Fungal Proteins: Sources, Production and Purification Methods, Industrial Applications, and Future Perspectives

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Single-cell protein is a type of protein derived from fungi and it has gained attention as a meat substitute due to its high protein content, low fat content, and specific texture. However, this type of protein can be used in the development of functional foods as well as additive to high-protein products, protein bars, shakes, snacks, dairy alternatives, etc.

Abstract

In recent years, there has been an increasing demand for new sources of protein, both for human and animal nutrition. In addition to alternative sources of protein, such as algae or edible insects, protein obtained from yeast and mold biomass is becoming more and more important. The main fungal protein producers are the yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida utilis, Yarrowia lipolytica, and the molds Fusarium venenatum, Aspergillus oryzae, and Monascus purpureus. The production of fungal protein has many advantages, including the ability to regulate the amino acid composition, high protein content in dry matter, the possibility of production in a continuous process, independence from climatic factors, and the possibility of using waste substrates as ingredients of media. One of the disadvantages is the high content of nucleic acids, which generates the need for additional purification procedures before use in food. However, a number of enzymatic, chemical, and physical methods have been developed to reduce the content of these compounds. The paper presents the current state of knowledge about fungal producers, production and purification methods, the global market, as well as opportunities and challenges for single-cell protein (SCP) production.

1. Introduction

The global population will reach almost 10 billion people by the year 2050, especially in regions such as Africa, Latin America, and Asia [1]. As the world’s population continues to grow, so is the need for alternative, sustainable and low-cost protein sources. The food industry is failing to meet the increasing demand for dietary protein, solely focusing on traditional agricultural practices. Those practices are generally considered to have a negative impact on the environment. Animal-derived proteins are closely linked to meat production, which is associated with a significant emission of greenhouse gases [2], and a considerable impact on the climate and water footprint [3,4,5].

Simultaneously, social awareness is becoming more widespread and there are more and more ‘conscious consumers’, who have decided to resign themselves from consuming meat, vegetarians [6], or even completely resign from animal products, known as veganism [7]. If a vegetarian or vegan diet is not well balanced it may cause lack of protein and exogenic amino acids, iron, zinc, fatty-acids, and vitamins deficiencies in human body, and it may become a risk factor for different diseases [8]. Therefore, to meet the demand for protein-rich food and finding vegan alternatives that will be reliable and sustainable is crucial [9]. Introducing microbial protein or single-cell protein into the human diet may be one possible solution [10]. The term single-cell protein (SCP) refers to the protein, which was extracted from dried microbial biomass [11]. Microorganisms considered for the production of SCP are divided into molds, yeast, bacteria, and algae (Figure 1) [12]. In 1966, Carol L. Wilson introduced the term “single-cell protein” (SCP). Initially, microbial protein was used as an animal feed. During the First World War, among German soldiers, yeast from the species Candida utilis was used in soup and sausage production. Currently, microbial protein may be used in the food industry as a source of protein in human nutrition [13].

Fungi are traditionally used in the food industry in fermentation processes. Among the products of fermentation with the use of yeast, the following products can be mentioned: alcohol, glycerol, and carbon dioxide. Also, citric acid, gluconic acid, antibiotics, vitamin B₁₂, and riboflavin are some of the products obtained from mold fermentation [14,15,16]. In addition to the traditional uses of these microorganisms, a significant increase in the interest of using microbial protein as a substitute for plant-based or animal-derived proteins can be observed [11]. In SCP production, it is crucial to provide a high protein content and rich amino acid profile to ensure the nutritional value of the finished product [13]. From a nutritional point of view, there are certain amino acids that are essential to the profile of a single-cell protein, such as arginine, histidine, lysine, leucine, isoleucine, phenylalanine, methionine, threonine, and valine. These amino acids are obtained on the level, which are required by FAO standards. Even though a single-cell protein is rich in lysine, it is still poor in methionine. Those amino acids are crucial, due to being the limiting amino acids [17,18,19,20].

This review will discuss the prospects and future applications of fungal proteins in the food industry as an alternative protein source as well as the opportunities and challenges to be faced by the use of SCP in the industry.

2. Characteristics of the Most Important SCP Producers

Nowadays, there are several fungi strains accepted in the EU for the production of SCP. Among them are Saccharomyces cerevisiae, known as Brewer’s yeast, or budding yeast, Quorn, the mycoprotein of the microfungus F. venenatum, successfully marketed as a meat alternative first in the UK and later on in EU countries. Furthermore, the biomass of Yarrowia lipolytica has been restricted to food supplements and is authorized via the Regulation (EU) 2017/2470. According to Regulation (EU) 2015/2283, microbial protein is considered a novel food [20,21]. Generally, microorganisms may be used in single-cell protein production, which are mentioned in the QPS list notified to EFSA, including the yeasts such as Debaryomyces hansenii, Candida kefyr, Wickerhamomyces anomalus, C. utilis, Hanseniaspora uvarum, Schizosaccharomyces pombe, Kluyveromyces lactis, and the previously mentioned S. cerevisiae and Y. lipolytica [22]. Representatives of the most important fungal species used to obtain SCP are described below, and their amino acid compositions are compared in Table 1.

2.1. Saccharomyces cerevisiae

S. cerevisiae is the cheapest and best source of protein obtained from cultivation, low-cost materials, or waste. Furthermore, the protein obtained from them is an attractive alternative to various types of meat or fish meals. During the beer manufacturing process, up to 49% of protein is obtained, while processing protein hydrolysates from chicken by-products yields 54% of the resulting protein content. For years, they have been used in the food industry, but also as a model object of research in laboratories. Because of this, they have been recognized by the U.S. Food and Drug Administration as safe for its intended use and have been granted GRAS status [23]. Their protein biomass is not only an ideal source of structural and enzymatic proteins, but also large amounts of endogenous amino acids (e.g., alanine, cysteine, glycine) and essential amino acids, especially lysine and threonine, which make S. cerevisiae biomass suitable for use as a supplement in cereals [24]. Protein derived from their biomass is able to replace allergenic protein sources such as dairy products [23,24,25,26,27].

2.2. Kluyveromyces marxianus

K. marxianus is a homothallic, haploid, hemi-ascomycetous, and thermotolerant yeast strain, naturally available in fruits and fermented dairy products. In addition, it has the ability to grow on various carbon sources, including agro-industrial wastes, among others. This feature of K. marxianus may be used in the production of protein-enriched livestock feeds. It is categorized as GRAS by the Food and Drug Administration (FDA). The possibility of using SCP from K. marxianus for the production of edible films, which can be used to extend the shelf life of cheese, has also been demonstrated. Some isolates have potentially probiotic effects. This yeast is capable of synthesizing β-galactosidase for industrial applications. The optimal parameters for growing conditions in relation to the highest protein yield for K. marxianus cultivated are a pH ranging from 4.4 to 5.8, and a temperature of 30 °C. K. marxianus biomass does not contain lysine and sulfur-containing amino acids [28,29,30,31,32,33].

2.3. Candida utilis

One of the best-known producers of microbial protein is C. utilis yeast. The Candida species has GRAS status, which means it has been recognized by the U.S. Food and Drug Administration as safe for its intended use. A protein preparation extracted from the yeast C. utilis has found use in feed additives. In addition, it can be used as a flavoring agent in vegetarian foods. C. utilis biomass has a high content of glutamic acid, so this species has been used to replace monosodium glutamate serving as a flavor enhancer. In addition, C. utilis biomass is rich in essential amino acids (especially lysine), B vitamins (riboflavin, folic acid, nicotinic acid), ergosterol (a precursor to vitamin D), and metal ions [19,34,35,36,37].

C. utilis shows the ability to utilize several types of substrates, including bamboo wastewater, rice bran, and molasses. By cultivating yeast in waste-containing media, it is possible to obtain valuable microbial protein, as well as effectively treat wastewater for total organic carbon and total nitrogen. The chemical oxygen demand reduction efficiency of the above wastewater can reach 48.3%, with the production of 0.68 g of protein/1 dm³ of wastewater. With its high protein synthesis ability, C. utilis can make good use of nitrogen and other organic substances in waste biomass to simultaneously reduce the environmental damage of wastewater and produce microbial protein [38].

2.4. Yarrowia lipolytica

Y. lipolytica is a non-pathogenic, oleaginous, strictly aerobic ascomycetous yeast [23]. This organism’s optimal growing conditions are mild acidic pH values (around 5.5) and temperatures varying between 28 and 30 °C. It is observed that the lipid-free biomass production, when the increase in the protein concentration occurs during the degradation of storage lipid bodies, depends on the nitrogen and magnesium level in the cultured medium. The amino acid profile of Y. lipolytica yeast is characterized by a distinguished lysine content. The Y. lipolytica biomass contains abundant protein in the range between 30.5 and 56.4%. The lowest amino acid levels are obtained when the yeast is grown on rye straw and rye or oat brans. Furthermore, the use of fatty substrates such as crude, industrial glycerol, or biofuel waste enhance the quantities of amino acids. Moreover, Y. lipolytica’s protein biomass is recognized as gluten-free [39]. The protein biomass of this yeast is also a good source of B-complex vitamins, such as vitamin B₁₂, as well as vitamin E. In nature, Y. lipolytica is often isolated from dietary products such as blue cheeses, milk, yoghurt, kefir, butter, cream, and meat products, to name a few. This yeast has a wide range of biotechnological applications through the synthesis of enzymes, e.g., lipolytic enzymes, biosurfactants, organic acids, aromatic compounds, and melanins. Several production processes based on this yeast were classified as GRAS by the Food and Drug Administration (FDA) [40,41,42,43,44].

2.5. Fusarium venenatum

F. venenatum is the most well-known mycoprotein producing fungi on the market. In the 1960s, the company Rank Hovis McDougall, Marlow, UK, investigated and compared 3000 different fungi, searching for a cheap and easy way to produce a meat substitute. The time taken to thoroughly examine the properties and structure of the mycoprotein makes it the most tested food product on the European market [45]. The mycoprotein produced by F. venenatum has been approved by the Ministry of Agriculture, Fishers and Food in the United Kingdom in 1984. Since then, it has been still available on the market under the name Quorn [46,47,48].

During the fermentation process, a relatively high percentage of protein can be achieved, within the range of 65–76%. The amino acid profile of F. venenatum biomass includes almost all the essential amino acids, lacking only the proline and aspartic acid. The sulfur-containing amino acids are relatively low. The mycoprotein produced by the fungi contains fiber, composed of one third chinin and two thirds beta-1,3 and 1,6 glucans originating from the myceliac walls. The lipid content of the material is usually measured between 2 and 3.5%. The fatty-acid content in the fungi biomass is much more similar to one of a plant, than the animal fat [49,50].

2.6. Aspergillus oryzae

A. oryzae is widely used and researched in the food and feed industries. For years, it has been used in Asian countries such as China and Japan to make beverages, including sake, vinegar, and food such as Koji, Oncom, and miso, or to ferment soybeans. Through its widespread use, it has been granted a GRAS status like many species in the genus Aspergillus [51]. A. oryzae has the ability to grow on a variety of substrates including wastewater [52] or high-fat dairy waste, where it is capable of degrading lipids to glycerol and fatty acids. The degradation produces large amounts of palmitic and myristic acid, which are most likely metabolized or stored in A. oryzae cells [53]. Its biomass produces large amounts of protein, such as on organosolv-pretreated brewers’ spent grain where the amount of protein produced reaches 44.8%. This protein can provide an alternative source of it in feed and food (such as soybean meal in fish feed) [54]. Their biomass is also a source of significant amounts of cell wall components, which act to stimulate the immune system [52], and elements such as calcium, potassium, sodium, magnesium, and sulfur [51].

2.7. Monascus purpureus

M. purpureus is a fungi mostly known for its ability to produce different pigments, ranging in color from bright yellow to deep red. It has been used as both coloring and flavoring agents in food and beverages, as in the production of red yeast rice and rice wine [55]. The addition of M. purpureus into unfermented heat-denatured soybean meal results in an increase in the amino acid content [56]. This observation indicates that the fermentation of Monascus results in the production of various amino acids, such as aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline, respectively. The amino acid content in the product is high, and the product has desirable nutritional value. A comparable change could be observed during the fermentation of rice [57] by M. purpureus. The fermented product contains more amino acids, such as L-methionine, L-serine, L-glutamic acid, L-phenylalanine, L-isoleucine, L-valine, L-alanine, L-arginine, and glycine. The antioxidant activities of the product similarly increased from 3.06 to 9.79%. The increased production of proteins observed during the fermentation could be a result of an increase in the extracellular protein production [55,56].

Table 1. Amino acids composition of SCP from C. utilis, F. venenatum, S. cerevisiae, A. oryzae, Y. lipolytica, K. marxianus.

	[g/100 g] of SCP
Amino Acids	Candida utilis [58]	Fusarium venenatum [50]	Saccharomyces cerevisiae [59]	Aspergillus oryzae [51]	Yarrowia lipolytica [23]	Kluyveromyces marxianus [60]
Alanine	7.59	2.41	6.89	2.37	8.00	3.63
Arginine	4.82	7.12	4.02	1.99	4.80	6.03
Cystine	10.16	2.11	1.24	0.28	1.10	1.11
Glycine	3.92	3.50	5.23	1.72	4.60	2.67
Histidine	3.78	7.22	2.78	0.75	2.60	2.66
Isoleucine	5.28	1.51	4.63	1.38	4.40	6.21
Leucine	6.73	1.90	8.75	2.46	6.80	9.57
Lysine	5.81	2.60	6.73	2.14	7.00	8.15
Methionine	1.67	4.21	3.12	0.57	1.20	2.48
Phenylalanine	4.62	3.01	5.57	1.42	4.00	5.61
Threonine	4.62	3.31	6.09	1.69	4.80	5.08
Valine	6.05	2.91	5.34	1.72	5.30	5.30

Table 1. Amino acids composition of SCP from C. utilis, F. venenatum, S. cerevisiae, A. oryzae, Y. lipolytica, K. marxianus.

	[g/100 g] of SCP
Amino Acids	Candida utilis [58]	Fusarium venenatum [50]	Saccharomyces cerevisiae [59]	Aspergillus oryzae [51]	Yarrowia lipolytica [23]	Kluyveromyces marxianus [60]
Alanine	7.59	2.41	6.89	2.37	8.00	3.63
Arginine	4.82	7.12	4.02	1.99	4.80	6.03
Cystine	10.16	2.11	1.24	0.28	1.10	1.11
Glycine	3.92	3.50	5.23	1.72	4.60	2.67
Histidine	3.78	7.22	2.78	0.75	2.60	2.66
Isoleucine	5.28	1.51	4.63	1.38	4.40	6.21
Leucine	6.73	1.90	8.75	2.46	6.80	9.57
Lysine	5.81	2.60	6.73	2.14	7.00	8.15
Methionine	1.67	4.21	3.12	0.57	1.20	2.48
Phenylalanine	4.62	3.01	5.57	1.42	4.00	5.61
Threonine	4.62	3.31	6.09	1.69	4.80	5.08
Valine	6.05	2.91	5.34	1.72	5.30	5.30

3. Bio-Utilization of Waste from the Agri-Food Industry during SCP Production

Due to population growth, the demand for food increases, and as a result, the amount of agri-food waste (FW) increases. Poor waste management may contribute to the formation of toxic compounds, providing a basis for the development of pathogens or constitute generally understood environmental pollution. Waste from the agri-food industry, e.g., peels, seeds, slaughterhouse wastewater, dairy factory wastewater, and edible oil factory wastewater, are rich in organic, bioactive compounds [61,62]. Food producers find a number of applications for FW, using them in the production of feed, fertilizers, as a substrate for the synthesis of many chemical compounds (e.g., citric acid, xanthan gum) or as a source of dyes, enzymes, and hydrocolloids [63,64].

One of the solutions to the increasing production of food wastes is to use it as a substrate for the production of SCP. The most commonly used waste for this purpose is waste from fruit and vegetable processing. However, it can also be liquid waste, e.g., from the dairy industry. Fruit and vegetable waste is an excellent source of bioactive compounds that are necessary for the growth of microorganisms growth. An important aspect that determines the use of waste as a compound of medium is its composition. Microorganisms choose carbohydrates as a source of carbon and energy. Potato peels, beetroot pomace, and carrot pomace, rich in carbohydrates, work well as a component of the fermentation medium for the production of SCP. The second important compound for biomass production is nitrogen. It has been shown that the addition of ammonium sulfate, ammonium nitrate, or sodium nitrate to the substrate has a beneficial effect on the growth of biomass [27,65]. In addition, among compounds that promote the production of SCP, compounds that inhibit the growth of microorganisms should be taken into account. Essential oils contained in citrus peels have proven antimicrobial properties [66], so the use of lemon, orange, or grapefruit waste may be associated with lower biomass production efficiency. The first step, after taking the industrial waste, is cleaning it with running water to remove all contaminants and pesticides (in the case of waste from the juice processing industry, Figure 2). After cleaning, the waste is dried separately at 50 °C for 48 h. The dried material is prepared in a grinder followed and sieved through a 80 mesh sieve [27]. The medium is prepared from a properly obtained powder (e.g., obtained from banana or potato peels) by adding a certain quantity of 10% hydrochloric acid and the heating it at 100 °C for an hour. After this period, it is necessary to filtrate the solution through Wattman filter paper and dilute it with distilled water, before bringing the solution to pH 4.5 using 2.5 M sodium hydroxide [67].

Mensah and Twumasi [68] conducted research on the production of SCP using pineapple waste. The raw materials for the production of the medium were peelings, stems and other unwanted parts of fruit. The waste was rinsed with distilled water, cut into smaller parts, pulverized and filtered. Seven media were prepared, differing in the concentration of the resulting filtrate, each containing 100 mL. The media contained 100, 90, 80, 70, 60, 50, and 40% of the extract of pineapple waste, respectively, and the remainder was distilled water. The media were inoculated with 1 mL of S. cerevisiae yeast culture. The aerobic fermentation process lasted 120 h at a temperature of 25 °C. Colony-forming units (CFU) were measured every 24 h. During the first 96 h, the highest increase in biomass was observed for the medium with a FW content of 50%. After 5 days of cultivation, the highest increase in biomass was observed in the medium with 60% FW content and it amounted to 3.03 × 10⁹ CFU/100 mL. Smaller growth was observed for the substrate with 50, 40, 70, and 80% FW content, respectively. In the case of 90 and 100% media, the number of cells dropped to 0 after 48 h. Pineapple waste is rich in sugars, and thus, a high concentration of this ingredient in the medium, and therefore high osmotic pressure, adversely affects the growth of biomass. Waste from pineapple processing has a high potential for the use as a medium for SCP. However, it is important to ensure the appropriate sugar concentration so that the efficiency of the fermentation process is as high as possible.

Rages et al. [64] used waste from olive oil production as a medium component for SCP production. The waste was subjected to alkaline hydrolysis to extract sugars. The process was carried out at different temperatures (50, 75, and 100 °C) and for different amounts of time (45, 60, and 90 min). Hydrolysis using 0.8 M NaOH at a temperature of 100 °C for 60 min allowed for the extraction of most sugars (12.5%). The waste prepared in this way was used to produce fermentation media for the yeast Candida lipolytica. The fermentation was carried out for 8 days at 30 °C. Starting from day 3, the content of biomass and protein was determined every day. The highest protein concentration was obtained on day 4. The protein content was just over 9 g/L and the protein to biomass ratio was 69.23%. In order to determine the optimal cultivation conditions, the influence of the pH of the medium and the content of peptone as a nitrogen source were also examined. The optimal conditions were set at a pH of 5.5 and a peptone content of 0.4%. Such a selection of parameters allowed us to obtain 11.24 g/L of protein and a protein-to-biomass ratio of 64.04%. The alkaline hydrolysis of waste, the selection of the appropriate pH, and the addition of peptone allow us to conclude that olive oil waste can be a good substrate for the production of breeding medium for SCP production [64].

Al-Farsi et al. [69] conducted research using date fruit processing waste as a raw material for the media for the production of SCP. The waste consisted mainly of date fibers and seeds. The media for SCP production consisted of 25% date waste, 2% agar, 0.3% ammonium sulfate, and the rest was water. Flasks with medium were inoculated with five fungal strains that belonged to the species Trichoderma reesei, F. venenatum, Thermomyces lanuginosus, A. oryzae, and Fusarium graminearum. For 120 h, the flask of T. lanuginosus was incubated at 45 °C, and the remaining species at 25 °C. The biomass and substrate were then dried and powdered, and their protein content was tested. The research showed that the highest increase in biomass and the highest protein content (7.63%) was obtained for the A. oryzae strain. Also, it was examined which compound among ammonium chloride, ammonium sulfate, and urea is the best source of nitrogen for the A. oryzae strain. The content of protein produced by the mold was 13.8% for ammonium sulfate, 13.2% for urea, and 13% for ammonium chloride, respectively. On this basis, ammonium sulfate was selected as a protein source, and in the next stage, the best amount of this compound was analyzed and selected, which affected the protein content. Substrates with the addition of 0.2, 0.4, 0.6, 0.8, and 1.0% were prepared, and for the medium with the smallest addition of ammonium sulfate, the protein content was 9.13%. Whereas the sulfate concentration increased, the protein content increased. The highest result (13.49%) was obtained for the medium with the 1% addition of a nitrogen source, but this result was not statistically different from those obtained for the medium with 0.8% addition. Thus, the 0.8% concentration of ammonium sulfate was chosen as the most favorable. In the next stage of the research, it was checked what amount of medium would allow one to achieve the highest protein content. The amounts of 45, 60, 75, and 90 g of substrates were used. The protein content for 75 and 90 g of substrate did not differ statistically and amounted to 15.5 and 15.27%, respectively. Thus, it was decided to use 75 g of substrate. The last factor examined was pH. The highest protein content was obtained for the medium with a pH of 5.5. In the final stage of the research, the amino acid content was checked. As a result, the ratio of essential amino acids to the total amino acids of the SCP produced was 46%. The optimal conditions for SCP production using A. oryzae were determined for 75 g of medium with 0.8% ammonium sulfate and at the pH of 5.5. Since date waste is a rich source of sugars, it does not require hydrolysis, and optimal growth conditions can be easily ensured, as it has the potential to be used on a larger scale.

In addition to culturing microorganisms in media containing waste materials, another opportunity to obtain microbial protein is the use of hydrogen-oxidizing bacteria. Such a group of microorganisms is characterized by the ability to use hydrogen as a source of electrons and convert carbon dioxide into microbial biomass with a high protein content. Through electrolysis, protein is produced, the production of which is characterized by significantly lower greenhouse gas emissions than traditional protein sources such as meat and plant protein, as well as a much lower water footprint. Protein extracted using hydrogen is characterized by its high quality. In addition, such a production is independent of the traditional agricultural system and sunlight. Moreover, its energy efficiency is very high compared to most other sunlight-independent food sources, such as crops grown in artificial [70]. Protein obtained from such cultures can contain up to 0.75 g/g d.m., which is much higher than the protein content of soybeans—0.46 g/g d.m. and wheat—0.15 g/g d.m. The concept that carbon dioxide can be converted into valuable feed protein, thus shortening current protein production processes, opens up new possibilities for anaerobic digestion as an important driver for the development of an economy that ensures sustainable feed production on site [71].

SCP can also be synthesized by solid state fermentation, with the simultaneous bio-utilization of waste from the agri-food industry. This fermentation process can be used to obtain enriched animal feed, but microbial protein preparations are not obtained in this way due to the difficulty in separating microbial cells from the fermented waste mass. It was found to be possible to use a medium containing orange pulp, molasses, potato pulp, whey, distilled water, and BSG for the cultivation of K. marxianus, in which the sum of fat and protein concentration (59.2% w/w dm) was obtained after culture [72]. The solid fermentation of guava and cashew by-products to obtain protein and then the inclusion of the fermented product as a protein ingredient in bars resulted in the creation of products with high consumer acceptability at reduced ingredient costs. During the fermentation of waste mass by S. cerevisiae, an 11-fold increase in protein content was observed [73]. It has also been shown that the use of acid-thermal hydrolysis pretreatment for waste such as tomato, capsicum, eggplant, and cucumber waste increases the protein content in the product fermented by S. cerevisiae and Candida tropicalis, which may contribute to reducing the costs of obtaining SCP through greater process efficiency [74].

There is great potential in the production of SCP using waste from the agri-food industry; however, to ensure optimal growth conditions, the appropriate preparation of the substrate and selection of raw material are necessary.

4. Preparation of Single-Cell Protein (SCP) for Use in Food

The nutritional characteristics of single-cell protein can vary with the species and the culture medium used. Currently, two forms of microbial protein extraction are used in the food industry: biomass is obtained from dried yeast or other species of microorganisms or protein extracted from extracted microbial biomass [75].

The biomass contains not only protein, but also cell wall, fat, carbohydrates, mineral salts, and nucleic acids [65,76]. Protein extracted from biomass contains protein, free amino acids, and significantly less nucleic acids compared to non-extracted biomass. The composition of biomass and extracted protein are presented in

Table 2. Composition of yeast biomass and extracted protein [%].

Component	Biomass (%) [65,76]	Extracted Protein (%) [77]
Protein	30–70	76
Lysine	35–60	8.78
Methionine	6.5–7.8	1.82
Lipids	1.5–1.8	6.30
Carbohydrates	34.88	8.78
Nucle acids	9.7	2.2
Ash	7.85	6.1

.

It is preferable to use microbial protein extracts rather than whole biomass. After fermentation, the yeast biomass is harvested and may be subjected to downstream processing steps such as washing, cell disruption, protein secretion, protein purification, and drying. The biomass should be extracted to reduce nucleic acids and destroy the cell wall (Figure 3). The cell wall of some microorganisms is difficult to digest and this makes the protein in their cells unavailable to the body. Furthermore, the cell wall of yeast and bacteria can also cause allergic reactions [78,79,80]. Allergies can occur in people allergic to molds caused by mycoproteins [81]. Factors may arise from the type of microorganism culture medium used.

4.1. Cell Disruption

For the extraction of biomass, various mechanical and non-mechanical methods can be used [82]. Each method has its unique advantages and limitations; therefore, the choice of method significantly affects the extraction efficiency, the quality of the final product, and both fixed and variable process costs [83].

Mechanical methods generally are characterized by the ease of scaling up and low operational cost. However, due to a non-selective disruption, they allow the simultaneous release of compounds from the cell wall, which may cause an increase in operation steps. One of the most applied mechanical methods is bead mill (also known as bead beating), in which cells are disrupted by shear forces during the rotatory movement of the cells and the glass, ceramic, or steel beads [82]. During high-pressure homogenization (HPH), a cell suspension is forced to pass through a narrow gap, also known as a homogenizing nozzle or a high-pressure valve, at high pressure. Cell disruption is mainly based on the fluid impingement through the high-pressure valve and the shear forces, and its effectiveness depends on the pressure, temperature, and the number of passes through the valve [82]. In the case of ultrasonication (US), bubble cavitation, which is assumed to be the main mechanism of cell disruption, is generated through the conversion of sonic into mechanical energy, in the form of intense elastic shockwaves [83]. Due to the operational and economical limitations of ultrasonication methods, such as amplitude and energy consumption, bead milling and HPH are widely favored at the industrial scale [83].

Another mechanical method that has been considered for industrial-scale applications is microwave extraction. It is perceived as a green method characterized by a short processing time and a low energy requirement. It involves the heating of polar solvents in contact with solid samples. The temperature and pressure increase caused by the heating contributes to the release of compounds from the cells. This method has an ability to effectively disrupt the cell wall with a short processing time and low-energy requirements [84]. Furthermore, Chew et al. [84] noted that microwave treatment combined with three-phase partitioning (TPP) can enhance the protein recovery yield from microalgae by more than 2.5 times in comparison to the TPP process alone.

The non-mechanical methods can be divided into four distinct groups: physical, electrical, chemical, and enzymatic methods. They are usually more selective, less severe towards the cells, and less energy-intensive in relation to mechanical strategies [82].

Freeze–thawing involves subjecting the microbial cells to slow freezing, during which large ice crystals inside the cell are formed, and this leads to its disruption, while in osmotic shock, the cell disruption occurs by submitting the cells at high osmotic pressure. Both methods are applicable only at the laboratory scale due to the high operating costs caused by considerable time and high energy consumption (freeze–thawing), or large volumes of water and a cooling system (osmotic shock). In turn, thermolysis, which consists of thermal decomposition, in which the reagent decays into at least two new substances, is easily scalable but the thermal degradation of selected bioactive compounds should be considered [85].

An interesting research area is the use of electrical methods that are based on the breakdown or disruption of the cell by applying electric fields. Both pulsed electric field (PEF) and high-voltage electrical discharge (HVED) techniques are considered low-temperature and energy-efficient, and thus environmentally friendly. PEF uses high-voltage pulses through an aqueous environment between two electrodes. The electric field generated by the voltage induces an increase in the cytoplasmatic membrane permeability (electroporation), facilitating the intracellular compounds release, whereas HVED produces an arc discharge through an intense electrical field from a needle electrode to one ground. Cell damage is caused by pressure shock waves, bubble cavitation, and high liquid turbulence [82,86]. Coustets et al. [87] demonstrated that PEF is an effective procedure for extracting proteins from Haematococcus pluvialis and C. vulgaris, Martínez et al. [88] applied PEF to release mannoproteins to the extracellular medium, and Ganeva et al. [89] extracted soluble proteins from S. cerevisiae. Furthermore, in various studies, HVED was indicated to be a more favorable technique than PEF due to its higher efficiency [90,91,92,93]. It may be explained by the fact that the PEF treatment affects the rupture of cell membranes, but HVED can cause more extensive damage by acting on both cell walls and membranes [89].

In chemical methods, different reagents (alkali or acid, organic solvents, deep eutectic solvents, detergents, and ionic liquids) can be utilized for the permeabilization or lysis of cells. These methods are based on the relative selectivity of chemicals with the specific components of the membrane, enabling proteins to pass through the cell wall. The type of chemical is determined by the target compound and its location in the microbial cell [82]. One of the most used chemical procedures to extract S. cerevisiae protein is alkaline precipitation. Zhang et al. [94] stated that pretreatment with lithium acetate (LiAc), described as a cell wall permeabilization enhancer, can improve the protein yields. It was also examined that the use of ionic liquids to destroy cell walls did not alter the properties of the extracted protein [95]. Furthermore, the combination of ultrasonication and alkali treatment revealed the higher cell disruption efficiency, and the total extracted proteins from microalgae (Chlorella sorokoniana and Chlorella vulgaris) in comparison to the use of each method separately [96]. However, in spite ease of scaling up and low energy inputs requirements, chemical treatments may cause the degradation of some intracellular compounds and introducing contamination to the system, resulting in more downstream operations. Additionally, some chemical substances are characterized by low selectivity, high cost, and toxicity, which makes them difficult to use [82].

Another promising mild disruption method is enzymatic degradation, which involves attacking the mannoprotein complex and glucan backbone of the cell wall by autolysis or lytic enzymes without destroying the cell integrity [97]. Enzymes provide a very gentle and specific means of disrupting cells thanks to their intrinsic catalytic activity and substrate specificity. Enzymatic methods enable one to prevent serious damage being inflicted upon the intracellular compounds, the use of toxic chemicals, or aggressive physical conditions such as high shear stress. In addition, they are effective, highly biologically selective, and operate at low temperatures with low energy demand. Enzymes are also easily controlled biological materials and commercially available, making the scale-up of this method relatively easy [86]. Moreover, the enzymatic degradation can be combined with other mechanical or non-mechanical methods. For example, Alavijeh et al. [98] developed a combination of enzymatic and bead-milling techniques for the extraction of proteins, lipids, and carbohydrates from microalgae C. vulgaris with the use of lipase, cellulase, protease, and phospholipase. In the study, 88% of lipids, 74% of carbohydrates, and 68% of proteins were recovered without loss of any products, reducing the residence time and avoiding corrosive solutions. Nevertheless, the main drawbacks to the enzymatic techniques compared to the mechanical and chemical methods are the longer processing time, possibilities of product inhibition, and lower production capabilities for cell disruption. The high price of enzymes can also limit their application for protein extractions [99].

4.2. Protein Secretion

At the cell wall disintegration stage, proteins are released into the solvent, leaving cell remains in the solution, e.g., cell walls, which must be removed by, e.g., centrifugation. Proteins that could be used in food technology must be precipitated. Many studies have been described in which these processes were carried out at a laboratory scale, but they are difficult to scale. The precipitation process can be carried out in several ways, among which the most common are heat treatment, pH change method, and the use of salts such as ammonium sulfate or non-ionic hydrophilic polymers (PEGs) [100,101,102,103]. Thermal methods are most often used, but thermal precipitation has the disadvantage that it changes the structural features of native proteins, which may affect their functional and nutritional value [104]. Attempts are made to extract the released protein using alkaline solubilization and precipitation at the isoelectric point. Yeast proteins are soluble in alkaline conditions (pH~12), and these conditions allow them to be separated from lipids and other insoluble fractions [105]. Subsequently, lowering the pH of the solution to a level close to the isoelectric point of the microproteins (pH~4.5) facilitated the precipitation of yeast proteins and the production of yeast protein isolates [80]. In turn, the use of an appropriate amount of sodium dodecyl sulfate (SDS) could inhibit the enzymatic digestion of proteins by endogenous enzymes, which effectively increased the amount of yeast protein extraction [105].

4.3. Protein Purification

Another important production process is the purification of the obtained protein. The most frequently used are chromatographic methods, which can be used even on an industrial scale [106]. The most commonly used methods are gel permeation, hydrophobic interaction, and affinity chromatography. Unfortunately, these methods require good process preparation, the selection of parameters and chemical reagents, such as solvents [79]. Another method for obtaining microbial protein may be the use of membrane processes (microfiltration and ultrafiltration). This process does not heat the solution, so the protein is not denatured. The disadvantage of this process is the difficulty in selecting a membrane with the appropriate pore size to enable protein purification on the one hand and be economical on the other [107,108]. In the case of plant proteins, aqueous alcohols (methanol, ethanol, isopropyl alcohol, and butanol), and organic solvents such as acetone are used on an industrial scale, but there are no reports of the use of these solvents in the production of fungi proteins [100].

4.4. Reducing the Nucleic Acid Content

Microbial protein must be purified primarily from nucleic acids. The Protein Advisory Group recommends that the daily intake of nucleic acids from SCP should not exceed 2 g. The metabolism of pyrimidines produces ammonia, water, and carbon dioxide, and purines produce uric acid. Excess uric acid in the human body leads to the development of gout or arthritis, and may contribute to the formation of kidney stones. This is due to the inability of humans to synthesize uricase [109]. Therefore, the nucleic acid content of SCPs must be reduced by enzymatic, chemical, or thermal methods [110].

A simple thermal method to reduce the amount of RNA was developed in QuornTM. In this process, Fusarium biomass is rapidly heated to 64 °C for 20–30 min to inhibit growth, destroy ribosomes, and activate endogenous RNAases. After this process, mycoprotein contains about 1% of nucleic acids, but there are also significant biomass losses (33–38%) [111]. For this reason, this method was improved by increasing the temperature to 72–74 °C and extending the incubation to 30–45 min (biomass losses of 30–33%) [110]. The heat shock method can also be successfully used to reduce the amount of nucleic acids in yeast biomass. Maul et al. [112] developed a three-step method that resulted in a reduction in the nucleic acid content in the biomass of the yeast C. utilis from 7 to 1.0–1.5%. The first stage of this process is a few seconds of thermal shock at a temperature of 54–70 °C, then cooling to 45 °C, and finally a 1 h incubation at 55 °C.

A significant reduction in the amount of nucleic acids can be achieved using alkali treatment. Alvarez and Enriquez (1988) [113] used a 4.5% NH₄OH solution and a temperature of 65 °C. After 30 min of incubation of the yeast biomass, the nucleic acid content in the K. marxianus biomass decreased from 12.08 to 1.2%, and in the case of the yeast S. cerevisiae, decreased from 7 to 1.4%. Zee and Simard (1975) [114] developed a method for reducing nucleic acids in the SCP of the yeast Rhodotorula glutinis using acidic treatment. After optimizing the parameters, it was found that the most efficient reduction in the nucleic acid content (from 6.5 to 1.2%) occurs after acidifying the medium to pH 2.0 and incubation at 90 °C.

4.5. Drying of Microbial Protein

The final stage of the process of preparing protein for use in the food industry is obtaining stable preparations. In the literature, many parameters for drying whole yeast cells can be found, which are treated as SCPs. However, drying microbial protein preparations is a very poorly understood technological process. Few reports describe spray drying, freeze drying, or supercritical drying [79]. Selecting drying parameters that will maintain protein stability, both chemical and microbiological, remains a challenge faced by scientists.

5. Products Containing Single-Cell Protein (SCP) on the Global Market

There are numerous products available on the world market that contain edible microorganisms’ biomass, produced from single-cell microorganisms. It can be consumed directly or used as a food ingredient. Most of the products that contain biomass are produced by S. cerevisiae, Y. lipolityca, C. utilis, and F. venenatum. Some of the products are presented in

Table 3. The products with SCP available on the market.

Type of Product	Name of the Product	Producer	Microorganisms	Ingredients	Nutritional Value	References
Meat substitute	Crispy nuggets	Quorn	F. venenatum	Fusarium venenatu, soy sauce, pea fiber	190 kcal/100 g	[120]
Meat substitute	Vegetarian chicken pieces	Quorn	F. venenatum	Mycoprotein (95%), hydrated free-range egg white, natural flavouring, firming agents: calcium chloride, calcium acetate	97 kcal/100 g	[121]
Fish substitute	Vegan fishless sticks	Quorn	F. venenatum	Rice flakes, wheat flour, mycoprotein (12%), water, natural flavouring, rapeseed oil, stabiliser: methylcellulose; yeast, salt, paprika, colouring: paprika extract	214 kcal/100 g	[122]
Paste	Paste with yeast extract	Vegemite (Mondelez)	S. cerevisiae	yeast extract (from barley, spelt), salt, malt extract (from barley), flavour enhancer (potassium chloride), colour (e150c), spice extract (contains seler), niacin, thiamin, riboflavin, folic acid	186 kcal/100 g	[123]
Paste	Yeast extract paste	Marmite	S. cerevisiae	Yeast extract (contains barley), salt, vegetable juice from concentrate, vitamins (thiamin, riboflavin, niacin, vitamin b12, folic acid), natural flavourings (contains celery)	250 kcal/100 g	[124]
Paste	Yeast extract dynamite	Marmite	S. cerevisiae	Yeast extract (contains barley, wheat, oat, rye), salt, vegetable juice concentrate, vitamins (thiamin, riboflavin, niacin, vitamin b12 and folic acid), natural chilli flavouring, natural flavouring (contains celery)	279 kcal/100 g	[125]
Snacks	Flatbreads	Marmite	S. cerevisiae	fortified wheat flour (wheat flour, calcium carbonate, iron, niacin, thiamin), marmite (18%) (yeast extract (contains barley, wheat, oats, rye), salt, vegetable juice concentrate, vitamins (thiamin, riboflavin, niacin, vitamin b12, folic acid), natural flavouring (contains celery)), cheddar cheese (milk) (16%), rapeseed oil, raising agent: ammonium bicarbonate	429 kcal/100 g	[126]
Paste	Crunchy peanut butter	Marmite	S. cerevisiae	peanuts (87%), yeast extract powder (9.5%), peanut oil, tocopherol extract (antioxidant), vitamins (thiamin, riboflavin, niacin, vitamin b12 and folic acid). may contain other nuts	574 kcal/100 g	[127]
Protein supplement	Yeast protein	Allnutrition	S. cerevisiae	Saccharomyces cerevisiae yeast protein, reduced fat cocoa, flavouring, salt, thickeners: cellulose gum, guar gum, xanthan gum, sweeteners: steviol glycosides from stevia, acesulfame k, sucralose	360 kcal/100 g	[128]
Food supplement	Yeast protein, vegan	Enkioo	S. cerevisiae	Yeast protein (derived from the yeast Saccharomyces cerevisiae) 100%	411 kcal/100 g	[129]

. One of the products using yeast biomass is Quorn^TM-meat substitute, in which the main ingredient is mycoproteins derived from the fungus F. venenatum. This product is marketed as a meat substitute, both as an intermediate product for further processing and as an ingredient in finished dishes. The company’s products Quorn^TM contain a high amount of protein, a low amount of fat, and generate little waste during production [115]. Quorn products represent a unique source of mycoprotein available in today’s food market, which has attracted the attention of many researchers. Research is currently being conducted into the potential use of Quorn in breakfast cereals and as a fat substitute in ice cream and yoghurt production [21,49]. The composition of mycoprotein, extracted from F. venenatum, includes high-value protein in the cytoplasm, dietary fiber in the cell walls, and polyunsaturated fatty acids in the cell membranes. Quorn products are distinguished by their low fat content, high biological value of protein comparable to that of skimmed milk, high amounts of fiber, and the presence of micronutrients and B vitamins. Due to the significant amount of fiber, these products can be labelled as a ‘source of fiber’ or ‘high fiber products’ [116]. Quorn products can play an important role in weight control through their low calorie density and their ability to reduce feelings of hunger, which is important in the prevention of obesity [117]. Formulations containing mycoproteins are often chosen by those seeking alternative sources of plant protein or looking for lower fat products. Mycoproteins, such as those from Quorn, are distinguished by their high nutritional value, high fiber content, and low fat content. They are therefore attractive to those who pay attention to healthy eating habits. Supplements with mycoproteins are available in various forms, such as powders, capsules, or ready-made protein drinks. Their popularity may be due to the growing interest in plant-based, vegetarian and vegan diets, as mycoproteins offer an alternative to traditional animal protein sources. Also, there is a European project, known as INGREEN, which uses the biomasses of selected strains of Y. lipolytica to produce substitutes of products in dairy industry, like Ricotta, Caciotta, and Squacquerone cheeses [118]. Attention to quality and balanced ration feed, taking into account the needs of animals, has led to the development of numerous innovative food additives of diverse composition, one of which is yeast, used for a long time in the nutrition of many groups of livestock. The use of preparations based on yeast contributes to improving the health and overall condition of animals. S. cerevisiae and Y. lipolytica are the main types of yeast used in feed production, which are sources of protein, B vitamins, bio-elements, and enzymes [119]. In 2019, EFSA recognized the biomass of Y. lipolytica as a novel food safe for use in accordance with Regulation (EU) 2015/2283 on dietary supplements. Yeast from this species has found use as an encapsulated dietary supplement with a high protein content. A review of the global market shows that single-cell proteins are finding increasing use in various food products, both in traditional and novel food alternative foods [22].

6. Opportunities and Challenges for the Single-Cell Protein (SCP) Market

Microbial protein is an alternative for protein of animal and plant origin. However, due to its valuable properties, it can also be used in other branches of the agri-food sector. Replacing conventional sources of protein will entail major environmental benefits. In particular, by restricting the cultivation of crops, it will be possible to convert land currently used for crop production to other uses, such as afforestation, which can be used to sequester carbon and counteract the loss of biodiversity. However, before microbial protein production becomes commercially feasible, a techno-economic assessment must be carried out and a pilot microbial culture must be carried out in reactors with higher production capacity [130].

Table 4. SWOT analysis of the use of single-cell protein SCP.

S Strengths	W Weaknesses	O Opportunities	T Threats
High vitamin content, e.g., riboflavin, thiamin, pyridoxine, niacin, choline, folic acid, pantothenic acid, biotin, and para-aminobenzoic acid	Risk of contamination microalgae protein with heavy metals such as cadmium, mercury or lead Limitation to the use of bacteria as SCP targets is the production of toxins by some of them	The potential use for food packaging Waste management for the SCP production	Not fully understood risk from SCP Potential allergenicity causing gastrointestinal problems, skin lesions and even lead to anaphylactic shock Challenges in increasing production scale from lab scale

presents a SWOT analysis that shows the benefits and threats resulting from the use of SCP in food, as well as what are the strengths and weaknesses of this product and what to pay attention to when trying to implement it as a food additive.

Food contact packaging is an essential element in ensuring consumer safety. In addition to ensuring microbiological safety, packaging protects food from changing physical and chemical properties. The most common materials used for food packaging are plastics, paper, glass, and steel. The European Environment Agency (EEA) has announced the need to move from a linear economy to a circular economy. In a linear economy, the main model for economic development is raw materials that are fully biodegradable [131]. Research is currently underway to obtain packaging that will not burden the environment. Microbial proteins, due to their ability to form a membrane, transparency, and barrier effect to gases, can be used in the production of packaging. The use of proteins in packaging materials comes with some limitations, such as a low barrier effect to water and fragility of the finished formulations. By growing microorganisms in media containing food industry waste, extracting the protein, and then producing packaging with microbial proteins, sustainable valorization and promotion of a closed-loop economy is possible [132].

Furthermore, the microbial protein can be used as an alternative source of organic fertilizer. By growing microorganisms in substrates containing agricultural waste, essential nutrients such as nitrogen, phosphorus and potassium are recovered, which are later applied to agricultural soils for bio-enrichment and consequently improved plant growth. However, further research is needed on the potential use of microbial protein as a nutrition for plants [133]. In addition to the use of agricultural waste, municipal wastewater can be a favorable substrate for microbial protein production. By using the organic fraction of municipal solid waste, it is possible to valorize it carried out in biorefineries [134].

There are many animal protein alternatives available on the market. When analyzing the composition of such products, it is important to pay attention not only to the protein content, but also to its quality. Mycoprotein preparations produced by F. venenatum have an actual protein digestibility of 86%. The natural structure of mycoprotein is porous, so proteases can diffuse through cell walls during the digestion phase in the small intestine. The extracted from Agaricus bisporus has a digestibility of 64%. In contrast, the application of alkaline extraction to Pleurotus ostreatus biomass contributes to preparation with a digestibility of 100%. In contrast, the total amino acid digestibility of Saccharomyces cerevisiae yeast is 97%. The digestibility values of such proteins of fungal origin contribute to the significant advantage of these products over plant proteins. The protein digestibility of the dry extruded mixture of chickpea and barley flour was 59%, while the digestibility of barley flour is only 33% [135].

Plant-based protein alternatives, in addition to their low digestibility, are low in micro and macronutrients. Vegan products should be supplemented with various nutrients, including iron, zinc, iodine, selenium, calcium, vitamin B₁₂, vitamin D, and vitamin B₂ [136]. Microbial proteins, on the other hand, are characterized by their high vitamin content. The most common vitamins present in microbial proteins are riboflavin, thiamin, pyridoxine, niacin, choline, folic acid, pantothenic acid, biotin, and para-aminobenzoic acid [137].

Single-cell proteins for which algae, fungi, and bacteria are used also carry several risks. The issue of food safety risks from proteins of microbial origin is not yet fully understood. There is a lack of specific research on this topic in the literature, and it is mostly only addressed theoretically. In the case of microalgae proteins, there is a risk of contamination with heavy metals such as cadmium, mercury, or lead. Toxins resulting from environmental contamination with toxinogenic cyanobacteria can also accumulate in them [138].

In products containing microalgae, there is an additional risk of microbial contamination with pathogenic bacteria or viruses. However, this can be prevented by the appropriate selection of culture media. Currently used in culture media, animal serum can bring with it many viruses. With the use of mycoproteins, the most significant risk is their potential allergenicity, especially in people with sensitive digestive systems. It can manifest as gastrointestinal problems, skin lesions, and even lead to anaphylactic shock. Allergic reactions are caused by certain mold species including Fusarium vasinfectum or Aspergillus fumigatus. Bacterial proteins, on the other hand, are characterized by the highest content of nucleic acids (15–16%). They include purines, which are metabolized to uric acid. Its increased accumulation in the body, on the other hand, can lead to many serious conditions such as gout or hyperuricemia. There is therefore a need to reduce the nucleic acid content of proteins of bacterial origin. This effect can be achieved by using the heat treatment in processing [81]. Another limitation to the use of bacteria as SCP targets is the production of toxins by some of them. Pseudomonas spp. and Methylomonas methanica are examples of this. These bacteria are capable of producing large amounts of protein; however, at the same time, they can also produce endotoxins that cause febrile reactions [110].

In the production of SCPs, waste materials from various industries are often used as substrates. An example is the wastewater of the brewing industry. In this case, it is extremely important to use systems that monitor the presence of chemical and biological hazards that can cause various health problems [13].

The topic of efficiency should also be touched upon. There are challenges in increasing the production scale. The laboratory scale is not an economical but an expensive process. Moving the laboratory scale to the industrial scale involves continuous fermentation to optimize the process itself, and therefore, its cost-effectiveness. Nonetheless, the progress made in recent years in developing new processes offers high hopes for SCP products [13,137,139].

7. Conclusions

The growth in the numbers of vegans and vegetarians causes the search for new sources of protein that will be acceptable to consumers on the one hand, and on the other hand, have adequate nutritional values. One type of such protein is the single-cell protein of microbial origin. Its amino acid composition is similar to animal protein, and breeding is more economically profitable. In addition, SCP can reduce the carbon and water footprint, and thus contribute to the creation of more ecological products. Many products with protein of mold origin have been developed, but the possibilities of economic production and the application of yeast protein preparations are still being sought.