Sequential co-culturing method for producing a vitamin- and protein-rich food product

Abstract

The present invention relates to a method for producing a vitamin- and protein-rich product, to a food product containing the vitamin- and protein-rich product, and to a nutrient medium appropriate for said method on the basis of agricultural tributaries or food tributaries.

Claims

1. A method for producing a vitamin- and protein-rich product, comprising the steps: a) Cultivating in a vessel, at least one species of the division of Basidiomycota submerged in a nutrient medium comprising at least one carbohydrate-containing agricultural waste or food waste to obtain a first cultivation product; b) Adding at least one vitamin B12-producing species of the genus Propionibacterium and/or of the genus Lactobacillus to the first cultivation product, wherein the at least one vitamin B12-producing species is selected from the group consisting of Propionibacterium freudenreichii sups. freudenreichii, Propionibacterium freudenreichii sups. shermanii, Lactobacillus reuteri, and combination thereof; and c) Cultivating the at least one species of the genus Propionibacterium and/or at least one species of the genus Lactobacillus in the first cultivation product to obtain a second cultivation product, wherein the at least one species of the division of Basidiomycota is selected from the group consisting of Agrocybe aegerita, Pleurotus roseus, Lentinula edodes, Laetiporus sulphureus, Pleurotus sapidus, Stropharia rugosoannulata, Wolfiporia cocos, and mixtures thereof; wherein the second cultivation product is the vitamin- and protein-rich product.

2. The method according to claim 1, wherein the first cultivation product has a total biomass in the range of 5 to 45 g/L based on dry mass.

3. The method according to claim 1, wherein the second cultivation product has a total biomass in the range of 10 to 50 g/L based on dry mass.

4. The method according to claim 1, wherein the at least one carbohydrate-containing agricultural waste or food waste is selected from the group consisting of apple pomace, aronia pomace, spinach pomace, pomegranate pomace, beet molasses, isomaltulose molasses, sunflower seed pomace, onion pomace, draff, grape mare, hay and whey.

5. The method according to claim 1, wherein the nutrient medium used in step a) has 5 to 25 g/L carbohydrates.

6. The method according to claim 1, wherein the nutrient medium used in step a) further comprises: at least one nitrogen source, wherein the nitrogen source is selected from the group consisting of L-asparagine, ammonium nitrate and yeast extract; at least one magnesium source; at least one potassium source and/or phosphate source; and trace elements, wherein the trace elements comprise iron(II), zinc(II), copper(II) and manganese(II) compounds.

7. The method according to claim 1, wherein in step b) the at least one vitamin B12-producing species of the genus Propionibacterium and/or of the genus Lactobacillus is added such that a total viable count of all added bacteria species is in the range of 10.sup.4 to 10.sup.10 CFU/ml in the first cultivation product.

8. The method according to claim 1, wherein the step c) of cultivating the at least one species of the genus Propionibacterium and/or the at least one species of the genus Lactobacillus is performed until reaching a vitamin B12-concentration in the range of 1 to 20 ng/mL culture.

9. The method according to claim 1, wherein the method further comprises the steps of: d) Harvesting the at least one species of the division of Basidiomycota and the at least one vitamin B12-producing species of the genus Propionibacterium and/or Lactobacillus from the second cultivation product; e) Drying the harvested at least one species of the division of Basidiomycota and the at least one vitamin B12-producing species of the genus Propionibacterium and/or Lactobacillus to obtain dried vitamin- and protein-rich product.

10. The method according to claim 1, wherein the method further comprises the step of: irradiating the at least one species of the division of Basidiomycota at least partially with a UV-light source, wherein the step of irradiating may be performed during the whole process.

11. The method according to claim 1, wherein in step b) at least one glutaminase-active bacteria species from the genus Lactobacillus is further added.

12. The method according to claim 1, wherein the method is performed without harvesting the at least one species of the division of Basidiomycota from the first cultivation product.

13. The method of claim 1, wherein the vitamin- and protein-rich product is a food product.

14. The method of claim 13, wherein the food product is edible mushrooms.

Description

3. SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1a shows the growth of bacteria of a bacteria culture consisting of P. freudenreichii subspecies shermanii and P. freudenreichii subspecies freudenreichii as n-fold increase of the initial added amount in CFU/mL.

(2) FIG. 1b shows the growth of bacteria of a bacteria culture consisting of P. freudenreichii subspecies shermanii and P. freudenreichii subspecies freudenreichii as the total biomass including Pleurotus sapidus (PSA) in g/100 ml determined by weighing the harvested and dried biomass.

(3) FIG. 2a shows the growth of bacteria of a bacteria culture consisting of P. freudenreichii subspecies shermanii and P. freudenreichii subspecies freudenreichii as n-fold increase of the initial added amount in CFU/mL.

(4) FIG. 2b shows the growth of bacteria of a bacteria culture consisting of P. freudenreichii subspecies shermanii and P. freudenreichii subspecies freudenreichii as the total biomass including Pleurotus sapidus (PSA) in g/100 ml determined by weighing the harvested and dried biomass.

(5) FIG. 3a shows the total biomass in g/100 ml of Pleurotus sapidus (PSA) cultures or co-cultures of PSA and vitamin B12-producing bacteria on M1-medium, determined by weighing the harvested and dried biomass.

(6) FIG. 3b shows the total biomass in g/100 ml of Pleurotus sapidus (PSA) cultures or co-cultures of PSA and vitamin B12-producing bacteria on M1-medium M2-medium, determined by weighing the harvested and dried biomass.

(7) FIG. 4a shows the growth of bacteria of a bacteria culture consisting of P. shermanii and P. freudenreichii as n-fold increase of the initial added amount in CFU/mL.

(8) FIG. 4b shows the growth of bacteria of a bacteria culture consisting of P. shermanii and P. freudenreichii as the total biomass including Pleurotus sapidus (PSA) in g/100 ml determined by weighing the harvested and dried biomass.

(9) FIG. 5 shows the growth of P. freudenreichii subspecies shermanii on optimum medium and on Pleurotus sapidus (PSA) culture supernatants.

(10) FIG. 6 shows the growth of E. coli, L. monocytogenes, S. enteritidis enteritis, and P. aeruginosa on optimum media and on Pleurotus sapidus (PSA) culture supernatants.

(11) FIG. 7 shows the increase in glutamate content in Pleurotus sapidus (PSA)-mycelium by cultivation with L. reuteri.

(12) FIG. 8 shows a vitamin B12 standard curve of the used ELISA assay for the determination of vitamin B12 in the co-cultures.

(13) FIG. 9 shows the quantification of the Pleurotus sapidus (PSA) proportion in the lyophilizate, cultivated on the agricultural tributary apple pomace, determined via the ergosterol-content (highlighted in black); the quantification of the protein content was performed according to the method of Kjeldahl (highlighted in grey).

(14) FIG. 10 shows the bulk elements of Pleurotus sapidus (PSA), cultivated on isomaltulose molasses (PM).

(15) FIG. 11 shows the essential trace elements of Pleurotus sapidus (PSA), cultivated on isomaltulose molasses (PM).

(16) FIG. 12 shows the composition of the substrate of the different agricultural tributaries used for cultivation.

(17) FIG. 13 shows the vitamin D2 concentration of a Pleurotus sapidus (PSA) culture on apple pomace after irradiation of the lyophilizate and the liquid submerged culture, respectively, which was subsequently lyophilized.

(18) FIG. 14 shows the total RNA content in percent of an untreated and heated Pleurotus sapidus (PSA) culture on isomaltulose molasses.

(19) FIG. 15 shows the composition of Pleurotus sapidus (PSA) on isomaltulose molasses.

(20) FIG. 16 shows the quantification of the single amino acids from Pleurotus sapidus (PSA) on isomaltulose molasses.

(21) FIG. 17 shows an optical comparison of vegetarian “bratwursts” fine (I to VI) and commercial bratwurst on meat basis (VII) and vegetarian “bratwursts” coarse (VIII to XIII).

(22) FIG. 18 shows a method according to the present invention for obtaining a product rich in proteins, vitamin B12 and vitamin D as well as containing glutamate.

(23) FIG. 19 shows the n-fold increase in biomass and vitamin B12 content of cultures consisting of Pleurotus sapidus (PSA) as well as cultures consisting of PSA, P. freudenreichii and L. reuteri under different cultivation conditions.

(24) FIG. 20 shows the n-fold increase in biomass and bacteria content (in CFU/ml) when using Palatinose and whey, respectively.

(25) FIG. 21 shows a test reactor after 62 h cultivation of a PSA pure culture.

(26) FIG. 22 shows a test reactor during cultivation of a PSA pure culture.

4. DETAILED DESCRIPTION OF THE INVENTION

(27) The present invention is based on the submerged co-cultivation of Basidiomycota species and vitamin B12-producing bacteria species in a nutrient medium. The nutrient medium preferably comprises agricultural tributaries or food tributaries, which comprise monosaccharides, disaccharides and/or oligosaccharides and/or which may be cellulose or starch-containing. Preferably, isomaltulose molasses from the production of isomaltulose, carotte pomace, apple pomace pomegranate pomace, spinach pomace and/or beet molasses are used as agricultural tributaries. Food tributaries may comprise draff, grape marc and/or whey. Preferably, cultivation of the Basidiomycota is performed aerobically at a temperature of between 20 and 28° C., until sufficient biomass is provided. Subsequently, in the nutrient medium, cultivation of vitamin B12-producing bacteria is performed, preferably of the genus Lactobacillus and/or Propionibacterium. The employed bacterial strains preferably are microorganisms which may already be safely used in food production and are considered as GRAS organisms (“generally recognized as safe”). For the cultivation of bacteria species, the temperature is raised to a temperature favorable for bacteria growth. Furthermore, anaerobe conditions may be realized.

(28) During cultivation of the Basidiomycota species, the cultivation of the bacteria species in the nutrient medium, during harvesting, and/or during lyophilization of the harvested species, vitamin D2 may be produced in the Basidiomycota by means of UV-B irradiation.

(29) Vitamin D is formed by the human body when exposed to sunlight or taken up with food. Thereby, vitamin D occurs in different forms, of which specifically vitamin D2 (ergocalciferol), occurring in fungi, and vitamin D3 (cholecalciferol), which is contained only in animal food products, are particularly relevant. Both compounds are converted in the liver to the pro-hormone 25-hydroxy cholecalciferol or 25-hydroxy ergocholecalciferol and subsequently converted to the vitamin D hormone 1α,25-dihydroxy cholecalciferol or 1α,25-dihydroxy ergocholecalciferol, respectively, in the kidney. Chanterelles contain, for example, 2.1 μg/100 g, mushrooms 1.9 μg/100 g vitamin D2. In addition, fungi have a high proportion of ergosterol, which is converted into vitamin D2 by irradiation with UV-B light sources. Thus, during the method of the invention, by irradiation causing a conversion of ergosterol to vitamin D2, a significant benefit may be achieved in the prepared product.

(30) The production of vitamin D2 may preferably performed via irradiation both in the submerged culture and the lyophilized variant of the mycelium. Hereby, the point in time of the irradiation may be chosen flexibly.

(31) Subsequent to cultivation of fungi and bacteria, the harvest of the obtained fermentation products takes place. These are subsequently processed to powders by drying technologies like lyophilization, milling or spray drying. The water content in the prepared product typically is in the range of 1 to 15 percent by weight. The fermentation product may, however, also be processed at high water content.

(32) Depending on the agricultural tributary or food tributary used, the powder appears light or brownish. Depending on the later application, different combinations of fungi and agricultural tributary/food tributary may be used. The protein powder has a neutral, nutty or fungus-like taste depending on the starting substrate used.

(33) Drying results in a product having a high protein and dietary fiber content, and, low fat content at the same time. When analyzing the technological properties, it turned out, that the water binding capacity, the oil binding capacity or the emulsifiability are comparable to those of plant proteins. Furthermore, the Maillard reaction is comparable to meat products. Additionally, in the fungal protein, the stickiness vis-à-vis plant proteins is increased. Likewise, parameters like hardness, chewability and gumminess are comparable, or, specifically regarding chewability, even better than for plant proteins. As to elasticity and stickiness when heated, there is likewise no difference to plant proteins. In total, the fungal protein has throughout better or almost as good as techno functional properties as plant proteins. In comparison to other food products, the biologic value is surprisingly high (e.g. Pleurotus sapidus cultivated on isomaltulose molasses: value of 73). As reference of a value of 100, hereby whole egg was used.

(34) Processing of the product may follow known recipes from sausage preparation.

(35) In context of the present invention, the following processes and measurement procedures are used:

(36) 1. Submerged Cultivation of Basidiomycota

(37) The submerged cultivation of Basidiomycota may be performed with different carbohydrate-containing agricultural tributaries or food tributaries, for example molasses from sugar production, cellulose-containing products from juice production such as carotte pomace and/or apple pomace or shells or press cakes from the oil production like sunflower seed shells and/or sunflower seed pomace or all further cellulose-containing agricultural tributaries or food tributaries.

(38) For cultivation, the Basidiomycota may be grown on malt extract agar (for example, 20 g/L malt extract, 15 g/L agar agar). For this, the agar plates are inoculated with an about 1 cm.sup.2 large piece of agar, vegetated with mycelium, sealed with parafilm, and cultivated in a incubator at 24° C., e.g. for 7 days. The plates vegetated to about 80% are stored at 4° C. and regularly over-inoculated according to the same procedure.

(39) For preparing a preculture, a 2 cm.sup.2 large malt extract agar piece, vegetated with Basidiomycota is added under sterile conditions to 200 mL of a 2% sterile malt extract medium (1 cm.sup.2/100 mL). The culture may be homogenized with a mixer; but this is not absolutely necessary. The incubation, for obtaining growth of Basidiomycota, may, for example, be carried out at 24° C., shaking (150 rpm) under the exclusion of light for 4-19 days (cf. Table 1).

(40) TABLE-US-00001 TABLE 1 Preculture periods of different Basidiomycota species in days [d]. Strain Preculture periods [d] AAE (Agrocybe aegerita) 11 LED (Lentinula edodes) 13 LSU (Laetiporus sulphureus) 13 PSA (Pleurotus sapidus) 6 PEO (Pleurotus roseus) 6 SRU (Stropharia rugosoannulata) 7 WCO (Wolfiporia cocos) 7

(41) For the Basidiomycota main culture, for example minimal medium M1 (4.5 g/L L-asparagine monohydrate; 2.4 g/L ammonium nitrate, 1.5 g/L potassium hydrogen phosphate, 0.5 g/L magnesium sulfate, 1 ml/L trace element solution (0.5 g/L iron (II) sulfate heptahydrate, 0.5 g/L zink sulfate heptahydrate, 0.002 g/L copper(II) sulfate pentahydrate, 0.002 g/L manganese(II) chloride tetrahydrate)) may be combined with a defined amount of substrate (cf. Table 2) in an Erlenmeyer flask (sealed with a stopper) and the obtained medium be adjusted to a pH value of 6 and sterilized in the autoclave for 20 min at 120° C.

(42) TABLE-US-00002 TABLE 2 Amounts of weighted agricultural tributary substrates in minimal medium M1 to obtain a uniform carbohydrate content of 15 g/L. Agricultural tributary substrate Concentration [g/L] Fresh apple pomace (AT) 112 Apple pomace (ATD) 24.6 Aronia pomace (ARO) 27.1 Lyophilized spinach 32.3 Pomegranate pomace 19.3 Beet molasses 27.2

(43) Subsequently, the Basidiomycota pre-culture is added to the medium mixture having a final concentration of 10% fungus pre-culture. Cultivation is carried out for 7-14 days at 24° C., shaking (150 rpm) under exclusion of light in the incubator. After full vegetation of the cultures, they are centrifuged for 10 min at 3283 g and the mycelium is washed three times with deionized water (DI water). In case of isomaltulose molasses as residue substrate, preferably M2 medium may be used for the cultivation of the Basidiomycota. The composition of the M2 medium is as follows: 3 g/L yeast extract, 1.5 g/L potassium dihydrogen phosphate, 0.5 g/L magnesium sulfate hydrate, 1.0 mL trace element solution. Per 100 ml M2 medium 10 ml Palatinose are added. The further procedure corresponds to the one described above.

(44) 2. Cultivation of the Basidiomycota in the 7.5 L Fermenter

(45) The fermenter is loaded with 5 L medium and a corresponding amount of substrate, the pH value is adjusted with 1M caustic soda to 6.0 and autoclaved. Inoculation is performed with 500 mL Basidiomycota pre-culture. The settings of the fermenters are: 150 rpm stirrer speed, 24° C. temperature and 0.3 vvm ventilation rate. The obtained fermentation products are subsequently processed by lyophilization at −70° C., 37 mbar pressure, until a specific water content of 8 to 12 weight-%.

(46) 3. Co-Cultivation of the Basidiomycota and Vitamin B12-Producers as Well as Glutaminase-Active Bacteria Illustrated by the Example of Pleurotus Rapidus (PSA) with Propionibacterium freudenreichii and Lactobacillus reuteri Cultivated on the Industrial Tributary Isomaltulose Molasses

(47) For co-cultivation of Basidiomycota and bacteria, first a pre-culture of Pleurotus sapidus (PSA) is grown. For this, a 2 cm.sup.2 large piece of malt extract agar vegetated with PSA is added under sterile conditions to 200 mL of a 2% sterile malt extract medium. The culture may be homogenized by means of a mixer (at maximum power level); this, however, is not absolutely necessary. Subsequently, incubation is carried out at 24° C., shaking (150 rpm) under exclusion of light for 7 days.

(48) Subsequent to successful growing the pre-culture, the main culture is inoculated. For this, 90 mL minimal medium M1 or M2 are combined with 10 mL isomaltulose molasses in an Erlenmeyer flask (sealed with a stopper), and the obtained medium is adjusted to a pH value of 6 and sterilized in the autoclave for 20 min at 120° C. Subsequently, the Basidiomycota pre-culture is added to the medium mixture having a final concentration of 10% fungus pre-culture. Cultivation is carried out for 7 days at 24° C., shaking (150 rpm) under exclusion of light in the incubator.

(49) In parallel, pre-cultures of Lactobacillus reuteri (L. reuteri DSM20016) and Propionibacterium freudenreichii (P. freudenreichii) subsp. freudenreichii (DSM20271) and shermanii (DSM4902) are made. For this, 10 μL of a common glycerol stock of the corresponding bacteria are added to 10 mL of the corresponding optimum medium (for L. reuteri: B12 assay medium (ready-made medium from Sigma Aldrich, St. Louis USA); for P. freudenreichii: Propionibacterium medium (5 g/l Caseinpepton, 10 g/l yeast extract, 16.8 g/L DL sodium lactate; pH value 6.7+/−0.2) and incubated over night at 30° C. (Propionibacterium) or 37° C. (L. reuteri) under anaerobe conditions (Wheaton tubes). The pre-cultures are adjusted to a bacteria concentration of about 6*10.sup.9 CFU/mL, to obtain a final concentration in the PSA culture of about 6*10.sup.7/100 mL. The initial OD.sub.595 nm in the fungus cultures with bacteria should be about 0.3 at the starting point.

(50) To the PSA culture aerobically cultivated for over 7 days, 1 mL of bacteria pre-culture (L. reuteri or mixture of P. freudenreichii subsp. shemanii and subsp. freudenreichii) is added and the cultures are incubated under anaerobe conditions in anaerobic pot with gaspack at 30° C. (culture with P. freudenreichii) or 37° C. (culture with L. reuteri) for 2 days. Subsequently, harvest of the protein pellet is carried out as explained in the following section. As negative control and for comparison of the Basidomycota growth without bacteria, a PSA culture without addition of the bacteria suspension is run along. The temperatures of the incubation period are maintained.

(51) The bacterial growth was determined by application of classical microbiological counting methods (L. reuteri on MRS agar; Propionibacterium on Propionibacterium agar) and, additionally, by determination of the OD at 595 nm at the point in time 0, i.e. directly after addition of the bacteria to the PSA main culture as well as at the end of the cultivation or before harvest.

(52) The total biomass was determined after harvest by weighing. First, wet weight was determined by weighing, subsequently the proteinpellet was dried at 80° C. and the weight was determined again. For this, the whole culture was put into a Büchner funnel which was equipped with a filter paper having a pore size of 7-12 μm. The Büchner funnel is first mounted on a suction bottle to which a vacuum pump is connected. By means of the vacuum pump, the whole liquid in the mycelium is filtrated by suction. The mycelium on the filter is put into an empty petri dish, of which the tara was defined and dried at 80° C. Subsequent to complete drying, the total biomass was determined in g on a fine balance.

(53) 4. Determination of the Vitamin B12 Content

(54) The determination of the vitamin B12 content in the different samples is conducted by means of an ELISA kit (Cloud-Clone Corp.). The micro titer plate contained in the kit is coated with a monoclonal antibody which specifically acts with cyanocobalamin (CNCbl). Biotin-tagged CNCbl functions as competitor to CNCbl from the samples and employed standards. Both compete for the antibodies on the plate. The binding occurs during a one-hour incubation phase, after which unbound conjugates are washed away. After several washing steps, an avidin-linked horse reddish peroxidase (HRP) is added. The avidin is bound by the biotin from the competitor and after a further incubation step the linked HRP forms, in combination with the substrate solution of the kit, a color complex. The color intensity in the wells is determined by means of measuring the optical density (OD) at 450 nm. It relates anti-proportionally to the CNCbl concentration present in the sample.

(55) Naturally occurring forms of cobalamin cannot be detected by means of the kit but must be converted to cyanocobalamin. A quantification of the cobalamin portions is carried out by employment of a standard series between 0 and 10.000 pg/mL cobalamin, which is run along with the experiments. The determined OD of the standard samples is plotted against the logarithm (log) of the standard concentration. A straight-line results, by which formula the logarithm of the sample OD may be calculated. An inversion of the logarithm provides the cobalamin content of the samples in pg/mL.

(56) The sample preparation for conversion of any cobalamin forms into cyanocobalamin is carried out as follows:

(57) The co-cultures are centrifuged for 10 min at 6.000 rpm, the supernatant rejected, the pellet washed with ddH.sub.2O one time. Subsequently, a defined amount of ddH.sub.2O is added and mixed with glass balls (diameter 0.25-0.55 mm) in a ratio of 1:2. The cell disruption is carried out by means of ultra sound using a sonicator (Sonifier 250 d Branson) for 10 min at 60% amplitude (1 min pulse, 1 min pause). The disrupted cultures are again centrifuged at 6000 rpm for 10 min. Subsequently the conversion into cyanocobalamin is carried out by addition of 10% KCN in a final concentration of 2% KCN in the corresponding culture followed by a 10-minute incubation at room temperature and subsequent storage on ice. Subsequently, ELISA is carried out according to manufacturer's specifications (ELISA KIT for Cyanocobalamin Cloud-Clone Corp.). As control for the conversion of cobalamin to cyanocobalamin, a defined amount of pure hydroxy cobalamin (5.000 pg/mL) is run along.

(58) 5. Determination of the Conversion of Glutamin into Glutamate

(59) In the amino acid profile of PSA, high glutamine contents occur, which may be converted into glutamate by using glutaminase-active bacteria. Lactobacillus rhamnosus and Lactobacillus brevis, for example, may be used as glutaminase-active bacteria. Moreover, it has been shown, that also Lactobacillus reuteri has a significant glutaminase activity. In addition to the mere confirmation of the glutaminase activity, it was analyzed subsequently, whether the addition of L. reuteri to lyophilized, milled PSA mycelium, cultivated on isomaltulose molasses, leads to conversion of the glutamine contained in the fungus mycelium to glutamate. The procedure for sample preparation as well as for determination of the conversion to glutamate is as follows:

(60) In Wheaton tubes, 0.1 g lyophilized, milled, heat-treated (30 min at 150° C.) PSA mycelium (cultivated with isomaltulose molasses as substrate) is weighted and 300 μl 1M sodium acetate buffer (pH 5.8), 10 μl alcalase 0.1% (v/v) and 800 μl flavourzyme 8% (v/v) are added and completed to 10 ml with H.sub.2O. Alcalase and flavourzyme are two enzyme mixtures containing different endo and exo proteases. These are for cleaving the proteins of the fungus mycelium, such that up-take by L. reuteri is facilitated. To one of the two batches 5 μL of an L. reuteri over-night culture is added, the other one serves a negative control. The incubation of both batches is carried out at 37° C. overnight. Subsequently, the cultures are centrifuged for 10 min at 6.000 rpm, the obtained supernatant rejected, and the pellet is washed with 50 mM sodium phosphate buffer with 1 mM PMSF final concentration and centrifuged for 5 minutes at 6.000 rpm. This procedure is repeated twice. 1 mL of 50 mM sodium phosphate buffer is added to the obtained pellet and the cells are disrupted by means of glass balls in a ratio of 1:2 w/v and vortexer for 10 min. Subsequently it is centrifuged for 10 minutes at 6.000 rpm and the obtained supernatant is transferred in a 1.5 mL reaction vessel. The samples are stored on ice until the analysis by means of a glutamate assay kit (abcam, Cambridge, UK). This measures free glutamate. The contained enzyme mix recognizes glutamate as specific substrate, leading to a proportional color development. This may by measured calorimetrically at an OD of 450 nm. These measurements are carried out with a plate reader Infinite 200 Pro from Tecan.

(61) 6. Determination of the Vitamin D Content

(62) By irradiation of the Basidiomycota mycelium with UV-B radiation (Arimed B 12 UV-lamps, JW Sales GmbH, Stuttgart) ergosterol located in the cell membrane may be converted into vitamin D2. Ergosterol and vitamin D2 are identified and quantified by means of HPLC-DAD (Absorption maxima: Ergosterol 282 nm, vitamin D2: 265 nm. The limits of detection and determination are determined according to DIN 32645 (calibration method) with n=7, significance level 99% and k=3. The limit of detection is 1.1 μg/ml, the limit of determination 4.0 μg/ml.

(63) The following system is used.

(64) Columns: Chromolith Performence Reserved/Phase-18 e 100-4.6 mm (length−diameter) (with precolumn) and EC 250/4 Nucleosil 100-5 C18, in series HPLC-DAD: La Chrom System L-7100/L-7200/D-7000/L-7455 from Merck Hitachi Eluents: Methanol, HPLC grade (A), acetonitrile HPLC grade (B) and 0.05% formic acid (C)

(65) TABLE-US-00003 Flow rate: 1 ml/min (gradient) Gradient: Time (min) % A % B % C 0 0 70 30 2 0 100 0 10 0 100 0 20 5 95 0 50 0 100 0 55 5 95 0 60 0 70 30

(66) Injection volume: 10 μl

(67) Software: HPLC System Manager HSM Manager, Version 4.1

(68) Standards: Ergocalciferol (≥98% Sigma), Cholecalciferol (99.9%, Supelco), Ergosterol (≥75.0%, Sigma) and 7-dehydrocholesterol (≥95.0%, Sigma)

(69) The quantification of vitamin D2 was carried out by ratios of peak areas analyte to internal standard of the calibration line and taking into account the sample preparation.
Vitamin D2 [μg(gDM).sup.−1]=(vitamin D2 [μg ml.sup.−1]*V.sub.MeOH (ml))/E[g])*(100/(100−% moisture)

(70) V.sub.MeOH: Volume of methanol, in which the residue was taken-up [ml]

(71) E: sample weight [g]

(72) % moisture: residual moisture determined by means of moisture analyzer

(73) Sample Preparation:

(74) The lyophilized fungus mycelium is milled in liquid nitrogen, about 2 g are weighted into a brown glass round bottom flask and saponificated with 50 mL ethanol, 4 mL sodium ascorbate solution (17.5 g in 100 ml 1M caustic soda), 10 ml KOH/H2=(50/50, w/w) and 0.5 ml internal standard vitamin D3 (200 μg/ml) for 1 h at 80° C. under reflux. Subsequent to addition of 50 ml DI water, cooling to room temperature and filtration, it is extracted with 50 ml diethyl ether, 50 ml n-pentane/10 ml ethanol, 50 ml n-pentane, 20 ml n-pentane. The organic phases were combined, washed trice with 50 mL 3% KOH in 5% ethanol and subsequently washed neutral with DI water, dried over sodium sulfate (overnight, 4° C.), filtered and the solvent reduced to dryness (40° C., rotary evaporator). The residue was taken-up in 1.5 mL methanol, dissolved by ultrasonication for 5 min and centrifuged (10 min, 18.000 g). Subsequent to membrane filtration (0.22 μm), the solution was used for quantification by means of HPLC.

(75) 7. Amino Acid Analytics

(76) The identification and quantification of the amino acids in the lyophilized fungus mycelium was carried out by means of amino acid analyzer. For this, the proteins were subjected to a total hydrolysis. Amino acid analyzer S 433 for protein hydrolysates from Sykam Columns: Separation column LCA K13 S/N, filter column LCA K04 S/N Eluents: Sodium citrate buffer solution pH 3.4 (A), Sodium citrate buffer solution, pH 10.85 (B), Regeneration solution (RegSol Na) Reagent: Ninhydrin, pH 10.85 Washing solution: Ethanol/isopropanol/water (250/250/500 v/v/v) Flow rate: 0.45 mL min-1 (gradient) Gradient:

(77) TABLE-US-00004 Time [min] A [%] B [%] RegSol Na [%] 0 100 0 0 5 100 0 0 11 95 5 0 13 80 20 0 25 70 30 0 29 30 70 0 31 20 80 0 33 10 90 0 41 0 100 0 49 0 0 100 49.1 0 0 100 52 0 0 100 52.1 100 0 0 Injection volume: 50 μL Software: Chromstar, Version 7 Standards: amino acid calibration solution (mixture of amino acids of known concentration for hydrolysates) (Sykam), L-Tryptophan (≥99.0%, Roth).

(78) For quantification of the amino acids a one-point calibration was carried out.

(79) The calculations are performed as follows:

(80) $m_{\left(\mathrm{factor}\right)}\left[\mathrm{mg}\mathrm{DM}\right]=\frac{E-\left[\mathrm{DM}-\left(\mathrm{DM}*\%\mathrm{moisture}\text{/}100\right]\right)}{V_{\left(\mathrm{HCl}\right)}}*V$$w_{\left(\mathrm{AS}\right)}\left[g{\left(100g\mathrm{DM}\right)}^{-1}\right]=\frac{c_{\left(\mathrm{AA}\right)}\left[\mathrm{mol}\right]*M_{\left(\mathrm{AA}\right)}\left[g{\mathrm{mol}}^{-1}\right]}{m_{\left(\mathrm{Factor}\right)}\left[g\right]}*100$$\mathrm{wherein}$$m_{\left(\mathrm{factor}\right)}\mathrm{Conversion}\mathrm{factor}\mathrm{from}\mathrm{nmol}{\mathrm{ml}}^{-1}\mathrm{to}\mathrm{nmol}{\mathrm{mg}}^{-1}$$\mathrm{DM}\mathrm{Dry}\mathrm{mass}\mathrm{with}\mathrm{residual}\mathrm{moisture}\mathrm{in}\mathrm{mg}$$\%\mathrm{moisture}\mathrm{Residual}\mathrm{moisture}\mathrm{determined}\mathrm{by}\mathrm{means}\mathrm{of}\mathrm{moisture}\mathrm{analyzer}\left[\%\right]$$E\mathrm{Sample}\mathrm{weight}\left[\mathrm{mg}\right]$$V_{\left(\mathrm{HCl}\right)}\mathrm{Volume}\mathrm{of}\mathrm{used}6M\mathrm{hydrochloric}\mathrm{acid}\left(3.4\mathrm{.10}\mathrm{.1}\right)$$V_{\left(\mathrm{Aliquot}\right)}\mathrm{Volume}\mathrm{of}\mathrm{evaporated}\mathrm{aliquot}$$w_{\left(\mathrm{AA}\right]}\mathrm{Mass}\mathrm{fraction}\mathrm{of}\mathrm{each}\mathrm{amino}\mathrm{acid}\mathrm{in}g{\left(100g\mathrm{DM}\right)}^{-1}$$c_{\left(\mathrm{AA}\right)}\mathrm{Molar}\mathrm{concentration}\mathrm{of}\mathrm{an}\mathrm{amino}\mathrm{acid}\mathrm{in}\mathrm{mol}{\mathrm{mL}}^{-1}$$M_{\left(\mathrm{AA}\right)}\mathrm{Molar}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{corresponding}\mathrm{amino}\mathrm{acid}$

(81) The sample preparation is carried out as described in the following.

(82) Acid hydrolysis for total amino acid determination:

(83) The lyophilized fungus mycelium is ground in a mortar, about 250 mg are weighted into a pyrex tube and 6 mL 6 M HCl (0.1% phenol) are added. For avoiding oxidation, oxygen is removed by introducing nitrogen. Hydrolysis is carried out for 24 and 48 h at 100° C. in a drying oven. Subsequent to cooling on ice, centrifugation (20 min, 4° C., 3.283 g) and membrane filtered (0.22 μm) are carried out. For separation of the acid, an aliquot (200 μL) is evaporated to dryness at 130° C. and taken-up in 1 mL sample dilution buffer (pH 2.20). Subsequent to diluting with sample dilution buffer (1:5), the solution is used for quantification by means of amino acid analyzer.

(84) Basic hydrolysis for the determination of tryptophan:

(85) The lyophilized fungus mycelium is ground in a mortar, about 250 mg are weighted into a pyrex tube and mixed with 6 mL 5 M NaOH (0.1% phenol). To avoid oxidation, the oxygen is removed by introducing nitrogen. Hydrolysis is carried out for 24 and 48 h at 100° C. in a drying oven. Subsequent to cooling on ice, centrifugation (20 min, 4° C., 3,283 g) and membrane filtration (0.22 μm) are carried out. An aliquot (200 μL) is evaporated to dryness at 130° C. and taken-up into 1 mL sample dilution buffer (pH 2.20). Subsequent to diluting with sample dilution buffer (1:5), the solution is used for quantification by means of amino acid analyzer.

(86) Oxidation before hydrolysis for cystein and methionin determination:

(87) The lyophilized fungus mycelium is ground in a mortar, about 250 mg are weighted into a pyrex tube and 5 mL 5 M oxidation solution (30% H2O2 in 98% formic acid and 0.1% phenol). The pyrex tubes are sealed and incubated for 16 h at 0° C. in an ice bath. By addition of sodium disulfite oxidation was quenched and 5 mL 6 M HCl (0.1% phenol) are added. Hydrolysis is carried out for 24 h at 100° C. in a drying oven. Subsequent to cooling on ice centrifugation is carried out (20 min, 4° C., 3.283 g) and membrane filtered (0.22 μm). The pH value is adjusted to 2.20 by means of 1 M sodium hydroxide. 200 μL are taken for evaporating and the residue is taken-up in 1 mL sample dilution buffer (pH 2.20). Subsequent to diluting with sample dilution buffer (1:5), the solution is used for quantification by means of amino acid analyzer.

(88) 8. Biological Value

(89) The biological value (BV) is the best-known method for estimating the quality of proteins in food. It is a measure of how much of an ingested food protein can be converted into proteins of the organism's body.

(90) The biological value results from the following equation:
BV=(retained nitrogen/absorbed nitrogen)*100

(91) The higher the biological value of the absorbed proteins, the less protein needs to be added to achieve a balanced protein and nitrogen balance. The most important criterion for the biological value is the composition of the amino acids in a food. The more proteinogenic amino acids are contained therein and the higher the content of essential amino acids, the higher the protein is classified as being of high value.

(92) Animal proteins generally have a higher biological value than plant proteins. As “reference protein” for the quality assessment of other dietary proteins, whole egg was selected, which is assigned a biological value of 100 or 1.0. The biological value of all other proteins is thus given in comparison to whole egg. However, the reference value “100” of whole egg does not correspond to a 100% conversion of the latter, which means that a value of 100 for the biological value can easily be exceeded, especially by combined foods.

(93) The biological value can be considerably increased by clever food combinations, as the amino acids of different foods complement each other, and deficits can be compensated (supplementary value). The combination of food proteins plays an important role especially in countries where the diet contains only few animal foods.

(94) By means of the following calculation, the biological value is calculated:

(95) $\mathrm{EAAi}\text{:}\sqrt[n]{\left(\frac{{\mathrm{Ile}}_T}{{\mathrm{Ile}}_R}*100\right)*\left(\frac{{\mathrm{Leu}}_T}{{\mathrm{Leu}}_R}*100\right)}*\left(\frac{{\mathrm{Lys}}_T}{{\mathrm{Lys}}_R}*100\right)$$\mathrm{EAAi}\text{:}\mathrm{Essential}\mathrm{Amino}\mathrm{Acid}\mathrm{Index}$$\mathrm{BV}=1.09*\mathrm{EAAi}-11.7$

(96) 9. Determination of Mineral Nutrients/Sugars

(97) The samples are digested with aqua regia in a micro wave system. Subsequently an ICP-MS (Inductively coupled plasma mass spectrometry) is carried out. An argon plasma is induced by a high-frequency current and the sample is heated to 5,000-10,000° C. The ions generated in the plasma are accelerated in the direction of the mass spectrometer's analyzer by an electric field, thus enabling the detection of elements and their isotopes.

(98) The determination of glucose and D-fructose and the detection of the conversion of D-glucose and D-fructose from the substrate is carried out enzymatically.

(99) 10. Determination of the Total Nitrogen Content According to Kjeldahl (Crude Protein)

(100) The total nitrogen content was quantified according to Kjeldahl (Kjeldahl 1883, modified according to Matissek et al. 2010), each in duplicate determination. The samples were weighted into nitrogen-free vellum boats and digested with a glass ball, half of a catalyst tablet and 15 mL concentrated sulfuric acid in a digestion flask at 400° C. for at least 3 h until the solution had turned into a greenish color. Subsequently a water vapor distillation was carried out. For this, caustic soda and some drops Sher indicator were added to the digestion flask. The formed ammonia was transferred into a boric acid and Sher indicator containing solution. Titration was carried out with 0.1 M hydrochloric standard solution.

(101) The crude protein content was calculated according to the following equation.

(102) $P\left[\%\mathrm{DM}\right]=\frac{a*1.4008*F}{E*10}*\frac{100}{100-\%\mathrm{moisture}}$$P\mathrm{Crude}\mathrm{protein}\mathrm{content}\left[\%\mathrm{DM}\right]$$a\mathrm{Consumption}\mathrm{of}0.1M\mathrm{HCl}\mathrm{standard}\mathrm{solution}\left[\mathrm{mL}\right]1.40081\mathrm{mL}0.1M\mathrm{HCl}\mathrm{corresponds}\mathrm{to}1.4008\mathrm{mg}\mathrm{nitrogen}$$F\mathrm{Conversion}\mathrm{factor}\mathrm{for}\mathrm{calculating}\mathrm{the}\mathrm{crude}\mathrm{protein}\mathrm{content}\left(\mathrm{comm}.\mathrm{factor}\mathrm{for}\mathrm{fungi}\text{:}4.38,\mathrm{for}\mathrm{PSA}\text{:}4.97,\mathrm{for}\mathrm{LED}\text{:}4.5\right)$$E\mathrm{Sample}\mathrm{weight}\left[g\right]$$10\mathrm{Conversion}\mathrm{factor}\mathrm{per}100g\mathrm{sample}\mathrm{and}\mathrm{mg}\mathrm{nitrogen}\mathrm{in}g$$\%\mathrm{moisture}\mathrm{Residual}\mathrm{moisture}\mathrm{determined}\mathrm{by}\mathrm{means}\mathrm{of}\mathrm{moisture}\mathrm{analyzer}\left[\%\right]$

(103) 11. Determination of Ash Content

(104) Subsequent to complete incineration at 550° C., the ash content was determined by means of difference weighing of the quartz crucibles. The calculation was carried out according to the following equation:

(105) $\mathrm{Ash}\left[\%\mathrm{DM}\right]=\frac{{\Delta }_{\mathrm{crucible}}*100}{E}*\frac{100}{100-\%\mathrm{moisture}}$$\Delta \mathrm{crucible}\mathrm{Difference}\mathrm{of}\mathrm{the}\mathrm{crucible}\mathrm{before}\mathrm{and}\mathrm{after}\mathrm{incineration}$$\mathrm{Ash}\mathrm{Ash}\mathrm{content}\left[\%\mathrm{DM}\right]$$E\mathrm{Sample}\mathrm{weight}\left[g\right]$$\%\mathrm{moisture}\mathrm{Residual}\mathrm{moisture}\mathrm{determined}\mathrm{by}\mathrm{means}\mathrm{of}\mathrm{moisture}\mathrm{analyzer}\left[\%\right]$

(106) 12. Determination of the Total Carbohydrate Content

(107) The total carbohydrate content of the substrates was determined by means of Orcinol-sulfuric acid assay. For this, 10 mg sample were hydrolyzed in 2 mL 2 M HCl (2 h, 100° C., 700 rpm) and, subsequently, membrane filtered. Subsequent to 1:50 dilution with purified water, 800 μL reagent solution (2 g L-1 Orcinol in conc. sulfuric acid) were added to 200 μL hydrolysate (or standard), shaken, and heated for 15 min at 80° C. Subsequent to cooling to room temperature, the total carbohydrate content was determined photometrically at 420 nm against water. Calibration was carried out with glucose (10-100 μg mL-1).

(108) 13. Determination of the Proportion of Fungus in the Lyophilizate Via Ergosterol

(109) When cultivating fungi on residual streams, substrate components may be present which are not or not completely degraded by the fungus and therefore still exist in the harvested mycelium. The proportion of fungus in this mycelium-substrate mixture can be determined via the ergosterol content, because ergosterol is found exclusively in fungi. To establish a calibration line, the corresponding fungus was cultivated in malt extract medium containing only soluble components and the biomass after cultivation, therefore, consists of 100% fungus mycelium. This was used for the calibration, in which the peak area ratio of ergosterol to 7-DHC (IST) was plotted against the mass of fungus mycelium [g DM].

(110) The sample preparation was carried out as in context of the vitamin D analytics. As internal standard 1 mL 7-dehydrocholesterol (7-DHC, 1 mg mL-1) was used. The absorption maximum of ergosterol and 7-DHC is at 282 nm. Subsequently, quantification was carried out at this wavelength.

(111) 14. Treatment of the Mycelium of the Fungus for Reduction of the RNA Proportion

(112) The mycelium of the fungus was subjected to a temperature treatment. For this, temperature ranges of 40-70° C. as well as incubation periods of 0-40 minutes were tested. Before and after the treatment, the crude protein content is determined, and the RNA is subsequently extracted by means of RNeasy® Plant Mini Kit. The quantification of the RNA concentration before and after the heat treatment was carried out by means of capillary gel electrophoresis.

(113) 15. Techno Functional Examinations

(114) Water binding capacity/oil binding capacity:

(115) An important parameter for the water-binding ability is the water-binding capacity (WBC). It indicates the mass of water that can be bound by one gram of the product. To determine this parameter, a defined mass of the sample material is saturated with water, whereby the water is added stepwise until the saturation point is reached. Just enough water is added to ensure that only a small aqueous supernatant is formed during centrifugation of the sample material. The advantage of adding an excess of water compared to the stepwise addition is a reduced amount of work. However, the disadvantage is that the excess water also separates soluble components of the sample material. This can lead to a significant falsification of the measurement result. For this reason, it was decided to add the water stepwise.

(116) The oil binding capacity (OBC) is to be considered analogous to the water binding capacity.

WORKED EXAMPLES

(117) The present invention is illustrated below by means of different exemplary processes and product examples which, however, are not to be regarded as limiting.

Example 1

Screening of Different Fungus-Substrate Combinations

(118) As selection criterion the growth [g DM L.sup.−1], the cultivation duration and the protein yield [g L.sup.−1], measured according to the method for determining the crude protein content according to Kjeldahl (cf. under 10. “Determination of the total nitrogen content according to Kjeldahl (crude protein)”) were used.

(119) Inter alia fresh apple pomace (Fischer) and apple pomace (Döhler) turned out to be highly suitable substrates for the production of Basidiomycota biomass in combination with Pleurotus sapidus (PSA) (culture period: 4 days, approx. 14 g DM L.sup.−1, approx. 3 g L.sup.−1 protein). Furthermore, also the isomaltulose molasses in combination with PSA (culture period: 4 days, approx. 11 g DM L.sup.−1, approx. 2.8 g L.sup.−1 protein), onion pomace in combination with PSA (culture period: 13 days, approx. 34.5 g DM L.sup.−1, approx. 3 g L.sup.−1 protein), and carotte pomace in combination with Lentinula edodes (LED) (culture period: 6 days, approx. 9 g DM L.sup.−1, approx. 2.3 g L.sup.−1 protein) turned out to be promising.

Example 2

Co-Cultivation of Pleurotus sapidus and Propionibacterium freudenreichii

(120) First, it was tested, which temperature ranges are tolerated by the used organisms. Thereby, it was found out that Basidiomycota show sufficient growth only until 27° C. and do hardly form any biomass after this raise in temperature even at optimum temperature. The used bacteria require higher temperatures for sufficient growth, preferably the optimum temperature for propioni bacteria is at 30° C.

(121) First, Pleurotus sapidus (PSA) in minimal medium M1, with 10% (v/v) isomaltulose molasses added, was cultivated for 24 h aerobically (10 ml PSA pre-culture and 90 ml M1/isomaltulose mixture) and, subsequently, different amounts of a bacteria pre-culture having about 6*10.sup.9 CFU/ml (P. freudenreichii subs. freudenreichii and subs. shermanii) were added. An incubation for 7 days under switching to anaerobe conditions was carried out at 30° C. Bacteria growth was observed at the end of the incubation period, specifically when using a 1 ml bacteria pre-culture. However, only little PSA growth/total biomass was observed (cf. FIG. 1).

(122) In further experiments, 10 ml PSA pre-culture in M1 medium and 10% isomaltulose molasses was aerobically incubated for 7 days, shaking (150 rpm) at 24° C. Subsequently, different amounts of bacteria pre-culture (approx. 6*10.sup.9 CFU/ml) consisting of P. shermanii and P. freudenreichii were added as well as switching to anaerobe conditions and a temperature shift to 30° C. and further incubation for 48 h were carried out. This variant, upon addition of 1 mL bacteria pre-culture, resulted in the greatest growth of bacteria and satisfying growth of fungus (determined optically) and sufficient total biomass. The control reaction with 0 μl bacteria pre-culture added and shifting the temperature to 30° C. yielded a total biomass, i.e. Basidiomycota biomass, of approx. 46 g/L, based on the dry mass (cf. FIG. 2).

(123) In a further series of experiments, the total biomasses of cultures of PSA as well as co-cultures of PSA and vitamin B12-producing bacterial strains, which were cultivated in different minimal media with isomaltulose molasses, were determined. Hereby, 10 ml PSA pre-culture were incubated for 5 days or 7 days in 90 ml M1 or M2 medium, each with 10% isomaltulose molasses added, aerobically at 24° C. and the total biomass was determined after harvest (cf. FIGS. 3a) and b), each 1. and 2. column). For further batches, the cultivation conditions were switched from aerobe to anaerobe after the 5-day incubation and incubation was continued at different temperature for 48 h (24° C., 30° C. or 37° C., cf. FIGS. 3a) and b), each 3. to 5. column). Subsequently, also the weight of the total biomass was determined. To further batches, after the 5-day incubation, bacteria pre-cultures (approx. 6*10.sup.9 CFU/ml) consisting of P. shermanii and P. freudenreichii or L. reuteri were added, it was switched to anaerobe conditions and a temperature shift to 30° C. or 37° C. was performed, and for 48 h cultivated under these conditions (cf. FIGS. 3a) and b), each 6. to 7. column). Subsequently, the determination of the total biomass was carried out as for all batches. In total, cultures in M2 medium showed over a period of 5 days a higher fungus biomass than cultures in M1 medium. However, the biomass could be further increased within 7 days incubation in M1 medium, while this was not the case for the M2 medium. Thus, both in the M1 as well as the M2 medium, total biomasses of up to 49 g/100 ml, based on the dry mass, could be achieved by cultivation of the Basidiomycota prior to addition of the bacteria species.

(124) Further, use of different amounts of PSA fungus pre-culture at unvaried bacteria amounts (1 ml, approx. 6*10.sup.9 CFU/ml) were tested. For this, 10 ml PSA pre-culture were incubated aerobically for 7 days in 90 ml M1 medium, with 10% isomaltulose molasses added. Subsequently, 1 ml of a pre-culture propionibacteria (approx. 6*10.sup.9 CFU/ml) was added, it was switched to anaerobe conditions and a temperature shift to 30° C. was performed as well as cultivated for 48 h under these conditions (cf. FIG. 4). Hereby, it could be shown that increasing amounts of PSA lead to higher total biomass but inhibit the growth of bacteria.

(125) In a further series of experiments, the growth of bacteria on Pleurotus sapidus (PSA) supernatants was determined (cf. FIG. 5). For this, PSA was cultivated on different agricultural tributaries and harvested. The culture on isomaltulose was kept in M2 medium for 5 days prior to harvest, the cultures on carotte pomace, onion pomace and apple pomace were kept in M1 medium over 4 days prior to harvest. In a further batch, the culture was kept in M1 medium on apple pomace for 2 days prior to harvest (cf. FIG. 5, “ATD 2d”). The media used for cultivation were, subsequent to the harvest of the fungus mycelium, used for cultivation of P. freudenreichii supspec. shermanii. In a control experiment, P. freudenreichii supspec. shermanii was cultivated on optimum medium (propioni bacteria medium). Hereby, it turned out that P. freudenreichii supspec. shermanii hardly grew on PSA culture supernatants. Also, further bacteria species showed only very little growth on PSA culture supernatants based on apple pomace as agricultural tributary after 5 days cultivation of the PSA mycelium and subsequent harvest (cf. FIG. 6, dotted lines) compared to growth on the corresponding optimum medium (cf. FIG. 6, straight lines).

(126) These results show that, specifically the form of co-cultivation with Basidiomycota, leads to particularly high bacteria division rates. Surprisingly, significantly lower bacteria division rates are achieved for the cultivation of Basidiomycota supernatants, i.e. on used media without Basidiomycota. Already after 2 days of PSA cultivation on apple pomace, the used media significantly inhibits growth of P. freudenreichii supspec. shermanii (cf. FIG. 5), whereas a significant bacteria growth is achieved in the co-culture also after 5 days of PSA cultivation prior addition of the bacteria (cf. FIG. 2-4).

Example 3

Conversion of the Glutamine Contained in the Pleurotus sapidus Mycelium into Glutamate by L. reuteri

(127) 1% lyophilized fungus mycelium was incubated with and without L. reuteri overnight at 37° C. and on the next day, the glutamate proportion was determined after digestion. The determination of the glutamate content on lyophilized, beforehand enzymatically digested Pleurotus sapidus (PSA) mycelium lead to the detection of an increased conversion of glutamine after fermentation by L. reuteri. Subsequent a triple determination, an average increase in glutamate content of 52% was determined (cf. FIG. 7).

Example 4

Determination of Vitamin B12 in Co-Cultures of Pleurotus sapidus and Vitamin B12-Producing Bacterial Strains as Well as in Bacteria Cultures Only

(128) 10 ml Pleurotus sapidus (PSA) pre-culture were incubated in 90 ml M1 medium, with 10% isomaltulose molasses added, for 7 days shaking at 24° C. Subsequently, 1 ml L. reuteri or P. freudenreichii supspec. freudenreichii and shermanii were added (1 ml, approx. 6*10.sup.9 CFU/ml) and incubation was continued for 48 h at 30° C. or 37° C. under anaerobe conditions. The cultures were centrifuged subsequent to incubation, the pellet was dissolved in H.sub.2O and sonicated for 10 min at 60% amplitude (1 min pulse, 1 min pause) together with glass balls in a ratio of 1:2 (glass balls/culture). Subsequently, the conversion of all cobalamins was carried out by addition of 10% potassium cyanide solution with a final concentration of 2%. After 10 min incubation at room temperature another centrifugation was carried out and the supernatant was analyzed by means of ELISA for the presence of cyanocobalamin.

(129) For the calculation of the amounts of vitamin B12 contained in the cultures, a standard curve of defined amounts of cyanocobalamin was prepared (cf. FIG. 8), which was used for determining the amounts of vitamin B12 contained in the cultures.

(130) In the following table, the present contents of vitamin B12 from the co-cultures are illustrated. Assuming that in the final product, e.g. vegan bratwurst, 25 g protein a used per 1 kg, the vitamin B12 content per 100 g bratwurst is about 0.3 μg.

(131) TABLE-US-00005 TABLE 3 Vitamin B12 content in co-cultures of Basidiomycota and bacteria. Amount of Amount dry co-culture mass for for covering covering the OD at μg Vit. B12 the daily daily Organisms of 450 Log (Vit. Conc. of Vit. in 100 ml requirement requirement of the co-culture nm B12-conc.) B12 (pg/ml) co-culture of Vit B12 (l) Vit B12 (g) PSA, L. reuteri 0.229 3.76 5738.25 0.057 5.22 29.8 PSA + 0.2521 3.73 5360.58 0.107 2.79 15.98 Propiobacterium freudenreichii sups. freudenreichii a. shermanii

(132) The vitamin B12 content in the bacteria cultures without Basidiomycota was also determined and was significantly higher (cf. Tab. 4). For this, the bacteria cultures were either anaerobically cultivated for 2 days and subsequently for 24 h aerobically or 3 days anaerobically at the corresponding optimum temperature of the bacterial strain. The bacteria content was approx. 6*10.sup.9 CFU/ml.

(133) However, such a production would be significantly more laborious and more prone to contamination, for which reason the approach of the co-culture is pursued.

(134) TABLE-US-00006 TABLE 4 Vitamin B12 content in differently cultured cultures of vitamin B12-producing bacterial strains. Amount of culture Amount of pellet Conc. Vit. B12 for covering the Conc. Vit. B12 in for covering the (ng/ml) daily requirement the pellet of daily requirement Bacterium Cultivation in culture of Vit B12 bacteria(μg/100 g) (g) L. reuteri 2 d anaerob, 12.4 4.03 n.a. n.a 1 d aerob L. reuteri 3 d anaerob 10.09 7.42 3   100 P. freudenreichii 2 d anaerob, 12.69 3.93 n.a. n.a. subs. 1 d aerob freudenreichii and shermanii P. freudenreichii 3 d anaerob 13.99 5.35 3.23 92.88 subs. freudenreichii and shermanii

Example 5

Quantification of the Fungus Proportion in the Lyophilizate Via Ergosterol Exemplified by the Cultivation of PSA on Apple Pomace

(135) The protein content and the proportion of Pleurotus sapidus (PSA) in the total biomass was measured for 6 days. Hereby, the proportion of PSA was determined via ergosterol measurement. The proportion of PSA as well as the protein content increase with the culture period. When determining the total weight of the mycelium, from day 4, a slight decreasing tendency was observable (cf. FIG. 9). The proportion of PSA in the lyophilizate was approx. 80% at the time of harvest.

Example 6

Results of the Determination of Mineral Nutrients and Sugars in PSA Mycelium, Cultivated on Isomaltulose Molasses as Well as Illustration of the Substrate Composition

(136) The conversion of glucose and fructose from the isomaltulose molasses (Palatinose molasses/PM) substrate by Pleurotus sapidus (PSA) as well as from the carotte pomace (K) by Lentinula edodes (LED) was quantified (cf. Tab. 5).

(137) TABLE-US-00007 TABLE 5 Conversion of D-glucose and D-fructose from the substrate. Basidiomycota Conversion D-glucose Conversion D-fructose species from the substrate [%] from the substrate [%] PSA - PM 75.2 ± 0.2 68.1 ± 0.3 LED - K 89.6 ± 0.3 98.9 ± 0.0

(138) The amount of glucose and fructose in the fungus mycelium as well as in the used substrate was determined (cf. Tab. 6).

(139) TABLE-US-00008 TABLE 6 Determination of D-glucose and D-fructose in the fungus mycelium and substrate. D-glucose D-fructose Sample [g (100 g DM).sup.−1] [g (100 g DM).sup.−1] PSA PM 4.9 ± 0.0 9.4 ± 0.2 Isomaltulose molasses 12.4 ± 1.7  19.0 ± 2.1  LED K 0.3 ± 0.0 0.1 ± 0.0 Carrot pomace 2.4 ± 0.1 7.0 ± 0.0

(140) TABLE-US-00009 TABLE 7 Composition of saccharides and heavy metal contamination in Pleurotus sapidus (PSA) cultivated on apple pomace (ATD), in the residue substrate ATD as well as saccharides composition in PSA cultivated on isomaltulose molasses (PM). Concentration Concentration PSA ATD [g/(100 g DM).sup.−1] ATD substrate [g/(100 g TM).sup.−1] Chitin 6.54 ± 1.79 Total glucan 9.22 ± 0.16 Total glucan 4.39 ± 0.97 Beta-glucan 5.59 Beta-glucan 3.42 Alpha-glucan 3.64 ± 0.09 Alpha-glucan 0.97 ± 0.05 Glucose 0.55 ± 0.02 Glucose 1.5 ± 0   Fructose 0.07 ± 0.01 Fructose 5.15 ± 0.02 Saccharose 0.34 ± 0.01 Saccharose 0.32 ± 0.06 Concentration Concentration PSA ATD [μg/(kg DM.sup.−1] ATD substrate [μg/(kg DM.sup.−1] Lead n.d. Lead n.d. Cadmium 13.3 ± 1.9 Cadmium 12.3 ± 1.9 Mercury n.d. Mercury n.d. Konzentration PSA PM [g/(100 g DM).sup.−1] Alpha-glucan 6.1 Beta-glucan 18.7 Total glucan 24.8

Example 7

Amino Acid Profile

(141) The total amino acid content of 29.41 g (100 g DM).sup.−1 was calculated as sum of the single amino acids. When comparing this content to the total crude protein content according to Kjeldahl (27.41 g (100 g DM).sup.−1), these values correlate well with each other. In sum, an amino acid spectrum of in total 18 amino acids results, amongst them the 8 essential as well as both the semi-essential amino acids arginine and histidine (cf. FIG. 16).

Example 8

Vitamin D2 Production

(142) In two series of experiments it was tested, whether there are differences in the irradiation of the already lyophilized fungus mycelium in comparison to the irradiation of the entire submerged culture in liquid state. The diameter of the crystallization dishes, in which light exposure was performed, was 9.5 cm in case of the lyophilizate (sample height: 0.8 cm) and 19.8 cm for the light exposure of the liquid culture (sample height: 1 cm). Already an exposure of only a few seconds was sufficient to produce so much vitamin D2 that the daily requirement of 5 μg is already exceeded by the consumption of 1 g dry fungus mycelium (cf. FIG. 13). Thus, for the future production, mycelium exposed and not exposed to light may be mixed to not exceed the daily requirement of vitamin D2. The irradiation of lyophilizate, which stems from the cultivation of PSA on isomaltulose and 10 min with UV light at a distance from the lamp of 10 cm, resulted in a vitamin D2 content of 0.2 μg/g dry mass.

Example 9

Decrease of the RNA Content

(143) By heat treatment of a PSA culture on isomaltulose molasses at the end of the incubation period, the RNA content could be significantly decreased in comparison with the untreated samples. Specifically, the combination of 40 minutes at 40° C. and 20 minutes at 70° C. lead to the significant decrease of the total RNA (18 S and 28 S RNA). Thus, a health benefit of the product can be achieved, which is particularly relevant for risk groups amongst the consumers (cf. FIG. 14).

Example 10

Techno Functional Examinations

(144) The following abbreviation are used in the following: “g” dried pomace (otherwise moist pomace); “[number] d”=number of days of the cultivation.

(145) Further, the following abbreviations are used for the Basidiomycota strains, the tributaries as well as the media:

(146) TABLE-US-00010 TABLE 8 Used strains and tributaries. Strain By-product stream AAE (Agrocybe aegerita) KTD (carotte pomace Döhler) LED (Lentinula edodes) KT (carotte pomace) LSU (Laetiporus sulphureus) AT (apple pomace) PSA (Pleurotus sapidus) ZT (onion pomace PEO (Pleurotus roseus) GA (pomegranate pomace) SRU (Stropharia rugosoannulata) Aro (aronia pomace) WCO (Wolfiporia cocos) BS (spinach) PM (isomaltulose molasses)

(147) TABLE-US-00011 TABLE 9 Liquid media used for media optimization. Na-Aspartate NH.sub.4NO.sub.3 (NH.sub.4).sub.2SO.sub.4 KH.sub.2PO.sub.4 MgSO.sub.4 SE-Sol. [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [mL L.sup.−1] M1 6.2 2.4 — 1.5 0.5 1.0 JA1 3.1 2.4 — 1.5 0.5 1.0 JA2.1 6.2 — 4.0 1.5 0.5 1.0 JA2.2 6.2 — 2.4 1.5 0.5 1.0 JA3 6.2 2.4 — 0.5 0.5 1.0 JA4 3.1 — 2.4 0.5 0.5 1.0 DI H.sub.2O with SE — — — — — 1.0 DI H.sub.2O — — — — — — SE: trace element solution, Na-Aspartate: L-aspartic acid monosodium salt monohydrate.

(148) TABLE-US-00012 TABLE 10 Liquid media used for media optimization.. Yeast Pepton extract (Soy) NH.sub.4NO.sub.3 (NH.sub.4).sub.2SO.sub.4 KH.sub.2PO.sub.4 MgSO.sub.4 SE-Sol. [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [g L.sup.−1] [mL L.sup.−1] M2 3.0 — — — 1.5 0.5 1.0 M2a 3.0 — 2.4 — 1.5 0.5 1.0 M2b 3.0 — — — 0.5 0.5 1.0 M3 — 3.0 — — 1.5 0.5 1.0 M3a 3.0 3.0 — — 1.5 0.5 1.0 M3b — 3.0 — — 0.5 0.5 1.0 SE: trace element solution

(149) Water Binding Capacity:

(150) The samples LSU_KTD_16T_M1_20.8.15, LSU_KTD_16T_M2_20.8.15, PSA_AT_M1_G_5.8.15, PSA_KT_G_5.8.15, PSA_KT_G_5.8.15_II, PSA_KTD_16T_M2_20.8.15 and PSA_ZT_20.2.15 all have a high water binding capacity. The influence of the agricultural by-product stream, however, is hereby not to be underestimated, because specifically fibers from carrots, apples or onions have high capillary forces, which also have a high water binding capacity. Considering the Basidomycete Pleurotus sapidus on isomaltulose molasses (PSA_PM), no functionality can be expected from the isomaltulose molasses used. The properties of mycelium from PSA_PM can be compared with pea protein isolate.

(151) TABLE-US-00013 TABLE 11 Water binding capacities. WBC/ WBC Protein Name Protein*100 [mL/g] [%] 112541_peaproteinisolate 3.50 3.2 90.1 113764_soyconcentrate 5.87 4.1 69.8 118770_soyisolate 7.45 6.7 90.2 AAE_AT_total6 30.45 5.1 16.7 AAE_ATD_G_10.8.15 31.10 5.8 18.6 AAE_ATD_G_10.8.15_II 35.73 6.6 18.6 AAE_BS_total2 12.37 3.2 26.1 AAE_GA_total7 27.56 5.1 18.6 AAE_KTD_16T_M1_20.8.15 23.04 5.0 21.8 AAE_KTD_16T_M2_20.8.15 28.42 5.5 19.2 AAE_P100_M1_16.2.15 8.43 1.1 13.3 LED_ATD_G_10.8.15 18.58 3.8 20.4 LED_ATD_G_10.8.15_II 22.20 4.5 20.4 LED_GA_16.4.15 19.94 3.2 16.3 LED_GA_16.4.15_II 21.50 3.5 16.3 LED_KTD_6T_M1_20.8.15 12.63 3.4 27.2 LED_KTD_M1_8.15 14.99 4.0 27.0 LED_P100_16T_M2_30.3.15 16.39 3.9 24.0 LSU_AT_total4 34.31 5.3 15.5 LSU_KTD_16T_M1_20.8.15 76.15 7.0 9.1 LSU_KTD_16T_M2_20.8.15 83.58 7.3 8.8 PSA_Aro_G_18.8.15 35.67 4.2 11.6 PSA_Aro_G_18.8.15_II 30.59 3.6 11.6 PSA_AT_total1 22.670 4.5 20.0 PSA_AT_M1_G_5.8.15 34.37 7.1 20.7 PSA_BS1_total 9 21.10 5.9 28.0 PSA_BS2_31.3.15 13.97 3.9 28.0 PSA_GA_total3 29.79 3.3 11.1 PSA_KT_G_5.8.15 28.91 7.3 25.1 PSA_KT_G_5.8.15_II 29.28 7.3 25.1 PSA_KTD_16T_M2_20.8.15 23.29 7.5 32.1 PSA_KTD_3T_M2b_29.9.15 13.20 3.1 23.5 PSA_KTD_4T_M2_14.9.15 7.24 2.2 30.7 PSA_KTD_6T_M2a_14.9.15 9.79 2.1 21.9 PSA_KTD_6T_M3_14.9.15 10.82 2.2 20.2 PSA_KTD_6T_M3a_21.9.15 10.51 1.9 18.5 PSA_KTD_9T_M3b_21.9.15 19.29 2.4 12.4 PSA_P100_M1M2_21.8.15 12.64 2.5 20.1 PSA_PM_M2 11.560 2.7 23.8 PSA_ZT_20.2.15 71.70 7.8 10.9 PSA_ZT_24.11.14 17.31 4.9 28.4 SRU_BS_total5 14.13 4.8 34.0 SRU_GA_total8 59.40 4.2 7.0 WCO_Aro_G_18.8.15 27.15 3.5 12.9 WCO_Aro_G_18.8.15_II 26.31 3.4 12.9 WCO_KTD_24T_M1_20.8.15 40.77 5.4 13.2 WCO_ZT_24.11.14 43.81 6.1 14.0 WCO_ZT_25.11.14_II 25.66 3.6 14.0

(152) Oil Binding Capacity:

(153) Some agricultural tributaries bind more oil than others (caused by the fiber proportion). Comparing the water binding capacity of Pleurotus sapidus on isomaltulose molasses (PSA_PM) to plant proteins, the Basidiomycota mycelium has a significantly higher oil binding capacity. This functionality may be explained by the formation of β-glucans and chitin by the basidiomycete.

(154) TABLE-US-00014 TABLE 12 Oil binding capacity. OBC/ OBC Protein Protein*100 [ml/g] [%] 112541_peaproteinisolate 1.09 0.9 90.1 113764_soyconcentrate 2.06 1.1 69.8 118770_soyisolate 1.14 0.9 90.2 AAE_AT_total6 35.43 5.1 16.7 AAE_ATD_G_10.8.15 41.30 6.9 18.6 AAE_ATD_G_10.8.15_II 46.49 7.8 18.6 AAE_BS_total2 14.56 3.1 26.1 AAE_GA_total7 38.15 6.3 18.6 AAE_KTD_16T_M1_20.8.15 32.18 6.2 21.8 AAE_KTD_16T_M2_20.8.15 35.22 6.0 19.2 AAE_P100_M1_16.2.15 24.95 2.5 13.3 LED_ATD_G_10.8.15 40.11 7.4 20.4 LED_ATD_G_10.8.15_II 50.49 9.5 20.4 LED_GA_16.4.15 29.08 3.9 16.3 LED_GA_16.4.15_II 28.98 3.9 16.3 LED_KTD_6T_M1_20.8.15 20.75 4.9 27.2 LED_KTD_M1_8.15 25.83 6.2 27.0 LED_P100_16T_M2_30.3.15 12.94 2.4 24.0 LSU_AT_total4 40.84 5.5 15.5 LSU_KTD_16T_M1_20.8.15 90.30 7.3 9.1 LSU_KTD_16T_M2_20.8.15 102.44 8.1 8.8 PSA_Aro_G_18.8.15 37.46 3.5 11.6 PSA_Aro_G_18.8.15_II 35.75 3.3 11.6 PSA_AT_total1 23.18 3.8 10.0 PSA_AT_M1_G_5.8.15 37.46 7.0 20.7 PSA_BS1_total 9 22.61 5.6 28.0 PSA_BS2_31.3.15 13.57 3.1 28.0 PSA_GA_total3 36.00 3.1 11.1 PSA_KT_G_5.8.15 32.70 7.5 25.1 PSA_KT_G_5.8.15_II 34.17 7.8 25.1 PSA_KTD_16T_M2_20.8.15 18.91 5.4 32.1 PSA_KTD_3T_M2b_29.9.15 27.97 5.8 23.5 PSA_KTD_4T_M2_14.9.15 14.97 3.9 30.7 PSA_KTD_6T_M2a_14.9.15 20.94 3.8 21.9 PSA_KTD_6T_M3_14-9.15 20.36 3.3 20.2 PSA_KTD_6T_M3a_21.9.15 23.90 3.6 18.5 PSA_KTD_9T_M3b_21.9.15 33.44 3.3 12.4 PSA_P100_M1M2_21.8.15 20.17 3.3 20.1 PSA_PM_M2 19.03 3.8 23.8 PSA_ZT_20.2.15 54.84 5.1 10.9 PSA_ZT_24.11.14 21.38 5.4 28.4 SRU_BS_total5 17.95 5.4 34.0 SRU_GA_total8 86.80 5.2 7.0 WCO_Aro_G_18.8.15 32.93 3.4 12.9 WCO_Aro_G_18.8.15_II 30.77 3.1 12.9 WCO_KTD_24T_M1_20.8.15 49.59 5.7 13.2 WCO_ZT_24.11.14 32.42 3.7 14.0

(155) Determination of the Biological Value:

(156) Determination of the biological value for Pleurotus sapidus, cultivated on isomaltulose molasses, results in 73. Compared to other food, this value is surprisingly high (cf. Tab. 13).

(157) TABLE-US-00015 TABLE 13 Comparison of the of the biological value for different food. Protein Biological value Egg 100 Pork 85 Beef 80 Poultry 80 Cow milk 72 Soy protein 81 Rye flour (82% milling) 78 Potatoes 76 Beans 72 Rice 66 Wheat flour (82% milling) 47

Example 11

Preparation and Recipe for a Vegan Bratwurst

(158) Browning and browning taste are formed in conjunction with reducing sugars and mycelium of Basidiomycota, obtained from submerged culture, with various carbohydrate-rich agricultural tributaries. For this purpose, 13 different bratwursts were prepared as fine (I to IV) or coarse (VII to XIII) or a fine commercial “meaty” bratwurst (VII) variant with the following designations:

(159) I: without protein

(160) II: pea protein isolate

(161) III: sunflower protein concentrate

(162) IV: soy protein isolate

(163) V: soy protein concentrate

(164) VI: Pleurotus sapidus cultivated on isomaltulose molasses (PSA PM)

(165) VII: normal bratwurst (meat protein)

(166) VIII: without protein

(167) IX: pea protein isolate

(168) X: sunflower protein concentrate

(169) XI: soy protein isolate

(170) XII: soy protein concentrate

(171) XIII: Pleurotus sapidus cultivated on isomaltulose molasses (PSA PM)

(172) The fine variation of the vegan bratwurst (I to VI) was prepared from 2 emulsions: a) Emulsion 1: 500 g emulsion 1 consisting of 0.85 g methylcellulose and 0.15 g corn flour with 13.3 g rape seed oil and 347 g ice water. The emulsion was prepared in the Thermomix (Year of manufacture 2010; TM31) on level 4 for 5 min. b) Emulsion 2: 500 g emulsion 2 consisting of 370 g ice water, 85 g rape seed oil, 4 g table salt, 1 g potassium chloride, 5 g citrus fiber, 20 g kappa-carrageen and 15 g pea starch instant. The emulsion was prepared in the Thermomix (see above) on level 6 for 5 min.

(173) Subsequently both emulsions were combined and emulsified in the Thermomix on level 5 for 2 min.

(174) Prior to the step of forming the emulsion, the following ingredients were added to 1,000 g emulsion consisting of 500 g emulsion 1 and 500 g emulsion 2:

(175) 5 g/kg Table salt

(176) 25 g/kg divers proteins or no additive: I: without protein II: pea protein isolate III: sunflower protein concentrate IV: soy protein isolate V: soy protein concentrate VI: Pleurotus sapidus on isomaltulose (PSA PM)

(177) and 30 g/kg seasoning having the following recipe:

(178) TABLE-US-00016 Dextrose fine 2.100 g Table salt 8.000 g Pepper white milled 2.500 g Mace milled 0.500 g Ginger milled 0.800 g Macis base 0.400 g Citric acid E330 0.700 g VM SMAK ® GOURMET WL 6.000 g Wurst flavor vegan 4.000 g Coarse broth w/o fe 4.900 g Lemon flavor (powder) 0.100 g

(179) The emulsion and all mentioned ingredients were blended for further 2 min at level 6 in the Thermomix (as before).

(180) The commercially available bratwurst (VII normal bratwurst) is prepared according to the following recipe:

(181) 200 g pork categorized according to GEHA SII

(182) 250 g pork leg meat

(183) 100 g pork cheeks

(184) 250 g pork bacon

(185) 200 g ice

(186) The following spices and additives were added to 1,000 g meat emulsion consisting of the above raw materials (in a total amount of 16 g/kg):

(187) TABLE-US-00017 Dextrose fine 1.254 g Table salt 1.95 g Phosphate E 450 1.05 g Emulsifier E471 0.675 g Emulsifier E472b 0.075 g Sodium carbonate E500 0.6 g Glucose syrup 1.15 g Citrate E331 1.75 g Pepper black base 2.8 g Silicic acid 0.008 g Macis Oleoresin liq 0.064 g Ginger Oleoresin liq 0.024 g Nutmeg base 0.16 g Cardamom milled 0.28 g Ginger de-oiled ground 2.96 g Seasoning 1.2 g

(188) The bratwurst is prepared in the cutter of the company Seydelmann (series K60) as follows:

(189) The meat is minced to the size of 3 mm using a MADO mincer (MEW613) and cut for 10 rounds with all ingredients at 3600 rpm. Then ⅓ of the ice is added and chopped for another 30 rounds at 3600 rpm. The lid of the cutter is cleaned from the inside, the remaining ice is added, and cutting is finished at 3600 rpm until the final temperature of 10° C. is reached.

(190) The coarse variation of the vegan bratwurst (VIII to XIII) was prepared from 2 emulsions and wheat texturate with a proportional water content: a) Emulsion 1: 500 g emulsion 1 consisting of 0.85 g methylcellulose and 0.15 g corn flour with 133 g rape seed oil and 347 g ice water. The emulsion was prepared in the Thermomix on level 4 for 5 min. b) Emulsion 2: 500 g emulsion 2 consisting of 370 g ice water, 85 g rape seed oil, 4 g table salt, 1 g potassium chloride, 5 g citrus fiber, 20 g kappa-carrageen and 15 g pea starch instant. The emulsion was prepared in the Thermomix on level 6 for 5 min.

(191) Subsequently both emulsions are combined.

(192) 900 g emulsion consisting of 450 g emulsion 1 and 450 g emulsion 2 and 100 g watered wheat texturate (33.35 g wheat texture dry and 66.65 g distilled water) were emulsified in the Thermomix at level 5 for 2 min.

(193) The following ingredients were added before the emulsion formation step.

(194) 5 g/kg table salt

(195) 25 g/kg diverse proteins or no additive: VIII: without protein IX: pea protein isolate X: sunflower protein concentrate XI: soy protein isolate XII: soy protein concentrate XIII: PSA PM

(196) And 30 g/kg seasoning according to the following recipe:

(197) TABLE-US-00018 Dextrose fine 2.1 g Table salt 8.0 g Pepper white milled 2.5 g Mace milled 0.5 g Ginger milled 0.8 g Macis base 0.4 g Citric acid E330 0.7 g VM SMAK ® Gourmet WL 6.0 g Wurst flavor vegan 4.0 g Coarse broth w/o fe 4.9 g Lemon flavor 0.1 g

(198) After thermal treatment for one hour at 85° C., the various bratwursts (FIG. 17) are cooled for 12 hours at 2° C. After a further 24 h, the bratwursts were tempered at room temperature for 3 h and subsequently thermally treated at 175° C. in the deep fryer for 3 min.

(199) Compared to plant proteins, the protein-rich Basidiomycota mycelium (PSA on isomaltulose molasses) achieved the best results when browning in bratwurst. A significantly browner color after frying increases consumer acceptance, as most other plant proteins do not show this browning.

(200) TABLE-US-00019 TABLE 14 Visual classification of the various bratwursts with different proteins referring to FIG. 14. Classification of the browning Without Pea Sunflower Soy Soy addition protein protein protein protein Basidiomycota Bratwurst/ of protein isolate concentrate isolate concentrate mycelium meat Without (−) (+) (+) (∘) (∘) (++) wheat texturate With (−) (+++) (++) (∘) (∘) (+++) (++) wheat texturate (−) = bad frying performance (+) = moderately good frying performance (++) = optimum good frying performance according to consumer expectation (+++) = excellent frying performance, time may be shortened (∘) = average frying performance

(201) TABLE-US-00020 TABLE 15 Lxaxb-measurement of the bratwurst after 3 min at 175° C. in the deep fryer. L [SD] a [SD] B [SD] Without wheat texturate Without protein 88.31 ±0.11 0.35 ±0.03 2.78 ±0.08 Pea protein isolate 87.55 ±0.12 0.30 ±0.05 3.04 ±0.09 Sunflower protein conc. 87.41 ±0.21 0.23 ±0.10 3.06 ±0.10 Soy protein isolate 87.50 ±0.14 0.14 ±0.02 2.80 ±0.07 Soy protein conc. 87.54 ±0.06 0.18 ±0.05 3.18 ±0.10 PSA_PM_15.07.2015 85.76 ±0.11 0.45 ±0.04 1.55 ±0.12 Normal bratwurst 87.31 ±0.02 0.20 ±0.05 2.99 ±0.03 With wheat texturate Without protein 86.35 ±0.11 0.15 ±0.05 1.79 ±0.10 Pea protein isolate 85.76 ±0.14 0.45 ±0.04 1.55 ±0.14 Sunflower protein conc. 86.03 ±0.17 0.42 ±0.08 2.14 ±0.31 Soy protein isolate 86.42 ±0.10 0.27 ±0.04 2.12 ±0.24 Soy protein conc. 86.49 ±0.24 0.22 ±0.06 2.08 ±0.26 PSA_PM_15.07.2015 85.73 ±0.12 0.35 ±0.04 1.57 ±0.07

Example 12

Co-Cultivation of Pleurotus sapidus, P. freudenreichii and L. reuteri

(202) Vitamin B12-producing L. reuteri or P. freudenreichii bacteria were added to a Pleurotus sapidus (PSA) pre-culture and further incubated aerobically or anaerobically for 24 h and 48 h, respectively, as described in Example 2. Glucose was additionally added in two of the cultures and 5,6-dimethylbenzimidazole (DMB), component of the vitamin B12 complex, was added in one of the cultures. The cultures were harvested after incubation and the increase in biomass and vitamin B12 content, respectively, was determined as described above. Specifically, by adding DMB the total biomass as well as the vitamin B12 content could be increased significantly.

Example 13

Co-Cultivation Using a Vegetarian Residue Substrate

(203) A fungus/bacteria co-cultivation according to the present invention was carried out as described above in Example 2 and the increase in total biomass and bacterial content (in CFU/ml) were determined. For this purpose, minimal medium mixed with Palatinose, as described above, on the one hand, and minimal medium mixed with the food tributary whey, on the other hand, was used.

(204) Both the biomass and the bacterial content could be increased by using whey as residual substrate compared to cultivations on Palatinose.

Example 14

Reactors for Co-Cultivation

(205) A Pleurotus sapidus (PSA) pure culture was cultivated in 4 L experimental reactors. Two exemplary designs of the experimental reactors are shown in FIGS. 20 and 21. High PSA biomasses could be achieved by cultivation in the experimental reactors, for example during 62 h, as shown in FIG. 20.