Process for industrial production of food-graded fungal biomass

Field of invention

The present invention relates to a process for industrially producing human food-graded mycoprotein and/or fungal biomass from grain crops. In particular, the present invention relates to a process for producing human food-graded fungal biomass rich in mycoprotein and/or essential amino acids, said process comprising cultivating Neurospora intermedia in liquid medium under aerobic conditions. The present invention also relates to a novel fungal biomass, rich in mycoprotein and/or essential amino acids, which is produced with the process of the present invention, for use as a nutritious source for human use.

The present invention further relates to food produced with and/or comprising the novel human food-graded Neurospora intermedia biomass produced with the process of the present invention.

Background of the invention

Poor nutrition is a chronic problem often linked to poverty, poor nutrition understanding and practices, and deficient sanitation and food security and its consequences are immense contributors to deaths and disabilities worldwide. With climate changes threatening the availability of land for rearing livestock and the growing awareness of the environmental impact of meat production, there is an ongoing search to provide the world with a nutritious, abundant, environmentally friendly protein that is not animal-based.

One alternative to animal proteins are mycoproteins. E.g. Quorn™ products first came to market in the 1980s and are produced from the microorganism Fusarium venenatum. Mycoproteins are an excellent source of high quality protein and contain all the essential amino acids for adults. Mycoproteins are high in dietary fibre, low in fat and saturated fats and contain less cholesterol or trans fats than animal proteins. Mycoproteins are also low in sodium.

Numerous attempts to create new non-animalia sources of protein, based on cultured microbes, and in particular on filamentous fungi have been made both prior to the discovery of Fusarium venenatum and thereafter. Still, it has been hard to find an organism that can provide a palatable biomass with a food-like texture and a high enough protein content and which can grow satisfactorily in continuous culture to produce the quantities needed for commercial success.

The classification Neurospora contains both yeasts and fungi. Neurospora is commonly known as orange bread mold. It first received major attention in 1843, when there was a Neurospora infestation in French bakeries. Neurospora was domesticated for research purposes in the 1920s. There are three species of Neurospora classified today, namely Neurospora crassa, Neurospora intermedia and Neurospora tetrasperma.

Neurospora is widely used in genetics as a model organism (especially N. crassa) because it is quickly reproducing, is easy to culture and can survive on minimal media (inorganic salts, glucose, water and biotin in agar). Neurospora intermedia var.

oncomensis is believed to be the only mold belonging to Neurospora which is used in food production, such as in the production of oncom.

Oncom is one of the traditional staple foods of West Javan (Sundanese) cuisine,

Indonesia. There are two kinds of oncom: red oncom and black oncom. Oncom is closely related to tempeh; both are solid foods fermented in room temperature, so called“solid state fermentation”.

Usually oncom is made from the by-products from the production of other foods: soy bean tailings (okara) left from making tofu, peanut press cake left after the oil has been pressed out, cassava tailings when extracting the starch ( pati singkong), coconut press cake remaining after oil has been pressed out or when coconut milk has been produced. Since oncom production uses by-products to make food, it increases the economic efficiency of food production. Black oncom is made by using Rhizopus oligosporus while red oncom is made by using Neurospora intermedia var. oncomensis.

In the production of oncom, sanitation and hygiene are important to avoid contaminating the culture with bacteria or other fungi like Aspergillus flavus (which produces aflatoxin). Neurospora intermedia var. oncomensis and Rhizopus oligosporus reduce the aflatoxin produced by Aspergillus flavus. However, aflatoxin-producing molds ( Aspergillus spp.) are often naturally present on peanut presscake. Furthermore, coconut presscake can harbor the very dangerous Burkholderia gladioli, which produces two highly toxic compounds - bongkrek acid and toxoflavin. While it is known that soybeans are the best substrate for growing R. oligosporus to produce tempeh, oncom has not been as thoroughly studied. Today, Neurospora intermedia var. oncomensis has been used to produce Oncom, low-salt miso by koji fermentation using soy-oncom and okara-oncom (9:1 ), (oncom miso, i.e. O-miso) as well as for processing colored quinoa seeds which were subjected to a modified oncom-type processing (precooking followed by solid-state fermentation with Neurospora intermedia).

All known use of Neurospora intermedia to produce food up until today is thus based on ’’solid state fermentation”, wherein the fungi is applied to the solid media that it is supposed to ferment. The fermentation takes place during several days at room- temperature and in open cultures, which is favorable under the natural climate conditions in the sub-tropic and tropic climates, wherein oncom is a traditional food product, i.e. at high temperatures and high humidity. Oncom is therefore less produced in other parts of the world. What is more, oncom does to a high degree consist of the material that is fermented and to a lesser degree of the fungus. Thus, the starting material dominates the nutritional value of oncom.

In the present application, a novel production process is disclosed which is suitable for producing a new food grade fungal biomass in particular on an industrial scale, rich in mycoprotein and essential amino acids. In said novel process, Neurospora intermedia is cultured in fluid suspensions which gives rise to a fungal biomass that can easily be harvested and used as a nutritional source for human consumption on its own.

Summary of the present invention

The present invention relates to a process for producing food-graded mycoprotein from grain crops, wherein said process comprises a single cultivation step consisting of cultivating filamentous fungi in liquid medium under aerobic conditions on an industrial scale, and wherein said filamentous fungi are selected from the group consisting of food- related strains of Neurospora intermedia. It will be appreciated that the process described herein is a process for producing food-graded mycoprotein and/or fungal biomass.

The present invention also provides a process for producing food-graded mycoprotein and/or fungal biomass from grain crops for human consumption, wherein said process comprises no yeast fermentation step and no more than one step of fungal cultivation of filamentous fungi, characterized by cultivating said filamentous fungi in liquid medium under aerobic conditions, wherein said filamentous fungi are selected from the group consisting of food-related strains of Neurospora intermedia.

A process for producing food-graded mycoprotein and/or fungal biomass from grain crops according to the present invention does not comprise and/or is devoid of any process step comprising fermentation and/or cultivation of yeast. Thus, a process of the present invention enables production of food-graded fungal biomass rich in mycoprotein and/or essential amino acids for human consumption which is free and/or at least essentially free from yeast-protein. In this document, the expressions’’essentially free” and’’substantially free” may be used interchangeably. Further, these expressions are understood to mean that a component referred to is absent, or is present in a very low amount such as an amount that does not affect the taste and/or function of a product into which said component is incorporated. Thus, the process for producing food-graded mycoprotein from grain crops as described herein provides mycoprotein which is substantially free from yeast, such as yeast-protein and/or yeast cells. Further, the process of the present invention enables production of food-graded fungal biomass rich in mycoprotein and/or essential amino acids for human consumption which is free and/or at least substantially free from yeast, such as yeast-protein and/or yeast cells. However, there might of course be residues of domestic and/or wild yeast in the harvested fungal biomass from earlier use(s) of the involved equipment and/or from people, such as staff, and/or environment involved in the process and/or from subsequent production steps which are added to a process according to the present invention.

A process according to the present invention is intended for industrial scale production (i.e. at least 10 m ³ cultivation volume) of human food and thus relates to a process for industrially producing food-graded mycoprotein and/or fungal biomass from grain crops. In particular, the present invention relates to a process for industrially producing food-graded fungal biomass which is rich in mycoprotein and/or essential amino acids for human consumption, said process comprises cultivating Neurospora intermedia on an industrial scale in liquid medium under aerobic conditions.

Consequently, the present invention also relates to a novel fungal biomass for use as a nutritious source for humans, rich in mycoprotein and/or essential amino acids, which is free and/or at least essentially free from yeast-protein and which is produced with and/or harvested from a process of the present invention. Thus, the present disclosure provides a novel fungal biomass for use as a nutritious source for humans, rich in mycoprotein and/or essential amino acids, which is free and/or at least substantially free from yeast, such as yeast-protein and/or yeast cells, and which is produced with and/or harvested from a process for production of fungal biomass and/or food-graded mycoprotein as described herein. The present invention also relates to a novel preparation of isolated mycoprotein, which is free and/or at least essentially free from yeast, such as yeast- protein and/or yeast cells, and which is produced with and/or harvested and/or isolated from a novel fungal biomass of the present invention. Thus, the isolated mycoprotein as described herein may be substantially free from yeast, such as yeast-protein and/or yeast cells.

The present invention further relates to food, such as human food product, and/or food supplement and/or a protein replacement intended for human consumption which is produced with a process according to the present invention and/or which comprises a novel fungal biomass and/or food-graded mycoprotein according to the present invention, for use as a nutritious source for humans.

The present invention relates to a process for producing food-graded mycoprotein and/or fungal biomass from grain crops according to the present invention, wherein said grain crops are selected from the group consisting of cereals and legumes, such as but not limited to wheat, rye, barley, oat, sorghum, corn, peas, and beans such as soybeans.

In a process for producing food-graded mycoprotein and/or fungal biomass from grain crops according to the present invention, the grain crops are typically provided as flour.

In a process for producing food-graded mycoprotein and/or fungal biomass from grain crops according to the present invention, the filamentous fungi are cultivated in liquid medium under agitation such as under constant agitation; said liquid medium is preferably water-based.

The present invention also relates to a process for producing food-graded mycoprotein and/or fungal biomass from grain crops, wherein the fungal cultivation step takes place in an airlift reactor and/or a bubble column.

The present invention also relates to a process for producing food-graded mycoprotein and/or fungal biomass from grain crops as described herein, wherein the food-graded mycoprotein and/or fungal biomass is killed. For instance, the food-graded mycoprotein and/or fungal biomass may be substantially killed. Furthermore, the present invention also relates to a process for producing food-graded mycoprotein and/or fungal biomass from grain crops, wherein the process produces between 1-30 g/L fungal biomass, such as between 1-30 g/L or 1-20 g/L, such as at least 1 , 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or 30 g/L fungal biomass.

A fungal biomass produced with a process according to the present invention comprises an average amount of 300-600 g mycoprotein/kilo dry weight fungal biomass, such as at least 300, 350, 400, 450, 500, 550, or 600 g mycoprotein/kilo dry weight fungal biomass, such as between 300-350, 350-450, 350-400, 400-450, 450-500, 400-500, 350-500, 500- 550, 500-600, or 550-600 g mycoprotein/kilo dry weight fungal biomass.

A fungal biomass comprising food-graded mycoproteins, produced with a process according to the present invention typically comprises between 0.3-0.5g/100g fungal biomass of phenylalanine, between 0.15-0.4g/100g fungal biomass of histidine, between 0.3-0.5g/100g fungal biomass of isoleucine, between 0.3-0.7g/100g fungal biomass of leucine, between 0.4-0.7g/100g fungal biomass of lysine, between 0.05-0.2g/100g fungal biomass of methionine, between 0.3-0.5g/100g fungal biomass of threonine, between 0.05-0.2g/100g fungal biomass of tryptophan, between 0.4-0.6g/100g fungal biomass of valine, and/or between 0.25-0.55g/100g fungal biomass of arginine.

A fungal biomass comprising food-graded mycoproteins, produced with a process according to the present invention is free and/or at least essentially free from yeast. Thus, the fungal biomass comprising food-graded mycoproteins, produced with a process described herein, is free or substantially free from yeast, such as yeast-protein and/or yeast cells.

A fungal biomass and/or food-graded mycoprotein and/or essential amino acids isolated and/or purified from said fungal biomass, produced with a process according to the present invention, is/are intended for human consumption and will thus be comprised in a human food product and/or food supplement or be presented as a purified ingredient. Consequently, the present invention further relates to a human food product and/or food supplement comprising and/or consisting of said fungal biomass and/or food-graded mycoprotein and/or essential amino acid(s) isolated and/or purified from said fungal biomass produced with a process according to the present invention.

A human food product and/or food supplement according to the present invention comprises said fungal biomass and/or food-graded mycoprotein and/or essential amino acids isolated and/or purified from said fungal biomass, produced with a process according to the present invention, in an essential amount. The amount of fungal biomass, food-graded mycoprotein and/or essential amino acids will vary according to the nature of the food, product and/or dish prepared with said ingredients. For example, if a fungal biomass according to the present invention is formed into nuggets, said food product will predominantly consist of the fungal biomass, whereas other foods, such as protein shakes can vary from 65-90% mycoprotein content.

Typically, a human food product and/or food supplement according to the present invention comprises said fungal biomass and/or food-graded mycoprotein and/or essential amino acids isolated and/or purified from said fungal biomass, produced with a process according to the present invention, in an amount of at least 50% (w/w), such as of at least 75%, 80%, 90% or 99% or 100% (w/w) of the total dry weight of the food product and/or food supplement. As used herein“% (w/w)” refers to the weight percent of the ingredient referred to of the total weight of the preparation, composition or product referred to. In this document, the expressions”% (w/w)”,“% w/w”,“per cent by weight” and“wt%” may be used interchangeably.

A fungal biomass and/or food-graded mycoprotein and/or essential amino acids isolated and/or purified from said fungal biomass, produced with a process according to the present invention, can be used as a substitute for and/or a supplement to a standard protein source of a human food product and/or food supplement according to the present invention.

The present invention also provides a human food product and/or food supplement as described herein for use as a substitute for, and/or a supplement to, a standard protein source. Thus, the present invention provides a human food product and/or food supplement as described herein as a substitute for, and/or supplement to, a standard protein source.

There is also provided a use of a food-graded mycoprotein and/or fungal biomass produced with the process described herein for the manufacture of a human food product and/or food supplement.

Further, there is also provided a use of a human food product and/or food supplement comprising or consisting of food-graded mycoprotein and/or fungal biomass produced with the process as described herein as a substitute for, or a supplement to, a standard protein source.

Figure legends

Table 1 in figure 1 : Experimental design carried out for starch as carbon source. The same experimental design was applied to the medium containing glucose or sucrose as carbon source.

Table 2 in figure 2: Production and yields of ethanol and biomass together with the final pH values after cultivation of N. intermedia in eight combinations of synthetic medium components using glucose as carbon source.

Table 3 in figure 3: Production and yields of ethanol and biomass together with the final pH values after cultivation of N. intermedia in eight combinations of synthetic medium components using sucrose as carbon source.

Table 4 in figure 4: Production and yields of ethanol and biomass together with the final pH values after cultivation of N. intermedia in eight combinations of synthetic medium components using starch as carbon source.

Figure 5: Ethanol concentration profiles during cultivation of N. intermedia in oat flour, wheat flour, field beans and field peas with or without addition of extra nitrate.

Figure 6: Data achieved from manual sampling and analyzed in HPLC.

Figure 7: Data achieved from manual sampling and analyzed in HPLC.

Figure 8: Data achieved from manual sampling analyzed in lab.

Figure 9: Data achieved from manual sampling and analyzed in HPLC.

Figure 10: Data achieved from manual sampling and analyzed in HPLC.

Figure 11 : Data achieved from manual sampling analyzed in lab.

Figure 12: Data achieved from manual sampling and analyzed in HPLC. Figure 13: Data achieved from manual sampling and analyzed in HPLC.

Figure 14: Data achieved from manual sampling analyzed in lab.

Detailed description of the invention

The present invention relates to a process for producing food-graded mycoprotein and/or fungal biomass from grain crops for human consumption, wherein said process comprises no more than one fungal cultivation stage of filamentous fungi, characterized by cultivating said filamentous fungi in liquid medium under aerobic conditions, and wherein said filamentous fungi are selected from the group consisting of food-related strains of

Neurospora intermedia.

Food-related strains of Neurospora intermedia

The present invention relates to a process comprising no more than one fungal cultivation stage, wherein the fungi introduced in the fungal cultivation stage are filamentous fungi. The filamentous fungi are selected from the group consisting of food-related strains of Ascomycetes ; in particular, the filamentous fungi are Neurospora intermedia.

In an example, the filamentous fungus may be Neurospora intermedia CBS 131.92 (Centraalbureau voor Schimmelcultures, The Netherlands).

Fungal biomass

The present invention relates to a fungal biomass produced by a process according to the present invention which can be either washed or non-purified. In particular, the present invention relates to a food-graded fungal biomass, wherein said fungal biomass is produced by a process according to the present invention. Thus, the fungal biomass is obtained by, or obtainable by, a process for producing food-graded mycoprotein as described herein.

A process for producing human food-graded fungal biomass according to the present invention typically yields and/or produces between 1-30 g/L fungal biomass from the cultivation, such as between 1-20 g/L, such as at least 5, 10, 15, 16, 17, 18, 19, or 20 g/L fungal biomass from the cultivation. For instance, the process described herein may produce between 1-30 g/L of human food-graded fungal biomass. In other words, the process described herein may produce human food-graded fungal biomass in the range of about 1 g/L to about 30 g/L.

A fungal biomass according to the present invention is rich in protein with an unusually favourable composition of amino acids. It typically has a crude protein content between 40-60% (w/w), i.e. in the range of from about 40 % (w/w) to about 60 % (w/w), such as approximately 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60% (w/w). The crude protein content of the fungal biomass may be at least 30, 35, 40, 45, 50, 55, or 60% (w/w).

A fungal biomass according to the present invention typically comprises between 3-7,5, 4- 6 or 4.5-5.5 g/L, such as at least 3.5, 4, 4.5, 5, or 5.5 g/L, of fungal biomass. For instance, the fungal biomass may comprise about 5 g/L of fungal biomass before drying. Further, the fungal biomass may comprise equal to or above about 3, such as about 4.5, such as about 5, such as about 5.5, such as about 6, such as about 6.5, or such as about 7 g/L of fungal biomass before drying.

A fungal biomass produced with a process as described herein may comprise an average amount of from about 300 to about 600 g mycoprotein/kilo dry weight fungal biomass, such as equal to or above about 300, about 350, about 400, about 450, about 500, about 550, or about 600 g mycoprotein/kilo dry weight fungal biomass, such as 300-350, 350- 450, 350-400, 400-450, 450-500, 400-500, 350-500, 500-550, 500-600, or 550-600 g mycoprotein/kilo dry weight fungal biomass.

Purifying the fungal biomass may comprise a variety of washing steps. Depending on the desired degree of purity of the fungal biomass, the person skilled in the art will know how to employ different standard procedures for purification.

A fungal biomass according to the present invention contains vitamins, unsaturated 18 carbon fatty acid compounds and chitosan. In particular, a fungal biomass according to the present invention comprises essential amino acids, omega-3 and/or omega-6 fatty acids.

The present invention relates to a process for producing food-graded fungal biomass comprising no more than one fungal cultivation stage, wherein the fungi introduced in the fungal cultivation stage are filamentous fungi selected from the group consisting of food- related strains of Ascomycetes, in particular Neurospora intermedia. The whole cell- biomass obtained by the process described herein may be killed and/or included into standard types of food, food supplement, or feed compositions. For instance, the fungal biomass as described herein may be killed, or at least be substantially killed. For instance, the food-graded mycoprotein as described herein may be killed, or at least be

substantially killed. For example, the food-graded mycoprotein and/or fungal biomass as described herein may be killed by a heat-treatment. For example, such heat-treatment may decrease the amount of RNA in the food-graded mycoprotein and/or fungal biomass as described herein.

Alternatively, the fungal biomass and/or parts of the fungal biomass as described herein is/are not killed. The fungal biomass produced according to the present invention can be used as probiotic(s). It has been found that active substances in the fungal biomass, such as Mannan-oligosaccharides and Beta-Glucans, may result in modulation of gut bacteria, stimulation of immune system and reduced risk of pathogen colonization to the gut wall.

Therefore, the fungal biomass according to the present invention is well suited as a nutrient source for humans. The present invention thus discloses a nutrient source comprising fungal biomass as described herein. Additionally or alternatively, the present disclosure provides a human food product and/or food supplement comprising and/or consisting of a nutrient source as described herein.

Human food needs to be safe for consumption by humans, and should be of a high nutritional value and also taste good. The fungal biomass according to the present invention comprises chitin and orange pigments. It has a high water content of between 15-30%, such as at least 15, 16, 17, 18, 19, 20, 25 or 30% which makes it particularly well suited for processing, reprocessing and/or refining. For instance, it may have a high water content in the range of from about 15% to about 30%, such as in the range of from about 15% (w/w) to about 30% (w/w). The fungal biomass according to the present invention can easily be formed into nuggets, mince, force or piecemeal, or any other form suitable for preparing meals, cooking and/or food transformation. During processing, it will easily take up alternative sources of fluid and/or ingredients and is thus easily spiced with e.g. soy, salt, oil, colorants, etc.

Food-graded mycoprotein

In the present context, the terms“food-graded” mycoprotein, or“human food-graded” mycoprotein, or mycoproteins are used interchangeably and define mycoprotein that is of a quality suitable for human consumption and/or for use in food production or storage.

Human Grade means‘food grade’,‘edible’ and/or fit for human consumption.

Proteins are the basis of many animal body structures (e.g. muscles, skin, and hair) and form the enzymes which catalyze chemical reactions throughout the body. Each protein molecule is composed of amino acids which contain inter alia nitrogen and sometimes sulphur. Essential amino acids

Proteins consist of amino acids in different proportions. The most important property and defining characteristic of protein from a nutritional standpoint is its amino acid

composition.

Amino acids which an animal cannot synthesize on its own from smaller molecules are deemed essential. The synthesis of some amino acids can be limited under special pathophysiological conditions, such as prematurity in the infant or individuals in severe catabolic distress and those are called conditionally essential.

An essential amino acid, or indispensable amino acid, must be supplied in diet. The nine amino acids humans cannot synthesize are phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine (i.e., F, V, T, W, M, L, I, K, H). Six other amino acids are considered conditionally essential in the human diet. These six are arginine, cysteine, glycine, glutamine, proline, and tyrosine (i.e., R, C, G, Q, P, Y).

In the present context, fungal biomass produced according to the present invention is rich in mycoproteins, essential amino acids and conditionally essential amino acids.

In one example, as exemplified in examples 4-6, fungal biomass produced according to the present invention comprises one or more of the following: between 0.3-0.5g/100g fungal biomass of phenylalanine, between 0.15-0.4g/100g fungal biomass of histidine, between 0.3-0.5g/100g fungal biomass of isoleucine, between 0.3-0.7g/100g fungal biomass of leucine, between 0.4-0.7g/100g fungal biomass of lysine, between 0.05- 0.2g/100g fungal biomass of methionine, between 0.3-0.5g/100g fungal biomass of threonine, between 0.05-0.2g/100g fungal biomass of tryptophan, between 0.4-0.6g/100g fungal biomass of valine, between 0.25-0.55g/1 OOg fungal biomass of arginine. For instance, the fungal biomass may comprise from 0.3 to 0.5 g phenylalanine per 100 g of the fungal biomass, i.e. between 0.3-0.5 g/100g fungal biomass of phenylalanine. In this document,“g” stands for“gram(s)” and“L” stands for liter.

Human consumption

The fungal biomass according to the present invention and/or food-graded mycoprotein according to the present invention is/are intended and suitable as a nutritious source for human use. In consequence, they are suitable for human consumption and can be distributed, sold and/or consumed as human food product and/or food supplement. In contrast, pet food is plant or animal material intended for consumption by pets.

Typically sold in pet stores and supermarkets, it is usually specific to the type of animal, such as dog food or cat food. Most meat used for nonhuman animals is a byproduct of the human food industry, and is not regarded as "human grade".

Grain crops

The present invention relates to a process for producing fungal biomass and/or food- graded mycoprotein from grain crops. Grains are small, hard, dry seeds, with or without attached hulls or fruit layers, harvested for human or animal consumption. "Grain crops" are grain seed producing plants. The two main types of commercial grain crops are cereals (e.g. but not limited to wheat, rye oat, corn (maize), barley, rye, sorghum, millet, triticale, fonio) and legumes (e.g. but not limited to peas, and beans such as soybeans).

In contrast, an industrial crop, also called a non-food crop, is a crop grown to produce goods for manufacturing, for example of fibre for clothing, rather than food for

consumption. In an example, the present invention relates to a process for producing food-graded mycoprotein from oat and/or wheat.

In the present context, a cereal is any grass cultivated for the edible components of its grain (botanically, a type of fruit called a caryopsis), composed of the endosperm, germ, and bran. Cereal grains are grown in greater quantities and provide more food energy worldwide than any other type of crop and are therefore staple crops. Edible grains from other plant families, such as buckwheat (Polygonaceae), quinoa (Amaranthaceae) and chia (Lamiaceae), are commonly referred to as pseudocereals. Crops from pseudocereals are in the present invention also enclosed in the term“cereal crops”.

In their natural form (as in whole grain), cereals are a rich source of vitamins, minerals, carbohydrates, fats, oils, and protein. When refined by the removal of the bran and germ, the remaining endosperm is mostly carbohydrate. The present invention relates to a process for producing food-graded mycoprotein from either whole grain and/or bran, germ, or endosperm of grain crops. Thus, in the present context, the term“grain crops” encompasses any one of whole grain, bran, germ or endosperm of cereals. The grain crops can be refined, milled and/or used as whole grain.

In an example, the present invention relates to a process for producing food-graded mycoprotein from grain crops, wherein the grain crops are provided as flour.

In a particular example, the present invention relates to a process for producing food- graded mycoprotein from grain crops, wherein the grain crops are provided as food grade oat flour as the main ingredient.

Process steps

The present invention relates to a process for producing fungal biomass and/or food- graded mycoprotein from grain crops, wherein said process comprises a single cultivation step consisting of cultivating filamentous fungi in liquid medium under aerobic conditions on an industrial scale, and wherein said filamentous fungi are selected from the group consisting of food-related strains of Neurospora intermedia.

Typically, and as described in further detail in the experimental section, the fungal cultivation is started with adding an amount of a pre-culture (an inoculum) of the filamentous fungi to a defined amount of medium in a large vessel. The pre-culture can have been grown on a medium comprising yeast, and/or yeast protein, but any yeast residue transferred into the single fungal cultivation step of the present process does not comprise any living yeast cells and the amount of yeast protein is insignificant in comparison to the amount of medium in the large-scale (industrial scale) single fungal cultivation step for producing the fungal biomass and/or food-graded mycoprotein of the present invention. Thus, the fungal biomass and/or food-graded mycoprotein as described herein is/are obtained from/obtainable by a process for producing fungal biomass and/or food-graded mycoprotein as described herein.

A process for producing food-graded mycoprotein from grain crops according to the present invention is thus free from any other fungal or other cultivation step and thus produces food-graded mycoprotein which is essentially free from yeast-protein and/or yeast cells. Thus, the food-graded mycoprotein produced by a process as described herein is substantially free from yeast, such as yeast-protein and/or yeast cells. In the present context, the expressions’’essentially free” and’’substantially free” may be used interchangeably. A process for producing food-graded mycoprotein and/or fungal biomass from grain crops according to the present invention comprising a single cultivation step enables a process which is less complex than a process comprising more than one cultivation steps.

Furthermore, such process as described herein may also be cost-efficient. For example, such process as described herein may involve lower investments costs and/or operational expenses.

A process for producing food-graded mycoprotein and/or fungal biomass from grain crops according to the present invention does not comprise and/or is devoid of any process step comprising fermentation and/or cultivation of yeast. Thus, the process of the present invention enables production of food-graded fungal biomass rich in mycoprotein and/or essential amino acids for human consumption which is free and/or at least essentially free from yeast, such as yeast-protein and/or yeast cells. Thus, the food-graded mycoprotein is free or substantially free from yeast, such as yeast-protein and/or yeast cells. However, there might of course be residues of domestic and/or wild yeast in the harvested fungal biomass from earlier use of equipment and/or from people such as staff and/or environment involved in the process and/or from subsequent production steps added to a process according to the present invention.

The process as described herein enables production of food-graded fungal biomass and/or food-graded mycoprotein which are well suited for human consumption since the taste of the food-graded fungal biomass and/or food-graded mycoprotein is good. For instance, the food-graded fungal biomass and/or food-graded mycoprotein may lack, or at least substantially lack, the taste of yeast. For instance, the food-graded fungal biomass and/or food-graded mycoprotein may lack the taste of yeast and its bi-products.

The present invention relates to a process for producing fungal biomass and/or food- graded mycoprotein from grain crops, wherein said process comprises no more than one (i.e. a single) fungal cultivation stage, characterized by cultivating filamentous fungi in liquid medium under aerobic conditions, wherein said filamentous fungi are selected from the group consisting of food-related strains of Neurospora intermedia.

Said fungi may be cultivated in liquid medium such as a liquid medium under constant agitation.

Said liquid medium is water-based, and can further comprise salts, minerals, vitamins, nitrogen and/or phosphates. Said liquid medium can further alternatively or in addition comprise ingredients selected from the group consisting of calcium chloride, magnesium chloride, potassium nitrate, phosphoric acid and any combination thereof.

The liquid medium can further comprise one or more enzyme(s), selected from the group consisting of industrial enzymes for food application, such as but not limited to hydrolases, isomerases, ligases, lyases, oxidoreductases, transferases, proteases, such as but not limited to lipases, nucleases, cellulases, amylases, collagenases, pectinases, epimerases, carboxylases, synthetases, decarboxylases, dehydrases, dehydrogenases, oxidases, reductases, kinases, transaminases, such as in particular but not limited to alpha amylase, bromelain, lactase, papain, ficin, and/or ananase.

The fungal cultivation step described herein may take place in an airlift reactor, a stirred tank reactor and/or a bubble column.

An aerobic organism or aerobe is an organism that can survive and grow in an

oxygenated environment. In contrast, an anaerobic organism (anaerobe) is any organism that does not require oxygen for growth. Some anaerobes react negatively or even die if oxygen is present. When an organism is able to survive in both oxygen and anaerobic environments, the use of the Pasteur effect can distinguish between facultative anaerobes and aerotolerant organisms. If the organism is using fermentation in an anaerobic environment, the addition of oxygen will cause facultative anaerobes to suspend fermentation and begin using oxygen for respiration. Aerotolerant organisms must continue fermentation in the presence of oxygen. Under aerobic conditions in the present context means that the cultivation takes place in the presence of oxygen, i.e. in an oxygenated environment and/or under aeration. The cultivation may be carried out at an aeration of 100 kg/h and elevated up to 500 kg/h over 9 h after inoculating, or of 100 kg/h and elevated up to 750 kg/h over 12 h after inoculating, or of 100 kg/h and elevated up to 750 kg/h over 10 h after inoculating. In this document,“kg” stands for“kilogram(s)” and“h” stands for“hour(s)”.

The cultivation step of the process described herein may be performed at a temperature of about 35°C ± 4.5°C. For instance, the cultivation step of the process described herein may be performed at a temperature between 30-40°C, 31-39°C, 32-38°C, 33-37°C, 34- 36°C, or 31.5-39.5°C. It will be appreciated that the expression“between .” such as “between 30-40°C” includes the end points of the range. Thus“between 30-40°C” intends “from 30°C to 40°C”.

In an example, the present invention relates to a process comprising fungal cultivation conditions at a pH of between 5-6, such as of 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6, preferably at a pH of 5.7. For instance, the fungal cultivation step may be performed at a pH in the range of from about 5 to about 6, such as about 5.7.

In an example, the present invention relates to a process comprising fungal cultivation conditions at a temperature of 35°C ± 4.5°C during cultivation, pH was 5.7 and aeration of 100 kg/h and elevated up to 500 kg/h over 9 h after inoculating.

In an example, the present invention relates to a process comprising fungal cultivation conditions at a temperature of 35°C ± 4.5°C during cultivation, pH was 5.5 and aeration of 100 kg/h and elevated up to 750 kg/h over 12 h after inoculating.

In yet another example, the present invention relates to a process comprising fungal cultivation conditions at a temperature of 35°C ± 4.5°C during cultivation, pH was 5.9 and aeration of 100 kg/h and elevated up to 750 kg/h over 10 h after inoculating.

In an example, the present invention relates to a process comprising fungal cultivation such as fungal cultivation under mechanical mixing such as fungal cultivation under mechanical mixing at low effect.

Industrial scale

The process according to the present invention is intended for industrial scale production (i.e. cultivation takes place in at least 10 m ³ cultivation volume(s), such as at least 20 m ³ , such as at least 30 m ³ cultivation volume(s)) of human food and thus relates to a process for industrially producing food-graded mycoprotein and/or fungal biomass from grain crops. In particular, the present invention relates to a process for industrially producing food-graded fungal biomass rich in mycoprotein and/or essential amino acids for human consumption, said process comprising cultivating Neurospora intermedia on an industrial scale in liquid medium under aerobic conditions.

In an example, the present invention relates to a process for producing food-graded fungal biomass on an industrial scale comprising a single fungal cultivation stage/step, wherein the fungal cultivation step takes place in an airlift reactor, a stirred tank reactor and/or a bubble column.

The cultivation of the filamentous fungi may take place in a fluid suspension (such as in at least 10 m ³ fluid suspension) and thus it is easy to harvest the fungal biomass from the cultivation media and also to wash the fungal biomass. The fungal biomass can thus be produced substantially pure and does not need to contain traces from the cultivation media or the grain crops used in the process. Thus, the fungal biomass may be produced substantially pure and contains no or substantially no traces from the cultivation media or the grain crops used in the process.

Human food and/or food supplement

A human food product and/or food supplement typically comprises a nutrient source according to the present invention, such as a fungal biomass and/or a food-graded mycoprotein according to the present invention, in an amount of at least 5% (w/w), such as at least 7.5% (w/w), such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% (w/w) of the total dry weight of the human food product and/or food supplement. For instance, the fungal biomass or food-graded mycoprotein may be present in an amount of equal to or above about 7.5% (w/w), such as about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99% (w/w) of the total dry weight of the human food product and/or food supplement. The human food product and/or food supplement may be used as a substitute for, or a supplement to a standard protein source. In this document, the term “standard protein source” refers to a source rich in protein, such a food product rich in protein. For example, the standard protein source may be meat, such as minced meat.

In an example, the human food product and/or food supplement typically comprises a nutrient source according to the present invention, such as a fungal biomass or a food- graded mycoprotein according to the present invention, in an amount equal to or less than 100% (w/w) of the total dry weight of the human food product and/or food supplement.

The human food product and/or food supplement may be used as a substitute for, or a supplement to a standard protein source.

The present invention provides a use of a fungal biomass and/or food-graded mycoprotein as described herein for the manufacture of a human food product and/or a food supplement. Further, the present invention provides a use of a human food product and/or food supplement comprising or consisting of food-graded mycoprotein and/or fungal biomass produced with a process as described herein as a substitute for, or a supplement to, a standard protein source.

Experiments Experiment 1

Pre-cultivation of Neurospora intermedia

1.1. Materials and Methods

1.1.1. Microorganism

The ascomycete Neurospora intermedia CBS 131.92 (Centraalbureau voor

Schimmelcultures, The Netherlands) was used throughout this study. The strain was maintained on potato dextrose agar (PDA) slants containing (in g/L): glucose 20, agar 15, and potato extract 4. The slants were renewed every six months. New PDA plates were prepared via two days incubation at 30 °C followed by storage at 4 °C. The spore solution was prepared by flooding the plates with 20 ml. sterile distilled water; a disposable plastic spreader was used to extract the spores. Spore number was determined by using a counting chamber.

1.1.2. Inoculum preparation

Neurospora intermedia was cultivated in two 250 ml. Erlenmeyer flasks with 100 ml of growth medium composed of glucose (20 g/L) and yeast extract (5 g/L). Four milliliters of spore solution (1.0><10 ⁷ spores/mL) were added to each flask followed by 24 h cultivation at 35 °C in a water bath shaking at 125 rpm.

1.1.3. Cultivation in a 26 L bubble column

The pre-grown inoculum was added to 20 L of cultivation medium containing (in g/L): sucrose 10, yeast extract 2.5, ZnSO ₄ .7H ₂ 0 0.9, MnCI ₂ .4H ₂ 0 0.19, CoCI ₂ .2H ₂ 0 0.06 and CUS0 ₄ .5H ₂ 0 0.06. The medium was adjusted to pH 5.0 with 2 M H ₂ S0 . Semi-continuous cultivations were carried out in a 2 m high, 15 cm diameter bubble column reactor with 26 L total volume (Bioengineering, Switzerland) at 1.5 vvm (air volume per culture volume per minute). The reactor was sterilized in situ with injection of steam (130 °C, 20 min). The cultivation temperature in the bubble column was maintained at 35 ± 0.4 °C. At every 12 h of cultivation, 75% of the grown medium (that was stored for further cultivation at larger scale) was replaced by fresh medium. Samples were taken before each medium change for HPLC analysis in order to monitor the concentration of ethanol, left sugars and organic acids.

1.1.4. High-performance liquid chromatography (HPLC)

A hydrogen-ion based ion-exchange column (Aminex HPX-87H, Bio-Rad, USA) at 60 °C and 0.6 mL/min 5 mM H ₂ S0 ₄ as eluent together with a refractive index (Rl) detector (Waters 2414) were used for analysis of acetic acid, ethanol, glycerol, lactic acid and sucrose. All supernatants for HPLC analysis were obtained after sample centrifugation for 10 min at 10,000xg.

Experiment 2

Cultivation of Neurospora intermedia in synthetic medium

2.1. Materials and Methods

2.1.1. Cultivation in shake-flasks

As a fist part of the project, an experimental design was carried out where the effect of different factors on growth of Neurospora intermedia in synthetic medium, containing glucose, sucrose or starch as carbon sources, was investigated. The factors studied included type of nitrogen source ((NH ₄ ) ₂ S0 ₄ and NaN0 ₃ ), C/N ratio (5 and 20), and presence vs absence of vitamins. An example of an experimental design carried out using starch as carbon source is presented in Table 1 (see figure 1 ).

The effect of the investigated factors was revealed by considering the highest amount of ethanol produced and by the fungal biomass dry weight at the end of the cultivation.

The main conclusions from the statistical analysis were as follows:

- the nitrogen source plays a significant role on biomass and ethanol production, where significant higher values were obtained when NaN0 ₃ was used as the nitrogen source;

- Vitamins do not play a significant role;

- C/N ratio was only found to have a significant role when starch was the carbon source. No effect of C/N ratio has been found if only data for NaN0 ₃ are considered. Production (in g/l) and yields (g/g) of ethanol and biomass together with the final pH values are presented in Tables 2, 3 and 4 (see figures 2, 3 and 4). As can be observed from the tables, a nitrate-based nitrogen source led to a significantly better output (ethanol and biomass) when added to the growth medium for N. intermedia. In addition, the pH values followed different trends when different nitrogen sources were added to the medium, where addition of NaN0 ₃ resulted in an increase in pH (the initial pH was 5.0- 5.5). It is hypothesized that higher yields of biomass could have been obtained if the harvesting had been applied earlier during cultivation.

Table 2 (figure 2) shows the production and yields of ethanol and biomass together with the final pH values after cultivation of N. intermedia in eight combinations of synthetic medium components using glucose as carbon source.

Table 3 (figure 3) shows the production and yields of ethanol and biomass together with the final pH values after cultivation of N. intermedia in eight combinations of synthetic medium components using sucrose as carbon source.

Table 4 (figure 4) shows production and yields of ethanol and biomass together with the final pH values after cultivation of N. intermedia in eight combinations of synthetic medium components using starch as carbon source.

Experiment 3

Cultivation of Neurospora intermedia in starchy substrates

3.1. Materials and Methods

3.1.1. Experiments in shake-flasks

Five different starch-containing substrates were used throughout the study, including oat flour, wheat flour, field beans, field peas and white beans. The starch content of each substrate is presented in Table 5a and 5b. Table 5a: Substrates used during the research with respective starch content and concentration in the medium for growth of N. intermedia.

Concentration Initial Starch

Substrate % Starch

used Concentration (g/l)

Wheat flour 78.4 ± 3.5 30 23.52

Oat flour 76.4 ± 0.1 34 25.98

Field beans 54.9 ± 3.0 50 27.45

Field peas 63.3 ± 7.7 50 31.65

White beans

Table 5b: Substrates used during the research with respective starch content and concentration in the medium for growth of N. intermedia.

Concentration Initial Starch

Substrate % Starch

used Concentration (g/l)

Wheat flour 74.1 ± 1.2 30 22.23

Oat flour 61.7 ± 0.6 34 20.98

Field beans 44.0 ± 0.1 50 22.00

Field peas 47.8 ± 0.1 50 23.90

White beans 35.3 ± 8.5 50 17.65

Fig. 5 shows the ethanol concentration profiles during cultivation of N. intermedia in oat flour, wheat flour, field beans and field peas with or without addition of extra nitrate.

The effect of nitrogen, salts (Mg, K, Ca) and trace metals addition to the different starchy substrates was studied in one-factor-at-a-time experiments. As can be observed in Fig. 5, the addition of nitrate did not lead to further improvements in the production of ethanol. Although only the effect of addition of nitrate is presented, no further improvements in ethanol production were found when supplementing further the medium with salts and trace metals. The same trends were found regarding final biomass concentrations.

Therefore, it seems that no extra supplementation of the medium is needed if the above substrates are used as cultivation medium for N. intermedia. The highest amounts of ethanol as well as the final biomass and their respective yields, obtained during cultivation of N. intermedia in the various starchy substrates, are presented in Tables 6a and 6b. The lowest biomass yield was observed when wheat flour was the cultivation medium, whereas the comparatively higher yields obtained when beans and/or peas were used as cultivation are more likely related to the entanglement of fungal filaments with medium particles.

Table 6a: Production (in g/l) and yields (in g/g) of ethanol and fungal biomass obtained from cultivation of N. intermedia in various starchy substrates without any

supplementation.

Ethanol Ethanol Biomass Biomass

Substrate

(g/i) (g/g) (g/i) (g/g)

Wheat flour 9.5 ± 1.9 0.39 ± 0.04 5.6 ± 0.8 0.24 ± 0.03

Oat flour 11.2 ± 0.3 0.43 ± 0.01 9.2 ± 3.5 0.35 ± 0.12 Field beans 10.2 ± 0.1 0.37 ± 0.00 11.3 ± 3.1 0.41 ± 0.10

Field peas 12.5 ± 0.6 0.39 ± 0.02 17.2 ± 1.8 0.54 ± 0.05 White beans

Table 6b: Yields (in g/g of flour) of ethanol and fungal biomass obtained from cultivation of N. intermedia in various starchy substrates without any supplementation.

Ethanol Biomass

Substrate

(g/g) (g/g)

Wheat flour 0.30 ± 0.03 0.13 ± 0.01

Oat flour 0.33 ± 0.01 0.18 ± 0.06

Field beans 0.20 ± 0.00 0.14 ± 0.03

Field peas 0.25 ± 0.01 0.22 ± 0.03

White beans 0.16 ± 0.00 0.17 ± 0.01

3.1.2. Cultivation in 4 I bench-scale airlift bioreactors using starchy materials

Cultivations in the various starchy substrates were also scaled up using 4 I bench-scale airlift bioreactors. Cultivations in oat flour were carried out at 0.4 and 1 vvm (volume of air per volume of liquid per minute) in order to investigate the aeration effect on ethanol and fungal biomass production (Table 7).

Table 7: Yields (in g/g of flour) of ethanol and fungal biomass obtained from cultivation of N. intermedia in various starchy substrates using 4 1 bench-scale airlift bioreactors.

Aeration Ethanol Biomass

Substrate

(vvm) (g/g) (g/g)

Oat flour 0.4 0.24 ± 0.07 0.17 ± 0.11

Oat flour 1 0.17 ± 0.08 0.25 ± 0.03

Wheat flour 1 0.07 ± 0.05 0.22 ± 0.02

Field beans 1 0.11 ± 0.00 0.21 ± 0.12

Field peas 1 0.14 ± 0.07 0.16 ± 0.04

As can be observed in Table 7, increasing the aeration rate from 0.4 to 1 vvm did not influence statistically the final yields of ethanol and biomass.

Experiment 4

Cultivation of 20 m ³ batches in an 80 m ³ reactor

Experiment 2 B1

In this experiment fungal biomass of the Ascomycete strain Neurospora intermedia was produced in an aerated 80 m ³ stirred tank reactor, to later be further processed to a protein rich biomass suitable for human consumption. Food grade oat flour was used as the main ingredient. 4.1.1. Materials and Methods

Prepare material in following order:

24 I (6.3 g dry matter/I liquid) viable biomass (N. intermedia ) in liquid was prepared according to Experiment 1 and used as inoculum in this experiment.

A stainless steel stirred tank reactor (80 m ³ ) cultivation vessel was prepared as following: Cleaning of cultivation vessel and attached piping and accessories with 70-80°C 3 % NaOH. Steam treatment of previously mentioned equipment for 5 h.

The reactor was first filled with 17 m ³ clean (hygienized by pasteurizing in 90°C) 70°C fresh water. Thereafter 20 g/l (340 kg) oat flour, acid (phosphoric acid) for pH adjustment (to pH 6.3), and industrial grade alpha amylase enzyme (0.5 I). Cooling of the medium started 3 h after enzyme addition and reached cultivation conditions after three more hours. The medium was mixed with mechanical stirring though-out the start-up process. The following cultivation aids and pH adjustment were added before inoculation of fungal biomass:

Cultivation conditions and process control

The inoculum (24 I liquid fungal biomass) was added to the broth after cultivation conditions were set as listed below.

- The temperature was kept at 35°C ± 4.5°C during cultivation

The pH was set to 5.7 and fluctuated according to Figure 8 throughout the cultivation

Aeration was set to 100 kg/h and elevated up to 500 kg/h over 9 h after inoculating Mechanical mixing was set to low effect Cultivation conditions and process parameters was monitored throughout the experiment and can be seen in Figure 6, Figure 7 and Figure 8. Temperature, volume, stirring and aeration was monitored online in the control system while pH, dissolved oxygen, ethanol, acetic acid, lactic acid, Dpn, Dp3, maltose glucose xylose, glycerol, dry matter and biomass was monitored by manual sampling and analysis. Anti-bacterial agent

(Fermasure XL) was added three times (7.5 I, 8 I, and 8 I at 13.5 h, 17.5 h and 22.5 h respectively). The experiment was terminated approx. 36 h after inoculation.

4.1.2. Results

A consumption of carbohydrates, represented in Figure 7, yielded in ethanol 4.0 g/l and fungal biomass 3.8 g dry matter/I (Figure 6, Figure 8). Experiment 5

Cultivation of 20 m ³ batches in an 80 m ³ reactor

Experiment B2 In this experiment fungal biomass of the Ascomycete strain Neurospora intermedia was produced in an aerated 80 m ³ stirred tank reactor, to later be further processed to a protein rich biomass suitable for human consumption. Food grade oat flour was used as the main ingredient. 5.1.1. Materials and Methods

Prepare material in following order:

30 I (3.0 g dry matter/I liquid) viable biomass ( N . intermedia ) in liquid was prepared according to Experiment 1 and used as inoculum in this experiment.

A stainless steel stirred tank reactor (80 m ³ ) cultivation vessel was prepared as following: Cleaning of cultivation vessel and attached piping and accessories with 70-80°C 3 % NaOH. Steam treatment of previously mentioned equipment for 5 h.

The reactor was first filled with 18 m ³ clean (hygienized by pasteurizing in 90°C) 70°C fresh water. Thereafter 19 g/l (340 kg) oat flour, acid (phosphoric acid) for pH adjustment (to pH 6.3), and industrial grade alpha amylase enzyme (0.7 I). Cooling of the medium started 2 h after enzyme addition and reached cultivation conditions after three more hours. The medium was mixed with mechanical stirring though out the start-up process. The following cultivation aids and pH adjustment was added before inoculation of fungal biomass:

Cultivation conditions and process control

The inoculum (30 I liquid fungal biomass) was added to the broth after cultivation conditions were set as listed below. The temperature was kept at 35°C ± 4.5°C during cultivation

The pH was set to 5.5 and fluctuated according to Figure 1 1 throughout the cultivation

Aeration was set to 100 kg/h and elevated up to 750 kg/h over 12 h after inoculating

Mechanical mixing was set to low effect

Cultivation conditions and process parameters was monitored throughout the experiment and can be seen in Figure 9, Figure 10 and Figure 1 1 . Temperature, volume, stirring and aeration was monitored online in the control system while pH, dissolved oxygen, ethanol, acetic acid, lactic acid, maltose (Dp2), maltotriose (Dp3), and other higher sugars (Dpn), maltose glucose xylose, glycerol, dry matter and biomass was monitored by manual sampling and analysis. Anti-bacterial agent Fermasure XL was added one time (9 I at 23 h). The experiment was terminated approx. 38.5 h after inoculation.

5.1.2. Results

A consumption of carbohydrates, represented in Figure 10, yielded in ethanol 2.5 g/l and fungal biomass 4.0 g dry matter/I (Figure 9, Figure 1 1 ).

Experiment 6

Cultivation of 20 m ³ batches in an 80 m ³ reactor

Experiment B5

In this experiment fungal biomass of the Ascomycete strain Neurospora intermedia was produced in an aerated 80 m ³ stirred tank reactor, to later be further processed to a protein rich biomass suitable for human consumption. Food grade oat flour was used as the main ingredient.

6.1.1. Materials and method

Prepare material in following order:

30 I viable biomass ( N . intermedia ) in liquid was prepared according to Experiment 1 and used as inoculum in this experiment.

A stainless steel stirred tank reactor (80 m ³ ) cultivation vessel was prepared as following: Cleaning of cultivation vessel and attached piping and accessories with 70-80°C 3 % NaOH. Steam treatment of previously mentioned equipment for 5 h. The reactor was first filled with 17 m ³ clean (hygienized by pasteurizing in 90°C) 70°C fresh water. Thereafter 20 g/l (334 kg) oat flour, acid (phosphoric acid) for pH adjustment (to pH 5.7), and industrial grade alpha amylase enzyme (0.6 I). Cooling of the medium started 3.5 h after enzyme addition and reached cultivation conditions after three more hours. The medium was mixed with mechanical stirring though out the start-up process. The following cultivation aids and pH adjustment was added before inoculation of fungal biomass:

Cultivation conditions and process control

The inoculum (30 I liquid fungal biomass) was added to the broth after cultivation conditions were set as listed below.

The temperature was kept at 35°C ± 4.5°C during cultivation

The pH was set to 5.9 and fluctuated according to Figure 14 throughout the cultivation. pH was adjusted with phosphoric acid and sodium hydroxide to stay close to 5.0-6.0 throughout the cultivation.

Aeration was set to 100 kg/h and elevated up to 750 kg/h over 10 h after inoculating

Mechanical mixing was set to low effect Cultivation conditions and process parameters was monitored throughout the experiment and can be seen in Figure 12, Figure 13 and Figure 14. Temperature, volume, stirring and aeration was monitored online in the control system while pH, dissolved oxygen, ethanol, acetic acid, lactic acid, Dpn, Dp3, maltose glucose xylose, glycerol, dry matter and biomass was monitored by manual sampling and analysis. Anti-bacterial agent Fermasure XL was added one time (7 I at 15 h). The experiment was terminated approx. 36 h after inoculation. 6.1.2. Results

A consumption of carbohydrates, represented in Figure 13, yielded in ethanol 3.0 g/l and fungal biomass 5.0 g dry matter/I (Figure 12, Figure 14). Summarized content of essential amino acids per 100g fungal biomass from experiments 4-6:

References

http://www.tandfonline.com/doi/abs/10.1081 /AL-120021552