CIRCULAR USE OF FOOD RESIDUES BY MICROBIAL FERMENTATION

Abstract

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

Claims

1. A method for producing microbial lipids, said method comprising the steps: a) providing a first substrate, wherein said first substrate is a food residue(s); b) cultivating a first microorganism selected from filamentous fungi and bacteria with said first substrate, and thereby allowing said first microorganism to produce at least one enzyme and an enzymatically treated first substrate; c) optionally, obtaining said at least one enzyme and pretreating a second substrate with said at least one enzyme, and thereby providing an enzymatically treated second substrate; wherein said second substrate is a food residue(s); d) cultivating a second microorganism, wherein said second microorganism is an oleaginous microorganism, with a medium comprising said enzymatically treated first substrate and/or, if step c) is present, with a medium comprising said enzymatically treated second substrate, and thereby allowing said oleaginous microorganism to produce microbial lipids; e) optionally, performing a purely enzymatic treatment of said cultivated second microorganism without any solvent-based extraction or chemicals-based demulsification, to make said microbial lipids produced in step d) amenable for subsequent harvesting; and f) harvesting said microbial lipids produced in step d).

2. The method according to claim 1, wherein said food residue(s), at each occurrence, is independently selected from food residue(s) comprising bakery food residue(s); fruit food residue(s); vegetable food residue(s); milling food residue(s); fish food residue(s); sea food residue(s); brewer's spent grain; cereal food residue(s); restaurant food residue(s); animal product food residue(s); supermarket food residue(s); and any combination thereof.

3. The method according to claim 1, wherein said filamentous fungi are selected from Ceratocystis sp.; Trichoderma sp.; Aspergillus sp.; Neurospora sp.; Fusarium sp.; Thermomyces sp.; Aureobasillium sp.; Ischnoderma sp.; Polyporus sp.; Pycnoporus sp.; Phanerochaete sp.; and Xylaria sp; wherein said bacteria are selected from Clostridium sp.; Halobacillus sp.; Halomonas sp.; Rhodothermus sp.; Streptomyces sp.; and Bacillus sp.; and/or wherein said microalgae are selected from Chlorella sp.; Scenedesmus sp.; Dunaliella sp.; Haematococcus sp.; Crypthecodinium sp.; Schizochytrium sp.; and Tetraselmis sp.

4. The method according to claim 1, wherein said second microorganism is an oleaginous microorganism selected from oleaginous yeasts, oleaginous fungi, oleaginous bacteria, and oleaginous microalgae; wherein said oleaginous yeasts are selected from Cutaneotrichosporon sp.; Trichosporon sp.; Rhodospirillum sp.; Rhodosporidium sp.; Rhodosporon sp.; Candida sp.; Cryptococcus sp.; Lipomyces sp.; Yarrowia sp.; Rhodotorula sp.; and Apiotrichum sp.; said oleaginous fungi are selected from Cunninghamella sp.; Aspergillus sp.; Neurospora sp.; Monascus sp.; Rhizopus sp.; Fusarium sp.; Mucor sp.; Mortierella sp.; said oleaginous bacteria are selected from Rhodococcus sp.; Acinetobacter sp.; and Bacillus sp.; and said oleaginous microalgae are 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.

5. The method according to claim 1, wherein said 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.

6. The method according to claim 1, wherein said step b) of said method comprises cultivating a first microorganism selected from filamentous fungi and bacteria with said first substrate, and thereby allowing said first microorganism to produce at least one enzyme, an enzymatically treated first substrate, and protein biomass and/or aroma compounds.

7. The method according to claim 6, wherein said method further comprises a step of harvesting said protein biomass and/or said aroma compounds.

8. The method according to claim 1, wherein said medium further comprises an additional carbon source, nitrogen source, trace metal, and/or vitamin.

9. The method according to claim 1, wherein said method further comprises a step of pretreating said first substrate and/or, if step c) is present, pretreating said second substrate, by mechanical pretreatment; dissolving said substrate(s) in a dissolvent; chemical hydrolysis of said substrate(s); thermal pretreatment of said substrate(s); fermentative pretreatment of said substrate(s); and/or enzymatic pretreatment of said substrate(s) using one or more enzymes; wherein said one or more enzymes are selected from proteases; and hydrolases.

10. The method according to claim 1, wherein said method comprises said step e) of performing said purely enzymatic treatment of said cultivated second microorganism without any solvent-based extraction or chemicals-based demulsification, wherein said purely enzymatic treatment of said cultivated second microorganism is a treatment of said microorganism with a hydrolase, alone, or in combination with/followed by a protease.

11. The method according to claim 1, wherein said method comprises 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 in the form of a liquid enzyme preparation obtained from culturing said first microorganism, or in the form of a freeze-dried enzyme preparation.

12. The method according to claim 1, wherein said at least one enzyme contains one or more activities selected from enzyme activities of cellulase; lichenase; xylanase; arabinanase; amylase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; hemicellulase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase.

13. The method according to claim 1, wherein said method is a method of producing microbial lipids and preparing foodstuff therefrom, wherein said method further comprises a step g) of preparing a foodstuff comprising the microbial lipid harvested in step f).

14. A method for the production of a foodstuff wherein said method comprises use of a microbial lipid produced using a method according to claim 1.

15. A composition comprising at least five enzymes selected from cellulase; lichenase; xylanase; arabinanase; amylase, e.g., -amylase; limit dextrinase; pullulanase; protease; galactanase; mannanase; rhamnogalacturonan hydrolase; rhamnogalacturonan lyase; xyloglucanase; amyloglucosidase; beta-glucosidase; pectinase; and laminarinase.

16. The method according to claim 2, wherein the food residue is bread food residue.

17. The method according to claim 4, wherein said second microorganism is an oleaginous yeast selected from Cutaneotrichosporon sp.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0150] The present invention is now further described by reference to the following figures.

[0151] All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.

[0152] FIG. 1 shows a schematic flowchart of the method of the invention which is a sustainable method for producing edible microbial lipids.

[0153] FIG. 2 shows a flow diagram of a method of the invention. A first substrate, particularly food residue(s) is used as a substrate for a first microorganism. The first microorganism produces an enzyme mix to digest said first substrate (said enzyme mix producing an enzymatically treated first substrate), and optionally further produces protein biomass and/or aroma compounds. Optionally, the first microorganism can be co-cultivated with microalgae which produce enzyme(s), proteins, and/or aroma compounds. The enzymatically treated first substrate can be used as or in a growth medium for cultivating a second microorganism which produces microbial oil. Alternatively or additionally, the enzyme mix is used to enzymatically treat a second substrate, which is preferably of the same type as the first substrate, thereby obtaining an enzymatically treated second substrate which can be used as or in a growth medium for cultivating a second microorganism which produces 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.

[0154] FIG. 3 shows a flow diagram of an embodiment of the method of the invention.

[0155] FIG. 4 shows a flow diagram of an embodiment of the method of the invention.

[0156] FIG. 5 shows a flow diagram of an embodiment of the method of the invention.

[0157] In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.

EXAMPLES

Example 1: Raw Material Selection and Composition Analysis-Bread Residue

[0158] An exemplary food residue(s) that can be used as first substrate and/or substrate used in a method for producing microbial lipids of the invention is bakery product food residue(s), such as bread residue. Today residual bread is commonly used in industry to get bread meal by grinding for bread production or as animal feed. Alternatively, the following example describes an exemplary process for selection of bakery supplied bread residues as feed stock for microbial fermentation and the composition analysis of this raw material. Fresh bread residues are collected and handled so as to avoid biological degradation and keep the food grade, which includes the steps of frequent residue pickup (e.g., at the same day after the residues determined unsaleable), drying fine sliced bread in a baking oven (e.g. starting temperature 160 C. without further heating overnight) and subsequently grinding to 4 mm particles with a stale 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. Bread residues are highly suitable as a substrate to be used in a method for producing microbial lipids of the invention, because bread residues are rich in nutrients, such as proteins, carbohydrates, fats, minerals, and vitamins (cf. Tables 1 and 2).

[0159] Table 1 shows an exemplary average nutrition value of wheat mixed bread [1]; Ingredients: wheat flour 54%, water, natural sourdough 12% (rye flour, water), rye flour, salt, rapeseed oil, yeast, acidity regulator sodium acetate.

TABLE-US-00002 nutritional value per 100 g (range) Total lipid (fat) 2.0 g (0.1-10.0 g) thereof saturated fatty acids 0.6 g (0.1-3.0 g) Carbohydrates 43.0 g (30.0-80.0 g) thereof sgars 2.5 g (0.1-10.0 g) Protein 7.5 g (1.0-20.0 g) Salt 1.38 g (0.1-2.0 g) Fibres 4.0 g (0.1-10.0 g)

[0160] Table 2 shows data of a composition analysis of wheat and rye bread in detail [2,3].

TABLE-US-00003 nutritional value per 100 g bread, wheat (range) per 100 g bread, rye (range) Protein 10.7 g (1.0-20.0 g) 8.5 g (1.0-20.0 g) Total lipid (fat) 4.5 g (0.1-10.0 g) 3.3 g (0.1-10.0 g) Carbohydrate. by difference 47.5 g (30.0-80.0 g) 48.3 g (30.0-80.0 g) Fiber. total dietary 4.0 g (0.1-10.0 g) 5.8 g (0.1-10.0 g) Sugars. total 5.73 g (0.1-10.0 g) 3.85 g (0.1-10.0 g) Starch 36.34 g (30.0-79.0 g) n/a (30.0-79.0 g) Minerals Calcium 125 mg (1-200 mg) 73 mg (0.1-200 mg) Iron 3.6 mg (0.1-10 mg) 2.8 mg (0.1-10 mg) Magnesium 41 mg (1-100 mg) 40 mg (1-100 mg) Phosphorus 129 mg (1-200 mg) 125 mg (1-200 mg) Potassium 141 mg (1-200 mg) 166 mg (1-200 mg) Sodium 473 mg (100-1000 mg) 603 mg (100-1000 mg) Zinc 1.04 mg (0.1-10 mg) 1.14 mg (0.1-10 mg) Copper 0.148 mg (0.01-1 mg) 0.168 mg (0.01-1 mg) Manganese 1.026 mg (0.1-10 mg) 0.824 mg (0.1-10 mg) Selenium 28.8 g (1-100 g) 30.9 g (1-100 g) Vitamins Vitamin C 0.2 mg (0.01-2 mg) 0.4 mg (0.01-2 mg) Thiamine (B1) 0.411 mg (0.01-2 mg) 0.434 mg (0.01-2 mg) Riboflavin (B2) 0.252 mg (0.01-2 mg) 0.335 mg (0.01-2 mg) Niacin (B3) 5.59 mg (0.1-10 mg) 3.81 mg (0.1-10 mg) Pantothenic acid (B5) 0.82 mg (0.01-2 mg) 0.44 mg (0.01-2 mg) Vitamin B6 0.111 mg (0.01-2 mg) 0.075 mg (0.01-2 mg) Folate (B9) 85 g (10-200 g) 110 g (10-200 g) Choline 18.7 mg (1-100 mg) 14.6 mg (1-100 mg) Vitamin E (alpha-tocopherol) 0.19 mg (0.01-2 mg) 0.33 mg (0.01-2 mg) Vitamin K (phylloquinone) 4.9 g (1-10 g) 1.2 g (1-10 g) Fatty acids Fatty acids. total saturated 0.697 g (0.1-2 g) 0.626 g (0.1-2 g) Fatty acids. total monounsaturated 0.612 g (0.1-2 g) 1.311 g (0.1-2 g) Fatty acids. total polyunsaturated 1.615 g (0.1-10 g) 0.799 g (0.1-10 g) Amino acids Tryptophan n/a (0.01 g-1 g) 0.096 g (0.01 g-1 g) Threonine n/a (0.01 g-1 g) 0.255 g (0.01 g-1 g) Isoleucine n/a (0.01 g-1 g) 0.319 g (0.01 g-1 g) Leucine n/a (0.01 g-1 g) 0.579 g (0.01 g-1 g) Lysine n/a (0.01 g-1 g) 0.233 g (0.01 g-1 g) Methionine n/a (0.01 g-1 g) 0.139 g (0.01 g-1 g) Cystine n/a (0.01 g-1 g) 0.173 g (0.01 g-1 g) Phenylalanine n/a (0.01 g-1 g) 0.411 g (0.01 g-1 g) Tyrosine n/a (0.01 g-1 g) 0.213 g (0.01 g-1 g) Valine n/a (0.01 g-1 g) 0.379 g (0.01 g-1 g) Arginine n/a (0.01 g-1 g) 0.325 g (0.01 g-1 g) Histidine n/a (0.01 g-1 g) 0.182 g (0.01 g-1 g) Alanine n/a (0.01 g-1 g) 0.299 g (0.01 g-1 g) Aspartic acid n/a (0.01 g-1 g) 0.442 g (0.01 g-1 g) Glutamic acid n/a (0.1 g-10 g) 2.603 g (0.1 g-10 g) Glycine n/a (0.01 g-1 g) 0.302 g (0.01 g-1 g) Proline n/a (0.1 g-10 g) 0.909 g (0.1 g-10 g) Serine n/a (0.01 g-1 g) 0.417 g (0.01 g-1 g)

Example 2: Production of Enzyme Solutions Via Cultivation of Fungi with Bread Residues as an Inducing SystemAspergillus

[0161] In this experiment the present inventors studied the production of an exemplary enzyme mix, e.g. enzyme solution, via submerged fermentation with Aspergillus niger van Tiegheim ATCC 10535 and bread residues as single media component. Therefore, appr. 100 g bread crumbs obtained as described before in example 1 where mixed with ddH.sub.2O to a total volume of 2 L (5% w/v) in a 5 L baffled Erlenmeyer flask in duplicate. After subsequent sterilisation 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 in a rotary shaker at 100 rpm at 28 C. Daily visual checks indicated good growth and enzymatic hydrolysis of the substrate because media was blacked by the fungi cells and liquefacting of bread crumbs was observed. The fermentation continued 3 weeks to stress the fungi and induce maximal hydrolase enzyme secretion. Filtration of the liquid culture with paper filter was applied to remove the vegetative cells subsequently. Spores were removed by centrifugation with a maximum of 75.600g (Beckman Coulter Avanti JXN-26) followed by bottle top or cross-flow filtration with 0.2 m pore size. 10 kDa Cross-flow filtration and buffer ex-changing (membrane made from regenerated cellulose was used with the following parameters: inlet pressure (P1) 4 bar, retentat pressure (P2) 1 bar, and the permeate was open to atmospheric pressure), was applied afterwards to concentrate, enrich, and purify the enzyme mix. The final enzyme solution was about 350 ml. For the enzyme mix first a qualitative enzyme activity pre-screen using colored carbohydrate polymers embedded in an agar matrix was proceeded. Extend of discoloration and the resulting diameter of the decolored halo around the applied enzyme solution is a measure of the present enzyme amount and relative activity correlated to the conversion of the substrates in this pre-screen test. Table 3 gives an overview of the substrates with the related enzyme activity.

TABLE-US-00004 TABLE 3 Overview of different substrates and respective enzyme activities. Substrate Enzyme Azo-CM-Cellulose endo-1,4-.Glucanase (Cellulase) AZCL-HE-Cellulose endo-1,4-.Glucanase (Cellulase) Azo-Alpha-Cellulase endo-1,4-.Glucanase (Cellulase) Azo-Avicel endo-1,4-.Glucanase (Cellulase) AZCL-Barley--Glucan Malt--Glucanase (end-Cellulase) and Lichenase AZCL-Xyloglucan Endo-Cellulase AZCL-Xylan endo-1.4--Xylanase AZCL-Arabinoxylan endo-1.4--Xylanase AZCL-Pachyman endo-1,3--Glucanase (Cellulase) AZCL-Dextran endo-1,6-alpha-Dextranase AZCL-Arabinan endo-1,5-alpha-Arabinanase Red-Arabinan endo-1,5-alpha-Arabinanase RedCL-Amylose Alpha-Amylase Red Starch Alpha-Amylase AZCL-Pullulan Malt limit-dextrinase and Pullulanase Red Pullulan Malt limit-dextrinase and Pullulanase Azo-Fructan endo-Fructanase AZCL-Chitosan Chitosanase Skim-milk platen Protease AZO-Casein Protease AZCL-Glalactan endo-1,4--Galactanase AZCL-Rhamnogalacturonan I Rhamnogalacturonan hydrolase and lyase AZCL-Galactomannan endo-1,4--Mannanase Olive oil Rhodamine B lipase Enzyme activity was evaluated from no activity (), activity (+), good activity (++), high activity (+++), very high activity (++++) up to substrate mostly converted (high). Results of the enzyme screening are shown in Table 4.

TABLE-US-00005 TABLE 4 Results of the screening for enzymatic activity. The enzyme mix produced by Aspergillus niger comprises various enzymes, as shown below. Substrate Enzyme Result 1 Azo-CM-Cellulose endo-1,4-.Glucanase (Cellulase) +++ 2 AZCL-HE-Cellulose endo-1,4-.Glucanase (Cellulase) ++++ 3 Azo-Alpha-Cellulase endo-1,4-.Glucanase (Cellulase) 4 Azo-Avicel endo-1,4-.Glucanase (Cellulase) 5 AZCL-Barley--Glucan Malt--Glucanase (endo-Cellulase) and Lichenase high 6 AZCL-Xyloglucan Endo-Cellulase high 7 AZCL-Xylan endo-1.4--Xylanase ++++ 8 AZCL-Arabinoxylan endo-1.4--Xylanase 9 AZCL-Pachyman endo-1,3--Glucanase (Cellulase) 10 AZCL-Dextran endo-1,6-alpha-Dextranase 11 AZCL-Arabinan endo-1,5-alpha-Arabinanase 12 Red-Arabinan endo-1,5-alpha-Arabinanase + 13 RedCL-Amylose Alpha-Amylase high 14 Red Starch Alpha-Amylase high 15 AZCL-Pullulan Malt limit-dextrinase and Pullulanase 16 Red Pullulan Malt limit-dextrinase and Pullulanase ++ 17 Azo-Fructan endo-Fructanase 18 AZCL-Chitosan Chitosanase 19 Skim-milk platen Protease +++ 20 AZO-Casein Protease +++ 21 AZCL-Glalactan endo-1,4--Galactanase ++ 22 AZCL-Rhamnogalacturonan I Rhamnogalacturonan hydrolase and lyase 23 AZCL-Galactomannan endo-1,4--Mannanase + 24 Olive oil Rhodamine B lipase

[0162] The enzyme screening showed high activity for the expected enzymes with bread residues as an inducing system. After this pre-screen, a targeted quantitative screen for the enzymes with high activity can be done subsequently.

[0163] The following method can be exemplified as the method for measuring the enzymatic activity of -amylases. A -amylase is allowed to act on endo-1,4--glucan bonds of soluble starch to generate dextrin and a mixture of reduced sugars (especially maltose). Dissolved 3,5-dinitrosalicylic acid (DNS) (by addition of 2M NaOH) is allowed to act on the generated reduced sugars in the presence of potassium-sodium tartrate-tetrahydrate at 95 C. to generate 3,5 amino nitro salicylic acid. Absorbance is measured at 540 nm, the amount of the 3-amino-5-nitrosalicylic acid is obtained by using a calibration curve and the enzymatic activity is calculated. The amount of the enzyme that liberates 1 mol of reducing sugar out of soluble starch in 1 minute at 25 C. and pH 6.9 is defined as 1 U (1 unit).

[0164] The following method can be exemplified as the method for measuring the enzymatic activity of amyloglucosidase. A amyloglucosidase is allowed to act on 1,4--glucan bonds of maltose to generate D-glucose. Glucose oxidase is allowed to act on the generated glucose as a substrate in the presence of oxygen to generate hydrogen peroxide. Peroxidase is allowed to act on the generated hydrogen peroxide in the presence of aminoantipyrine and phenol to generate a quinoneimine dye. Absorbance is measured at 500 nm, the amount of the quinoneimine dye is obtained by using a calibration curve and the enzymatic activity is calculated. The amount of the enzyme that oxidises 1 mol of maltose in 1 minute at 25 C. and pH 4.3 is defined as 1 U (1 unit).

[0165] The following method can be exemplified as the method for measuring the enzymatic activity of hemicellulase. A hemicellulase is allowed to act on xylose to generate reduced sugars. Dissolved 3,5-dinitrosalicylic acid (DNS) (by addition of 2M NaOH) is allowed to act on the generated reduced sugars in the presence of potassium-sodium tartrate-tetrahydrate at 95 C. to generate 3,5 amino nitro salicylic acid. Absorbance is measured at 540 nm, the amount of the 3-amino-5-nitrosalicylic acid is obtained by using a calibration curve and the enzymatic activity is calculated. The amount of the enzyme that liberates 1 mol of reducing sugar out of xylose in 1 minute at 40 C. and pH 4.5 is defined as 1 U (1 unit).

[0166] The following method can be exemplified as the method for measuring the enzymatic activity of proteases. A protease is allowed to act on casein to generate amino acids. Reaction is stopped by adding trichloroacetic acid. The test solution is filtered by using a 0.45 m polyethersulfone syringe filter subsequently and Folin & Ciolcaltea's, or Folin's Phenol reagent is allowed to act on the amino acids after filtration to generate a measurable color change that will be directly related to the activity of proteases. Absorbance is measured at 660 nm, the amount of the amino acid is obtained by using a tyrosine calibration curve and the enzymatic activity is calculated. The amount of the enzyme that produces amino acid equivalent to 10 g tyrosin out of casein in 1 minute at 37 C. and pH 8.0 is defined as 1 U (1 unit).

[0167] The following method can be exemplified as the method for measuring the enzymatic activity of proteases. A protease is allowed to act on casein to generate amino acids. Reaction is stopped by adding trichloroacetic acid. The test solution is filtered by using a 0.45 m polyethersulfone syringe filter subsequently and Folin & Ciolcaltea's, or Folin's Phenol Reagent is allowed to act on the amino acids after filtration to generate a measurable color change that will be directly related to the activity of proteases. Absorbance is measured at 660 nm, the amount of the amino acid is obtained by using a tyrosine calibration curve and the enzymatic activity is calculated. The amount of the enzyme that produces amino acid equivalent to 10 g tyrosin out of 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 mix, i.e. an exemplary composition of the invention, comprises cellulose, lichenase, xylanase, arabinanase, amylase e.g. -amylase, limit dextrinase, pullulanase, protease, galactanase, and mannanase.

Example 3: Production of Enzyme Solutions Via Cultivation of Fungi with Bread Residues as an Inducing SystemCeratocystis

[0168] In this experiment the present inventors studied the production of an enzyme solution via submerged fermentation with Ceratocystis paradoxa CBS 374.83 and bread residues as single media component. Therefore, cultivation and downstream processing was done as described in example 2. The final enzyme solution was about 30 ml. Results of the enzyme pre-screen proceeded as described in example 2 are shown in Table 5.

TABLE-US-00006 TABLE 5 Results of the screening for enzymatic activity. The enzyme mix produced by Ceratocystis paradoxa comprises various enzymes, as shown below. Substrate Enzyme Result 1 Azo-CM-Cellulose endo-1,4-.Glucanase (Cellulase) high 2 AZCL-HE-Cellulose endo-1,4-.Glucanase (Cellulase) high 3 Azo-Alpha-Cellulase endo-1,4-.Glucanase (Cellulase) 4 Azo-Avicel endo-1,4-.Glucanase (Cellulase) 5 AZCL-Barley--Glucan Malt--Glucanase (endo-Cellulase) and Lichenase 6 AZCL-Xyloglucan Endo-Cellulase 7 AZCL-Xylan endo-1.4--Xylanase high 8 AZCL-Arabinoxylan endo-1.4--Xylanase high 9 AZCL-Pachyman endo-1,3--Glucanase (Cellulase) 10 AZCL-Dextran endo-1,6-alpha-Dextranase 11 AZCL-Arabinan endo-1,5-alpha-Arabinanase high 12 Red-Arabinan endo-1,5-alpha-Arabinanase high 13 RedCL-Amylose Alpha-Amylase high 14 Red Starch Alpha-Amylase high 15 AZCL-Pullulan Malt limit-dextrinase and Pullulanase 16 Red Pullulan Malt limit-dextrinase and Pullulanase ++++ 17 Azo-Fructan endo-Fructanase 18 AZCL-Chitosan Chitosanase 19 Skim-milk platen Protease 20 AZO-Casein Protease 21 AZCL-Glalactan endo-1,4--Galactanase ++++ 22 AZCL-Rhamnogalacturonan I Rhamnogalacturonan hydrolase and lyase ++++ 23 AZCL-Galactomannan endo-1,4--Mannanase ++++ 24 Olive oil Rhodamine B lipase

[0169] The enzyme screening showed high activity for the expected enzymes with bread residues as an inducing system. Examples of methods for a targeted quantitative screen of enzymes with high activity are given in example 2. As shown in table 5, an exemplary enzyme mix, i.e. an exemplary composition of the invention, comprises cellulase, xylanase, arabinanase, amylase e.g. -amylase, limit dextrinase, pullulanase, galactanase, mannanase, rhamnogalacturonan hydrolase, and rhamnogalacturonan lyase.

Example 4: Hydrolysis of Food ResiduesBread

[0170] Saccharides, proteins and other nutrients contained in bread residues as described in example 1 are suitable for fermentation. Microorganisms convert it into profitable products such as microbial oil, useful product for food industry. Pretreating of substrate as bread residue can be applied before fermentation to get the nutrients easily accessible for microorganisms. One of typical pretreatment method is enzymatic hydrolysis. Bread residues contain high concentration of starch (more than 70% on dry matter) and proteins (up to 14% on dry matter) and treatment with -amylases, glucoamylases and proteases easily lead to the release of compounds available for microbial growth. Hydrolysis of contained starch as main constituent of the bread dry weight comprise two stages. In the first stage -amylase enzyme is used to liquefy the starch giving a solution including products of destrins and small amounts of glucose. The liquefied starch is subject to saccharification stage by using glucoamylase enzyme to obtain maximum terminal glucose conversion. Both stages have been found to be interrelated and there has been an optimal degree of liquefaction needed for adequate saccharification in the subsequent step. The following method is an exemplary enzymatic pretreatment of said substrates using one or more enzymes, for example a hydrolysis of starch, e.g. starch contained in bread residues. Homogenized bread residues in water suspension is prepared for the experiment. Size of bread particles are less than 4 mm. For liquefaction, -amylase is added (e.g. SUG-001, Creative Enzymes, Shirley, NY; USA). Liquefaction is proceeded in a thermostat or water bath with specific pH (e.g. pH 6) and under specific temperature (e.g. 80 C.). The pH of the suspension is adjusted by e.g. 1% (w/v) solution of H.sub.2SO.sub.4 and 1% (w/v) solution of NaOH. The liquefaction is ended by freezing the suspension. Time of liquefaction is about 180 minutes. The saccharification is performed in liquefied suspension by addition of glucoamylase (e.g. SUG-002; DIS-1013, Creative Enzymes, Shirley, NY; USA). Saccharification is proceeded in a tempered shaker with specific pH (e.g. pH 4.2) and under specific temperature (e.g. 60 C.) and shaking (e.g. 130 rpm). Enzyme activity is ended by heating of the suspension at 80 C. for 5 minutes. Time of saccharification is at least 90 minutes. Hemicellulases (e.g. DIS-1023 Creative Enzymes, Shirley, NY; USA) and proteases (e.g. Neutrase, Novozymes, Denmark) are added in the saccharification step to yield the highest content of converted sugars and amino acids. Instead of using commercial available enzymes, enzyme mixes produced as described in example 2 and 3 or any combination of the foregoing are suitable. For example, the hydrolysis can be performed in only one step with elongated incubation time (e.g. 24 h) with specific pH (e.g. pH 5.5) specific temperature (e.g. 55 C.) and shaking (e.g. 130 rpm). An adaption of the procedure to a bioreactor is also possible.

[0171] After the incubation, the mixture is centrifugated (e.g. 14.000 rpm for 20 min) to separate not hydrolyzed solid residues and supernatant comprising the nutrients is collected. At this point the supernatant can be characterised as bread residue hydrolysate. This hydrolysate obtained from the protocol could be freeze-dried to assess the suitability of the nutrients to be rehydrated. For fermentation the hydrolysate could be freshly prepared or rehydrated applied as is or by optional addition of supplements singly or in mixture.

Example 5: Production of Microbial Food IngredientsMicrobial Lipids

[0172] Oleaginous microorganisms such as oleaginous yeasts represent interesting microbial lipid factories. Their rapid growth, along with the ability to utilize a wide variety of raw materials and their easy cultivation in large fermenters, make them highly suitable candidates for bio refinery processes. Oleaginous microorganisms such as oleaginous yeasts are able to grow and accumulate high levels of lipids. The strain ATCC 20509 Cutaneotrichosporon oleaginosus (C. oleaginosus) has been studied for its ability to accumulate TAGs by up to 70% of DCW, and advantageously has a fatty acid composition very similar to the fatty acid compositions of cocoa butter or palm oil. C. oleaginosus has the ability to use highly diverse carbon sources, e.g. 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 a growth medium for oleaginous microorganisms. For example, the inventors have successfully converted food residue(s) into nutrients using filamentous fungi and bacteria, and these nutrients can be used as growth medium for oleaginous microorganisms. Particularly, enzymatically treated food residue(s) can be used as nutrients, e.g. carbon source, for producing microbial lipids with oleaginous microorganisms e.g. C. oleaginosus. Hydrolysate obtained as described in example 4 is considered as a potential alternative carbon source for producing microbial lipids with C. oleaginosus fermentation.

[0173] Conventional lipid production with oleaginous organisms is a 2-step process, where the first step provides for biomass formation under non-limiting conditions (exponential growth phase), the second lipid induction step (nutrient limitation phase) affords high intracellular lipid accumulation at stagnant cell counts. It is also possible to do a C. oleaginosus co-fermentation with organic acid and carbon source leading to concurrently high biomass and lipid yields (84.9% of DCW) without the need of nutrient restriction that enables simultaneous assimilation of e.g. bread residue hydrolysate and acetic acid. A mild downstream processing is also favorable for producing edible microbial lipids. Therefore, performing e.g. a purely enzymatic treatment of C. oleaginosus without any solvent-based extraction or chemicals-based demulsification is suitable, to make the produced microbial lipids amenable for subsequent harvesting. Harvesting of the produced microbial lipids is done by a density-based separation method e.g. with a semi-continues centrifuge. Refining the microbial lipid is the last step of processing to get edible oil proceed e.g. physical refining. FIG. 1 shows a method that can be exemplified as a sustainable method for producing edible microbial lipids.

[0174] The following method can be exemplified as the method for analysing the fatty acid profile of the final product using gas chromatography with flame ionization detector (GC-FID) after methylation. The methylation is briefly done by incubating approx. 1 mg of oil with 1 ml of NaOCH.sub.3 (for 20 minutes at 80 C.). Then 1 ml of HCl (37% in methanol) is added and the mixture is incubated again for 20 minutes at 80 C. Resulted fatty acid methyl esters (FAMEs) are extracted by hexane in injected GC-FID. The triglycerol C19:0 is used as an internal standard. An exemplary microbial lipid composition of C. oleaginosus is shown in Table 6.

TABLE-US-00007 TABLE 6 An exemplary microbial lipid composition produced using C. oleaginosus. Fatty Acid Relative % C14:0 (myristic acid) 2.0 C16:0 (palmitic acid) 24.0 C16:1 (palmitoleic acid) 1.0 C18:0 (stearic acid) 10.0 C18:1 (oleic acid) 40.0 C18:2 (linoleic acid) 20.0 C18:3 (linolenic acid) 3.0

Example 6: Production of Microbial Food IngredientsMicrobial Proteins

[0175] The inventors have demonstrated that food residue(s), such as freeze-dried bread, can be used as a substrate to produce microbial protein biomass. Protein biomass is produced as a side product when cultivating the first microorganism.

Example 7: Production of Microbial Food IngredientsAroma Compounds

[0176] The inventors have surprisingly found that microorganisms, such as Ceratocystis species, produce aroma compounds when cultivated on food residue(s). For example, Ceratocystis species e.g. Ceratocystis fimbriata and Ceratocystis moniliformis produce a wide range of complex aroma such as: peach, banana, pear, rose or citrus depending on the strain and environmental conditions. The advantage of using these fungi consist of their relatively rapid growth and the variety of complex aroma mixtures synthesized. The inventors found that Ceratocystis paradoxa CBS 374.83 produced aroma compounds during the fermentation experiment as described in example 2 detected by a strong fruity smell of the supernatant. The supernatant comprised at least one aroma compound. Thus, the method of the invention surprisingly does not only provide microbial lipids, but also aroma compounds. To obtain the aroma compounds, a mild separation technique was used to provide an extract with sensory characteristics as close as possible to the complete product. For example, organophilic pervaporation (O-PV) which is a membrane-based technique was used for the mild recovery of natural aroma compounds, as well as solid-phase micro extraction (SPME) which is a non-exhaustive extraction technique in which a fiber coated with sorbent materials is exposed to the sample. The inventors have found that it is advantageous to maintain the ingredient functionality of the aroma during processing by using a mild separation technique.

[0177] The inventors further performed aroma fractionation to recover a particular aroma or a group of aroma compounds from a mixture of aroma compounds. Furthermore, the inventors analysed the extracted aroma compounds by capillary gas chromatography (GC) alone by comparing the retention indices of the sample with those of the corresponding authentic reference compound or via a GC-olfactometry (GC-O). GC-O enables the correlation between a volatile organic compound and its perception using the human nose as a detector. Therefore, the outlet of the GC capillary column is installed into a column flow splitter. The column flow splitter possesses two outlets directing the gas flow into a destructive detector e.g. flame ionization detector (FID) or a mass spectrometer (MS) and into a nondestructive olfactory detection part (ODP). With this equipment it is possible to directly assign an odor impression to a MS spectrum or FID peak.

Example 8: Usage of Microbial Components in Food IndustryMicrobial Lipids in Bakery Products

[0178] A broad spectrum of applications to microbial components in food industry is possible. The inventors exemplary used the microbial lipids obtained with a method of the invention in different bakery products:

a) Kaiser roll

Ingredients:

[0179] 100% flour type 550 or 650 [0180] 2-3% buttermilk [0181] 3-4% baker's yeast [0182] 10-15% microbial lipid [0183] 2-3% baking agent [0184] 1.8-2.2% salt [0185] 55-63% water

Kneading:

[0186] Starter dough [0187] Mixing of 20% flour, 2% buttermilk, 10% water and 1% baker's yeast to get a smooth dough using a dough kneader at lowest level 3 to 4 minutes followed by 2 to 3 minutes of rapid mixing. Covering the starter dough subsequently with cling film and rest it in a fridge (4 C.) for 6 to 24 hours. [0188] Main dough [0189] Slowly kneading of 80% flour, 3% baking agent, 2% salt, 15% microbial lipid, 3% baker's yeast and starter dough thoroughly with a spiral kneader 5 to 7 minutes followed by 5 to 8 minutes fast kneading till the dough is mouldable.

[0190] The dough temperature ranges between 23 and 26 C.

[0191] After removing the dough out of the spiral kneader the dough sits for up to 10 minutes.

Processing:

[0192] Portions of 1800 to 2100 g are rounded and covered with cling film subsequently. [0193] Let it rest for 15 to 20 minutes so that the dough can rise briefly. [0194] Moulding of the portions followed by rounding. [0195] Put it on a food carrier and let it rise in a proofing chamber (Temperature 30-35 C. with a relative humidity of 75-85%) for 50 to 75 minutes.

Baking:

[0196] Oven temperature: 230-240 C. [0197] Steam input: standard [0198] Damper: closed [0199] Baking time: approx. 20 minutes

[0200] Kaiser rolls were successfully produced with microbial lipids produced with a method of the invention. The bread rolls produced using microbial lipid had the same smell, taste, and texture as conventional Kaiser rolls.

b) Pretzel

Ingredients:

[0201] 100% wheat flour type 550 [0202] 2-3% bakery malt [0203] 1.8-2.2% salt [0204] 10-15% microbial lipid [0205] 1.5-2% baker's yeast [0206] 45-48% water

Kneading:

[0207] Starter dough [0208] Mixing of 20% flour, 0.2% baker's yeast and 12% water (10 C.) using a dough kneader at lowest level 3 to 4 minutes followed by 3 minutes of rapid mixing at highest level. Covering the starter dough subsequently with cling film and rest it at room temperature for 1 hour. Then leave it for rising for 12 to 14 hours. [0209] Main dough [0210] Slowly kneading of 80% flour, 2% bakery malt, 2% salt, 1.5% baker's yeast, 10% microbial lipid, 10% water (10 C.) and starter dough with a spiral kneader 4 minutes at low level followed by 5 minutes fast kneading hat highest level. [0211] The dough temperature is approx. 24 C. [0212] After removing the dough out of the spiral kneader the covered dough sits for approx. 20 minutes.

Processing:

[0213] Portions of 1800 to 2100 g are rounded and covered with cling film subsequently. [0214] Let it rest for 15-20 minutes. [0215] Moulding of the portions. [0216] Roll dough in portions on a floured work surface to strands with a length of approx. 60 cm so that the largest diameter of the strands is in the center and the strands become narrower towards the outside with rounded tips. [0217] Twisting every straight rope of dough to form it into pretzel shape. [0218] Put the dough pieces on a baking tray covered with baking linen. [0219] Covering of portions with cling film subsequently and let it rest for 1 to 2 hours at room temperature so that the dough has risen to double volume. [0220] Provide the pretzel lye and put on eye protection and protective gloves. [0221] Submerge the pretzels in lye for approx. 5 seconds, remove them and put them on a baking tray covered with baking paper. [0222] Cut the pretzels at the bulbous part and sprinkle them with some salt.

Baking:

[0223] Oven temperature: preheated 230 C. top and bottom heat [0224] Baking time: 12 to 14 minutes till gold brown [0225] Pretzels are cooled on a wire rack subsequently.

[0226] Pretzels were successfully produced with microbial lipids produced with a method of the invention. The Pretzels produced using microbial lipid had the same smell, taste, and texture as conventionally produced Pretzels.

c) Bismarck

Ingredients:

[0227] 100% flour type 550 or 405 [0228] 20-30% milk [0229] 5-8% baker's yeast [0230] 20% eggs [0231] 8% egg yolk [0232] 10-15% microbial lipid [0233] 10% sugar [0234] 1.8-2.2% salt [0235] 60 g water [0236] Aroma: vanilla and lemon zest

Kneading:

[0237] Starter dough [0238] Mixing of 30% flour, 20% milk, 5% baker's yeast and 60 g water (10 C.) to get a smooth dough using a dough kneader at lowest level 3 to 4 minutes. Covering the starter dough subsequently with cling film and rest it at room temperature for 30 minutes. [0239] Main dough [0240] Slowly kneading of 70% flour, 20% eggs, 8% egg yolk, 10% microbial lipid, 10% sugar, 7% milk, 2% salt, aroma and starter dough thoroughly with a spiral kneader 5 to 7 minutes followed by 10 to 12 minutes fast kneading till the dough is mouldable. [0241] After removing the dough out of the spiral kneader the dough sits between 20 and 30 minutes.

Processing:

[0242] Portions of 1200 to 1500 g are rounded and covered with cling film subsequently. [0243] Let it rest for 15 to 20 minutes so that the dough can rise briefly. [0244] Moulding of the portions followed by rounding. [0245] Put it on a food carrier and let it rise in a proofing chamber (Temperature 30-35 C. with a relative humidity of 60-70%) for 50 to 75 minutes.

Stiffening:

[0246] The proofing of the dough pieces is interrupted so that the surface gets completely dry and solid.

Boiling:

[0247] Fill in microbial lipid in a chip pan and heat it up to 150-175 C. [0248] Check temperature. [0249] Bake on the dough pieces with the round part upwards. [0250] Turn them afterwards and press them down with a grid towards the end of baking time. [0251] Baking time: 6 to 8 minutes with optimum intervals of 3 min./2 min./30 seconds and 30 seconds.

Filling:

[0252] Filling like apricot jam is placed in the center of the bismarcks.

Topping:

[0253] Still hot bismarcks are dusted with e.g. sugar or icing sugar.

[0254] Bismarcks (German doughnut with no central hole) were successfully produced with microbial lipids produced with a method of the invention. The bismarcks produced using microbial lipid had the same smell, taste, and texture as conventionally produced bismarcks.

d) Fruit loaf

Ingredients:

[0255] 30% wheat flour type 550 [0256] 20-30% milk [0257] 1.5% baker's yeast [0258] 8.5% water [0259] 13% microbial lipid [0260] 3% sugar [0261] 2% marzipan [0262] 1.8-2.2% salt [0263] 0.3% sour cream [0264] 0.3% spice [0265] 1% milk powder [0266] 0.3% rum aroma [0267] 0.3% vanilla aroma [0268] 0.3% almonds bitterly [0269] 0.3% lemon zest [0270] 19% sultanas [0271] 0.4% almonds baton-cut [0272] 0.4% candied orange and lemon peel [0273] 4.5% Overseas rum

Kneading:

[0274] Starter dough [0275] Mixing of 10% wheat flour, 1.5% baker's yeast, 7% water and 0.3% salt with a dough kneader at lowest level for 2 minutes followed by 3 to 4 minutes fast kneading to get a cool (dough temperature is 24 C.) and semisolid starter dough. Covering the starter dough subsequently with cling film and rest it at room temperature for 30 minutes. After removing the dough out of the spiral kneader rest it in a fridge (5 C.) for 10 to 15 hours. [0276] Main dough [0277] Mixing of all other ingredients of the main dough except flour to get a smooth dough. Add starter dough and flour subsequently and mix it slowly for 5 minutes followed by 3 minutes fast kneading. After a briefly dough relaxation the fruit loaf mixture is filled.

Processing:

[0278] Weight portions are rounded [0279] 15 minutes dough relaxation and elongation subsequently. [0280] Cover it and do the proofing at room temperature. [0281] Moulding of the portions followed by rounding. [0282] Put it on a food carrier and let it rise in a proofing chamber (Temperature 30-35 C. with a relative humidity of 60-70%) for 50 to 75 minutes.

Baking:

[0283] Oven temperature: start 210 C. decreasing to 200 C. [0284] Steam input: none [0285] Damper: opened after 5 minutes baking time [0286] Baking time: approx. 50 minutes for 780 g dough pieces [0287] Still hot coat it with liquid butter and turn it in vanilla sugar subsequently. [0288] After cooling the fruit loaf is dusted with sweet snow.

[0289] Fruit loaf was successfully produced with microbial lipids produced with a method of the invention. The fruit loaf produced using microbial lipid had the same smell, taste, and texture as conventionally produced fruit loaf.

Example 9: Use of Microbial Components in Food IndustryChocolate Products

[0290] It is possible to produce numerous food products using microbial lipids produced with a method of the invention. For example, the following products can be produced:

TABLE-US-00008 Chocolate spread Sugar 10-70% Microbial lipids 5-60, Plant lipids (eg. palm oil and/or cocoa butter): 0-20 hazelnuts (0-20%), skim milk powder (0-10%) whey powder (0-10%) cocoa (including cocoa products or any product (5-25%), that is cocoa alternative that have cocoa taste-and smell-like Additions (Including: non-fat milk solids, 1-5% emulsifier (lecithin INS 322), flavour (vanillin) contains added flavour (nature identical flavouring substance - vanillin). Chocolate bar Sugar, 10-70% Microbial lipids 10-50, Plant lipids (eg. palm oils and/or cocoa butter): 0-40 Skimmed milk powder: 0-15% Cocoa: 20-70%. Additions (Including: hazelnuts non-fat milk 1-10% solids, other nuts and wheat, whey powder, soy components emulsifier (lecithin INS 322), flavour (vanillin) contains added flavour (nature identical flavouring substance - vanillin). Dark Chocolate bar Sugar, 1-20% Microbial lipids 10-50, Plant lipids (eg. palm oils and/or cocoa butter): 0-40 Skimmed milk powder: 0-15% Cocoa: 50-80%. Additions (Including: hazelnuts non-fat milk 1-10% solids, other nuts and wheat, whey powder, soy components emulsifier (lecithin INS 322), flavour (vanillin) contains added flavour (nature identical flavouring substance - vanillin). MARGARINE Microbial Lipids 1-80% Plant Lipids (Palm oil, rapeseed oil, Sunflower oil 0-50% and/or olive oil) Salt: 1-5% Water: 10-30% Additions [Such as emulsifier (eg. E475; E471; 1-3% lecithins); acid (e.g. citric acid); natural flavour; colour (carotenes)]

Example 10: Changing the Triglycerides Content Over Modeling the Process Parameters

[0291] Changing the triglycerides content over modeling the process parameters. TAG composition of Yeast oil (%)

TABLE-US-00009 Yeast Yeast Yeast Yeast Oil 1 Oil 2 Oil 3 Oil 4 Unknown 0.5 1.7 1.7 9.4 Other Liquid TAGs 8.7 8.1 9.0 22.3 PLiP 2.6 0.8 0.7 8.4 MOP/PPoP 1.1 0.2 0.2 1.3 OOO 3.5 6.6 7.6 5.9 POO 17.0 13.9 15.6 32.9 PLiS 2.5 1.8 1.7 1.4 POP 19.5 26.6 25.9 11.6 PPP 0.2 0 0 0.0 SOO 7.1 13.2 14.4 4.0 SLiS 0.4 0.2 0.2 0.2 POS 21.2 5.1 4.8 1.6 SOS 4.2 11.1 8.8 0.2 SSS 0.5 0.9 0.8 0 PSS 0 0.1 0.1 0 Other high melting TAGs 1.6 1.2 0.3 0.0 P; palmitic, St; stearic, O; oleic, Li; linoleic,

REFERENCES

[0292] [1] Lieken. (2021 Apr. 12). Lieken Brot-und Backwaren GmbH. Retrieved from www.lieken-urkorn.de: https://www.lieken-urkorn.de/produkte/produkt/bauernmild-500g [0293] [2] USDA1. (2021 Apr. 12). USDA, Agricultural Research Service, Fooddata central. Retrieved from https://fdc.nal.usda.gov/fdc-app.html #/food-details/172686/nutrients [0294] [3] USDA2. (2021 Apr. 12). USDA, Agricultural Research Service, Fooddata central. Retrieved from https://fdc.nal.usda.gov/fdc-app.html #/food-details/172684/nutrients

[0295] The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.