Recycling food waste through microbial fermentation

Abstract

The present invention relates to a method for producing microbial lipids, optionally producing microbial lipids and protein biomass and / or aromatic compounds.The present invention further relates to uses of microbial lipids.The present invention also relates to a composition comprising at least five enzymes.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for producing microbial lipids, optionally a method for producing microbial lipids and protein biomass and / or aroma compounds.The present invention further relates to uses of microbial lipids.The present invention also relates to a composition comprising at least five enzymes. [Background technology]

[0002] 2. Background of the Invention Edible oils and fats are known to be essential nutritional requirements for humans. A healthy adult requires approximately 5 g per day of linoleic acid and unsaturated fatty acids, which the body cannot manufacture. In 2018, the world production of edible oils reached 204 million tons. Palm oil, for example, accounted for 36% of the total manufactured oils, and its consumption reached approximately 69 million tons. According to the Food and Agriculture Organization (FAO), the demand for palm oil is expected to reach 156 million tons by 2050. Considering the potential adverse effects of plant-based production of triglyceride oils due to excessive land use, monoculture and removal of biologically diverse habitats in favor of a single species, such an increasing demand for foods such as palm oil is an environmental risk.

[0003] In particular, a growing world population leads to an inevitable increase in the demand for food, such as edible fats. Furthermore, the increasing demand for such food has been associated with an increasing amount of waste per person and environmental impact. The Food and Agriculture Organization of the United Nations has estimated that, on average, about one-third of the food produced worldwide for human consumption is lost or wasted.

[0004] According to a WWF study, bakery waste is one of the most disposed food products: in 2015, approximately 4.5 million tons of German bakery products generated 1.7 million tons of waste, thus resulting in 398,000 hectares of agricultural land being used unnecessarily and 2.46 million tons of greenhouse gases being produced.

[0005] There is a need to reduce food residue(s), e.g. food waste. There is also a need to provide ways to produce food and food ingredients in an environmentally friendly way. Furthermore, there is a need for measures to promote sustainable food value chains. Summary of the Invention

[0006] The elements of the present invention are described below. These elements are listed with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine two or more of the explicitly described embodiments, or combine one or more of the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0007] In a first aspect, the present invention relates to a method for producing microbial lipids, optionally microbial lipids and protein biomass and / or aroma compounds, said method comprising the steps: a) providing a first substrate, wherein said first substrate is food residue(s), preferably food grade food residue(s); b) culturing a first microorganism selected from filamentous fungi and bacteria with said first substrate, thereby allowing said first microorganism to produce at least one enzyme and an enzymatically treated first substrate, and optionally further to produce protein biomass and / or aroma compounds; optionally co-culturing one or more microalgae with said first microorganism, thereby allowing said one or more microalgae to produce protein biomass, microbial lipids, and / or aroma compounds; c) optionally obtaining said at least one enzyme and pre-treating a second substrate with said at least one enzyme, thereby providing an enzymatically treated second substrate; wherein said second substrate is food residue(s), preferably food grade food residue(s); and wherein preferably said first substrate and said second substrate are of the same type; d) culturing a second microorganism, wherein said second microorganism is an oleaginous microorganism, in a medium containing said enzymatically treated first substrate and / or, if step c) is present, in a medium containing said enzymatically treated second substrate, thereby allowing said oleaginous microorganism to produce microbial lipids; e) optionally carrying out a purely enzymatic treatment of the cultivated second microorganism, without any solvent-based extraction or chemical-based demulsification, to make the microbial lipids produced in step d) suitable for subsequent recovery; and f) recovering the microbial lipids produced in step d), preferably by density-based separation methods. Includes.

[0008] In one embodiment, the food residue(s) may be, at each occurrence, bakery food residue(s), such as bread, rolls, biscuits, muffins, cookies, or cakes; fruit food residue(s), such as fruit pulp, fruit peel, or fruit juice; vegetable food residue(s), such as vegetable peel, vegetable pulp, or vegetable juice; milling food residue(s), such as bran or bran meal; fish food residue(s), such as fish processing residue; seafood residue(s), such as seafood processing residue; brewer's use food residue(s) comprising or consisting of: dried cereals; cereal food residue(s), such as rice, wheat, millet, or corn; restaurant food residue(s), such as restaurant leftovers; animal food residue(s), such as milk or cheese; supermarket food residue(s), such as expired food; or any combination thereof; preferably comprising or consisting of bakery food residue(s), more preferably bread food residue(s).

[0009] In one embodiment, the filamentous fungus is selected from the group consisting of Ceratocystis sp., such as Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa, preferably Ceratocystis paradoxa; Trichoderma sp., such as Trichoderma reesei and Trichoderma harzianum; Aspergillus sp., such as Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Fusarium sp., such as Fusarium venenatum; Thermomyces sp.; Penicillium sp.; Aureobasillium sp.; Ischnoderma sp., such as Ischnoderma benzoinum; Polyporus sp., such as Polyporus durus; Pycnoporus sp., such as Pycnoporus cinnabarinus; Phanerochaete sp., such as Phanerochaete chrysosporium; and Xylaria sp; preferably selected from Ceratocystis sp., Trichoderma sp., Aspergillus sp., and Fusarium sp.; the bacterium is selected from Clostridium sp., such as Clostridium stercorarium, Clostridium beijerinckii, and Clostridium acetobutylicum; Halobacillus sp., such as Halobacillus trueperi and Halobacillus karajensis; Halomonas sp., such as Halomonas meridiana and Halomonas elongata; Rhodothermus sp., such as Rhodothermus marinus; Streptomyces sp., such as Streptomyces griseus, Streptomyces olivochromogenes, Streptomyces griseorubens, and Streptomyces matensis; and Bacillus sp., such as Bacillus nato, Bacillus subtilis, Bacillus licheniformis, and Bacillus stearothermophilus; and / or The microalgae are selected from Chlorella sp., such as Chlorella vulgaris, and Chlorella protothecoides; Scenedesmus sp., such as Scenedesmus obliquus; Dunaliella sp., such as Dunaliella salina; Haematococcus sp., such as Haematococcus pluvialis; Crypthecodinium sp., such as Crypthecodinium cohnii; Schizochytrium sp., such as Schizochytrium limacinum; and Tetraselmis sp., such as Tetraselmis chui; preferably selected from Chlorella sp., and Scenedesmus sp.

[0010] In one embodiment, the second microorganism is an oleaginous microorganism selected from oleaginous yeast, oleaginous fungi, oleaginous bacteria, and oleaginous microalgae; Here, preferably, The oleaginous yeasts include Cutaneotrichosporon sp., such as Cutaneotrichosporon oleaginosus; Trichosporon sp., such as Trichosporon oleaginosus, Trichosporon capitatu, and Trichosporon asahii; Rhodospirillum sp.; Rhodosporidium sp., such as Rhodosporidium toruloides; Rhodosporon sp.; Candida sp., such as Candida viswanathii and Candida freyschussii; Cryptococcus sp., such as Cryptococcus curvatus; Lipomyces sp., such as Lipomyces starkeyi; Yarrowia sp., such as Yarrowia lipolytica; Rhodotorula sp., such as Rhodotorula graminis, Rhodotorula gracilis, and Rhodotorula glutinis; and Apiotrichum sp., such as Apiotrichum curvarum; preferably selected from Cutaneotrichosporon sp., more preferably Cutaneotrichosporon oleaginosus; The oleaginous fungus is selected from Cunninghamella sp., such as Cunninghamella echinulate; Aspergillus sp., such as Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Fusarium sp., such as Fusarium venenatum; Mucor sp., such as Mucor moelleri; Mortierella sp., such as Mortariella isabellina and Mortierella alpine, preferably Mortierella alpine; and Humicola sp.; The oleaginous bacteria is selected from Rhodococcus sp.; Acinetobacter sp.; and Bacillus sp.; and The oleaginous microalgae is selected from Chlorella sp., Pseudochlorococcum sp., Nannochloris sp., Nannochloropsis sp., Isochrysis sp., Tribonema sp., Dunaliella sp., Ankistrodesmus sp., Botryococcus sp., Pavlova sp., Scenedesmus sp., Skeletonema sp., and Nitzschia sp.; More preferably, said second microorganism is an oleaginous yeast selected from Cutaneotrichosporon sp., such as Cutaneotrichosporon oleaginosus.

[0011] In one embodiment, the second microorganism is an oleaginous yeast selected from Cutaneotrichosporon oleaginosus, Trichosporon oleaginosus, Trichosporon capitatu, Trichosporon asahii, Lipomyces starkeyi, Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula graminis, Rhodotorula gracilis, Rhodotorula glutinis, Apiotrichum curvarum, Cryptococcus curvatus, Candida viswanathii, and Candida freyschussii; preferably Cutaneotrichosporon oleaginosus.

[0012] In one embodiment, said step b) of said method comprises culturing a first microorganism selected from filamentous fungi and bacteria with said first substrate, thereby allowing said first microorganism to produce at least one enzyme, an enzymatically treated first substrate, and protein biomass and / or aroma compounds; optionally further comprising co-culturing one or more microalgae with said first microorganism, thereby allowing said one or more microalgae to produce protein biomass, microbial lipids, and / or aroma compounds.

[0013] In one embodiment, the method further comprises the step of recovering said protein biomass and / or said aroma compounds, preferably using any of centrifugation, filtration, organophilic pervaporation, solid phase microextraction, distillation, and combinations thereof.

[0014] In one embodiment, the medium further comprises additional carbon sources, nitrogen sources, trace metals, and / or vitamins.

[0015] In one embodiment, the method further comprises: - mechanical pre-treatment, preferably by milling, mixing, chopping and / or sieving said substrate(s); - dissolving said substrate(s) in a solvent, preferably in water; - chemical hydrolysis of said substrate(s), preferably using an acid, such as sulfuric acid; - a thermal pretreatment of said substrate(s), preferably at a temperature of between 50°C and 200°C, for between 10 and 240 minutes, more preferably at a temperature of between 80°C and 170°C, for between 30 and 90 minutes; - Fermentation pretreatment, preferably anaerobic fermentation, of said substrate(s); and / or - enzymatic pretreatment of said substrate(s) using one or more enzymes, optionally commercially available enzymes; and / or, if step c) is present, pretreating the second substrate by wherein the one or more enzymes are selected from proteases, such as endopeptidases and exopeptidases, serine endopeptidases, subtilisin A, and pepsin; and hydrolases, preferably glycoside hydrolases, more preferably α-amylase, amyloglucosidase, cellulase, hemicellulase, xylanase, xyloglucase, galactanase, arabinase, mannanase, lipase, glucoamylase, and pectinase.

[0016] In one embodiment, the method comprises said step e) of carrying out said purely enzymatic treatment of said cultured second microorganism without any solvent-based extraction or chemical-based demulsification, wherein said purely enzymatic treatment of said cultured second microorganism is treatment of said microorganism with a hydrolytic enzyme, alone or in combination / followed by a protease.

[0017] In one embodiment, the method comprises a step c) of obtaining said at least one enzyme and of pretreating said second substrate with said at least one enzyme, said pretreating comprising contacting said second substrate with said at least one enzyme in the form of a liquid enzyme preparation, preferably directly obtained from culturing said first microorganism, or in the form of a lyophilized enzyme preparation, optionally reconstituted in solution.

[0018] In one embodiment, the at least one enzyme is selected from the enzymatic activity of cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase; Optionally, it contains one or several activities selected from the enzymatic activities of cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; and rhamnogalacturonan lyase.

[0019] In one embodiment, the method is a method for producing microbial lipids and preparing food products therefrom, the method further comprising step g) of preparing a food product comprising the microbial lipids recovered in step f), preferably a bakery product, such as bread, rolls, biscuits, muffins, cookies or cakes; a confectionery product, such as flour confectionery or sugar confectionery; a dairy product, such as ice cream or baby milk; a spread, such as margarine, mayonnaise or soft cheese; an instant food, such as instant noodles, pizza, pizza dough or sauces; a drink; a vegetarian or vegan food, such as meat analogues and dairy analogues; a sweet, such as cocoa-free chocolate; and / or a chocolate product; Optionally, the method further comprises a step h) of recycling the food residue(s) of the food product by using said food residue(s) as the first substrate and / or, if step c) is present, as the second substrate.

[0020] In a further aspect, the present invention relates to the use of microbial lipids, preferably produced using the method defined above, in the production of food products, preferably bakery products, such as bakery products such as bread, rolls, biscuits, muffins, cookies or cakes; confectionery products, such as flour confectionery or sugar confectionery; dairy products, such as ice cream or baby milk; spreads, such as margarine, mayonnaise or soft cheese; instant foods, such as instant noodles, pizza, pizza dough or sauces; beverages; vegetarian or vegan foods, such as meat analogues and dairy analogues; sweets, such as cocoa-free chocolate; and / or chocolate products.

[0021] In a further aspect, the present invention relates to a composition comprising at least five, preferably at least six, more preferably at least seven enzymes selected from cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase, preferably a composition produced in step b) of the process defined above, wherein, optionally, the composition comprises: cellulase; amylase; hemicellulase; limit dextrinase, such as malt limit dextrinase; and pectinase; or cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; and mannanase; or These include cellulases; xylanases; arabinanases; amylases, such as α-amylases; limit dextrinases; pullulanases; galactanases; mannanases; rhamnogalacturonan hydrolases; and rhamnogalacturonan lyases.

[0022] In a further aspect, the present invention relates to a method for producing microbial lipids and protein biomass and / or aroma compounds, said method comprising the method for producing microbial lipids as defined above, wherein in step b) said first microorganism further produces protein biomass and / or aroma compounds and / or, if step b) comprises co-cultivating one or more microalgae with said first microorganism, said microalgae produce protein biomass and / or aroma compounds.

[0023] In one embodiment, in step d), said second microorganism produces microbial lipids and further produces protein biomass and / or aroma compounds.

[0024] In one embodiment, the method includes the step of recovering the protein biomass and / or aroma compounds.

[0025] In one embodiment, the method of producing microbial lipid, the microbial lipid, the protein biomass, the aroma compound, the first microorganism, the co-cultivating, the microalgae, the second microorganism, and the recovering are as defined above.

[0026] In a further aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) providing microbial lipids using a method for producing microbial lipids as defined above; optionally further providing protein biomass and / or aroma compounds, preferably using a method for producing microbial lipids and protein biomass and / or aroma compounds as defined above; b) preparing a food product comprising said microbial lipids and, optionally, further comprising said protein biomass and / or aroma compounds. The present invention relates to a method for preparing a food product, comprising:

[0027] In one embodiment, the food product, the microbial lipid, the method for producing the microbial lipid, the protein biomass, the aroma compounds, and the method for producing the microbial lipid and protein biomass and / or aroma compounds are as defined above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description It is an object of the present invention to provide a method for the sustainable production of food products, such as microbial lipid and protein biomass and / or aroma compounds. In one embodiment, such microbial lipid and protein biomass can be directly consumed by humans or animals or can be incorporated into food products, such as bakery products. Furthermore, it is an object of the present invention to reduce food waste, for example by using food residues to prepare value products, such as microbial lipid and protein biomass and / or aroma compounds. In particular, it is an object of the present invention to recycle food residues into the food value chain. Recycling such food residues makes it possible to provide a sustainable food value chain.

[0029] The method of the present invention is advantageous in transforming the food value chain from a linear value chain with food residues and / or food losses to a circular value chain that recycles food residues as substrates for the production of valuable products, such as microbial oils, protein biomass, and / or aroma compounds. The method of the present invention improves the efficiency of resource use, e.g. the use of food raw materials. Furthermore, the method of the present invention allows for an increase in the market value of food residues. That is, the economic, environmental and social costs of food waste are successfully reduced by the method of the present invention.

[0030] The term "microbial lipids" as used herein relates to lipids produced by oleaginous microorganisms, such as yeast oil, bacterial oil, and / or fungal oil. In one embodiment, the term "microbial lipids" is used interchangeably with "single cell oil" or "microbial oil". Typically, microbial lipids are rich in unsaturated fatty acids. In one embodiment, the microbial lipids are edible microbial lipids. Such microbial lipids can be used to prepare food products that contain microbial lipids. For example, microbial lipids can be used to replace fats with high saturated fatty acid content and / or environmentally unfriendly fats such as palm oil. Microbial lipids have a fatty acid composition similar to those of vegetable oils, but they are considered to be more sustainable. Indeed, the production of microbial lipids is not affected by the season, they can be produced in large quantities and with reduced space requirements, and they can be produced by oleaginous microorganisms from a wide range of carbon sources. Microorganisms are defined as oleaginous as a result of their ability to accumulate lipids, for example as much as 20% of their dry cell weight (DCW). For example, oleaginous microorganisms include several eukaryotic microorganisms such as fungi, yeasts, algae and some bacterial species that can accumulate lipids in the form of triglycerides (TAGs) and free fatty acids (FAs). In one embodiment, the composition of the microbial lipids produced by the methods of the present invention can be customized by one of skill in the art by selecting the appropriate microorganism and / or substrate.

[0031] In one embodiment, the term "protein biomass" as used herein relates to biomass with a high protein content, for example a protein content of up to 60% or more by weight of the dry biomass. In one embodiment, the protein biomass has a protein content of at least 20% by weight or at least 50% by weight, preferably at least 60% by weight or at least 80% by weight, more preferably at least 90% by weight or at least 95% by weight, even more preferably at least 99% by weight, and / or consists of protein. In one embodiment, the protein biomass comprises or consists of mycoprotein. In one embodiment, the protein biomass, for example a protein biomass comprising or consisting of mycoprotein, is produced as a by-product of the method of producing microbial lipid of the invention. In one embodiment, the protein biomass comprises or consists of mycoprotein, for example mycoprotein produced by Fusarium venenatum, Neurospora intermedia, and / or Aspergillus oryzae. In one embodiment, the protein biomass is an edible protein biomass. For example, such protein biomass can be used to prepare food products comprising said protein biomass. Additionally, microbial protein biomass can be further processed into protein-rich feed and food supplements. In one embodiment, said protein biomass is enzymatically hydrolyzed or used directly for further processing, e.g., into food. In one embodiment, the protein biomass comprises a protein(s) selected from cell wall proteins, structural proteins, and membrane proteins. In one embodiment, the protein biomass, e.g., the protein biomass produced as a by-product of the method of producing microbial lipids of the present invention, comprises mycoproteins, e.g., mycoproteins produced by Fusarium venenatum, Neurospora intermedia, and / or Aspergillus oryzae.In one embodiment, the first microorganism comprises Fusarium venenatum, Neurospora intermedia, and / or Aspergillus oryzae. In one embodiment, the first microorganism does not comprise or consist of Aspergillus oryzae. In one embodiment, the protein biomass is produced by Fusarium venenatum, Neurospora intermedia, and / or Aspergillus oryzae. In one embodiment, the protein biomass, preferably the protein biomass produced by F. venenatum, has an amino acid content ranging from 4 to 6.5% by weight, such as about 5.25% by weight alanine; 5.5 to 6.5% by weight, such as about 6.13% by weight arginine; 5.0 to 6.0% by weight, such as about 5.75% by weight aspartic acid; 2.0 to 4.5% by weight, such as about 3.25% by weight cystine; 12.0 to 14% by weight, such as about 13.13% by weight glutamic acid; 3.5 to 5.5% by weight, such as about 4.75% by weight glycine; 2.0 to 4% by weight, such as about 3.00% by weight histidine; 3.5 to 5.5% by weight, such as about 4.25% by weight isoleucine; 2.0 to 3.5% by weight, such as about 2.75% by weight leucine; 6.0 to 8.5% by weight, such as about 7.25% by weight lysine; 2.0 to 4.0% by weight, such as about 3.00% by weight methionine; 3.0 to 6.0% by weight, such as about 4.50% by weight phenylalanine; 1.5 to 3.5% by weight, such as about 2.25% by weight proline; 5.0 to 6.5% by weight, such as about 5.75% by weight serine; 2.5 to 4.5% by weight, such as about 3.50% by weight threonine; 3.0 to 6.0% by weight, such as about 4.50% by weight tyrosine; and / or 4.0 to 6.0% by weight, such as about 5.00% by weight valine. In one embodiment, the protein biomass produced in step b) is a) Protein (g) 11.5 Total Carbohydrates (g) 1.7 Total Fat (g) 2.9 Dietary Fiber (NSP) (g) 6.0 Salt (mg) 4, Biomass produced, for example, by Fusarium venenatum; b) Protein: 91% by weight Lipid 2% by weight Glucan 1% by weight, Biomass produced, for example, by Trichoderma reesei; or c) Protein: 23.1% by weight Fat 19% by weight Chitin 20% by weight Glucan 35% by weight For example, biomass produced by Aspergillus niger It comprises or consists of:

[0032] In one embodiment, in step b), the first microorganism produces protein biomass and / or the first microorganism is co-cultured with one or more microalgae that produce protein biomass. In one embodiment, the method comprises obtaining the protein biomass produced in step b), for example by centrifugation, filtration, sedimentation, coagulation, flotation, and combinations thereof, preferably by centrifugation, filtration, and combinations thereof. In one embodiment, in step d), the second microorganism further produces protein biomass as a by-product of the production of microbial lipids. In one embodiment, the method comprises obtaining the protein biomass produced in step d), for example by centrifugation, filtration, sedimentation, coagulation, flotation, and combinations thereof, preferably by centrifugation, filtration, and combinations thereof. In one embodiment, the terms "recovering protein biomass" and "obtaining protein biomass" are used interchangeably. In one embodiment, the method is a method of producing microbial lipids and preparing a food product therewith, wherein preparing the food product further comprises incorporating the protein biomass into the food product. In one embodiment, the method of the invention is a method of producing microbial lipids and protein biomass, and the first microorganism is preferably a microorganism that produces at least 30% of its biomass as protein.

[0033] Advantageously, the method of the present invention allows microbial protein biomass to be produced as a by-product of the method of producing microbial lipids. That is, an alternative to the intensive raising of livestock to produce protein is the cultivation of microorganisms to produce edible microbial protein biomass. In one embodiment, the protein biomass produced using the method of the present invention can be consumed as direct biomass or as a supplement to increase the protein content of food. The advantage of the method of the present invention is that the production of protein biomass is economically viable, i.e., it can compete successfully with more established meat substitutes, such as tofu and other soybean derivatives, as well as meat itself. A further advantage is that the first and second microorganisms of the present invention can be cultivated in large-scale cultures and provide large amounts of microbial lipids and protein biomass and / or aroma compounds.

[0034] In one embodiment, the term "aroma compound" as used herein relates to a compound having a smell or odor, preferably a pleasant smell or odor, such as an odorant, aroma, fragrance or flavor. In one embodiment, the aroma compound is a fragrance compound. In one embodiment, the aroma compound is produced as a by-product of the process of the present invention. In one embodiment, the aroma compound is benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol, geranyl acetate, methyl formate, methyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, propyl acetate, isobutyl acetate, isoamyl acetate, pentyl butyrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, hexyl acetate, myrcene, geraniol, nerol, citral. , citronellal, citronellol, linalool, nerolidol, ocimene, limonene, camphor, menthol, carvone, terpineol, alpha-ionone, thujone, eucalyptol, jasmone, ethyl caproate, isoamyl alcohol, 3-methyl-1-butanol, 1-octen-3-ol, and combinations thereof; preferably selected from citronellol, geraniol, ethyl acetate, propyl acetate, isobutyl acetate, isoamyl acetate, and combinations thereof. In one embodiment, the aroma compound is ethyl acetate, wherein the aroma compound is produced by a filamentous fungus selected from Ceratocystis sp., e.g. Ceratocystis fimbriata, using a substrate, e.g. coffee husks. In one embodiment, the aroma compounds are selected from ethyl caproate, isoamyl alcohol, 3-methyl-1-butanol, 1-octen-3-ol, and / or ethyl acetate, wherein the aroma compounds are produced by a filamentous fungus selected from Neurospora sp. using a substrate that is rice, e.g., pregelatinized rice.

[0035] In one embodiment, aroma compounds are obtained using any of distillation, organophilic permeation evaporation, solid phase microextraction, and combinations thereof. In one embodiment, aroma compounds are secreted into the culture medium, and said aroma compounds are obtained from said culture medium by distillation, such as distillation under vacuum with supercooling, by organophilic permeation evaporation, by solid phase microextraction, and combinations thereof. Aroma is one of the most important attributes of food and is directly related to product acceptance by consumers. The method of the present invention allows such aroma compounds to be produced in an efficient manner as a by-product of the method of producing microbial lipids. Advantageously, such aroma compounds produced by biotechnology are sustainable aroma compounds and are useful, for example, as additives for the food industry.

[0036] The term "first substrate" as used herein preferably relates to food residue(s), such as carbohydrates, proteins, fats, and minerals, that contain nutrients for the growth, metabolism, and / or activity of microorganisms, such as the first microorganism and / or the second microorganism. In one embodiment, the first substrate is a material that provides substances that allow the growth of microorganisms. In one embodiment, the first substrate is a bakery food residue(s), such as bread, rolls, biscuits, muffins, cookies, or cakes; fruit food residue(s), such as fruit pulp, peel, or fruit juice; vegetable food residue(s), such as vegetable peel, vegetable pulp, or vegetable juice; milling food residue(s), such as bran or bran meal; fish food residue(s), such as fish processing residue; seafood residue(s), such as seafood processing residue; brewer's food residue(s), such as malt processing residue; food residue(s) selected from: spent grains; cereal food residue(s), such as rice, wheat, millet, or corn; restaurant food residue(s), such as restaurant leftovers; animal food residue(s), such as milk or cheese; supermarket food residue(s), such as expired food; or any combination thereof; preferably comprising or consisting of bakery food residue(s), more preferably bread food residue(s). In one embodiment, the first microorganism produces at least one enzyme in response to the presence of the first substrate. For example, the at least one enzyme is an adaptive enzyme(s) configured to act enzymatically on the components of the substrate. In one embodiment, by contacting the first microorganism with the first substrate, the first microorganism is stimulated to produce an enzyme suitable for acting enzymatically on the components of the substrate, for example for digesting the substrate and / or for using the components of the substrate for growth. In one embodiment, the first substrate is an induction system for the first microorganism, in particular for the production of an enzyme specific for the substrate. In one embodiment, the first substrate is used to prepare at least one enzyme, preferably an enzyme mixture, which is adapted to pretreat the second substrate to provide therefrom a growth medium.In one embodiment, the second substrate is pretreated, e.g. digested with said at least one enzyme, preferably an enzyme mixture, to obtain a medium containing nutrients for the second microorganism. In one embodiment, the enzymatically treated first substrate and / or the enzymatically treated second substrate is a medium for growing the second microorganism.

[0037] In one embodiment, the first substrate is an induction system for the first microorganism to stimulate the first microorganism to produce an enzyme that is adjusted to digest the substrate. Such an adjusted enzyme system is then used to digest a second substrate, preferably of the same type as the first substrate. For example, both the first substrate and the second substrate are the same type of food residue(s), such as bakery food residue(s). In one embodiment, the enzyme obtained from the induction system, i.e. the cultivation of the first microorganism with the first substrate, is prepared separately and used as a liquid preparation or as a freeze-dried preparation that is optionally reconstituted in a solution for further use.

[0038] In one embodiment, the term "food residue(s)" as used herein refers to any food material, raw or cooked, that is intended or required to be disposed of. The term "residue(s)" as used herein includes the singular "residue(s)" and the plural "residue(s)". In many of the embodiments, when the term "residue(s)" is used in combination with a verb that is singular, the combination of the verb and the term "residue(s)" should be understood as referring to both the singular and the plural, even if the verb is singular. Similarly, when the term "residue(s)" is used in combination with a verb that is plural, the combination of the verb and the term "residue(s)" should be understood as referring to both the singular and the plural, even if the verb is plural. Food residue(s) further relates to uneaten food and food preparation residue(s) from residential and commercial establishments such as grocery stores, restaurants, farm stands, institutional cafeterias and kitchens, and industrial sources such as employee lunch rooms. For example, food residue(s) is food that is left uneaten or unused, such as food residue(s) from food production, food distribution, food storage, food retail, and consumer food residue(s), such as from households, restaurants, and catering.In one embodiment, the food residue(s) are selected from the group consisting of bakery food residue(s), such as bread, rolls, biscuits, muffins, cookies, or cakes; fruit food residue(s), such as fruit pulp, peel, or fruit juice; plant food residue(s), such as vegetable peel, vegetable pulp, or vegetable juice; milling food residue(s), such as bran or bran meal; fish food residue(s), such as fish processing residues; seafood residue(s), such as seafood processing residues; brewer's spent grains; cereal food residue(s), such as The food residue(s) may be independently selected from food residue(s) comprising or consisting of: rice, wheat, millet, or corn; restaurant food residue(s), such as restaurant leftovers; animal food residue(s), such as milk or cheese; supermarket food residue(s), such as expired food; or any combination thereof; preferably selected from food residue(s) comprising or consisting of bakery food residue(s), more preferably bread food residue(s). In one embodiment, the food residue(s) are food grade food residue(s). In one embodiment, the food residue(s) are not spoiled and / or rotten food. In one embodiment, the food residue(s) are free of inedible mold. In one embodiment, the food residue(s) are free of any ingredients that are toxic and / or harmful to human and / or animal health. For example, the fruit or vegetable food residue(s) are residues of juice preparations, instant food preparations, and / or canning processes of fruits or vegetables, such as the flesh or skin of carrots, pears, apples, potatoes, bananas, or lychees. In one embodiment, the food residue(s) have a food grade. In one embodiment, the food residue(s) do not contain or consist of meat. In one embodiment, the food residue(s) are meat-free. In one embodiment, the term "food residue(s)" includes food waste and food losses, such as food losses along the food supply chain from recovery, such as losses after recovery.In one embodiment, said food residue(s) comprise or consist of food grade food residue(s), preferably said food residue(s) do not contain chemical contaminants, infectious agents and / or pathogens. In a preferred embodiment, the food residue(s) are bakery product food residue(s), such as bread food residue(s). In one embodiment, the terms "bakery food residue(s)" and "bakery product food residue(s)" are used synonymously. In one embodiment, bakery food residue(s) relate to any food residue(s) occurring at any stage throughout the entire supply chain, from initial agricultural production to final domestic consumption. In one embodiment, the food residue(s) used as the first substrate and the food residue(s) used as the second substrate are the same type of food residue(s), for example the first substrate and the second substrate are both bakery product food residue(s), for example both bread residue(s). In one embodiment, the first substrate is an induction system in which a first microorganism produces said at least one enzyme adapted to degrade said first substrate, preferably said first substrate and second substrate. In one embodiment, said food residue(s) is unspoiled food.

[0039] In one embodiment, the term "food grade food residue(s)" as used herein relates to materials that are safe for consumption, e.g. human consumption and / or animal consumption, and / or safe for further processing into consumable food / feed products. For example, such food grade food residue(s) are food residue(s) that are not spoiled and / or not rotten. In one embodiment, the food grade food residue(s) are edible food residue(s). In one embodiment, the food grade food residue(s) are food residue(s) that are not spoiled. In one embodiment, the food grade food residue(s) comprise or consist of edible ingredients. In one embodiment, the food grade food residue(s) do not contain ingredients that are toxic to humans and / or animals. In one embodiment, the food grade food residue(s) consist of non-toxic ingredients or non-toxic concentrations of ingredients. In one embodiment, the food grade food residue(s) do not comprise food waste. In one embodiment, the food grade food residue(s) do not contain chemical contaminants, infectious agents, and / or pathogens. In one embodiment, the food grade food residue(s) do not comprise chemical contaminants and / or pathogens. In one embodiment, the food residue(s), preferably the food grade food residue(s), do not comprise uncooked food, spoiled food, expired food, and leftovers from dishes such as customer's dishes. In one embodiment, the food residue(s), preferably the food grade food residue(s), consist of food. In one embodiment, the food residue(s), preferably the food grade food residue(s), do not comprise non-food ingredients.

[0040] The term "first microorganism" as used herein refers to a microorganism selected from filamentous fungi and bacteria. In one embodiment, the first microorganism can produce at least one enzyme, preferably an enzyme mixture, adapted to the first substrate, for example adapted to digest the first substrate. In one embodiment, the first microorganism produces at least one enzyme that can be subsequently used to digest a second substrate to provide a growth medium for the second microorganism, providing an enzymatically treated substrate that can be used as a growth medium for the second microorganism. In one embodiment, in step b), the first microorganism produces at least one enzyme, preferably an enzyme mixture, that can be used to pretreat the second substrate in step c), thereby providing an enzymatically treated second substrate that can be used as a growth medium for the second microorganism.

[0041] In one embodiment, the first microorganism is selected from filamentous fungi and bacteria, where the filamentous fungi are selected from Ceratocystis sp., such as Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa, preferably Ceratocystis paradoxa; Trichoderma sp., such as Trichoderma reesei and Trichoderma harzianum; Aspergillus sp., such as Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Fusarium sp., such as Fusarium venenatum; Thermomyces sp.; Penicillium sp.; Aureobasillium sp.; Ischnoderma sp., for example Ischnoderma benzoinum; Polyporus sp., for example Polyporus durus; Pycnoporus sp., for example Pycnoporus cinnabarinus; Phanerochaete sp., for example Phanerochaete chrysosporium; and Xylaria sp; preferably selected from Ceratocystis sp., Trichoderma sp., Aspergillus sp., and Fusarium sp; and The bacterium is selected from Clostridium sp., such as Clostridium stercorarium, Clostridium beijerinckii, and Clostridium acetobutylicum; Halobacillus sp., such as Halobacillus trueperi and Halobacillus karajensis; Halomonas sp., such as Halomonas meridiana and Halomonas elongata; Rhodothermus sp., such as Rhodothermus marinus; Streptomyces sp., such as Streptomyces griseus, Streptomyces olivochromogenes, Streptomyces griseorubens, and Streptomyces matensis; and Bacillus sp., such as Bacillus nato, Bacillus subtilis, Bacillus licheniformis, and Bacillus stearothermophilus. In one embodiment, the first microorganism is selected from the filamentous fungi and the bacteria, wherein the first microorganism is not Aspergillus awamori and / or is not Aspergillus oryzae. In one embodiment, the first microorganism is selected from the filamentous fungi and the bacteria, wherein the filamentous fungi do not include Aspergillus awamori and / or Aspergillus oryzae.

[0042] In one embodiment, the Aspergillus is Aspergillus niger, such as Aspergillus niger van Tiegheim ATCC 10535, or Aspergillus terreus, such as Aspergillus terreus CBS 117.37. In one embodiment, when the first microorganism is Aspergillus sp., the Aspergillus sp. is selected from Aspergillus niger, such as Aspergillus niger van Tiegheim ATCC 10535, and Aspergillus terreus, such as Aspergillus terreus CBS 117.37. In one embodiment, the Aspergillus niger is Aspergillus niger van Tiegheim. In one embodiment, the Aspergillus niger is Aspergillus niger van Tiegheim ATCC 10535. In one embodiment, the Aspergillus is not Aspergillus awamori. In one embodiment, when the first microorganism is an Aspergillus sp., the Aspergillus sp. is not Aspergillus awamori and / or is not Aspergillus oryzae. In one embodiment, the first microorganism is not Aspergillus awamori and / or is not Aspergillus oryzae.

[0043] In one embodiment, the Ceratocystis paradoxa is Ceratocystis paradoxa CBS 374.83 or Ceratocystis paradoxa DSM 63054. In one embodiment, the first microorganism is selected from Aspergillus sp., preferably Aspergillus niger, more preferably Aspergillus niger van Tiegheim ATCC 10535; and Ceratocystis sp., preferably Ceratocystis paradoxa, more preferably Ceratocystis paradoxa CBS 374.83. In one embodiment, the Aspergillus sp. does not include Aspergillus awamori and / or Aspergillus oryzae. In one embodiment, the first microorganism is not Aspergillus awamori. In one embodiment, the first microorganism is not Aspergillus oryzae.

[0044] In one embodiment, the first microorganism is an organism capable of producing at least one enzyme and further capable of producing protein biomass, such as a microorganism selected from Aspergillus sp., such as Aspergillus oryzae; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Trichoderma sp.; and Fusarium sp., such as Fusarium venenatum; preferably Fusarium venenatum. In one embodiment, the first microorganism is a microorganism that produces at least one enzyme and protein biomass, said microorganism being selected from Aspergillus sp., such as Aspergillus oryzae; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Trichoderma sp.; and Fusarium sp., such as Fusarium venenatum; preferably Fusarium venenatum.

[0045] In one embodiment, the first microorganism is an organism capable of producing at least one enzyme and further capable of producing an aroma compound and is selected from Ceratocystis, such as Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa; Aspergillus sp., such as Aspergillus oryzae, Aspergillus tubingensis, and Aspergillus niger, preferably Aspergillus niger; Ischnoderma sp., such as Ischnoderma benzoinum; Polyporus sp., such as Polyporus durus; Pycnoporus sp., such as Pycnoporus cinnabarinus; Phanerochaete sp., such as Phanerochaete chrysosporium. In a preferred embodiment, the first microorganism is an organism capable of producing at least one enzyme and further capable of producing an aroma compound and is selected from Aspergillus niger, Ceratocystis fimbriata, Ceratocystis moniliformis, Ceratocystis paradoxa, Ischnoderma benzoinum, Polyporus durus, Pycnoporus cinnabarinus, and Phanerochaete chrysosporium; preferably Ceratocystis paradoxa or Aspergillus niger. In one embodiment, the first microorganism is a microorganism that produces at least one enzyme and an aroma compound, and the microorganism is selected from Ceratocystis sp., e.g. Ceratocystis paradoxa, such as Ceratocystis paradoxa CBS 374.83 or Ceratocystis paradoxa DSM 63054; and Aspergillus sp., e.g. Aspergillus niger, such as Aspergillus niger van Tieghem.In one embodiment, the first microorganism is an organism capable of producing at least one enzyme and further capable of producing protein biomass and aroma compounds, preferably selected from Aspergillus sp., more preferably Aspergillus oryzae.

[0046] In one embodiment, the first microorganism is an organism capable of producing at least one enzyme and further capable of producing aroma compounds and protein biomass, such as Aspergillus sp., such as Aspergillus oryzae. In one embodiment, the first microorganism is co-cultured with an organism producing protein biomass, such as microalgae, such as Chlorella microalga. In one embodiment, in said step b) of culturing the first microorganism, at least one enzyme and an enzymatically treated first substrate are produced; optionally, protein biomass, microbial lipids, and / or aroma compounds, preferably protein biomass and / or aroma compounds are produced.

[0047] In one embodiment, culturing a first microorganism comprises subjecting said first microorganism to suitable growth conditions. In one embodiment, the terms "culturing" and "growing" are used interchangeably. In one embodiment, culturing a first microorganism comprises contacting said first microorganism with said first substrate. In one embodiment, the first substrate is a growth substrate for the first microorganism. In one embodiment, in said step b) of culturing a first microorganism, said first microorganism produces at least one enzyme, preferably an enzyme mixture, having an enzymatic activity specific for the first substrate, and optionally further produces protein biomass and / or aroma compounds. In one embodiment, the first microorganism is co-cultured with microalgae that produces protein biomass, microbial lipids, and / or aroma compounds. In one embodiment, when the method of the present invention is a method for producing microbial lipids and protein biomass and / or aroma compounds, the first microorganism is selected so that the first microorganism can produce protein biomass and / or aroma compounds in addition to producing the at least one enzyme, and / or the first microorganism is co-cultured with one or more microalgae that can produce protein biomass and / or aroma compounds.In one embodiment, when referring to a microorganism that produces an enzymatically treated substrate, it means that the microorganism produces one or more enzymes, preferably an enzyme mixture, that enzymatically treat the substrate; for example, it means that the microorganism indirectly produces the enzymatically treated substrate by producing the at least one enzyme, preferably an enzyme mixture, that enzymatically treats the substrate.In one embodiment, the terms "at least one" and "one or more" are used interchangeably.

[0048] As used herein, "filamentous fungi" refers to fungi having filamentous structures, especially hyphae. In one embodiment, the filamentous fungus is selected from the group consisting of Ceratocystis sp., for example Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa, such as Ceratocystis paradoxa CBS 374.83 or Ceratocystis paradoxa DSM 63054, preferably Ceratocystis paradoxa; Trichoderma sp., for example Trichoderma reesei and Trichoderma harzianum; Aspergillus sp., for example Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger, such as Aspergillus niger van Tiegheim ATCC 10535; Neurospora sp., for example Neurospora intermedia; Monascus sp., for example Monascus purpureus; Rhizopus sp., for example Rhizopus oryzae; Fusarium sp., for example Fusarium venenatum; Thermomyces sp.; Penicillium sp.; Aureobasillium sp.; Ischnoderma sp., for example Ischnoderma benzoinum; Polyporus sp., for example Polyporus durus; Pycnoporus sp., for example Pycnoporus cinnabarinus; Phanerochaete sp., for example Phanerochaete chrysosporium; and Xylaria sp; preferably selected from Ceratocystis sp., Trichoderma sp., Aspergillus sp., and Fusarium sp.In one embodiment, the filamentous fungus is selected from Aspergillus sp., preferably Aspergillus niger, more preferably Aspergillus niger van Tiegheim ATCC 10535; and Ceratocystis sp., preferably Ceratocystis paradoxa, more preferably Ceratocystis paradoxa CBS 374.83.

[0049] The term "bacteria" as used herein in terms of step b) relates to any bacterium capable of producing at least one enzyme when cultured with a first substrate. In one embodiment, the bacterium is selected from Clostridium sp., such as Clostridium stercorarium, Clostridium beijerinckii, and Clostridium acetobutylicum; Halobacillus sp., such as Halobacillus trueperi and Halobacillus karajensis; Halomonas sp., such as Halomonas meridiana and Halomonas elongata; Rhodothermus sp., such as Rhodothermus marinus; Streptomyces sp., such as Streptomyces griseus, Streptomyces olivochromogenes, Streptomyces griseorubens, and Streptomyces matensis; and Bacillus sp., such as Bacillus nato, Bacillus subtilis, Bacillus licheniformis, and Bacillus stearothermophilus.

[0050] The term "at least one enzyme" as used herein relates to one or more enzymes, preferably a mixture of enzymes. In one embodiment, said at least one enzyme has an activity targeting said first substrate and / or second substrate and / or at least one component of said first substrate and / or second substrate. In one embodiment, said at least one enzyme can enzymatically process a substrate to provide a growth medium and / or a component for a growth medium. In one embodiment, said at least one enzyme can enzymatically process a substrate to convert said substrate into nutrients and / or release nutrients from said substrate, preferably nutrients for the growth of a first microorganism and / or a second microorganism. In one embodiment, said at least one enzyme acts on said substrate to make it a suitable growth medium for said first microorganism and / or a second microorganism. In one embodiment, the at least one enzyme is selected from the enzymatic activities of cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase; and optionally contains one or several activities selected from the enzymatic activities of cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; and rhamnogalacturonan lyase.In one embodiment, the at least one enzyme is any of cellulase, lichenase, xylanase, arabinanase, amylase, such as α-amylase, limit dextrinase, pullulanase, protease, galactanase, mannanase, rhamnogalacturonan hydrolase, rhamnogalacturonan lyase, xyloglucanase, hemicellulase, amyloglucosidase, beta-glucosidase, pectinase, laminarinase, and combinations thereof. In one embodiment, the at least one enzyme is an enzyme mixture. In one embodiment, the enzyme mixture comprises at least two or three enzymes, preferably at least five, more preferably at least six, and even more preferably at least seven. In one embodiment, the enzyme mixture comprises at least two or three, preferably at least five, more preferably at least six, even more preferably at least seven enzymes selected from cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase. In one embodiment, the at least one enzyme is adapted to the substrate, i.e., by contacting the first microorganism with the substrate, the first microorganism produces an enzyme that acts specifically on the substrate. For example, when bread residue(s) are used as the substrate, the at least one enzyme produced in step b) of the method of the invention and / or the composition of the invention is adapted to the substrate. cellulase; amylase; hemicellulase; limit dextrinase, e.g., malt limit dextrinase; and pectinase; and / or cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; and mannanase; and / or Cellulases; xylanases; arabinanases; amylases, such as α-amylases; limit dextrinases; pullulanases; galactanases; mannanases; rhamnogalacturonan hydrolases; and rhamnogalacturonan lyases. may include.

[0051] In one embodiment, the composition of the invention is the at least one enzyme produced in step b) of the method of the invention. In one embodiment, the at least one enzyme produced in step b) of the method of the invention is a composition of the invention. In one embodiment, said at least one enzyme has a food grade. In one embodiment, said at least one enzyme is a food grade enzyme or a food grade enzyme composition. In one embodiment, said at least one enzyme comprises or consists of at least one food grade enzyme. In one embodiment, when said first substrate is food grade food residue(s), said at least one enzyme is a food grade enzyme or a food grade enzyme composition.

[0052] In one embodiment, the term "enzymatically treated first substrate" as used herein refers to a first substrate contacted with at least one enzyme produced by the first microorganism. In one embodiment, the enzymatically treated first substrate, or a component thereof, is used as a growth medium for the second microorganism or as a supplement(s) in a growth medium for the second microorganism. In one embodiment, the medium used in step d) comprises or consists of the enzymatically treated first substrate and / or the enzymatically treated second substrate, and / or comprises or consists of components, preferably nutrients, of the enzymatically treated first substrate and / or the enzymatically treated second substrate. In one embodiment, the enzymatically treated first substrate is a hydrolysate of the first substrate.

[0053] The term "microalgae" as used herein in terms of step b) relates to any microalgae capable of producing protein biomass, microbial lipids, and / or aroma compounds.In one embodiment, the microalgae used in step b) are selected from Chlorella sp., such as Chlorella vulgaris and Chlorella protothecoides; Scenedesmus sp., such as Scenedesmus obliquus; Dunaliella sp., such as Dunaliella salina; Haematococcus sp., such as Haematococcus pluvialis; Crypthecodinium sp., such as Crypthecodinium cohnii; Schizochytrium sp., such as Schizochytrium limacinum; and Tetraselmis sp., such as Tetraselmis chui; preferably selected from Chlorella sp. and Scenedesmus sp. In one embodiment, step b) comprises co-cultivating the first microorganism with a microalga to obtain the protein biomass, microbial lipids, and / or aroma compounds produced by the microalgae in addition to the at least one enzyme and the enzymatically treated first substrate produced by the first microorganism. Such protein biomass, microbial lipids, and / or aroma compounds produced by the microalgae can be used to prepare a food product comprising the protein biomass, microbial lipids, and / or aroma compounds. In one embodiment, the microalgae used in step b) to produce protein biomass are selected from Chlorella vulgaris, Dunaliella salina, Haematococcus pluvialis, and Scenedesmus obliquus. In one embodiment, the microalgae capable of producing protein biomass are selected from Chlorella vulgaris, Dunaliella salina, Haematococcus pluvialis, and Scenedesmus obliquus.In one embodiment, the microalgae used in step b) to produce aroma compounds are selected from Crypthecodinium cohnii, Schizochytrium limacinum, Tetraselmis chui, Chlorella vulgaris, and Chlorella protothecoides.In one embodiment, the microalgae capable of producing aroma compounds are selected from Crypthecodinium cohnii, Schizochytrium limacinum, Tetraselmis chui, Chlorella vulgaris, and Chlorella protothecoides.

[0054] In one embodiment, culturing the first microorganism in step b) is carried out at a temperature ranging from 25° C. to 37° C. In one embodiment, culturing the first microorganism in step b) is carried out at a pH ranging from pH 4 to pH 8. In one embodiment, culturing the first microorganism in step b) is carried out for 2 to 12 days, preferably 3 to 10 days. In one embodiment, culturing the first microorganism in step b) is carried out at a pO2>20%. In one embodiment, culturing the first microorganism in step b) is carried out under agitation, preferably under stirring at a rotation speed ranging from 100 rpm to 800 rpm. For example, a paddle stirrer may be used. In one embodiment, culturing said first microorganism in step b) is carried out at a temperature ranging from 25° C. to 37° C., at a pH ranging from pH 4 to pH 8, for a period of 3 to 10 days, with a pO2>20%, under agitation, preferably under stirring at a rotation speed ranging from 100 rpm to 800 rpm.

[0055] In one embodiment, culturing the first microorganism in step b) comprises culturing the first microorganism for 2 to 12 days, preferably 3 to 10 days, at a temperature in the range of 25° C. to 37° C., at a pH in the range of pH 4 to pH 8, and / or at a dissolved oxygen (pO2) pO2>20%.

[0056] In one embodiment, step b) of the method of the invention comprises culturing a first microorganism selected from filamentous fungi and bacteria with said first substrate, thereby allowing said first microorganism to produce at least one enzyme, an enzymatically treated first substrate, and a protein biomass and / or aroma compounds; optionally further comprising co-culturing one or more microalgae with said first microorganism, thereby allowing said one or more microalgae to produce a protein biomass, microbial lipids, and / or aroma compounds.

[0057] In one embodiment, the term "co-cultivating one or more microalgae with said first microorganism" as used herein refers to a combined culture of said first microorganism with said microalgae, for example in the same container. In one embodiment, the co-cultivation comprises subjecting said first microorganism and said microalgae to culture conditions suitable for both said first microorganism and said microalgae. In one embodiment, if the culture conditions suitable for the first microorganism are different from the culture conditions suitable for the microalgae, said co-cultivation in step b) comprises subjecting said first microorganism and microalgae, if present, subsequently to culture conditions suitable for the first microorganism and culture conditions suitable for the microalgae, or vice versa.

[0058] In one embodiment, the method comprises obtaining the protein biomass and / or aroma compounds produced by the first microorganism in step b); and optionally obtaining the protein biomass, microbial lipids, and / or aroma compounds produced by the one or more microalgae. In one embodiment, when step b) comprises co-cultivating one or more microalgae with the first microorganism, the method comprises obtaining the protein biomass, microbial lipids, and / or aroma compounds produced by the one or more microalgae. In one embodiment, the terms "obtaining" and "recovering" are used interchangeably. In one embodiment, recovering the protein biomass and / or the aroma compounds is carried out using any of centrifugation, filtration, distillation, organophilic permeation evaporation, solid phase microextraction, and combinations thereof. In one embodiment, recovering the microbial lipids produced by the microalgae in step b) includes centrifugation, solvent extraction, such as with hexane, solvent-free extraction, such as enzymatic hydrolysis of the cell walls of the microalgae followed by centrifugation, such as semi-continuous centrifugation of the broken cells, cold pressing, demulsification, and any combination thereof; preferably includes centrifugation, solvent-free extraction, such as enzymatic hydrolysis of the cell walls of the microalgae followed by centrifugation, such as semi-continuous centrifugation of the broken cells, cold pressing, and any combination thereof. In one embodiment, recovering the microbial lipids produced by the microalgae in step b) is free of solvent-based extraction and chemical-based demulsification. For example, protein biomass is recovered using centrifugation and / or filtration. For example, aroma compounds are recovered using distillation, organophilic permeation evaporation, solid-phase microextraction, and combinations thereof.

[0059] In one embodiment, the term "pre-treating a second substrate" as used herein relates to contacting said second substrate with said at least one enzyme, preferably enzymatically treating said second substrate with said at least one enzyme. In one embodiment, by pre-treating said second substrate with at least one enzyme, said second substrate is converted and / or releases compounds, preferably nutrients, that allow a second microorganism to grow, e.g. the second substrate is converted into and / or into a component of a growth medium for said second microorganism. In one embodiment, pre-treating said second substrate with said at least one enzyme comprises contacting said second substrate with said at least one enzyme, preferably an enzyme mixture, for 6-72 hours, preferably 12-48 hours, more preferably 20-30 hours; e.g. at a temperature in the range of 20-80°C, preferably 30-60°C, and / or at a pH in the range of pH 3 to pH 9, preferably pH 4 to pH 7. In one embodiment, pre-treating the second substrate with said at least one enzyme comprises or consists of enzymatically treating said second substrate with said at least one enzyme.

[0060] In one embodiment, if step c) is present, pretreating said second substrate in step c) is carried out at a temperature in the range of 30° C. to 80° C. In one embodiment, pretreating said second substrate in step c) is carried out at a pH in the range of pH 4 to pH 9. In one embodiment, pretreating said second substrate in step c) is carried out under agitation, preferably under stirring at a rotation speed in the range of 200 rpm to 800 rpm. In one embodiment, pretreating said second substrate in step c) is carried out at a temperature in the range of 30° C. to 80° C., at a pH in the range of pH 4 to pH 9, and under agitation, preferably under stirring at a rotation speed in the range of 200 rpm to 800 rpm. In one embodiment, pre-treating said second substrate in step c), if present, comprises treating said second substrate with said at least one enzyme, preferably an enzyme mixture, optionally a composition of the invention, for 6 to 72 hours, preferably 12 to 48 hours, more preferably 20 to 30 hours; for example at a temperature in the range of 20° C. to 80° C., preferably 30° C. to 60° C., and / or at a pH in the range of pH 3 to pH 9, preferably pH 4 to pH 7. In one embodiment, pre-treating said second substrate in step c), if present, comprises treating said second substrate with said at least one enzyme in a liquid, for example a medium comprising a buffer system.

[0061] In one embodiment, the method comprises a step c) of obtaining said at least one enzyme and pretreating said second substrate with said at least one enzyme, wherein said pretreating comprises contacting said second substrate with said at least one enzyme obtained, preferably directly obtained, from culturing said first microorganism, in the form of a liquid enzyme preparation or in the form of a lyophilized enzyme preparation, optionally reconstituted in a solution. In one embodiment, said obtaining said at least one enzyme in step c) comprises obtaining said at least one enzyme in liquid form or in the form of a lyophilized enzyme preparation. In one embodiment, such a lyophilized enzyme preparation of said at least one enzyme is dissolved prior to said pretreating said second substrate with said at least one enzyme.

[0062] In one embodiment, the term "enzymatically treated second substrate" as used herein refers to a second substrate contacted with at least one enzyme produced by the first microorganism. In one embodiment, the enzymatically treated second substrate, or a component thereof, is used as a growth medium for the second microorganism or as a supplement(s) in a growth medium for the second microorganism. In one embodiment, the enzymatically treated second substrate is a hydrolysate of the second substrate.

[0063] In one embodiment, the term "same type" as used herein refers to a first substrate and a second substrate that comprise or consist of the same or substantially the same type of food residue(s), for example, both the first substrate and the second substrate are bakery product food residue(s). For example, the first substrate and the second substrate of the same type are from the same source, for example, the same batch of food residue(s), and / or have the same or similar composition. In one embodiment, when the first substrate and the second substrate are of the same type, the first substrate and the second substrate are identical. In one embodiment, the composition of the first substrate and the composition of the second substrate are at least 50%, preferably at least 70%, more preferably at least 90% similar; i.e., the ingredients, and optionally their concentrations, have at least 50%, preferably at least 70%, more preferably at least 90% similarity. In one embodiment, when the first substrate and the second substrate are of the same type, the first substrate and the second substrate are both bakery food residue(s), such as bread, rolls, biscuits, muffins, cookies, or cakes; fruit food residue(s), such as fruit pulp, fruit peel, or fruit juice; plant food residue(s), such as vegetable peel, vegetable pulp, or vegetable juice; milling food residue(s), such as bran or bran meal; fish food residue(s), such as fish processing residue; seafood residue(s), such as seafood processing residue; brewer's used grains; cereal food residue(s), such as rice, wheat, millet, or corn; restaurant food residue(s), such as restaurant leftovers; animal food residue(s), such as milk or cheese; supermarket food residue(s), such as expired food; or any combination thereof. In a preferred embodiment, when the first substrate and the second substrate are of the same type, said first substrate and said second substrate are both bakery food residue(s), preferably bread food residue(s).

[0064] The term "second microorganism" as used herein refers to an oleaginous microorganism, preferably selected from oleaginous yeast, oleaginous fungus, oleaginous bacteria, and oleaginous microalgae.Oleaginous microorganisms are known to those skilled in the art.Oleaginous yeast, oleaginous fungus, oleaginous bacteria, and oleaginous microalgae are typically microorganisms that can produce microbial lipids.

[0065] In one embodiment, the oleaginous yeast is selected from the group consisting of Cutaneotrichosporon sp. Cutaneotrichosporon oleaginosus; Trichosporon sp., such as Trichosporon oleaginosus, Trichosporon capitatu, and Trichosporon asahii; Rhodospirillum sp.; Rhodosporidium sp., such as Rhodosporidium toruloides; Rhodosporon sp.; Candida sp., such as Candida viswanathii and Candida freyschussii; Cryptococcus sp., such as Cryptococcus curvatus; Lipomyces sp., such as Lipomyces starkeyi; Yarrowia sp., such as Yarrowia lipolytica; Rhodotorula sp., such as Rhodotorula graminis, Rhodotorula gracilis, and Rhodotorula glutinis; and Apiotrichum sp., such as Apiotrichum curvarum. In a preferred embodiment, the oleaginous yeast is selected from Cutaneotrichosporon sp., preferably Cutaneotrichosporon oleaginosus. In one embodiment, the oleaginous yeast is not Rhodosporidium toruloides. In one embodiment, the Rhodosporidium sp does not include Rhodosporidium toruloides. In one embodiment, when the second microorganism is Rhodosporidium sp., the Rhodosporidium sp. is not Rhodosporidium toruloides. In one embodiment, when the second microorganism is Rhodosporidium sp., the Rhodosporidium sp. is not Rhodosporidium toruloides. In one embodiment, when the oleaginous yeast is Rhodosporidium sp., the Rhodosporidium sp. is not Rhodosporidium toruloides.

[0066] In one embodiment, the oleaginous fungus is selected from Cunninghamella sp., such as Cunninghamella echinulate; Aspergillus sp., such as Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Fusarium sp., such as Fusarium venenatum; Mucor sp., such as Mucor moelleri; Mortierella sp., such as Mortariella isabellina and Mortierella alpine, preferably Mortierella alpine; and Humicola sp.

[0067] In one embodiment, the oleaginous bacteria is selected from Rhodococcus sp.; Acinetobacter sp.; and Bacillus sp.

[0068] In one embodiment, the oleaginous microalgae is selected from Chlorella sp., Pseudochlorococcum sp., Nannochloris sp., Nannochloropsis sp., Isochrysis sp., Tribonema sp., Dunaliella sp., Ankistrodesmus sp., Botryococcus sp., Pavlova sp., Scenedesmus sp., Skeletonema sp., and Nitzschia sp.

[0069] In one embodiment, the second microorganism is an oleaginous yeast, preferably selected from Cutaneotrichosporon oleaginosus, Trichosporon oleaginosus, Trichosporon capitatu, Trichosporon asahii, Lipomyces starkeyi, Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula graminis, Rhodotorula gracilis, Rhodotorula glutinis, Apiotrichum curvarum, Cryptococcus curvatus, Candida viswanathii, and Candida freyschussii; e.g., Cutaneotrichosporon oleaginosus. In one embodiment, the second microorganism is not Rhodosporidium toruloides. For example, the second microorganism may be an oleaginous yeast selected from Cutaneotrichosporon oleaginosus, Trichosporon oleaginosus, Trichosporon capitatu, Trichosporon asahii, Lipomyces starkeyi, Yarrowia lipolytica, Rhodotorula graminis, Rhodotorula gracilis, Rhodotorula glutinis, Apiotrichum curvarum, Cryptococcus curvatus, Candida viswanathii, and Candida freyschussii. In a preferred embodiment, the second microorganism is an oleaginous yeast, preferably selected from Cutaneotrichosporon sp., more preferably Cutaneotrichosporon oleaginosus. According to one embodiment of the present invention, the oleaginous microorganism used in the method according to the present invention is selected from oleaginous yeast, oleaginous fungi, oleaginous bacteria, and oleaginous microalgae; preferably, it is an oleaginous yeast.In a preferred embodiment, the oleaginous microorganism / yeast is Cutaneotrichosporon oleaginosus (C. oleaginosus).

[0070] The term "medium containing said enzymatically treated substrate" as used herein relates to a medium containing an enzymatically treated first substrate and / or an enzymatically treated second substrate and / or a component of said enzymatically treated first substrate and / or second substrate; for example a medium containing or consisting of a hydrolysate of the first substrate obtained in step b) and / or a hydrolysate of the second substrate obtained in step c). In one embodiment, said enzymatically treated first substrate and / or said enzymatically treated first component is a hydrolysate obtained in step b), preferably a hydrolysate of said first substrate. In one embodiment, said enzymatically treated second substrate and / or said enzymatically treated second component is a hydrolysate obtained in step c), preferably a hydrolysate of said second substrate. In one embodiment, the medium used in step d) comprises a hydrolysate of said first substrate and / or a hydrolysate of said second substrate. In one embodiment, said medium is a growth medium for said second microorganism. In one embodiment, the terms "culture medium" and "growth medium" are used interchangeably. In one embodiment, the medium comprises components / nutrients that allow, for example, an organism, for example, a second microorganism, to grow. In one embodiment, the medium comprises at least components of an enzymatically treated first substrate and / or an enzymatically treated second substrate, and optionally further comprises additional carbon sources, nitrogen sources, organic acids, such as acetic acid, trace metals, and / or vitamins. In this context, "additional" means that the medium may comprise carbon sources, nitrogen sources, organic acids, trace metals, and / or vitamins derived from the enzymatically treated first substrate and / or the enzymatically treated second substrate, and in addition, further carbon sources, nitrogen sources, organic acids, trace metals, and / or vitamins that are not derived from the enzymatically treated first substrate and / or the enzymatically treated second substrate. In one embodiment, the carbon source is a food-grade carbon source.In one embodiment, the term "medium containing an enzymatically treated substrate" relates to a medium containing an enzymatically treated substrate, such as the enzymatically treated first substrate of step b) or the enzymatically treated second substrate of step c), and / or a medium containing components of the enzymatically treated substrate(s). For example, the components of the enzymatically treated substrate may be recovered, said components may be incorporated into the medium, or the enzymatically treated substrate may be used directly as a medium. For example, the enzymatically treated first substrate obtained in step b) and / or the enzymatically treated second substrate obtained in step c) may be used directly as a medium used in step d). The enzymatically treated first substrate and the enzymatically treated second substrate may be mixed to provide a medium for step d). In one embodiment, the medium / mediums used in step b) and / or c) contain the enzymatically treated first substrate and the second substrate, respectively, and are used as a medium for culturing the second microorganism in step d). In one embodiment, the medium used in step d) comprises or consists of the medium used in step b) containing the enzymatically treated first substrate and / or comprises or consists of the medium used in step c) containing the enzymatically treated second substrate. In one embodiment, the terms "enzymatically treated first substrate" and "hydrolysate of first substrate" are used interchangeably. In one embodiment, the terms "enzymatically treated second substrate" and "hydrolysate of second substrate" are used interchangeably. In one embodiment, such hydrolysates, such as hydrolysates of first substrate and / or hydrolysates of second substrate, are used as medium for growing a second microorganism in step d). In one embodiment, the cell culture broth of steps b) and / or c) containing the enzymatically treated substrate is used as medium for step d). In one embodiment, after step b) or, if step c) is present, after step c), the second microorganism is cultivated in step d) in the medium used in step b) or step c), respectively.For example, the second microorganism may be added to a vessel, such as a cell culture vessel, in which step b) and / or step c) have been performed.

[0071] In one embodiment, the enzymatically treated first substrate used in step d) comprises the culture medium used in step b); optionally, said culture medium has been filtered and / or centrifuged. In one embodiment, the enzymatically treated second substrate used in step d) comprises a liquid, e.g., culture medium, used in step c); optionally, said liquid has been filtered and / or centrifuged. In one embodiment, the culture medium used in step d) is the culture medium used in step b) and / or step c); optionally, said culture medium has been filtered and / or centrifuged. In one embodiment, the liquid, e.g., culture medium, used in step b) and / or c) is transferred to step d) and used as culture medium in step d).

[0072] In one embodiment, the enzymatically treated first substrate produced in step b) is centrifuged before using the enzymatically treated first substrate in the medium in step d). In one embodiment, the growth medium used in step b) containing the enzymatically treated first substrate produced is centrifuged, and the supernatant of such centrifugation is used as the medium for step d) or as a component of the medium for step d). In one embodiment, the enzymatically treated second substrate produced in step c) is centrifuged before using the enzymatically treated second substrate in the medium in step d). In one embodiment, the medium used in step c) containing the enzymatically treated second substrate produced is centrifuged, and the supernatant of such centrifugation is used as the medium for step d) or as a component of the medium for step d). For example, such centrifugation can be in the range of 5,000 rpm to 20,000 rpm, for example 14,000 rpm, for 1 minute to 60 minutes, for example 20 minutes. In one embodiment, such centrifugation can be used to recover protein biomass by separating it from the supernatant containing the enzymatically treated substrate.

[0073] In one embodiment, the medium further comprises additional carbon sources, nitrogen sources, organic acids, trace metals, and / or vitamins. In one embodiment, the medium comprises carbohydrates such as monosaccharides, preferably pentoses or hexoses, more preferably glucose, xylose, mannitol, arabinose; and oligosaccharides; protein hydrolysates such as oligoamino acids and amino acids or other peptide hydrolysates; fatty acids; organic acids, preferably acetic acid; minerals; vitamins and trace elements, and any combination thereof. In one embodiment, the additional carbon source is selected from sugars, glycerol, sugar alcohols, sugar acids, fatty acids, fatty alcohols, and fatty esters. In one embodiment, the nitrogen source is selected from urea, protein hydrolysates, amino acids, and inorganic nitrites. In one embodiment, the trace metals are selected from V, Mo, Cu, and Fe. In one embodiment, the vitamins are selected from vitamin C, vitamin B, vitamin A, and vitamin E. In one embodiment, the additional carbon sources, nitrogen sources, organic acids, trace metals, and / or vitamins are food grade.

[0074] In one embodiment, the carbon source is selected from the group comprising carbohydrates: amino acids; fatty acids; preferably monosaccharides, preferably pentose or hexose, more preferably glucose, xylose and / or mannitol; oligosaccharides; hydrolysates of animal tissue, plant tissue or microorganisms; and any combination of the foregoing, where more preferably the carbon source is glucose. In one embodiment, the concentration of the carbon source in the medium ranges from 50 mM to 400 mM, preferably from 200 mM to 300 mM. The glucose can be used alone or in combination with a suitable hydrolysate, such as peptone, tryptone, etc. In one embodiment, the hydrolysate is a marine biomass hydrolysate, such as an algae hydrolysate, a lignocellulosic hydrolysate, a plant hydrolysate, a marine macroalgae and microalgae hydrolysate, a corn hydrolysate, a wheat hydrolysate or other hydrolysate. In some embodiments of the method according to the invention which include a recycling step, the hydrolysate may also be a microbial hydrolysate, e.g. a yeast hydrolysate derived from the hydrolysis of an oleaginous microorganism, e.g. the yeast itself, preferably after such a hydrolysate has been used to produce lipids.

[0075] In one embodiment, the weight ratio of carbon to nitrogen (C:N) in the medium is (100-200):1, especially when it is a nitrogen-limited medium. In another embodiment, the weight ratio of carbon to nitrogen (C:N) in the medium is (10-100):1, preferably (10-50):1, especially when it is not a nitrogen-limited medium. In one embodiment, the medium is a nitrogen-rich medium. The weight ratios shown above and further below are the weight ratios in the starting medium, i.e. when step d) begins. The term "nitrogen-rich medium" as used herein refers to a medium that is not a nitrogen-limited medium. In one embodiment, "nitrogen-rich medium" refers to a medium having a carbon to nitrogen weight ratio (C:N) of <100, preferably ≦80, more preferably 25-80.

[0076] In one embodiment, the medium used in step d) is a nitrogen-rich medium. In one embodiment, the medium used in step d) further comprises a nitrogen source, preferably in the form of a protein hydrolysate, such as peptone, tryptone or other peptide hydrolysate, preferably wherein said peptide hydrolysate comprises animal tissue, plant tissue and / or components of said oleaginous microorganism. In one embodiment, the nitrogen source is selected from protein hydrolysates, such as peptone, tryptone or other peptide hydrolysates, preferably wherein said peptide hydrolysate comprises animal tissue, plant tissue and / or components of said oleaginous microorganism. Without wishing to be bound by any theory, the inventors believe that the presence of such an additional nitrogen source allows the production of lipids and biomass to be increased overall.

[0077] In one embodiment, the organic acid is selected from the group including acetic acid, malonic acid, oxalic acid, citric acid, propionic acid, valeric acid, acrylic acid, crotonic acid, butyric acid, isobutyric acid, isovaleric acid, 3-hydroxybutyric acid, 3-hydroxypropionic acid, 2-hydroxybutyric acid, lactic acid, and the respective salt(s) of such acids, as well as mixtures of any of the aforementioned organic acids. Preferably, the organic acid is acetic acid or an acetate salt. In one embodiment, the organic acid is only acetic acid or an acetate salt; in another embodiment, the organic acid is a combination of acetic acid or an acetate salt with any of the other aforementioned organic acids. It should be noted that the term "organic acid" as used herein is meant to encompass the respective organic acid regardless of its degree of protonation, i.e., to encompass the acid in its protonated state(s) as well as in its deprotonated state(s), for example when it is in an aqueous solution at a pH at which it is protonated or deprotonated, respectively, depending on its respective pKa value(s). The term "organic acid" as used herein is also meant to include the salt(s) of organic acid, such as the respective metal salts of such organic acid. Examples of such metal salts are alkali or alkaline earth salts of the respective organic acid. The salts may be in their dissociated or undissociated form. The term "mixture of organic acids" as used herein is meant to include mixtures of organic acids in their respective acid forms, i.e., with other organic acids, mixtures of organic acids in their acid forms with other organic acids in their salt forms, and mixtures of salts of organic acids with salts of other organic acids. It should also be noted that the term "organic acid" as used herein does not include fatty acids or amino acids. The phrase "carbon source and organic acid" as used herein implies that the "carbon source" is different from the "organic acid". Thus, the two entities are chemically different. In one embodiment, the concentration of said organic acid ranges from 20 mM to 200 mM, preferably from 30 mM to 100 mM. In one embodiment the medium used in step d) is supplemented with an organic acid.

[0078] In one embodiment, said culturing of the second microorganism in step d) is carried out at a temperature ranging from 10° C. to 37° C., preferably from 18° C. to 33° C. In one embodiment, said culturing of the second microorganism in step d) is carried out at a pH ranging from pH 3 to pH 9. In one embodiment, said culturing of the second microorganism in step d) is carried out for 1 to 12 days, preferably for 2 to 10 days. In one embodiment, said culturing of the second microorganism in step d) is carried out at a pO2>20%. In one embodiment, said culturing of the second microorganism in step d) is carried out under agitation, preferably under stirring at a rotation speed ranging from 20 rpm to 1000 rpm, for example from 50 rpm to 800 rpm. For example, a paddle stirrer may be used. In one embodiment, said culturing of the second microorganism in step d) is carried out at a temperature ranging from 10° C. to 37° C., preferably from 18° C. to 33° C., at a pH ranging from pH 3 to pH 9, for 1 to 12 days, preferably from 2 to 10 days, at a pO2>20%, under agitation, preferably under stirring at a rotation speed ranging from 20 rpm to 1000 rpm, for example from 50 rpm to 800 rpm. In one embodiment, said culturing of the second microorganism in step d) comprises culturing the second microorganism for 1 to 12 days, preferably from 2 to 10 days, at a temperature ranging from 10° C. to 37° C., preferably from 18° C. to 33° C., at a pH ranging from pH 3 to pH 9, and / or at a dissolved oxygen (pO2) pO2>20%, preferably in a medium comprising any of the enzymatically treated first substrate or a component thereof, the enzymatically treated second substrate or a component thereof, YPD, YNB, Sabouraud broth, and any combination thereof.

[0079] In one embodiment, the process is suitable for producing lipids having a content of unsaturated fatty acids >60% relative to the total fatty acid content and / or a pour point <10° C., for example in the range of 9° C. to 5° C., such as for example around 5° C. This is especially the case if the following parameters are selected for step d): The oleaginous microorganisms are grown in a medium containing an enzymatically treated substrate, such as an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: temperature 10° C.-20° C., dissolved oxygen content 10%-30% and / or addition of crotonic acid (0%-10% of total organic acids), and thereby allowing the oleaginous microorganisms to produce microbial lipids characterized by a "pour point" in the range of 9° C. to 5° C., such as at <10° C., e.g., about 5° C., and / or an unsaturated fatty acid content of >60% relative to the total fatty acid content. As used herein, "pour point" is typically determined according to any of the following standards: DIN 51597, DIN EN 23015:1994-05, DIN ISO 3015:1982-10, DIN ISO 3016:1982-10, ASTM D97, ASTM D5985, preferably using DIN ISO 3016.

[0080] During the cultivation of microorganisms for the production of microbial oils, there is a trade-off in either lipid productivity or biomass productivity for the production of microbial oils. Conventionally, lipids are produced in a two-phase system, including a first step for biomass production under non-limiting conditions, followed by a nutrient-limited phase in which biomass growth stops and only lipids accumulate. Under these conditions, lipid productivity does not exceed 70% w / w. Surprisingly, this invention discloses a completely new lipid production route that can achieve biomass growth and lipid accumulation simultaneously. This has led to the development of a 200g L ... -1 It provides options for biomass and lipid yield over biomass.

[0081] In one embodiment, the oleaginous microorganism is grown in a medium comprising said enzymatically treated substrate, and optionally further comprising any of a carbon source, a nitrogen source, an organic acid, trace metals, and / or vitamins. Depending on the pH at which the medium is, the organic acid dissociates / deprotonates, in which case it exists as a salt / carboxylate anion form, or it is undissociated, in which case it exists as an organic acid in its protonated form. In one embodiment, the medium in which the oleaginous microorganism grows in step d) comprises said enzymatically treated substrate. In one embodiment, the terms "cultivating" and "growing" are used interchangeably. In one embodiment, when referring to an "enzymatically treated substrate", such term relates to an "enzymatically treated first substrate" and / or an "enzymatically treated second substrate". Without wishing to be bound by any theory, the inventors believe that the presence of an organic acid, such as acetic acid / acetate, allows lipid productivity to be increased even further, while the presence of a carbon source allows an increase in total biomass. It should be noted that the "carbon source" and organic acid, as defined above, are two different entities that are distinct from each other.

[0082] In one embodiment, the term "purely enzymatic treatment of the cultured second microorganism without solvent-based extraction or chemical-based demulsification" is meant to refer to an enzymatic treatment of the oleaginous microorganism without a) any extraction using one or several solvents or b) any demulsification using one or several (suitable) chemical reagents or c) both a) and b). Preferably, such term is meant to refer to an enzymatic treatment without any exposure to an extraction solvent and without any exposure to a demulsifying chemical reagent. The term is also meant to exclude the implementation of any other pretreatment of the grown oleaginous microorganism. It should be noted that in an embodiment of the present invention, "purely enzymatic treatment" excludes the implementation of any pretreatment of the grown oleaginous microorganism, which pretreatment can be chemical (using one or several chemical reagents to which the grown oleaginous microorganism will be exposed) or physical (such as changes in physical conditions, e.g. temperature, pressure, ultrasound, light, irradiation with electromagnetic radiation, etc.).

[0083] In one embodiment, the purely enzymatic treatment of the grown oleaginous microorganisms is a treatment with hydrolases of the microorganisms, alone or in combination with / followed by a protease. In one embodiment, the hydrolases are obtained from fungi, preferably filamentous fungi, more preferably from the genera Trichoderma, Aspergillus, Penicillium, Aureobasilium and Fusarium. In one embodiment, the hydrolases are obtained from fungi cultured in the presence of an induction system, preferably, the induction system being a component of the oleaginous microorganism, more preferably one or several cell wall components of the oleaginous microorganism used to produce the microbial lipids, to obtain a hydrolase preparation allowing the dissolution of the cell wall of the oleaginous microorganism. In one embodiment, the induction system is the residual biomass of the oleaginous microorganism produced during steps e) and / or f) or is a part or component of the residual biomass.

[0084] In one embodiment, the hydrolases obtained from the fungus are prepared separately (from carrying out steps d) to f) of the invention) and used in step e) either as a liquid preparation obtained directly from culturing the fungus or as a lyophilized preparation that is subsequently reconstituted in the solution used in step e). In one embodiment, the hydrolases contain one or several activities, for example, selected from, but not limited to, cellulase, xyloglucanase, beta-glucosidase, mannanase, xylanase and laminarinase enzyme activities. In one embodiment, the proteases, if present, are selected from proteases produced by Aspergillus sp., Streptomyces sp. or Bacillus sp. In one embodiment according to the invention, the "purely enzymatic treatment" further comprises treatment of the microorganism with a protease (but still excluding pretreatment or solvent-based extraction or chemical-based demulsification). It should be noted that such proteases, if and when used, follow or are combined with the treatment using the hydrolases described above. Without being bound by any theory, the inventors believe that the treatment involving proteases allows the separation of the lipids produced from any proteins associated with them, thus aiding in the release of the lipids. In one embodiment, the protease is selected from the group of proteases produced by Aspergillus sp., Streptomyces sp. or Bacillus sp.

[0085] In one embodiment, in step e), if present, the "purely enzymatic treatment of the cultured second microorganism without solvent-based extraction" is a treatment with a hydrolytic enzyme of the microorganism. Preferably, such hydrolytic enzyme is obtained from another microorganism, preferably from a fungus, more preferably from a filamentous fungus. In one embodiment, the fungus is selected from the genera Trichoderma, Aspergillus, Penicillium, Aureobasillium and usarium. In a more preferred embodiment, the filamentous fungus is Trichoderma reesei, since it has been shown to produce particularly efficient hydrolytic enzymes that allow the lysis of cell walls of oleaginous microorganisms. In one embodiment, the hydrolytic enzyme is obtained from a fungus, preferably a filamentous fungus, cultured in the presence of an induction system, wherein preferably, the induction system is one or several cell wall components of the oleaginous microorganisms used to produce microbial lipids, in order to obtain a hydrolytic enzyme preparation that allows the lysis of a component of the oleaginous microorganism, preferably the cell wall of the oleaginous microorganism. In one embodiment, said induction system is said residual biomass, or a component thereof, that may be produced during steps e) and f). Preferably, such hydrolytic enzymes are produced by culturing said filamentous fungi in the presence of cell wall fragments of said oleaginous microorganism. Without wishing to be bound by any theory, the inventors believe that exposure of filamentous fungi, such as T. reesei, to the presence of such cell wall components of said oleaginous microorganism allows said filamentous fungi to produce precisely the enzyme(s) suitable for completing the lysis of the cell wall of the oleaginous microorganism. In a particularly preferred embodiment, a filamentous fungus from the genus Trichoderma is used, such as Trichoderma reesei, and in a particularly preferred embodiment, a mutant of Trichoderma reesei is used, such as the mutants with ATCC deposit number(s) 56765 and 13631.Once the filamentous fungi are cultured, the resulting culture may be further processed, such as concentrated, to remove the fungal biomass, and the resulting supernatant may be used in such form, or it may be lyophilized and kept for storage and subsequent reconstitution in an appropriate aqueous solution. Again, without wishing to be bound by any theory, the inventors believe that the hydrolases so produced may represent a combination of various enzymatic activities, such as cellulases, xyloglucanases, β-glucosidases, mannanases, xylanases, and laminarinases, and / or others.

[0086] In one embodiment, "suitable for subsequent recovery" as used herein relates to facilitating recovery of microbial lipids, for example by making the microbial lipids produced accessible for subsequent recovery, such as accessible for subsequent recovery from the culture vessel. For example, by performing a purely enzymatic treatment of oleaginous microorganisms, the microbial lipids become accessible for subsequent recovery. In one embodiment, the terms "suitable" and "accessible" are used interchangeably.

[0087] In one embodiment, purely enzymatic treatment of oleaginous microorganisms without solvent-based extraction or chemical-based demulsification renders the microbial lipids suitable for subsequent recovery, such as by density-based separation methods, which in one embodiment are selected from gravity-based separation, gravity-assisted phase separation, and centrifugation, each of which is performed alone or in combination with decantation, aspiration, or other mechanical recovery methods.

[0088] Growth of oleaginous microorganisms in a suitable medium containing an enzymatically treated substrate allows said oleaginous microorganisms to produce microbial lipids. However, typically, these microbial lipids are still contained within the cells of the oleaginous microorganisms and therefore need to be made available for subsequent recovery. An embodiment of the present invention thus provides an enzymatic treatment of the grown oleaginous microorganisms that renders the produced microbial lipids suitable or accessible for subsequent recovery from the culture vessel. It should be noted that the method according to the present invention includes a purely enzymatic treatment of the grown oleaginous microorganisms without having to resort to any pretreatment step, such as a chemical pretreatment step or subsequent or simultaneous extraction of said lipids using a solvent. Examples of such chemical pretreatment steps or subsequent or simultaneous extraction steps are solvent-based extraction or chemical-based demulsification. Such excluded pretreatment steps can however also be physical steps such as temperature change, pressure change, centrifugation, sonication, irradiation, etc. The term "chemical-based demulsification" as used herein refers to the exposure of the grown biomass to a demulsifier that allows the breaking of any emulsions (that may have formed).

[0089] According to an embodiment of the present invention, the method does not include solvent-based extraction, chemical-based demulsification, or other treatments such as temperature shock, chemical treatment, or high pressure homogenization or ultrasonic homogenization. These would potentially increase the cost or hazardous characteristics of the method and are avoided by the present invention. The advantage of such absence of solvent-based extraction and chemical-based demulsification is that the downstream products, especially the microbial oil, are suitable for further processing into food products. In particular, the microbial oil produced by the method of the present invention can be directly used to prepare food products, for example, due to the absence of solvent and / or chemical residues in the microbial oil. The inventors have surprisingly found that such harsh processing steps are not strictly necessary.

[0090] In an embodiment according to the present invention, the microbial lipids produced are recovered using density-based separation.In its simplest form, such density-based separation can be a process in which the culture of oleaginous microorganisms is simply left for a period of time, resulting in the lipid phase separating from the aqueous phase due to different densities and / or solubility in water.This can be combined with subsequent decantation, suction or other mechanical removal of the lipid phase from the culture.In other embodiments, separation can occur by centrifugation, gravity-assisted phase separation, alone or in combination with subsequent mechanical removal or lipid phase, for example decantation or suction.

[0091] In one embodiment, the method comprises step e) of carrying out the purely enzymatic treatment of the cultured second microorganism without solvent-based extraction or chemical-based demulsification, wherein the purely enzymatic treatment of the cultured second microorganism is a treatment with the hydrolases of the microorganism, alone or in combination with / followed by proteases. In one embodiment, steps d) and e) are carried out in the same reaction vessel. This is also referred to herein as a "one-pot method" or "one-pot process". In this embodiment, the method according to the present invention can be considered as a one-pot process for the production of microbial lipids, in particular food-grade microbial lipids.

[0092] In one embodiment, steps e) and / or f) result in a lipid phase and a hydrolysate of the oleaginous microorganism. In some cases, steps e) and / or f) may also result in a residual biomass of the oleaginous microorganism, the residual biomass being different from the lipid phase and the hydrolysate. In one embodiment, the method comprises repeated performance of steps d) to f), and wherein the hydrolysate of the oleaginous microorganism resulting from steps e) and / or f) is reused / recycled to perform step d). In such an embodiment, the microbial hydrolysate resulting from steps e) and / or f) may be re-fed to the medium used in step d) and act as an additional carbon source (in addition to the carbon source present in the enzymatically treated substrate). The embodiment of reusing / recycling by-products in steps d) to f), for example reusing / recycling the product resulting from steps e) and / or f) in step d), avoids the generation of waste. Such an embodiment may therefore also sometimes be referred to herein as a "waste-free process" or a "waste-free method". In one embodiment, steps d) to f) are carried out 2 to n times, where n is an integer selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. In one embodiment, steps d) to f) are carried out 2 to 3 times. The repeated recycling allows for efficient use of the various media involved and avoids the production of excessive waste, since the products resulting from the process, such as hydrolysates of oleaginous microorganisms, are reused as starting media for growing such oleaginous microorganisms.

[0093] In one embodiment, the method of the invention further comprises one or more steps of recovering said protein biomass and / or said aroma compounds, for example using any of centrifugation, filtration, distillation, organophilic permeation evaporation, solid phase microextraction, sedimentation, coagulation, flotation, and combinations thereof, preferably using any of centrifugation, filtration, distillation, organophilic permeation evaporation, solid phase microextraction, and combinations thereof. In one embodiment, the method of the invention is carried out in fed-batch, semi-continuous or continuous mode, wherein optionally, said method comprises repeated addition of an organic acid.

[0094] In one embodiment, the method of the present invention comprises: - mechanical pre-treatment, preferably by milling, mixing, chopping and / or sieving said substrate(s); - dissolving said substrate(s) in a solvent, preferably in water; - chemical hydrolysis of said substrate(s), preferably using an acid, such as sulfuric acid; For example, an acid, preferably sulfuric acid, is used at a concentration ranging from 0.1 to 10% by volume at a temperature ranging from 20° C. to 260° C., preferably from 80° C. to 140° C., for a period of 1 second to 24 hours, preferably from 30 minutes to 3 hours. - a thermal pretreatment of said substrate(s), preferably at a temperature of between 50°C and 200°C, for between 10 and 240 minutes, more preferably at a temperature of between 80°C and 170°C, for between 30 and 90 minutes; - Fermentation pretreatment, preferably anaerobic fermentation, of said substrate(s); and / or - enzymatic pretreatment of said substrate(s) using one or more enzymes, optionally commercially available enzymes, enzymatic pretreatment, wherein the one or more enzymes are proteases, such as endopeptidases and exopeptidases, serine endopeptidases, subtilisin A, and pepsin; and hydrolases, preferably glycoside hydrolases, more preferably glycoside hydrolases selected from α-amylase, amyloglucosidase, cellulase, hemicellulase, xylanase, xyloglucase, galactanase, arabinase, mannanase, lipase, glucoamylase, and pectinase. and / or, if step c) is present, pre-treating the second substrate by

[0095] In one embodiment, the first substrate and / or the second substrate are pretreated using any combination of the mechanical pretreatment, the lysis, the chemical hydrolysis, the heat pretreatment, the fermentation pretreatment, and the enzymatic pretreatment. In one embodiment, such a step of pretreating the first substrate, if present, is carried out before culturing the first microorganism with the first substrate. In one embodiment, the step of pretreating the second substrate using any of the mechanical pretreatment, the lysis, the chemical hydrolysis, the heat pretreatment, the fermentation pretreatment, the enzymatic pretreatment, or any combination thereof, is a further pretreatment in addition to the pretreatment of the second substrate with the at least one enzyme in step c). For example, the step of pretreating the second substrate using any of the mechanical pretreatment, the lysis, the chemical hydrolysis, the heat pretreatment, the fermentation pretreatment, the enzymatic pretreatment, or any combination thereof, is carried out before and / or subsequent to the pretreatment of the second substrate with the at least one enzyme in step c). In one embodiment, such a step of pretreating the second substrate, if present, is carried out before culturing the second microorganism with the second substrate, preferably before pretreating the second substrate with the at least one enzyme of step c). In one embodiment, the fermentation pretreatment is an anaerobic fermentation using an anaerobic microorganism. In one embodiment, the fermentation pretreatment comprises treatment of the substrate at a pH in the range of pH 2.5 to pH 5.5, preferably pH 3 to pH 4, at a temperature in the range of 20° C. to 50° C., preferably 25° C. to 40° C., at a pO2 in the range of 0 to 30%, preferably 5 to 10%, for 1 to 5 days, preferably 1 to 3 days, and / or with agitation of 50 rpm to 500 rpm.

[0096] In one embodiment, the pretreatment of the first substrate and / or the second substrate is an enzymatic pretreatment, which is hydrolysis. In one embodiment, the step of pretreating the first substrate and / or the second substrate comprises an enzymatic pretreatment, which is hydrolysis. Hydrolysis, for example of starch contained in the food residue(s), can comprise two stages; in a first stage, starch is liquefied using α-amylase, thereby providing a solution containing dextrins and glucose; in a second stage, the liquefied starch is subjected to glucoamylase. In one embodiment, the step of pretreating the first substrate and / or the second substrate comprises an enzymatic pretreatment, which comprises treatment with α-amylase and glucoamylase. In one embodiment, the method of the invention comprises a step of purifying the microbial lipids recovered in step f), for example by physical purification.

[0097] In one embodiment, the ability of the first or second microorganism to produce enzymes, protein biomass, microbial lipids, and / or aroma compounds is further improved by genetic modification, for example genetic modification by non-targeted or targeted gene modification.In one embodiment, the non-targeted gene modification comprises chemically induced mutation or radiation induced mutation.In one embodiment, the radiation induced mutation comprises UV, ionizing, gamma radiation, X-ray, and neutron radiation.In one embodiment, the targeted gene modification comprises CRISPR-CAS system, bacterial transformation, and / or plasmid-based insertion.

[0098] In one embodiment, the method of the present invention is a method for producing microbial lipids and preparing a food product therewith, wherein the method comprises a step, e.g. step g) of preparing a food product, preferably a bakery product, such as bread, rolls, biscuits, muffins, cookies or cakes, confectionery products, such as flour confectioneries or sugar confectioneries, dairy products, such as ice cream or baby milk, spreads, such as margarine, mayonnaise or soft cheese, instant foods, such as instant noodles, pizza, pizza dough or sauces, beverages, vegetarian or vegan foods, such as meat analogues and dairy analogues, sweets, such as cocoa-free chocolate, and / or chocolate products, comprising the microbial lipids recovered in step f); wherein optionally the method further comprises a step, e.g. step h) of recycling the food residue(s) of the food product by using the food residue(s) as the first substrate and / or, if step c) is present, as the second substrate. In one embodiment, the method is a method for recycling food residues, i.e. food residue(s) are converted into food-grade products, such as microbial oil, protein biomass, and / or aroma compounds, which are further processed into food. Such food residue(s), such as food residues, can be subsequently converted into food-grade products using the method of the present invention. Advantageously, the method of the present invention increases the efficiency of the food chain and the use of food resources. An advantage of the method and use of the present invention is that food residue(s) and / or food loss are reduced by converting such food residue(s) into edible raw materials, such as microbial oil and optionally protein biomass and / or aroma compounds. In one embodiment, when referring to preparing a food product "with" or "from" in terms of a product such as microbial lipid, it means preparing a food product that includes said product.

[0099] The advantage of the method of the present invention is that the microbial food ingredients produced, in particular the microbial lipids and protein biomass and / or aroma compounds, allow the production of food products containing said microbial food ingredients, since the microbial food ingredients are food grade ingredients. For example, the microbial lipids produced by fermentation of bread residue hydrolysates can replace palm oil in bakery applications, such as bread, praline production, ice cream, instant noodles, margarine, chocolate, pizza dough and cookie production. The aroma compounds can be used to flavor food, preferably dairy products, and confectionery products according to Commission Implementing Regulation (EU) No 872 / 2012. The protein biomass produced in the method of the present invention, preferably as a by-product of the method of producing microbial lipids, can be used as a meat substitute for any minced or formed meat application, such as burger patties, chili, nuggets, meat sauce and sausages. In one embodiment, the food grade properties of the ingredients involved are ensured during the entire process of the method of the present invention. That is, the method of the present invention allows to achieve a sustainable use of food resources and reduces food waste and food loss. In particular, the method of the invention unexpectedly allows the use of food residue(s), preferably food-grade food residue(s), to produce valuable raw materials such as microbial lipids, protein biomass and / or aroma compounds, which can subsequently be used to produce food; the residue(s) from the food so produced can in turn be recycled and used as substrate in the method of the invention to produce further food. Conclusively, and advantageously, the method of the invention can be used as a cycle.

[0100] A further advantage of the method of the present invention is that it does not require the cultivation of oleaginous microorganisms in a two-step process, where the first step is under non-limiting conditions for biomass formation and the second step is under new trend-restricting conditions for new lipid production induction. Instead, the method of the present invention allows for a cultivation in which lipids are directly accumulated. In particular, by providing a medium containing the enzymatically treated first substrate and / or the enzymatically treated second substrate to the second microorganism in step d), a high accumulation of intracellular lipids is achieved. In particular, by providing the medium to the second microorganism, a high protein biomass and lipid yield is achieved. For example, a lipid yield of 84.9% of dry cell weight has been achieved. Furthermore, a protein biomass yield of up to 60% of dry fungal biomass has been achieved. Advantageously, a step of nutrient limitation is not required to prepare microbial oil with the oleaginous microorganism. A further advantage of the method of the present invention is that only mild downstream processing is required, e.g. a purely enzymatic treatment can be carried out to make the microbial lipids suitable for subsequent recovery, i.e. no hazardous chemicals are involved; the recovered microbial lipids are edible, i.e. free of hazardous residues.

[0101] In one embodiment, the term "use of microbial lipids in the manufacture of food" as used herein relates to producing a food comprising microbial lipids, preferably microbial lipids obtained by the method according to the invention. In one embodiment, the use of microbial lipids in the manufacture of food comprises replacing fat(s) in the recipe of the food with said microbial lipids. For example, microbial lipids may replace fats such as palm oil in food, e.g. bakery products. In one embodiment, the use of microbial lipids in the manufacture of food further comprises using protein biomass and / or aroma compounds, preferably protein biomass and / or aroma compounds obtained by the method according to the invention, in the manufacture of said food. For example, a food comprising said microbial lipids and said protein biomass and / or aroma compounds may be provided. In one embodiment, the terms "foodstuff", "food product" and "food" are used interchangeably. In one embodiment, the use according to the invention comprises a method of producing microbial lipids as defined herein and preparing food therefrom. In one embodiment, the use of the present invention comprises using microbial lipid produced using the method of producing microbial lipid as defined herein in the manufacture of a food product.

[0102] In one embodiment, the term "composition comprising at least 5, preferably at least 6, more preferably at least 7 enzymes" as used herein refers to an enzyme mixture. In one embodiment, said composition can be obtained by carrying out step b) of the method of the invention. In one embodiment, step b) of the method of the invention results in the production of said composition by said first microorganism. In one embodiment, the composition is tailored to said first substrate. In one embodiment, the composition comprises enzymes selected from cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase. In a preferred embodiment, the composition comprises an enzyme selected from cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; and rhamnogalacturonan lyase. In one embodiment, the composition comprises an enzyme selected from cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; and mannanase; or cellulase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; galactanase; mannanase; rhamnogalacturonan hydrolase; and rhamnogalacturonan lyase. In a preferred embodiment, the composition, e.g. the composition produced in step b) of the method of the present invention using bread residue as the first substrate, comprises an enzyme selected from cellulase; amylase; hemicellulase; limit dextrinase, e.g. malt limit dextrinase; and pectinase.In one embodiment, the composition of the present invention comprises or consists of cellulase, amylase, hemicellulase, limit dextrinase, e.g., malt limit dextrinase, and pectinase. In one embodiment, the composition is a food grade composition.

[0103] In one embodiment, the composition is produced in step b) of the method for producing microbial lipids as defined herein, where preferably the composition comprises or consists of the at least one enzyme produced in step b) of the method for producing microbial lipids as defined herein. In one embodiment, the composition is producible in step b) of the method for producing microbial lipids as defined herein, where preferably the composition comprises or consists of the at least one enzyme producible in step b) of the method for producing microbial lipids as defined herein. In one embodiment, the at least one enzyme produced in step b) of the method for producing microbial lipids as defined herein comprises or consists of the composition of the invention. In one embodiment, the composition is a food grade composition when the first substrate in step b) of the method for producing microbial lipids is food grade food residue(s).

[0104] In one embodiment, the composition is obtained in step c) of the method for producing microbial lipids as defined herein, wherein preferably the composition comprises or consists of the at least one enzyme obtained in step c) of the method for producing microbial lipids as defined herein. In one embodiment, the composition is obtainable in step c) of the method for producing microbial lipids as defined herein, wherein preferably the composition comprises or consists of the at least one enzyme obtainable in step c) of the method for producing microbial lipids as defined herein. In one embodiment, the at least one enzyme obtained in step c) of the method for producing microbial lipids as defined herein comprises or consists of the composition of the invention. In one embodiment, the at least one enzyme obtained in step c) of the method for producing microbial lipids as defined herein is a composition of the invention. In one embodiment, the composition of the invention is a by-product of the method for producing microbial lipids as defined herein.

[0105] In one embodiment, said culturing of the second microorganism of step d) is carried out at a temperature ranging from 18° C. to 37° C., preferably from 18° C. to 33° C. In one embodiment, said culturing of the second microorganism of step d) is carried out at a pH ranging from pH 3 to pH 9. In one embodiment, said culturing of the second microorganism of step d) is carried out for 1 to 12 days, preferably for 3 to 10 days. In one embodiment, said culturing of the second microorganism of step d) is carried out at a pO2>20%. In one embodiment, said culturing of the second microorganism of step d) is carried out under agitation, preferably under stirring at a rotation speed ranging from 50 rpm to 800 rpm. In one embodiment, said culturing of the second microorganism in step d) is carried out at a temperature in the range of 10° C. to 37° C., preferably 18° C. to 33° C., at a pH in the range of pH 3 to pH 9, for 1 to 12 days, preferably 2 to 10 days, at a pO2>20%, under agitation, preferably under stirring at a rotation speed in the range of 50 rpm to 800 rpm. In one embodiment, said culturing of the second microorganism in step d) comprises culturing said second microorganism for 2 to 12 days, preferably 3 to 10 days, at a temperature in the range of 18° C. to 37° C., preferably 18° C. to 33° C., at a pH in the range of pH 3 to pH 8, and / or at a dissolved oxygen (pO2) pO2>30%.

[0106] In one embodiment, the method is particularly suitable for producing lipids with a content of unsaturated fatty acids >60% relative to the total fatty acid content and / or a pour point <10° C., for example in the range of 9° C. to 5° C., such as for example around 5° C. This is especially the case if the following parameters are selected for step d): The oleaginous microorganisms are grown in a medium containing an enzymatically treated substrate, such as an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: a temperature of 10° C. to 20° C., a dissolved oxygen content of 10% to 30%, and / or the addition of crotonic acid (e.g., 0% to 10% of total organic acids), thereby enabling the oleaginous microorganisms to produce microbial lipids characterized by a "pour point" of <10° C., e.g., in the range of 9° C. to 5° C., such as, for example, about 5° C., and / or an unsaturated fatty acid content of >60% relative to the total fatty acid content. As used herein, "pour point" is typically determined according to any of the following standards: DIN 51597, DIN EN 23015:1994-05, DIN ISO 3015:1982-10, DIN ISO 3016:1982-10, ASTM D97, ASTM D5985, preferably using DIN ISO 3016.

[0107] In one embodiment, the method is suitable for producing lipids having a content of saturated fatty acids of 20% to 65% relative to the total fatty acid content and / or a triglyceride POP C18:1-C16:0), POS (C16:0-C18:1-C18:0) and SOS (C18:0-C18:1-C18:0) content of 40% to 85% relative to the total triglyceride content. In one embodiment, the method is suitable for producing lipids that are equivalent to cocoa butter-like microbial oils. This is especially true if the following parameters are selected for step d): Growing the oleaginous microorganism in a medium containing an enzymatically treated substrate, e.g., an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: a temperature of 20°C to 33°C, a dissolved oxygen content of 10% to 30%, a pH of 5 to 8, and / or the addition of butyric acid and propionic acid (e.g., 1% to 60% of total organic acids); optionally further comprising genetic modifications of fatty acid synthase, delta 9 desaturase, and / or delta 12 desaturase; and thereby enabling the oleaginous microorganism to produce microbial lipids characterized by a high content of triglyceride POP, POS, and SOS content of 40% to 85% relative to the total triglyceride content.

[0108] In one embodiment, the method is suitable for producing lipids having a content of saturated fatty acids of 20% to 55% relative to the total fatty acid content and / or a triglyceride PPP, PPS, PSS and SSS content of 1% to 20% relative to the total triglyceride content. In one embodiment, the method is suitable for producing lipids that are equivalent to palm stearin-like microbial oils and / or palm-like microbial oils. This is especially the case when the following parameters are selected for step d): Growing the oleaginous microorganism in a medium containing an enzymatically treated substrate, e.g., an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: temperature 22°C-33°C, dissolved oxygen content >10%-40%, pH 5-8, addition of butyric acid and propionic acid (e.g., 1%-50% of total organic acids); optionally further comprising a genetic modification of either fatty acid synthase, delta 9 desaturase, and / or delta 12 desaturase; and thereby enabling the oleaginous microorganism to produce microbial lipids characterized by a content of triglyceride PPP, PPS, PSS, and SSS content of 1%-20% relative to the total triglyceride content.

[0109] In one embodiment, the method comprises obtaining a fatty acid content of 20% to 55% of saturated fatty acids based on the total fatty acid content and / or a fatty acid C content of 1% to 60% based on the total triglyceride content. 10 , C 12 , and / or C. 14 In one embodiment, the method is suitable for producing lipids having a palm kernel oil-like microbial oil content. This is especially true when the following parameters are selected for step d): Growing the oleaginous microorganism in a medium containing an enzymatically treated substrate, such as an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: temperature 25°C-30°C, dissolved oxygen content >30%, pH 6-8; optionally further comprising genetic modifications of any of fatty acid synthase, thioesterase, delta 9 desaturase, and / or delta 12 desaturase; and thereby enabling the oleaginous microorganism to produce microbial lipids characterized by a content of triglyceride PPP, PPS, PSS, and SSS contents of 1%-20% relative to the total triglyceride content.

[0110] In one embodiment, the method is suitable for producing lipids having a polyunsaturated fatty acid content of 10% to 45% relative to the total fatty acid content and / or a fatty acid content of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and / or docosapentaenoic acid (DPA) of 10% to 45% relative to the total fatty acid content, in particular if the following parameters are selected for step d): Growing the oleaginous microorganism in a medium containing an enzymatically treated substrate, such as an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: temperature 15° C.-28° C., dissolved oxygen content >40%, pH 6-8; optionally further comprising genetic modifications of any of fatty acid synthase, thioesterase, delta 4, 5, 6, 8, 9, 12, and / or 15 desaturase, and / or delta 5, 6, and / or 9 elongase; and thereby enabling the oleaginous microorganism to produce microbial lipids characterized by a fatty acid content of 10%-45% alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and / or docosapentaenoic acid (DPA) relative to the total fatty acid content.

[0111] In one embodiment, the method is particularly suitable for producing lipids with a content of unsaturated fatty acids of >50% based on the total fatty acid content and / or a triglyceride POO, OOS and OOO content of 50% based on the total triglyceride content.In one embodiment, the method is particularly suitable for producing lipids that are equivalent to palm olein-like microbial oils, sunflower-like microbial oils, rapeseed-like microbial oils.This is especially true when the following parameters are selected for step d): Growing the oleaginous microorganism in a medium comprising an enzymatically treated substrate, e.g., an enzymatically treated first substrate and / or an enzymatically treated second substrate, under the following conditions: a temperature of 10°C to 30°C, a dissolved oxygen content of 10% to 30%, and / or the addition of crotonic acid (e.g., 0% to 10% of total organic acids), thereby enabling the oleaginous microorganism to produce microbial lipids characterized by a "pour point" of <10°C, e.g., in the range of 9°C to 5°C, such as, for example, about 5°C, and / or an unsaturated fatty acid content of >60% relative to the total fatty acid content.

[0112] As used herein, the terms "of the invention," "in accordance with the invention," "according to the invention," and the like are meant to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0113] The term "comprising" as used herein should be interpreted as embracing both "including" and "consisting of," with both meanings specifically intended and both meanings therefore being separately disclosed embodiments according to the present invention. When used herein, "and / or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be taken as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, exactly as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature. The terms typically indicate deviations from the indicated numerical value at ±20%, ±15%, ±10%, and for example ±5%. As would be recognized by a person skilled in the art, the specific such deviations for the numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than those for man-made or engineered technical effects. When an indefinite or definite article such as "a", "an" or "the" is used when referring to a singular noun, this includes the plural of that noun unless something else is specifically stated. [Brief description of the drawings]

[0114] The invention will now be further described with reference to the following figures. All methods referred to in the following figure legends were performed as detailed in the Examples. [Figure 1] FIG. 1 shows a schematic flow chart of the process of the present invention, which is a sustainable method for producing edible microbial lipids. [Diagram 2] FIG. 2 shows a flow diagram of the method of the invention. A first substrate, in particular food residue(s), is used as a substrate for a first microorganism. The first microorganism produces an enzyme mixture for digesting said first substrate (said enzyme mixture produces an enzymatically treated first substrate), and optionally further produces protein biomass and / or aroma compounds. Optionally, the first microorganism can be co-cultured with microalgae that produces enzyme(s), protein, and / or aroma compounds. The enzymatically treated first substrate can be used as or in a growth medium for culturing a second microorganism that produces a microbial oil. Alternatively or additionally, the enzyme mixture is used to enzymatically treat a second substrate, preferably of the same type as the first substrate, thereby obtaining an enzymatically treated second substrate that can be used as or in a growth medium for culturing a second microorganism that produces a microbial oil. The enzymatically treated first substrate and the enzymatically treated second substrate can also be combined to provide a growth medium for said second microorganism. [Diagram 3] FIG. 3 shows a flow diagram of one embodiment of the method of the present invention. [Figure 4] FIG. 4 shows a flow diagram of one embodiment of the method of the present invention. [Diagram 5] FIG. 5 shows a flow diagram of one embodiment of the method of the present invention. EXAMPLES

[0115] In the following, reference is made to examples which are given to illustrate, but not to limit, the invention.

[0116] Example 1: Selection of raw material and composition analysis - bread residue An exemplary food residue(s) that can be used as the first substrate and / or substrate used in the method for producing microbial lipids of the present invention is bakery product food residue(s), such as bread residue. Today, bread residue is generally used in the industry to obtain bread meal by grinding for bread production or as animal feed. Alternatively, the following example describes an exemplary process for the selection of bread residues provided by bakeries as feedstock for microbial fermentation and the compositional analysis of this raw material. Fresh bread residues are collected and processed to avoid biological deterioration and to keep food grade, which includes frequent residue collection (e.g., on the same day after the residues are determined to be unsalable), drying the finely sliced ​​bread in a baking oven (e.g., starting temperature 160°C without further heating overnight) and subsequently grinding to 4 mm particles in an old bread mill. These bread crumbs are stored at room temperature for up to 2 weeks or frozen at -20°C for up to 3 months. Because bread residues are rich in nutrients, such as proteins, carbohydrates, fats, minerals, and vitamins (see Tables 1 and 2), bread residues are highly suitable as a substrate for use in the method of producing microbial lipids of the present invention.

[0117] [Table 1]

[0118] [Table 2]

[0119] Example 2: Production of enzyme solution through cultivation of fungi using bread residue as induction system - Aspergillus In this experiment, we investigated the production of an exemplary enzyme mixture, e.g., enzyme solution, via submerged fermentation using Aspergillus niger van Tiegheim ATCC 10535 and bread residue as a sole medium component. Thus, about 100 g of bread crumb obtained as described above in Example 1 was mixed with ddH2O in duplicate in a 5 L baffled Erlenmeyer flask to a total volume of 2 L (5% w / v). After subsequent sterilization by autoclaving at 121°C for 20 min, the inoculum (spore suspension of Aspergillus niger van Tiegheim ATCC 10535) was added. The culture was incubated at 100 rpm and 28°C in a rotary shaker. Daily visual inspection indicated good growth and enzymatic hydrolysis of the substrate, as the medium was blackened by fungal cells and liquefaction of the bread crumb was observed. The fermentation was continued for 3 weeks to stress the fungus and induce maximum hydrolytic enzyme secretion. Subsequently, filtration of the liquid culture with filter paper was applied to remove vegetative cells. Spores were removed by centrifugation at up to 75.600×g (Beckman Coulter Avanti JXN-26) followed by bottle-top filtration or cross-flow filtration with 0.2 μm pore size. 10 kDa cross-flow filtration and buffer exchange (a membrane made of regenerated cellulose was used with the following parameters: inlet pressure (P1) 4 bar, residence pressure (P2) 1 bar, and the filtrate was open to atmospheric pressure) were then applied to concentrate, enrich and purify the enzyme mixture. The final enzyme solution was about 350 ml. The enzyme mixture was first subjected to a qualitative enzyme activity pre-screening using a colored carbohydrate polymer embedded in an agar matrix. The extent of discoloration around the applied enzyme solution and the diameter of the resulting decolorization halo are measures of the current enzyme amount and relative activity that correlate to the conversion of the substrate in this pre-screening test. Table 3 gives an overview of substrates with associated enzymatic activity.

[0120] [Table 3]

[0121] Enzyme activity was assessed from no activity ("-"), activity ("+"), good activity ("++"), high activity ("+++"), very high activity ("+++") to almost complete conversion of substrate ("High"). The results of the enzyme screening are shown in Table 4.

[0122] [Table 4]

[0123] Enzyme screening showed high activity for the predicted enzymes using bread residue as an induction system. After this pre-screening, quantitative screening of the target for enzymes with high activity can be carried out subsequently.

[0124] The following method can be exemplified as a method for measuring the enzyme activity of α-amylase. α-amylase acts on the endo-1,4-α-glucan bond of soluble starch to produce a mixture of dextrins and reducing sugars (especially maltose). Dissolved 3,5-dinitrosalicylic acid (DNS) (by adding 2M NaOH) acts on the reducing sugars produced in the presence of potassium sodium tartrate tetrahydrate at 95°C to produce 3,5 aminonitrosalicylic acid. The absorbance is measured at 540 nm, and the amount of 3-amino-5-nitrosalicylic acid is obtained by using a calibration curve to calculate the enzyme activity. The amount of enzyme that releases 1 μmol of reducing sugars from soluble starch in 1 minute at 25°C and pH 6.9 is defined as 1 U (1 unit).

[0125] The following method can be exemplified as a method for measuring the enzyme activity of amyloglucosidase. Amyloglucosidase acts on the 1,4-α-glucan bond of maltose to produce D-glucose. Glucose oxidase acts on the produced glucose as a substrate in the presence of oxygen to produce hydrogen peroxide. Peroxidase acts on the produced hydrogen peroxide in the presence of aminoantipyrine and phenol to produce quinoneimine dye. The absorbance is measured at 500 nm, and the amount of quinoneimine dye is obtained by using a calibration curve to calculate the enzyme activity. The amount of enzyme that oxidizes 1 μmol of maltose in 1 minute at 25° C. and pH 4.3 is defined as 1 U (1 unit).

[0126] The following method can be exemplified as a method for measuring the enzyme activity of hemicellulase. Hemicellulase is allowed to act on xylose to produce reducing sugars. Dissolved 3,5-dinitrosalicylic acid (DNS) (by adding 2M NaOH) is allowed to act on the produced reducing sugars at 95°C in the presence of potassium sodium tartrate tetrahydrate to produce 3,5 aminonitrosalicylic acid. The absorbance is measured at 540 nm, and the amount of 3-amino-5-nitrosalicylic acid is obtained by using a calibration curve to calculate the enzyme activity. The amount of enzyme that releases 1 μmol of reducing sugars from xylose in 1 minute at 40°C and pH 4.5 is defined as 1 U (1 unit).

[0127] The following method can be exemplified as a method for measuring the enzyme activity of proteases. Proteases are allowed to act on casein to produce amino acids. The reaction is stopped by adding trichloroacetic acid. The test solution is subsequently filtered by using a 0.45 μm polyethersulfone syringe filter, and Folin & Ciolcaltea's or Folin's phenol reagent is allowed to act on the filtered amino acids to produce a measurable color change that will be directly related to the activity of the proteases. The absorbance is measured at 660 nm, and the amount of amino acids is obtained by using a tyrosine calibration curve, and the enzyme activity is calculated. The amount of enzyme that produces amino acids equivalent to 10 μg of tyrosine from casein in 1 minute at 37° C. and pH 8.0 is defined as 1 U (1 unit).

[0128] The following method can be exemplified as a method for measuring the enzyme activity of proteases. Proteases are allowed to act on casein to produce amino acids. The reaction is stopped by adding trichloroacetic acid. The test solution is subsequently filtered by using a 0.45 μm polyethersulfone syringe filter, and Folin & Ciolcaltea's or Folin's phenol reagent is allowed to act on the filtered amino acids to produce a measurable color change that will be directly related to the activity of the proteases. The absorbance is measured at 660 nm, and the amount of amino acids is obtained by using a tyrosine calibration curve, and the enzyme activity is calculated. The amount of enzyme that produces amino acids equivalent to 10 μg of tyrosine from casein in 1 minute at 37° C. and pH 8.0 is defined as 1 U (1 unit). As shown in Table 4, an exemplary enzyme mixture, i.e., an exemplary composition of the present invention, includes cellulose, lichenase, xylanase, arabinanase, amylase, such as α-amylase, limit dextrinase, pullulanase, protease, galactanase, and mannanase.

[0129] Example 3: Production of enzyme solution through cultivation of fungus - Ceratocystis using bread residue as induction system In this experiment, we investigated the production of enzyme solution via submerged fermentation using Ceratocystis paradoxa CBS 374.83 and bread residue as the sole medium component. Thus, the cultivation and downstream processing were carried out as described in Example 2. The final enzyme solution was about 30 ml. The results of the enzyme prescreening carried out as described in Example 2 are shown in Table 5.

[0130] [Table 5]

[0131] Enzyme screening showed high activity for the expected enzymes using bread residue as an induction system. An example of a method for quantitative screening of enzyme targets with high activity is given in Example 2. As shown in Table 5, an exemplary enzyme mixture, i.e., an exemplary composition of the present invention, includes cellulase, xylanase, arabinanase, amylase such as α-amylase, limit dextrinase, pullulanase, galactanase, mannanase, rhamnogalacturonan hydrolase, and rhamnogalacturonan lyase.

[0132] Example 4: Hydrolysis of food residues - bread The sugars, proteins and other nutrients contained in the bread residue described in Example 1 are suitable for fermentation. Microorganisms convert it into profitable products such as microbial oil, a product useful for the food industry. Pretreatment of the substrate as bread residue can be applied before fermentation to obtain nutrients that are easily accessible to microorganisms. One of the typical pretreatment methods is enzymatic hydrolysis. Bread residue contains high concentrations of starch (more than 70% on dry matter) and protein (up to 14% on dry matter), and treatment with α-amylase, glucoamylase and protease easily leads to the release of compounds available for microbial growth. The hydrolysis of starch contained as the main component of the dry weight of bread includes two stages. In the first stage, the starch is liquefied using α-amylase enzyme, giving a solution containing the product of destrin and a small amount of glucose. The liquefied starch is subjected to a saccharification stage by using glucoamylase enzyme to obtain maximum terminal glucose conversion. Both steps were found to be interrelated, with an optimal degree of liquefaction required for proper saccharification in the subsequent steps. The following method is an exemplary enzymatic pretreatment of the substrate, such as the hydrolysis of starch, for example starch contained in bread residue, using one or more enzymes. A homogenized bread residue in water suspension is prepared for the experiment. The size of the bread particles is less than 4 mm. For liquefaction, α-amylase is added (for example SUG-001, Creative Enzymes, Shirley, NY; USA). The liquefaction proceeds in a thermostat or water bath with a specific pH (for example pH 6) and at a specific temperature (for example 80° C.). The pH of the suspension is adjusted, for example, by a 1% (wt / vol) solution of H2SO4 and a 1% (wt / vol) solution of NaOH. The liquefaction is terminated by freezing the suspension. The liquefaction time is about 180 minutes. Saccharification is carried out in the liquefied suspension by addition of glucoamylase (e.g. SUG-002; DIS-1013, Creative Enzymes, Shirley, NY; USA).Saccharification proceeds in an enhanced shaker with a specific pH (e.g. pH 4,2) and at a specific temperature (e.g. 60°C) and with shaking (e.g. 130 rpm). Enzyme activity is terminated by heating the suspension at 80°C for 5 minutes. The duration of saccharification is at least 90 minutes. Hemicellulase (e.g. DIS-1023 Creative Enzymes, Shirley, NY; USA) and protease (e.g. Neutrase, Novozymes, Denmark) are added in the saccharification step to produce the highest content of converted sugars and amino acids. Instead of using commercial enzymes, an enzyme mixture prepared as described in Examples 2 and 3 or any combination of the aforementioned is suitable. For example, hydrolysis can be carried out in only one step at a specific pH (e.g. pH 5,5), a specific temperature (e.g. 55°C) and a long incubation time (e.g. 24 hours) with shaking (e.g. 130 rpm). Adaptation of the procedure to a bioreactor is also possible.

[0133] After incubation, the mixture is centrifuged (e.g., 14,000 rpm for 20 minutes) to separate the unhydrolyzed solid residue and collect the supernatant containing nutrients. At this point, the supernatant can be characterized as bread residue hydrolysate. This hydrolysate obtained from the protocol could be freeze-dried to evaluate the suitability of the nutrients to be rehydrated. For fermentation, the hydrolysate could be freshly prepared or could be rehydrated and applied as is or with the optional addition of supplements, either alone or in a mixture.

[0134] Example 5: Production of Microbial Food Ingredients - Microbial Lipids Oleaginous microorganisms such as oleaginous yeasts represent interesting microbial lipid factories. Their rapid growth, together with their ability to utilize a wide variety of feedstocks and their easy cultivation in large fermenters, make them highly suitable candidates for biorefinery processes. Oleaginous microorganisms such as oleaginous yeasts can grow and accumulate high levels of lipids. The ATCC 20509 Cutaneotrichosporon oleaginosus (C. oleaginosus) strain has been studied for its ability to accumulate up to 70% TAG of DCW, advantageously with a fatty acid composition very similar to that of cocoa butter or palm oil. C. oleaginosus has the ability to use highly diverse carbon sources, such as carbon sources from food residue(s). The inventors have surprisingly found that food residue(s), such as bread residue, can be used as a substrate for obtaining nutrients for the growth medium for oleaginous microorganisms. For example, the present inventors have successfully converted food residue(s) into nutrients using filamentous fungi and bacteria, and these nutrients can be used as growth media for oleaginous microorganisms. In particular, enzymatically treated food residue(s) can be used as a carbon source for producing nutrients, such as microbial lipids, using oleaginous microorganisms, such as C. oleaginosus. The hydrolysate obtained as described in Example 4 is considered as a potential alternative carbon source for producing microbial lipids with C. oleaginosus fermentation.

[0135] Conventional lipid production using oleaginous organisms is a two-stage process, where the first stage provides biomass formation under non-limiting conditions (exponential growth phase) and the second lipid induction step (nutrient-limited phase) gives high intracellular lipid accumulation at stagnant cell numbers. It is also possible to carry out co-fermentation of C. oleaginosus with organic acids and carbon sources, leading to high biomass and lipid yields (84.9% of DCW) simultaneously without the need for nutrient limitation, allowing for the simultaneous accumulation of, for example, bread residue hydrolysates and acetic acid. Mild downstream processing is also favorable for producing edible microbial lipids. Thus, for example, carrying out a purely enzymatic treatment of C. oleaginosus without any solvent-based extraction or chemical-based demulsification is suitable to make the produced microbial lipids suitable for subsequent recovery. Recovery of the produced microbial lipids is carried out by density-based separation methods, for example using semi-continuous centrifuges. Purifying the microbial lipids is the last step of processing to obtain edible oils, for example physical purification. Figure 1 shows a method that can be exemplified as a sustainable method for producing edible microbial lipids.

[0136] The following method can be exemplified as a method for analyzing the fatty acid profile of the final product after methylation using gas chromatography with a flame ionization detector (GC-FID). Methylation is performed simply by incubating 1 mg of oil with 1 ml of NaOCH3 (20 min at 80°C). Then, 1 ml of HCl (37% in methanol) is added and the mixture is incubated again at 80°C for 20 min. The resulting fatty acid methyl esters (FAMEs) are extracted by hexane in the injected GC-FID. Triglycerol C19:0 is used as an internal standard. An exemplary microbial lipid composition of C. oleaginosus is shown in Table 6.

[0137] [Table 6]

[0138] Example 6: Production of microbial food ingredients - microbial proteins The inventors have demonstrated that food waste(s), such as freeze-dried bread, can be used as a substrate for producing microbial protein biomass, which is produced as a by-product when culturing a first microorganism.

[0139] Example 7: Production of Microbial Food Ingredients - Aroma Compounds The inventors have surprisingly found that microorganisms, such as Ceratocystis species, produce aroma compounds when cultivated on food residue(s). For example, Ceratocystis species, such as Ceratocystis fimbriata and Ceratocystis moniliformis, produce a wide range of complex aromas, such as peach, banana, pear, rose or citrus, depending on the strain and the environmental conditions. The advantage of using these fungi consists of their relatively rapid growth and the various complex aroma mixtures synthesized. The inventors have found that Ceratocystis paradoxa CBS 374.83 produces aroma compounds, which are detected by the strong fruity aroma of the supernatant during the fermentation experiments described in Example 2. The supernatant contained at least one aroma compound. That is, the method of the present invention surprisingly not only provides microbial lipids, but also aroma compounds. To obtain the aroma compounds, gentle separation techniques were used to provide an extract with sensory properties as close as possible to the finished product. For example, organophilic pervaporation (O-PV), a membrane-based technique, as well as solid-phase microextraction (SPME), a non-exhaustive extraction technique in which a sorbent-coated fiber is exposed to the sample, have been used for gentle recovery of natural aroma compounds. The inventors have found that it is advantageous to maintain the functionality of aroma components during processing by using gentle separation techniques.

[0140] We further carried out aroma fractionation to recover specific aromas or groups of aroma compounds from a mixture of aroma compounds. Furthermore, we analyzed the extracted aroma compounds by capillary gas chromatography (GC) alone by comparing the retention index of the sample with that of the corresponding authentic reference compounds, or via GC-olfactometry (GC-O). GC-O allows the correlation between volatile organic compounds and their perception, using the human nose as a detector. Thus, the outlet of the GC capillary column is mounted in a column flow splitter. The column flow splitter is equipped with two outlets that direct the gas flow into a destructive detector, such as a flame ionization detector (FID) or a mass spectrometer (MS), and into a non-destructive olfactory detector (ODP). With this device, it is possible to directly assign odor impressions to MS spectra or FID peaks.

[0141] Example 8: Use of microbial ingredients in the food industry - microbial lipids in bakery products A wide range of applications for microbial ingredients in the food industry is possible. The inventors have exemplarily used the microbial lipids obtained by the method of the invention in different bakery products.

[0142] a) Kaiser Roll component: - Flour type 550 or 650 100% - Buttermilk 2~3% - Baker's yeast 3~4% - Microbial lipids 10-15% - Baking agent 2~3% - Salt 1.8~2.2% - Water 55~63%

[0143] Kneading: - Starter dough Mix 20% flour, 2% buttermilk, 10% water and 1% baker's yeast in a dough mixer at the lowest level for 3-4 minutes, then mix rapidly for 2-3 minutes to obtain a smooth dough. Cover the starter dough with cling film and leave it in the refrigerator (4°C) for 6-24 hours. - Main fabric 80% flour, 3% baking agent, 2% salt, 15% microbial lipid, 3% baker's yeast and starter dough are kneaded thoroughly in a spiral kneader for 5 to 7 minutes slowly, then knead at high speed for 5 to 8 minutes until the dough is shapeable. The dough temperature ranges between 23°C and 26°C. After removing the dough from the spiral mixer, the dough is allowed to rest for up to 10 minutes.

[0144] processing: - Roll 1800g to 2100g portions into balls and continue covering with cling film. - Leave it for 15 to 20 minutes so that the dough can rise easily. - Shaping of the part and subsequent rounding. - Place it on a food carrier and ferment it in a fermentation room (temperature 30℃~35℃, Ferment for 50 to 75 minutes (with a relative humidity of 75% to 85%).

[0145] Baking: Oven temperature: 230℃~240℃ Steam input: Standard Damper:Closed Baking time: Approximately 20 minutes

[0146] Kaiser rolls were successfully made using the microbial lipids produced using the methods of the present invention. The rolls made using the microbial lipids had the same aroma, taste, and texture as conventional Kaiser rolls.

[0147] b) Pretzel component: - Wheat flour type 550 100% - Bakery malt 2~3% - Salt 1.8~2.2% - Microbial lipids 10-15% - Baker's yeast 1.5~2% - Water 45~48%

[0148] Kneading: - Starter dough 20% flour, 0.2% baker's yeast and 12% water (10°C) are mixed using a dough mixer at the lowest level for 3-4 minutes, followed by rapid mixing at the highest level for 3 minutes. The starter dough is then covered with cling film and left at room temperature for 1 hour. It is then left to rise for 12 to 14 hours. - Main fabric 80% flour, 2% bakery malt, 2% salt, 1.5% baker's yeast, 10% microbial lipids, 10% water (10°C) and starter dough are kneaded slowly in a spiral kneader at low level for 4 minutes and then at high speed for 5 minutes at the highest level. The temperature of the dough is approximately 24°C. After removing the dough from the spiral kneader, the covered dough is allowed to rest for approximately 20 minutes.

[0149] processing: - Roll 1800g to 2100g portions into balls and continue covering with cling film. - Leave it on for 15 to 20 minutes. - Part molding. - Roll the dough into strands approximately 60 cm long on a lightly floured work surface. The largest diameter of the strand is in the center, with the strands having rounded tips and becoming thinner towards the outside. - Twist every straight rope of dough and form it into a pretzel shape. - Place the pieces of dough on a baking tray covered with baking linen. - Then cover the portion with cling film and leave it at room temperature for 1 to 2 hours. The dough was allowed to rise to double its volume. - Provide pretzel lye and wear safety glasses and gloves. - Dip the pretzels in the lye for approximately 5 seconds, remove them and place them on a baking tray covered with baking paper. - Cut the pretzels into bulbous pieces and sprinkle them with some salt.

[0150] Baking: Oven temperature: Preheat at 230℃ using double heat. Baking time: 12 to 14 minutes until golden brown. Continue cooling the pretzels on a wire rack.

[0151] Pretzels were successfully made using the microbial lipids produced using the methods of the present invention, and the pretzels made using the microbial lipids had the same aroma, taste, and texture as pretzels made using conventional methods.

[0152] c) Bismarck component: - Flour type 550 or 405 100% - Milk 20~30% - Baker's yeast 5~8% - Eggs 20% - Egg yolk 8% - Microbial lipids 10-15% - Sugar 10% - Salt 1.8~2.2% - 60g water - Aroma: Vanilla and Lemon Peel

[0153] Kneading: - Starter dough Mix 30% flour, 20% milk, 5% baker's yeast and 60g water (10°C) and use the dough mixer at the lowest level for 3 to 4 minutes to obtain a smooth dough. Then cover the starter dough with cling film and leave it at room temperature for 30 minutes. - Main fabric 70% flour, 20% eggs, 8% egg yolks, 10% microbial lipids, 10% sugar, 7% milk, 2% salt, aromatics and starter dough is kneaded thoroughly in a spiral kneader for 5 to 7 minutes slowly, then knead at high speed for 10 to 12 minutes until the dough is moldable. After removing the dough from the spiral mixer, the dough is allowed to rest for between 20 and 30 minutes.

[0154] processing: - Roll into 1200g to 1500g portions and continue covering with cling film. - Leave it for 15 to 20 minutes so that the dough can rise easily. - Shaping of the part and subsequent rounding. - Place it on a food carrier and ferment it in a fermentation room (temperature 30℃~35℃, Ferment for 50 to 75 minutes (with a relative humidity of 60% to 70%).

[0155] Curing: The fermentation of the dough pieces is stopped so that the surface becomes completely dry and hard.

[0156] boiling: - Fill the microbial lipids into a chip pan and heat it to 150°C-175°C. - Check the temperature. -Bake the pieces of dough, round side up. - Rotate them later and press them down on the grill towards the end of the baking time. - Baking time: 3min / 2min / 30sec and 6 to 8min with optimal intervals of 30sec.

[0157] filling: Place the apricot jam-like filling in the center of the Bismarck.

[0158] topping: Sprinkle the still hot Bismarck with, for example, sugar or powdered sugar. Bismarcks (German doughnuts without any hole in the center) were successfully made using the microbial lipids produced using the methods of the present invention. The Bismarcks made using the microbial lipids had the same aroma, taste, and texture as the traditionally made Bismarcks.

[0159] d) Fruit loaf component: - Wheat flour type 550 30% - Milk 20~30% - Baker's yeast 1.5% - Water 8.5% - Microbial lipids 13% - Sugar 3% - Marzipan 2% - Salt 1.8~2.2% - Sour cream 0.3% - Spices 0.3% - Milk powder 1% - Rum aroma 0.3% - Vanilla Fragrance 0.3% - Almond bitter (bitterly) 0.3% - Lemon peel 0.3% - Sultana 19% - Almond Baton Cut 0.4% - Candied orange and lemon peel 0.4% - Imported rum 4.5%

[0160] Kneading: - Starter dough 10% flour, 1.5% baker's yeast, 7% water and 0.3% salt are mixed with a dough mixer at the lowest level for 2 minutes, followed by rapid mixing for 3 to 4 minutes to obtain a cold (dough temperature is 24°C) semi-solid starter dough. The starter dough is then covered with cling film and left at room temperature for 30 minutes. After removing the dough from the spiral mixer, it is left in the refrigerator (5°C) for 10 to 15 hours. - Main fabric All other ingredients of the main dough except the flour are mixed to obtain a smooth dough. Then add the starter dough and flour, which are mixed slowly for 5 minutes, then kneaded rapidly for 3 minutes. After a brief dough relaxation, fill with the fruit loaf mixture.

[0161] processing: - Roll up the weights - Relax the dough for 15 minutes and continue stretching. - Cover it and let it ferment at room temperature. - Shaping of the part and subsequent rounding. - Place it on a food carrier and ferment it in a fermentation room (temperature 30℃~35℃, Ferment for 50 to 75 minutes (with a relative humidity of 60% to 70%).

[0162] Baking: Oven temperature: Start at 210°C, reduce to 200°C Steam Input: None Damper: Opened after 5 minutes baking time. Baking time: approximately 50 minutes for a 780g piece of dough Then coat it with hot liquid butter, followed by vanilla sugar. After cooling, sprinkle the fruit loaf with "sweet snow."

[0163] Fruit loaves were successfully produced using the microbial lipids produced using the methods of the present invention, and the fruit loaves produced using the microbial lipids had the same aroma, taste, and texture as the conventionally produced fruit loaves.

[0164] Example 9: Use of microbial ingredients in the food industry - chocolate products The microbial lipids produced using the methods of the present invention can be used to produce a number of food products. For example, the following products can be produced: JPEG2024540960000008.jpg157158

[0165] JPEG2024540960000009.jpg135158

[0166] Example 10: Varying triglyceride content while modeling process parameters. Varying triglyceride content while modeling process parameters. TAG composition (%) of yeast oil JPEG2024540960000010.jpg131158

[0167] References [1] Lieken. (2021, April 12). Lieken Brot- und Backwaren GmbH. Retrieved from www.lieken-urkorn.de: https: / / www.lieken-urkorn.de / produkte / produkt / bauernmild-500g [2] USDA1. (2021, April 12). USDA, Agricultural Research Service, Fooddata central. Retrieved from https: / / fdc.nal.usda.gov / fdc-app.html# / food-details / 172686 / nutrients [3] USDA2. (2021, April 12). USDA, Agricultural Research Service, Fooddata central. Retrieved from https: / / fdc.nal.usda.gov / fdc-app.html# / food-details / 172684 / nutrients

[0168] The features of the invention disclosed in the specification, the claims and / or in the accompanying drawings may, both individually and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. 1. A method for producing microbial lipids, optionally a method for producing microbial lipids and protein biomass and / or aroma compounds, said method comprising the steps of: a) providing a first substrate, wherein said first substrate is food residue(s), preferably food-grade food residue(s); b) culturing a first microorganism selected from filamentous fungi and bacteria with said first substrate, thereby allowing said first microorganism to produce at least one enzyme and an enzymatically treated first substrate, and optionally further to produce protein biomass and / or aroma compounds; optionally co-cultivating one or more microalgae with said first microorganism, thereby allowing said one or more microalgae to produce protein biomass, microbial lipids, and / or aroma compounds; c) optionally obtaining said at least one enzyme and pre-treating a second substrate with said at least one enzyme, thereby providing an enzymatically treated second substrate; wherein said second substrate is food residue(s), preferably food-grade food residue(s); and wherein preferably said first substrate and said second substrate are of the same type; d) culturing a second microorganism, wherein said second microorganism is an oleaginous microorganism, in a medium containing said enzymatically treated first substrate and / or, if step c) is present, said enzymatically treated second substrate, thereby allowing said oleaginous microorganism to produce microbial lipid; e) optionally carrying out a purely enzymatic treatment of the cultivated second microorganism without any solvent-based extraction or chemical-based demulsification to make the microbial lipid produced in step d) suitable for subsequent recovery; and f) recovering the microbial lipids produced in step d), preferably by density-based separation methods. A method comprising:

2. The food residue(s) may, at each occurrence, be bakery food residue(s), such as bread, rolls, biscuits, muffins, cookies, or cakes; fruit food residue(s), such as fruit pulp, peel, or fruit juice; plant food residue(s), such as vegetable peel, vegetable pulp, or vegetable juice; milling food residue(s), such as bran or bran meal; fish food residue(s), such as fish processing residues; seafood residue(s), such as seafood processing residues; brewer's grains; cereal food residues.

2. The method of claim 1, wherein the food residue(s) are independently selected from: residue(s), such as rice, wheat, millet, or corn; restaurant food residue(s), such as restaurant leftovers; animal food residue(s), such as milk or cheese; supermarket food residue(s), such as expired food; or any combination thereof; preferably comprising or consisting of bakery food residue(s), more preferably bread food residue(s).

3. The filamentous fungus may be selected from Ceratocystis sp., such as Ceratocystis fimbriata, Ceratocystis moniliformis, and Ceratocystis paradoxa, preferably Ceratocystis paradoxa; Trichoderma sp., such as Trichoderma reesei and Trichoderma harzianum; Aspergillus sp. Aspergillus sp., for example Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger; Neurospora sp., for example Neurospora intermedia; Monascus sp., for example Monascus purpureus; Rhizopus sp., for example Rhizopus oryzae; Fusarium sp., for example Fusarium venenatum; Thermomyces sp.; Penicillium sp.; Aureobasillium sp. Ischnoderma sp., for example Ischnoderma benzoinum; Polyporus sp., for example Polyporus durus; Pycnoporus sp., for example Pycnoporus cinnabarinus; Phanerochaete sp., for example Phanerochaete chrysosporium; and Xylaria sp.; preferably selected from Ceratocystis sp., Trichoderma sp., Aspergillus sp., and Fusarium sp.; The bacterium may be selected from the group consisting of Clostridium sp., such as Clostridium stercorarium, Clostridium beijerinckii, and Clostridium acetobutylicum; Halobacillus sp., such as Halobacillus trueperi and Halobacillus karajensis; Halomonas sp., such as Halomonas meridiana and Halomonas elongata; Rhodothermus sp., such as Rhodothermus marinus; Streptomyces sp. Streptomyces sp., such as Streptomyces griseus, Streptomyces olivochromogenes, Streptomyces griseorubens, and Streptomyces matensis; and Bacillus sp., such as Bacillus nato, Bacillus subtilis, Bacillus licheniformis, and Bacillus stearothermophilus; and / or The microalgae may be selected from the group consisting of Chlorella sp., for example Chlorella vulgaris, and Chlorella protothecoides; Scenedesmus sp., for example Scenedesmus obliquus; Dunaliella sp., for example Dunaliella salina; Haematococcus sp., for example Haematococcus pluvialis; Crypthecodinium sp., for example Crypthecodinium cohnii; Schizochytrium sp. , for example Schizochytrium limacinum; and Tetraselmis sp., for example Tetraselmis chui; preferably selected from Chlorella sp., and Scenedesmus sp., 3. The method according to claim 1 or 2.

4. 3. The method of claim 1 or 2, wherein the second microorganism is an oleaginous microorganism selected from oleaginous yeast, oleaginous fungi, oleaginous bacteria, and oleaginous microalgae; Here, preferably, The oleaginous yeast may be selected from the group consisting of Cutaneotrichosporon sp., such as Cutaneotrichosporon oleaginosus; Trichosporon sp., such as Trichosporon oleaginosus, Trichosporon capitatu, and Trichosporon asahii; Rhodospirillum sp.; Rhodosporidium sp., such as Rhodosporidium toruloides; Rhodosporon sp.; Candida sp. Cryptococcus sp., such as Cryptococcus curvatus; Lipomyces sp., such as Lipomyces starkeyi; Yarrowia sp., such as Yarrowia lipolytica; Rhodotorula sp., such as Rhodotorula graminis, Rhodotorula gracilis, and Rhodotorula glutinis; and Apiotrichum sp. , for example Apiotrichum curvarum; preferably Cutaneotrichosporon sp., more preferably Cutaneotrichosporon oleaginosus; The oleaginous fungi include Cunninghamella sp., such as Cunninghamella echinulate; Aspergillus sp., such as Aspergillus oryzae, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus niger; Neurospora sp., such as Neurospora intermedia; Monascus sp., such as Monascus purpureus; Rhizopus sp., such as Rhizopus oryzae; Fusarium sp. selected from Mucor sp., for example Fusarium venenatum; Mucor sp., for example Mucor moelleri; Mortierella sp., for example Mortierella isabellina and Mortierella alpine, preferably Mortierella alpine; and Humicola sp.; The oleaginous bacterium is selected from Rhodococcus sp.; Acinetobacter sp.; and Bacillus sp.; and The oleaginous microalgae is selected from Chlorella sp., Pseudochlorococcum sp., Nannochloris sp., Nannochloropsis sp., Isochrysis sp., Tribonema sp., Dunaliella sp., Ankistrodesmus sp., Botryococcus sp., Pavlova sp., Scenedesmus sp., Skeletonema sp., and Nitzschia sp.; More preferably, the second microorganism is an oleaginous yeast selected from Cutaneotrichosporon sp., such as Cutaneotrichosporon oleaginosus.

3. The method according to claim 1 or 2.

5. The second microorganism is selected from the group consisting of Cutaneotrichosporon oleaginosus, Trichosporon oleaginosus, and Trichosporon capitatu, Trichosporon asahii, Lipomyces starkeyi, Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula graminis, Rhodotorula gracilis, Rhodotorula glutinis, Apiotrichum curvarum, Cryptococcus curvatus, Candida viswanathii, and Candida freyschussii; preferably Cutaneotrichosporon 3. The method of claim 1 or 2, wherein the yeast is an oleaginous yeast selected from the group consisting of Saccharomyces cerevisiae, ...

6. 3. The method according to claim 1 or 2, wherein step b) of the method comprises culturing a first microorganism selected from filamentous fungi and bacteria with the first substrate, thereby allowing the first microorganism to produce at least one enzyme, an enzymatically treated first substrate, and protein biomass and / or aroma compounds; and optionally further comprising co-culturing one or more microalgae with the first microorganism, thereby allowing the one or more microalgae to produce protein biomass, microbial lipids, and / or aroma compounds.

7. 7. The method of claim 6, wherein the method further comprises recovering the protein biomass and / or the aroma compounds, preferably using any of centrifugation, filtration, organophilic pervaporation, solid phase microextraction, distillation, and combinations thereof.

8. The method of claim 1 or 2, wherein the culture medium further comprises additional carbon sources, nitrogen sources, trace metals, and / or vitamins.

9. The method further comprises: - mechanical pretreatment, preferably by milling, mixing, chopping and / or sieving said substrate(s); - dissolving said substrate(s) in a solvent, preferably in water; - chemical hydrolysis of said substrate(s), preferably using an acid, for example sulfuric acid; - thermal pretreatment of said substrate(s), preferably at a temperature of between 50°C and 200°C for a period of between 10 and 240 minutes, more preferably at a temperature of between 80°C and 170°C for a period of between 30 and 90 minutes; - Fermentation pretreatment, preferably anaerobic fermentation, of said substrate(s); and / or - enzymatic pretreatment of said substrate(s) using one or more enzymes, optionally commercially available enzymes; and / or, if step c) is present, pretreating the second substrate by 3. The method of claim 1 or 2, wherein the one or more enzymes are selected from proteases, such as endopeptidases and exopeptidases, serine endopeptidases, subtilisin A, and pepsin; and hydrolases, preferably glycoside hydrolases, more preferably α-amylase, amyloglucosidase, cellulase, hemicellulase, xylanase, xyloglucase, galactanase, arabinase, mannanase, lipase, glucoamylase, and pectinase.

10. 3. The method of claim 1 or 2, wherein the method comprises step e) carrying out the purely enzymatic treatment of the cultured second microorganism without any solvent-based extraction or chemical-based demulsification, wherein the purely enzymatic treatment of the cultured second microorganism is treatment of the microorganism with a hydrolase, alone or in combination with / followed by a protease.

11. 3. The method according to claim 1 or 2, wherein the method comprises a step c) of obtaining the at least one enzyme and pretreating the second substrate with the at least one enzyme, wherein the pretreating comprises contacting the second substrate with the at least one enzyme obtained from culturing the first microorganism, preferably in the form of a liquid enzyme preparation obtained directly, or in the form of a lyophilized enzyme preparation, optionally reconstituted in solution.

12. the at least one enzyme is selected from the enzymatic activity of cellulase; lichenase; xylanase; arabinanase; amylase, e.g., α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase; 3. The method of claim 1 or 2, optionally containing one or several activities selected from the enzymatic activities of cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; and rhamnogalacturonan lyase.

13. the method is a method for producing microbial lipids and preparing food products therefrom, the method further comprising step g) of preparing a food product comprising the microbial lipids recovered in step f), preferably a bakery product, such as bread, rolls, biscuits, muffins, cookies or cakes; a confectionery product, such as flour confectionery or sugar confectionery; a dairy product, such as ice cream or baby milk; a spread, such as margarine, mayonnaise or soft cheese; an instant food, such as instant noodles, pizza, pizza dough or sauce; a drink; a vegetarian or vegan food, such as meat analogues and dairy analogues; a sweet, such as cocoa-free chocolate; and / or a chocolate product; 3. The method of claim 1 or 2, optionally further comprising step h) of recycling the food residue(s) of the food product by using the food residue(s) of the food product as the first substrate and / or, if step c) is present, as the second substrate.

14. Use of microbial lipids, preferably microbial lipids produced using the method of claim 1 or 2, in the production of food products, preferably bakery products, such as bakery products such as bread, rolls, biscuits, muffins, cookies, or cakes; confectionery products, such as flour confectionery or sugar confectionery; dairy products, such as ice cream or baby milk; spreads, such as margarine, mayonnaise, or soft cheese; instant foods, such as instant noodles, pizza, pizza dough, or sauces; drinks; vegetarian or vegan foods, such as meat analogues and dairy analogues; sweets, such as cocoa-free chocolate; and / or chocolate products.

15. 1. A composition comprising at least five, preferably at least six, and more preferably at least seven enzymes selected from cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase, wherein, optionally, the composition comprises: cellulases; amylases; hemicellulases; limit dextrinase, e.g., malt limit dextrinase; and pectinases; or cellulase; lichenase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; protease; galactanase; and mannanase; or A composition comprising cellulase; xylanase; arabinanase; amylase, such as α-amylase; limit dextrinase; pullulanase; galactanase; mannanase; rhamnogalacturonan hydrolase; and rhamnogalacturonan lyase.

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