		T. aestivum		EPS (1.80 g/L), Lipids (6.51 g/L)
	Black rice bran hydrolysate	L. edodes	Shake flasks	Bioprocessed polysaccharide	2013	[225]
	Expired wheat cereal hydrolysate	L. edodes	Shake flasks	EPS (0.98 g/L), Lipids (0.45 g/L)	2020	[25]
		M. vulgaris		EPS (0.59 g/L), Lipids (0.60 g/L)
		M. elata		EPS (1.34 g/L), Lipids (1.73 g/L)
		T. aestivum		EPS (1.25 g/L), Lipids (3.06 g/L)
	Wheat Bran	A. chaxingu	Shake flasks	Oligosaccharides (35.4 μM)	2013	[226]
	Wheat bran	M. esculenta	Shake flasks	EPS (2.91 g/L)	2010	[227]
	Wheat bran	P. ostreatus	Shake flasks	Laccase (12.124 U/L)	2014	[228]
	Wheat straw	I. obliquus	Shake flasks	Flavonoids (ECG 374.1 mg/g, EGCG 447.2 mg/g), Antioxidant activity (DPPH IC50 30.96 mg/L)	2021	[229]
	Wheat straw	G. lucidum	Shake flasks	Ganoderic acid (1.7 mg/L)	2019	[222]
	Starch processing waste	C. militaris	Shake flasks	Biomass (1.91 g/L/day)	2016	[230]
	Soybean residue	M. esculenta	Shake flasks	EPS (36.22 g/L)	2013	[231]
	Soybean curd residues	F. velutipes	Shake flasks	EPS (59.15 mg/g)	2012	[232]
	Groundnut shell	P. pulmonarius	Shake flasks	EPS (5.6 g/L)	2020	[199]
	Coconut coir			EPS (3.6 g/L)
	Walnut husk			EPS (2.1 g/L)
	Walnut leaves	C. unicolor	Shake flask	Protein (1.44 g/L), Lectins	2011	[200]
	Walnut pericarp	C. unicolor	Shake flasks	Protein (1.78 g/L), Lectins
Animal industry	Ram Horn Hydrolysate	A. bisporus	Shake flasks	Protein (47.1%), EPS	2004	[233]
	Chicken feather hydrolysate	M. esculenta	Shake flasks	EPS (4.60 g/L)	2011	[197]

sums up the findings of 52 publications in which 56 species of macrofungi were grown in substrates containing 47 different agro-industrial wastes and residues in submerged cultivation and successfully produced various fungal products.

Fruit and vegetable production creates streams of residues from peels, leaves, kernels, and pomace. Pleurotus sp. produced significant amounts of the enzyme laccase (6493 U/mL) when cultured in substrates containing mandarin peels [194], while the Pleurotus mycelial biomass protein content was 25.4% and 55% when apple pomace [24] and yam dextrose [202] were used as substrates, respectively. M. esculenta was utilized for the valorization of loquat kernels through the production of considerable quantities of EPS (5.4 g/L) [197], in contrast with Pleurotus sp., which produced much smaller quantities of EPS (0.54 g/L) when cultured on yam dextrose [202].

Olive mill wastewater with various pretreatments and dilutions has been exploited for the production of substantial amounts of laccase by T. ochracea (1490 U/mL) [206], C. unicolor (112.8 U/mL), and P. ostreatus (23.4 U/mL) [204]. Satisfying amounts of glucans were produced by P. ostreatus (21 g/L), G. lucidum (19.7 g/L), and P. citrinopileatus (18.8 g/L), cultured in a 2.5 L bioreactor [207]. According to a recent study, P. pulmonarius produced an impressive 32.76 g/L of biomass that was rich in intracellular polysaccharides (4.38 g/L), indicating that OMW is a promising substrate for the biosynthesis of high-added value bioactive compounds [27].

Winemaking residues such as grape marc have been valorized for the production of fungal protein by L. edodes (19.9%) and G. lucidum (19.7%) [209]. The beer industry generates enormous amounts of lignocellulosic residues known as brewer’s spent grains (BSG), which are suitable to produce mycelial biomass and enzymes. H. erinaceus cultures in BSG produced 7.7 g/L of biomass rich in the valuable secondary metabolite erinacine C (175 mg/g) [212], and T. versicolor generated high laccase activity (560 U/L) and biomass with total polyphenols content of 3% [213]. Spent coffee grounds have been valorized in liquid cultures of C. sinensis, resulting in biomass with satisfying glucosamine content (140.3 μg/mL) and antioxidant activity [215].

Side streams from the sugar industry, cereals, legumes, and nuts value chains are also very promising feedstock materials for liquid cultures of macrofungi. Indicatively, P. pulmonarius produced 5.6 g/L of EPS in groundnut shells and 2.1 g/L in walnut husks [199], whereas M. rotunda, M. vulgaris, and M. conica cultures in beet molasses generated 3.94, 3.93, and 3.18 g/L EPS and 3.34, 4.24, and 4.8 g/L of IPS, respectively [28]. M. elata cultured in expired wheat cereal hydrolysate generated 1.34 g/L EPS [25], while M. esculenta generated 2.91 g/L in wheat bran. The lipid content of the mycelial biomass produced by Morchella species in beet molasses and cereal side streams is generally low and ranges from 0.28 g/L up to 5.91 g/L [25,28].

Extensive research has been conducted on the solid-state cultivation of macrofungi using substrates containing agro-industrial wastes and residues [16,18,26,45,47]. Solid wastes that have been successfully used in solid-state cultivations can be ground into powder in order to be used in submerged liquid fermentation and exploit the advantages of liquid fermentations [29,60,199]. Industrial-level application of submerged cultures utilizing agro-industrial residues to produce bioactive metabolites by using macrofungi can be an eco-friendly solution for waste management and at the same time produce added revenue [199]. Successful adoption of such biotechnological applications will depend on the initial cost and the expected return on investment. Applied research is crucial for the optimization of liquid/submerged cultures, increased product yields, and the creation of novel biotechnological production systems that will make such solutions attractive to the industry [88].

7. Challenges and Future Perspective

Submerged liquid cultivation is mostly investigated in flasks and lab-scale bioreactors, and there is not enough research on the behavior of such systems in upscaled industrial applications for the commercial production of fungal biocompounds. There is not enough knowledge of how macrofungi and the substrates might behave on such a scale. Although Basidiomycetes and Ascomycetes are highly valued as sources of bioactive compounds, only a few species have been thoroughly studied, and many still remain chemically unexplored [152]. Practical application of the biotechnological processes using mycelial cultures depends on the development of industrial technologies for large-scale submerged cultivation and downstream processing to ensure commercial success. Physiological and biochemical mechanisms that regulate the biosynthesis and secretion of bioactive substances as well as the techno-functional properties of fungi and their products need to be further studied and understood if optimized production is to be achieved [17]. Of course, the existing research on yeast and mold behavior in large-scale fermentations can provide useful insights and strategies for macrofungi liquid cultures. Furthermore, the use of engineering tools such as mathematical models and optimization techniques has not been properly utilized and integrated into the process of submerged cultures yet. Tools such as computational fluid dynamics and the implementation of novel sensors in the bioreactors hold promise for calculating how the morphology of broth rheology and mass transfer affect the overall process [172]. Deep learning, machine learning, and the use of AI in general are constantly gaining popularity in all areas of industrial production and will certainly have an impact on the improvement of secondary metabolite biosynthesis from fungi [234]. Molecular biology, genetic engineering, bioinformatics, next-generation sequencing, and multi-omics have an important role in increasing the production potential of macrofungi on a commercial scale [235,236].

8. Conclusions

The medicinal use of mushrooms has a very long tradition in Asia and has been gaining ground in western countries. During the last decades, products containing macrofungi mycelium in the form of capsules, powders, and tinctures are being utilized for their therapeutic effects. Clinical data, although incomplete, support the potential beneficial effects of fungal compounds in the prevention and treatment of many life-threatening diseases. Fungal mycelium can find many uses in food, pharmaceuticals, and nutraceuticals. The submerged/liquid culture of macrofungi is a scalable cost-effective way to produce added-value secondary, targeted, and massive metabolites in a short amount of time, not to mention that mycelia and culture media many times give better results than the fruiting bodies. In addition, it seems to be the only way to receive the biomass and relevant metabolites from wild-type mushrooms that cannot be cultivated artificially. Utilizing liquid cultures to produce mycelium with high content of potent bioactive compounds could prove an efficient green way to valorize the ever-present abundant and harmful agri-food residues.