Another fungal SCP product is PEKILO (mycoprotein from
Paecilomyces variotii), which was used as feed for poultry and fish. It was first developed in the 1960′s to valorize pulp and paper industry side streams
[29]. Until 1991, PEKILO was commercially produced and presently, the PEKILO production is possessed by a start-up company eniferBio (
https://www.eniferbio.fi/, accessed on 6 May 2021). Though PEKILO was first produced to be used as protein rich feed, its potential use as food ingredient was also studied
[29].
2. Selected Fermented Foods of Fungal Origin
Fermented foods are produced nearly by every ethnic group. They vary based on food ingredients, microbes that induce the fermentation processes, the containers used in fermentation and storage, fermentation time and temperature, and such. Apparently, the main purpose of the fermentation processes is preserving foods and getting a new taste and aroma. However, in fact, the early understanding for the fermentation as a process has been broader. Foods have been fermented to get ethanol, which is one of the main chemical compounds used in mummification by the ancient Egyptians, due to its dehydration capability, antimicrobial activity, and volatility. At the same time, alcohol had been used as a solvent and as an extracting enhancer for the active ingredients of the medicinal plants. Fermentation produces stability in the foods that have been fermented, which means for early humans, a positive and unique approach. In their minds, the process that keeps food unspoiled should involve elements, factors, or even energy that will keep them healthy.
Modern science has a new explanation based on the experimentation and analysis of food ingredients. Thus, there are established facts about fermentation in today’s life, the most important of them is that fermentation increases the nutritional value of raw ingredients and that fermented foods improve our digestion. However, such early beliefs keeps many of fermented foods available until now. Many of them are consumed during religious celebrations and on particular days in the year. The old civilization believes in the combination of elements. For example, they believe in consuming the whole plant that contains active ingredients rather than extracting these active ingredients. Fermented foods gave early humans what they need; fermented foods have different ingredients in an unspoiled mixture and are available in the form of foods that are digestible, healthy, tasty, and cheap. Due to their highly positive properties, nearly every meal contained a dish made from fermented foods.
Due to the excessive modernization, such habits started to disappear slowly. However, this has led to discover, what such foods have given us, and why our grandfathers for generations keep them on their tables. Some of such foods have been treated with great care and kept as a secret for hundreds of years such as kefir. Some Caucasians believe that kefir has a miracle power, and if any foreigner discovers it, this power will disappear. It took an effort from the Kaiser of RUSSA to know the secret and to get a sample of it. This sample opened minds about the science of probiotics and kefir has been used for years as a treatment for every illness.
For some ethnic groups around the world, fermented foods are not just a tradition or a medicine but a matter of survival. Non-food material could be changed to be food only after being fermented. Nowadays, fermented foods—particularly yeasts and filamentous fungi-based fermented foods—can save humanity from the hunger.
Another important criterion of fermented foods is that some of the wild microbes with the ability to ferment foods can colonize our gastrointestinal system to digest food inside our intestine. Others cannot but can survive for days and also do their activities and help us in food digestion. For that and during hundreds of years, human blindly learns that some fermented foods should be eaten daily, weekly or even once a year.
Nowadays, some diseases have been prevented by the consumption of fermented foods. For example, the intake of fermented foods was reported to be a crucial step toward successful management of Alzheimer’s disease
[30]. The explanation for this effect might be given to the increase in particular nutrients such as protein and vitamins. In contrast, unwanted compounds or those not good for our health such as the anti-nutritional chemicals including phytates, tannins and polyphenols might disappear due to the fermentation process or at least their amounts are reduced. For more details, refer to Kumar et al. (2022) and the references within
[30].
The fermentation of food is often defined as the manufacture of foods employing the action of microorganisms and their enzymes. In Africa (e.g., Mozambique and Uganda), the use of mycelial fungi for fermentation is exemplified by their use in detoxification of bitter cassava roots
[31]. There are a significant number of the fungal genera involved in fermented foods production. For more details, refer to
[32][33][34] and the references within.
There are hundreds of beneficial yeasts, and the most famous is
S. cerevisiae. It is used to produce bread, beer, and wine.
Saccharomyces yeasts also form symbiotic relation with bacteria to form kefir
[35]. Yeast can be found and isolated from different environments, particularly from the sugary mediums. There is a respective number of fermented foods that contain yeast
[34][36].
The fungal fermented foods and products are common in Asia and Europe
[15][37]. Filamentous fungi are used traditionally as starter cultures in Asia. They have several contributions, such as saccharification, and ethanol production. In Europe, they are used in developing different dairy products particularly in the ripening processes of various types of cheese (e.g., Roquefort, Camembert) and for enzyme production
[34][38][39]. Within the genus Aspergillus, there are important species such as
Aspergillus acidus,
A. oryzae,
A. niger,
A. sojae,
A. sydowii,
A. versicolor and
A. flavus, which are used in traditional fermented foods production such as soya sauce fermentations, Miso, sake, awamori liquors, and Puerh tea
[37][40][41][42].
2.1. Kefir
Kefir is fermented by both of bacteria and yeast. It is a fermented milk drink (cow, goat, or sheep milk with kefir grains) similar to a thin yogurt or ayran
[43][44][45][46][47][48][49][50][51][52]. It had been coined in Caucasus in the 1900s. The grains are composed of colonies of living Lactobacilli and yeast. They live together in a symbiotic relationship and ferment milk at room temperature. Traditional kefir has been made in goatskin bags that have been hung near a doorway. The bag has been knocked by anyone passing by to keep milk and kefir grains well mixed. Such a practice is usually also done by traveler Bedouin in the Middle East. The kefir grains are initially created by auto-aggregations of
Lactobacillus kefiranofaciens and
Saccharomyces turicensis. They are biofilm producers. In addition, they can adhere multiply to the surface to become a three-dimensional micro-colony
[53][54]. Yeasts found in kefir include
Candida kefir,
S. cerevisiae.
Kluyveromyces marxianus,
Kluyveromyces lactis,
Saccharomyces fragilis,
Torulaspora delbrueckii, and
Kazachstania unispora [55]. During the fermentation process, the lactose is digested, which makes kefir ideal for those persons that are with lactose intolerance. Kefir possesses natural antibiotics and rich in the B1 and B12 vitamins, calcium, folic acid, phosphor, and K vitamin.
2.2. Tempeh
Tempeh has been firstly produced in Indonesia for thousands of years. It remains the most important staple food there and an inexpensive source of dietary protein. It is being spread to other countries such as Malaysia and the Netherlands. Tempeh is made from partially cooked fermented soybeans. It is especially popular on the island of Java. Indonesian tempeh, of Javanese, is in form of soybean cakes. It is prepared by making a natural culture and process of controlled fermentation.
Rhizopus oligosporus is used in this fermentation process (known as tempeh starters)
[56][57]. The main step to make tempeh is the fermentation of soybeans.
Rhizopus ssp. is used to inoculate the soybeans. Traditionally, a previously fermented tempeh is mixed. It contains the spores of
Rhizopus oligosporus or
Rhizopus oryzae. The mixture then spread to a form of a thin layer and allowed to ferment for 24 h at a temperature around 30 °C. The soybeans must get cold to allow the germination of the spore. Typically, tempeh is ripened after 48 h. The fermented product has distinguishable whitish color, firm texture, and nutty flavor. Fermented soybeans in tempeh contain B12 vitamin, a byproduct of the fermentation process. Tempeh enhances the body’s absorption of the isoflavones. The fermentation process also removes the enzyme inhibitors that occur naturally in soybeans. Tempeh is high in fiber, beneficial bacteria, enzymes, and manganese. It is a good digestible protein source and a great meat substitute (high in protein). It is also a good source of calcium and contains all essential amino acids. Sometimes, it is made from a blend of grains, beans, or other vegetables.
2.3. Miso
A product of fermented soybeans, which is produced and consumed in the Far East. Miso came during an early time in China and Korea. However, Japan nowadays is the main producer and consumer. It is estimated that 5k/person/year are consumed in Japan
[58]. In traditional practice, miso is prepared using a seed culture of the other previously produced miso. A seed culture usually contains some yeast and bacteria. The most common yeast is
Z. vousir and
C. versetilis. Miso is also prepared with salt and koji (mold
Aspergillus oryzae). Sometimes, rice, barley, algae, or other ingredients are added. In Japan, miso of fish had been manufactured since the Neolithic time
[59]. Miso is rich in protein, vitamins, and minerals. It can be produced as very salty or very sugary. Miso fleshy fermented dough is made by grinding some soybeans, malted rice or barley and salt together, then used specially to make soups and sauces. The natural miso is a living food that contains many beneficial microorganisms
[60].
Tetragenococcus halophilus is an example. Those microbes can be killed by overcooking.
3. Safety of Fungal Proteins
Fungal proteins as any other product used for food or feed need to be safe to produce and use. Regulations exist in most regions to ensure that human food or animal feed are safe for consumption and these regulations differ depending on whether products are expected to be food (providing nutrition along with pleasant taste and aroma) or will be marketed as additives for food (texture modifiers, preservatives, etc.) or applied as feed and additives for feed
[61]. The pleasant taste and aroma of the products made of fungal proteins will help in raising demand for them and boosting the appeal of these novel protein sources in a crowded marketplace. As discussed above, three crude protein products from fermentation (by-) products from yeast and filamentous fungi are approved as feed by Commission Regulation (EU) No 68/2013. Additionally, three SCP fungal strains are accepted for food use in EU countries, only one of them (the yeast
Yarrowia lipolytica) has been authorized via the Novel Food Regulation ((EU) 2017/2470), and its use is restricted to food supplements. Molitorisová et al. (2021) mapped the regulatory environment that governs mushrooms and mycelium products (MMP) in EU countries—food law provisions applicable to MMP produced or marketed in the EU
[62]. They found that the sector is still in the developing phase, and regulatory framework application to MMP comprises numerous legal doubts. The law classifies MMP as foods or medicines based on the proposed use. Novel MMP could be classified as medicines. This classification can exclude provisions of food law. Operators of food business that work with borderline products (food/medicine) should consider their claims. Mushrooms and mycelia, along with products derived thereof, can be subjected to the common agricultural policy rules. The classification of MMP as novel foods is challenging. As an example, regardless of a long history of some fruiting bodies consumption, products derived from mycelium of the same species may be subjected to novel foods regulation ((EU) 2015/2283). This is similarly the case of species that are not consumed commonly. The Novel Food Regulation obligates important regulatory requirements on applicants, who applying for authorizations of novel food. These regulatory requirements include safety proof via robust and solid scientific evidence, such as several studies of toxicity, animal models, or human data. MMP are generally classified as “vegetarian” or “vegan” in EU countries and bear claims of sustainability.
The name of the raw materials or substrates used in SCP production represents the main safety hazard. For example, the possible presence of carcinogenic hydrocarbons in n-paraffin or gas oil or presence of heavy-metal contamination in the mineral salts and solvents after extraction. Quorn is produced in a chemically defined medium from glucose (hydrolyzed starch) in a well-defined process which meets international standards
[28]. It is a privilege to use a standard medium, since the fungal nutritional composition differs based on the changes in the medium composition
[63]. Meanwhile, the economic production of fungal proteins in some cases (e.g., the production of edible mushrooms) favors the use of agriculture wastes. Nevertheless, such wastes should be free from any toxic or harmful compounds (e.g., pesticides).
Dried and heat-killed yeast biomass used as a protein source must be safe for both human and animal nutrition in accordance with the current food and feed safety regulation. Additionally, the concentration of heavy metals in yeast biomass should be low and must not exceed the EU threshold values
[64]. In certain cultivation and growth conditions, fungi could produce specific secondary metabolites. For that reason, product quality assurance, good manufacturing practice (GMP) as well as monitoring fungal molecular properties should be performed to ensure that fungal products are consistently produced and controlled according to quality standards
[65]. In 2018, King et al. performed genomic analysis (using shotgun sequencing) to differentiate between
F. venenatum (Quorn fungus) and
F. graminearum (closely related phytopathogen), comprising genes that code for different mycotoxin types (type A trichothecene mycotoxin TRI5 cluster in case of
F. venenatum and type B trichothecene mycotoxin TRI5 cluster in case of
F. graminearum)
[66]. They identified differences between the genomes of the two fungal species that could participate in their contrasting lifestyles and highlighted
F. graminearum-specific candidate genes potentially required for pathogenesis. So far, Quorn fungus is not known to produce mycotoxins in the used processing conditions, even though regular monitoring is still performed to ensure that the final product contains none of these toxins
[67].
It is wise to simplify the production methods for certain fungal species (e.g., mushrooms) to be in-house, reliable, and applicable for the farmers. Even so, the end product quality and safety should be investigated
[68]. In addition, the use of waste-derived substrates for SCP production needs public acceptance of waste-derived foods in addition to the safety regulation. The public acceptance can help countries to set their own safety standards, nevertheless, those standards should be science-based
[69]. Products with a short shelf life, particularly products from viable fungi such as fresh mushrooms, which are prone to spoilage should be continuously checked and properly stored in the markets.
Vital safety concerns of SCP include microbial toxins either from SCP producing microorganisms or contaminants, SCP RNA content, potential allergy reactions, and harmful substances derived from production raw materials. Srividya et al. (2013) and Ukaegbu-Obi, (2016) highlighted that the suitability of SCP as a source of edible protein should be considered individually because, such as any food, it might cause different allergic reactions based on the individual sensitivity to the fungal protein
[70][71]. In addition, the inactivation or killing of the viable fungal cells is so important. EFSA (2008) reported that subjects who are exposed to viable yeast inhalation are particularly at health risk
[72].
The key concern of food safety associated with mycoprotein is allergens
[73]. Data are limited on this aspect, but adverse effects to mycoprotein consumption were reported in patients with a history of allergies to molds. Type I hypersensitivity reactions were found in a 27-year-old mold allergic female patient within a few minutes of consuming Quorn burger
[74]. Likewise, Sandhu and Hopp (2009) reported that 15-year-old male patient with a history of allergies to several molds showed type I hypersensitivity reaction to ingestion of meatless chicken (Quorn)
[75]. In 2018, Jacobson and DePorter analyzed self-reported adverse reactions related to Quorn-brand containing foods from 1752 individuals and found that most of these reactions involved allergies such as anaphylaxis and hives or gastrointestinal symptoms as diarrhea and vomiting
[76].
Yeast is most appropriate for SCP production due to its superior nutritional quality. Cereals supplemented with SCP derived from yeast have been proved to be as good as animal proteins
[77]. Yeast for use as SCP is characterized by the absence of toxic and carcinogenic compounds biosynthesized by yeast from the substrates or formed during processing. In addition, about 100 pounds of yeast will produce 250 tons of proteins per day. However, the use of yeast for human and animal consumption may be limited by a high nucleic acid content, which is mostly metabolized to yield uric acid possibly at high levels leading to renal stones, and low cell-wall digestibility
[78].
Cautiously choosing SCP producing organisms (e.g., fungi that produce mycotoxins cannot be chosen as they may cause allergic reactions, carcinogenesis, or even death)
[79], the process conditions, and the product formulation will overcome toxins challenge. SCP RNA content could be decreased to acceptable levels. The consumption of yeast protein with high DNA content might cause gut and kidney stone for those who have purine metabolism malfunction
[80][81]. Only 1% for short time are recommended in feed or food
[80]. SCP product that contains DNA higher than 1% is allowed only as a feed to short life-span animals
[82].
The safety concerns of fungal proteins including SCP could be summarized in the following points:
-
A need to specify the unique properties for fungus to be claimed/and used as a protein producer or as SCP;
-
Using internationally standard fungal strains in protein production or as SCP and suitable culture conditions for maintaining fungi;
-
Good manufacturing practice rules must be followed and controlled by the quality control lab (QC) and quality assurance lab (QA), labeling (e.g., avoidance instruction for some health conditions), as well as defining minimum/maximum consumed amount/day. Other effectors must be controlled such as the shelf life conditions;
-
The accepted amount that could be consumed concerning sex/age/weight and such;
-
Consumer feedback concerning any adverse effects or health problems;
-
It is preferred to use chemically defined media as well as a well-defined process that meets international standards to achieve consistent production of fungal proteins; Globalization of GMP, QC, QA, market practices, feedback, consumer service, etc. is necessary;
-
Restricted governmental laws for safety regulation should be available;
-
Inactivation of the used fungi in the end product is a crucial step and must be applied;
-
In case of waste usage (e.g., agriculture wastes in mushrooms cultivation), they should be free from any toxic components or heavy metals (and such) to guarantee safe final products;
-
The effect of cultivation conditions (other than media) on the microbes should be under control to avoid the production of any undesired secondary metabolites;
-
The nucleic acid content should not exceed 1%. In addition, the product that contains more than 1% nucleic acids should be fed to short life-span animals;
-
Products with short shelf life, particularly products from viable fungi such as fresh mushrooms, which are prone to spoilage should be continuously checked and properly stored in the markets;
-
Mycotoxins are known fungal products. Some safe fungal production processes under certain changes in the production conditions might lead to production of mycotoxins or other types of toxins. Only experts should decide if the proposed changes in the production conditions are accepted or not, therefore the new changes should be under investigations concerning the end product quality. One should not neglect any physical, chemical or biological measures;
-
The production process is not the end point. The end point will be when consumers utilize fungal products safely and for a long time. For that, investigating the effect of products on consumers (either human or animals) should not stop;
-
GM fungi products should be labeled and should be deactivated from any genetic elements.