SE545256C2 - Liquid dairy replacement product containing fungi biomass and methods for producing the liquid dairy replacement product - Google Patents

Abstract

The present disclosure relates to a liquid dairy replacement product intended for human consumption, containing fungi biomass or protein derived from fungi biomass, with a protein content between 0.5 and 20 g / 100mL. The present disclosure also relates to methods for producing the liquid dairy replacement product.

Description

Consumption of dairy products has been shown to have an environmental impact that is not sustainable with an increasing world population. This is mainly due to the high impact and low resource-efficiency of growing cattle for milk production. Replacement of dairy products by plant-based equivalents has been a rising trend among consumers for both health, environmental and ethical reasons, and several sources of plant-based milk replacement beverages are today available in the market.

The most common plant materials used for manufacturing of plant-based milk replacement products include soybeans, almonds, oats, rice and coconut. Even though all these sources are environmentally beneficial compared to milk, there are still some sustainability concerns when it comes to large scale supply of these. Many of these crops still consume large amounts of water to produce, and as such cannot be considered the definite solution to milk's problems. Additionally, there are health and nutritional concerns to consumption of some plant-based dairy replacements. These plant-based sources often contain large amounts of antinutrients such as Phytic acid, which will encumber absorption of particular nutrients by the body. Phytic acid in specific inhibits the absorption of important minerals such as Zinc, lron and Calcium, which can be already present in lower levels in plant-based diets. Other concerns relate to the allergenicity of sources such as soy and gluten-containing crops or claimed presence of hormonal analogues for example in soybeans.

Filamentous fungal mycelia, often referred to as Mycoprotein, has been reported to be a high-quality protein. lt is also considered a non-allergen, contains a healthy amount of fibres and carbohydrates, and additionally the amount of phytic acid in mycoprotein is low. lts neutral taste is also an advantage to other plant-based sources, since it reduces the need to add sugar and flavours in order to mask unpleasant taste notes. However, mycoprotein is a fibrous, resistant food product usually applied in meat replacements due to its natural form in a mycelial structure. ln view of the above, the object of the present disclosure to provide an improved dairy replacement products and method for producing the same.

SUMMARY OF THE INVENTION One or more of the above objects may be achieved with liquid dairy replacement product in accordance with claim 1, a method for producing the liquid dairy replacement product in accordance with claim claim and/or claim Further embodiments are set out in the dependent claims and in the following description.

A liquid dairy replacement product intended for human consumption according to the present disclosure contain a fungi biomass or protein derived from fungi biomass and has a protein content between 0.5 and 13 g / 100mL.

There is a rising concern for the environmental impact with the use of dairy product. ln view of the plant-based milk replacement products, such as soybeans, almonds, oats, rice and coconut have been developed to replace the use of dairy products. However, even though all these sources are environmentally beneficial compared to milk, there are still some sustainability concerns when it comes to large scale supply of these. Many of these crops still consume large amounts of water to produce, and as such cannot be considered the definite solution to milk's problems. Additionally, there are health and nutritional concerns to consumption of some plant-based dairy replacements. ln view of this, there has been developed a liquid dairy replacement product intended for human consumption according to the present disclosure contain a fungi biomass or protein derived from fungi biomass. Filamentous fungal mycelia, often referred to as mycoprotein, has been reported to be a high-quality protein, in which this protein contains all the essential amino acids, and has been found to have muscle building properties as good or better than milk. Hence, the liquid dairy replacement product according to the present disclosure provides both environmental and health benefits compared to the products currently on the market.

The liquid dairy replacement product is free from dairy products, such as milk, and is liquid in room temperature.

The phytic acid content may be between 0 and 0.5 g/100 mL. A problem with plant based liquid dairy replacement products is the presence of phytic acid. Phytic acid is the primary vtfay phosptiortss is stored in many ptanis, inciuding beahs, Seeds, and nuts. Wiien phytic acid is consurned, it binds to ether ntinerals to create phytates and thus prevent access to these minerais for the body.

The liquid dairy replacement product may be a solution or suspension of fungi biomass particles with a concentration of biomass bet\Neen 1% and 10%.

The product may be an oil in Water emulsion comprising oil droplets and said fungi biomass being in an aqueous solution or suspension, the oil in water emulsion having a fat content within the range of from 1 to 30 mL/ 100mL. This has been found to give a smooth and thicker mouthfeel, and higher concentrations become important in the formulation of cream-like products.

The oil droplets may have droplet sizes within the range of from 1 to 100 um. This enables a stable emulsion and benefits in terms of taste and consistency.

The liquid dairy replacement product may have a foaming capacity within the range of from 50% to 250% height of foam volume compared to height of initial liquid, as measured according to the foaming capacity test disclosed herein. The high foaming capacity is desired in products such as cappuccino or other frothed dairy drinks.

The liquid dairy replacement product may have a foaming stability of at least 250 min before collapse of foam, as measured according to the foaming stability method as disclosed herein.

The liquid dairy replacement product may have an emulsification capacity of from >0 tom2/mg of fungal biomass powder, preferably within the range of from 0.1 to 0.6 m2/mg powder, as measured according to the emulsification capacity method as disclosed herein.

The liquid dairy replacement product may have an emulsification index between 20% and 70%, preferably between 25% and 50%.

A method for preparing a liquid dairy replacement product according to the present disclosure comprises the steps of a) cultivating fungi of in a liquid fermentation in an aerated bioreactor, to obtain a fungi biomass; b) vacuum dehydration or by freeze drying; and drying the fungi biomass obtained in step a) either by controlled low c) suspending the dried fungi biomass obtained in step b) in an aqueous solution to form a suspension containing between 1 and 20 g/L of dry biomass content and to obtain the liquid dairy replacement product.

The method may include mixing of the dried fungi biomass suspension, such as in a high- shear homogeniser, with a vegetable oil to obtain an oil in water emulsion. ln step c), the aqueous solution or the suspension obtained may be adjusted to a pH within the range of from 5 to The aqueous solution in step c) may have a viscosity within the range of from 2 mPa-s to 400 mPa-s. This may be obtained by including viscosity increasing additives, such as xanthan gum. This may enhance the suspension and prevent sedimentation of the dried fungi biomass.

The fungi biomass suspension may be mixed with the vegetable oil at least 1 min at 20,000 rpm or more. This has been seen to provide a stable emulsion with suitable oil droplet sizes.

A method for producing a liquid dairy replacement product according to the present disclosure comprises the steps of; a) cultivating fungi in a liquid fermentation in an aerated bioreactor, to obtain a fungi biomass; b) c) providing the fungi biomass obtained in step a) or b), i.e. in fresh, frozen or optionally drying or freezing the fungi biomass obtained in step a) powdered form, in an aqueous solution to form a suspension containing between 1 and 20 g/L of dry biomass content; adjusting the pH of said suspension to a value within the range of from 10 and 13; subjecting the suspension from step d) to a high-shear mixing step, optionally with a subsequent high pressure homogenization step, while keeping the pH within the range of from 10 to 13, thereby extracting protein from the fungi mycelium; and f) adjusting the pH to to a value within the range of from 5.5 to 7.5 and thereby obtaining the liquid dairy replacement product.

Such method for preparing a liquid dairy replacement product according to the present disclosure has been found to provide the liquid dairy replacement product with a high protein content, such as between 0.5 and 20g/100 mL.

The suspension obtained in step f) may be mixed, such as in a high-shear homogeniser, with a vegetable oil to obtain an oil in water emulsion.

A method for producing a liquid dairy replacement product according to the present disclosure comprises the steps of; a) cultivating fungi in a liquid fermentation in an aerated bioreactor, to obtain a fungi biomass; b) optionally drying or freezing the fungi biomass obtained in step a) c) providing the fungi biomass obtained in step a) or b), i.e. in fresh, frozen or powdered form, in an aqueous solution to form a suspension containing between 1 and 20 g/L of dry biomass content; d) adjusting the pH of said suspension to a value within the range of from 10 and 13; e) subjecting the suspension from step d) to a high-shear mixing step optionally with a subsequent high pressure homogenization step while keeping the pH within the range of from 10 to 13, thereby extracting protein from the fungi mycelium; and f) adjusting the pH to a value within the range of from 3.5 to 4.5 to promote protein precipitation; 9) h) collecting and drying the precipitates, optionally with freeze drying or spray drying; and suspending the dried obtained in step in an aqueous solution to obtain the liquid dairy replacement product.

Such method for preparing a liquid dairy replacement product according to the present disclosure has been found to provide the liquid dairy replacement product with a high protein content, such as between 0.5 and 13g/100 mL.

The suspension obtained in step h) may be mixed, such as in a high-shear homogeniser, with a vegetable oil to obtain an oil in water emulsion. The suspension may be mixed with the vegetable oil at least 1 min at 20,000 rpm or more.

The viscosity of the final preparation may be adjusted to a value within the range of frommPa-s to 400 mPa-s, optionally with addition of xanthan gum, to provide a suitable viscosity of the final preparation.

BRIEF DESCRIPTION OF THE DRAWINGS Fig.

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Fig.is a graph illustrating the results from a phytic acid analysis of fungi biomass and plant-based dairy replacement products; is a graph showing the water holding capacity (WHC) of fungi biomass after being dried using different drying methods; is a graph showing the result from Turbidity measurements on fungi biomass comprising different additives and suspended in water having different pH values; is a graph showing result from Turbidity measurements on fungi biomass suspensions with varying viscosity; shows images obtained from a light microscope of oil droplets in emulsions according to the present disclosure; is a graph illustrating particle size distribution in emulsions prepared with freeze-dried fungal biomass at different concentrations; is a graph illustrating oil droplet size of emulsions stabilized with fungal biomass according to the present disclosure using a high shear mixing and a high-pressure homogenizer; Fig.

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Fig.is a graph showing the extraction of fungi protein from fungi biomass at different pH; is a graph showing amount soluble protein from different emulsion formulations; shows the oil droplet size of emulsions comprising freeze dried fungi protein isolate (left) and spray-dried fungi protein isolate (right); shows the particle size distributions of emulsions with 128 mg powder/mL oil. The left image shows the emulsion mixed in high shear mixer and the right image illustrates the emulsion mixed with high-pressure homogeniser, FDB; freeze-dried biomass, HPH; high-pressure homogeniser, FDPI; freeze- dried protein isolate, SDPI; spray-dried protein isolate; shows the emulsifying capacity of different emulsions according to the present solution, with FDB; freeze-dried biomass, FDPI; freeze-dried protein isolate, SDPI; spray-dried protein isolate shows the foaming properties of fungi biomass powder vs protein isolate. FDB; freeze-dried biomass, FDPI; freeze-dried protein isolate, SDPI; spray- dried protein isolate shows the effect of pH in the protein extraction from fungi biomass using different extraction methods; shows emulsification index for different fungal biomass stabilised emulsions according to the present disclosure; shows emulsification index for different fungal biomass stabilised emulsions according to the present disclosure compared to commercial emulsifiers; shows foaming properties of liquid dairy replacement product according to the present disclosure; shows foaming properties of fungi biomass in a liquid dairy replacement product according to the present disclosure compared to commercial plant- based substances; shows a comparison of the environmental impact of a liquid dairy replacement product according to the present disclosure and other plant- based liquid dairy replacement products.

DETAILED DESCRIPTION The invention describes the creation of mycoprotein-based drinks through different methods. These drinks are composed by 0.5% to 13% w/v protein content and can be milk or dairy replacements, as well as shakes, liquid meals or other similar products. For creating this product, fungal biomass from the mycelium of a fungal species with a protein content between 45% and 65% in dry weight may be used so that the resulting drink contains the desired protein content. Between 1% and 10% of fungal biomass can be used to achieve this necessary protein content. Different methods can be used to extract and dissolve or resuspend the fungal protein into a liquid phase.

The first method to create such product relies on drying of the fungal biomass to a fine powder and resuspending such powder in water or solution to create a drink. For this, the drying may be done at low temperatures, such as -20°C to 15°C, using freeze drying or variations of controlled low vacuum dehydration technologies so that the water affinity and the ability to rehydrate of the powder are not compromised and dissolution is possible. For such formulation, different solutions, such as buffer solutions, hydrocolloid solutions or emulsifiers can be used to produce a quality product such as increasing the viscosity of the solution in order to avoid a too fast precipitation of the powder. ln another method, the cellular contents from the fungi mycelium including protein are extracted to a liquid media, which is then directly used for drinks formulations. For this extraction, a pH between 10-13 is used in conjunction with a mechanical method to break the cell walls in order to ensure maximum protein extraction. The pH is then normalized to food acceptable values such as pH 5.5 to 7.5, optionally to pH 6.0 to 7.0, for product formulation. ln a third method, protein is extracted from fungal biomass and concentrated into a powder with a protein concentration between 70% and 80%. Extraction of protein is based on using pH values between 10-13 in conjunction with a mechanical method to break the cell walls for ensuring maximum protein extraction. The solution is then shifted to pH 3.5- 4.5 to promote protein precipitation. The precipitate is collected and dry. Drying can be done with different powder drying methods such as freeze drying and spray drying.

Dairy replacements and drinks can then be formulated by using any of the methods above and promoting mixture of oils using emulsification techniques. Different liquid vegetable oils, such as canola (rapeseed) oil can be used, while the water phase can be pure water or for example a food graded buffer solution, such as a phosphate buffer. The water phase of the emulsion can be fortified with different vitamins or minerals for further nutritional benefits. The fat content of the emulsion can be up to third of the volume, depending on intended use. The fungal proteins are able to act as an emulsifier for the oil- in-water emulsions, promoting formation of small oil droplets, less than 100 um in diameter. Small oil droplets are beneficial for emulsion stability, resulting in less phase separation in the end product. Here it is shown that fungal biomass can create emulsions with an emulsification capacity between 0.1 and 0.6 m2/mg powder, indicating the ability to create small, stable oil droplets with low amount of powder. Size can be measured using laser light scattering. Emulsion stability can be achieved using the fungal proteins without further additives. The fungal emulsion drinks are naturally white in colour.

This emulsification property is also related to foaming properties, in which fungal proteins are here shown to create larger volumes of foam than other proteins and stabilize this foam phase without other additives. High foaming properties is of interest in for example cream or egg replacer applications, but also for making foamed coffee drinks such as cappuccino.

Example 1. Fungi biomass production and suitability for beverage applications Production of fungi biomass A fungal spore suspension was prepared by flooding a PDA plate culture with 10-20 mL of sterile water and spores scraped off the surface with a disposable, sterile spreader. Spores were counted in a hemocytometer under a light microscope, and used directly as inoculum for liquid cultivations. Fungi cultures were cultivated in Erlenmeyer flasks (volumes 100-2000 mL) with or without baffles, filled with liquid growth medium to a maximum of 20% of the total flask volume. 1 mL of spore suspension (10 ^7 spores/mL) per 100 mL of growth media was added to each flask, followed by incubation at 30-35°C for 18-24h under shaking (100-150 rpm).

Sterilisation of the liquid in the bioreactor was done by heating up the liquid with steam (via the bioreactor's double jacket) to 121 °C and 1 bar overpressure for 20min. Upon sterilization, A volume of 30 L of fungi culture obtained from a 16-24h rich media preculture was used to inoculate 300 L of media in a 400 L stirred-tank bioreactor using the media composition described previously. The pH was adjusted to 4.0-5.5 with 5M NaOH. Fermentation conditions were kept at pH 4.0 using NH3 as a base for pH titration, an air flow of 120 L/min (0.6 vvm) and a temperature of 30-35°C were kept constant with a stirring of 200 rpm. The fermentation process was carried for 24h and biomass was harvested after this period. 50L from this culture was used to inoculate a volume of 500 L in a 600 L bioreactor and the process was repeated for an additional 24h.

Macronutrient and mineral analysis Biomass from two different fermentation batches were used for experiments, where both samples prior and after heat treatment were collected. The dry matter of fungal biomass harvested from bioreactor cultivations was determined by first pressing all water down using a centrifuge and then drying samples at 105°C overnight and measuring the weight of the sample before and after drying, to determine the mass of water evaporated. Protein content was determined with Dumas combustion method (FlashEA 1112 Element Analyzer, Thermo Finningan, US) where nitrogen content was determined and converted to protein content with factor 6.25. Additional nutritional composition of the fungal biomass was analysed in detail by an external accredited laboratory (ALS Scandinavia AB). One representative example of the nutritional composition of the dry biomass showed the following values (per 100 g): 340 kcal (or 1400 kJ), 60.29 g protein, 3.74 g carbohydrates, 5.97 g fat, 12.30 g fibre. One representative example of the nutritional composition of the wet biomass showed the following values (per 100 g): 85 kcal 13 (or 350 kJ), 15.07 g protein, 0.94 g carbohydrates, 1.49 g fat (of which 0.34 g saturated fat, 0.40 g monounsaturated, 0.68 g polyunsaturated fat), 3.08 g fibre.

Regarding micronutrients, the following values were obtained for one representative measurement of the dry Rhizopus biomass (per 100 g): 509.1 mg calcium, 1004.5 mg potassium, 116.5 mg magnesium, 148.6 mg sodium, 238.5 mg sulfur, 2550.2 mg phosphorus, 14 10.8 mg iron, 15.5 mg zinc, 5.2 mg copper.

Phytic acid analysis Phytic acid was measured using a Phytic acid (phytase) /Total phosphorous colorimetric quantification kit (Megazyme, Wicklow, Ireland) according to manufacturer's indications. Shortly, Phytic acid was extracted using 0.66M hydrochloric acid and mixed for 3h. The sample was then centrifuged, and the supernatant transferred and neutralized. This sample was then used in an enzymatic dephosphorylation reaction composed of a first reaction with a phytase solution, and then secondly with an alkaline phosphatase solution, and then addition of a trichloroacetic acid solution. Phosphorous was then determined by colourimetry in a spectrophotometer at 655 nm, using a phosphorous calibration curve.

The results of the Phytic acid analysis is illustrated in figure Example 2. Creating a fungi biomass powder as protein concentrateDehydration methods Fungal biomass samples to be used in dehydration were first dewatered as much as possible using dewatering techniques such as centrifugation and decanting, resulting in a dry matter level between 25% and 75% depending on parameters and equipment.

Fungal biomass obtained from example 1 was dehydrated using conventional convection air drying. The fungal mycelium biomass was dried at 50°C or 70°C for 6h or overnight (until there was no significant change in sample weight). Samples dried through hot air drying resulted in a dark-coloured, compact and extremely hard mass.

The fungal biomass from example 1 was also freeze dried. For this, the biomass was cut in cubes of 1cm and frozen for 24h at -20°C. After frozen, samples were placed in an Alpha 1-4 LSCplus freeze dryer set to shelf temperature of -10°C, vacuum between 1 and 3 mbar using a rotary vacuum pump, and condenser temperature at -86°C. The product temperature was monitored and the drying was deemed complete when the product did not show a cooling from ongoing sublimation. The time to a dry product averaged at 64h. Products from freeze dry showed a bright white colour similar to the fresh product, with an intact structure similar to the original product.

For controlled low temperature vacuum dehydration, the fungal biomass from example 1 was subjected to a customized vacuum process at low temperature. The samples were chilled in a fridge to a stable temperature of 10°C. The samples were then placed in a vacuum chamber with shelves regulated to be kept at 10°C. The samples were spread among the shelves so that all pieces would be in contact with the regulated surface. The chamber was also connected to a condenser with a temperature between -50°C and - 86°C. The chamber was subjected to a vacuum pressure of 4 mbar. Samples were collected every hour and water content was calculated by measuring the original weight of the sample and the dry weight by drying at 105°C overnight.

Water holding capacity Dehydrated fungal mycelium biomass obtained from chilled vacuum dehydration at 10°C, freeze drying and convective oven drying at 50°C was grinded through a mill in a "fine" particle setting. 1g of powder was hydrated with excess water for 5 minutes and filtered. The wet mass was weighted and the water holding capacity (WHC) was calculated as: WHC = (weight of rehydrated biomass - weight dry biomass) /weight dry biomass. Figure 2 illustrates the water holding capacity of the fungal mycelium biomass dehydrated with different drying methods.Example 3. Use of fungi biomass powder for creation of liquid emulsions, suspensions and solutions Resuspension of fungi biomass Fungi biomass was added to water at pH 7.5 in a 1% w/v concentration. Alternatively, the fungi biomass was resuspended in water at pH 1.0 or pH 12.0 and solutions of 5% NaCl, 10% Sucrose, 5% CaCl2 or 5% KH2PO4. Turbidity was measured over time by measuring the solution absorbance value at a wavelength of 600 nm and normalizing absorbance at t=0 as 100%. The results for turbidity over time for the fungi biomass dispersion in the different solutions is shown in Figure 3 showing how NaCl and Sucrose can be added to solutions without affecting the stability of fungal biomass suspensions, but Ca2+ addition promotes a higher rate of precipitation of the fungal biomass powder.

Solutions of xanthan gum were also prepared in order to increase the viscosity of the solution and a suspension of biomass was prepared and measured in the same way. Concentration of xanthan gum has been directly and linearly correlated in literature with increase in viscosity values. Figure 4 shows that the biomass suspension is highly stabilized by the addition of small amounts of xanthan gum, in which the effect of reduction in the precipitation rate is observed with lowest concentrations, in this case of 0.025%, and having a maximum effect at 0.1%, which is not improved with further concentrations of xanthan gum. 0.1% Xanthan gum solution has a viscosity aroundmPa-s, while a 0.2% solution has a viscosity of 400 mPa-s (CPKelco, 2008).

Creating oil-in-water emulsions using fungi biomass powder Fungal biomass was freeze dried for 6 days to create a powder which was evaluated as an emulsifier by creating oil-in-water emulsions according to method described in Östbring et al., 2020. Emulsions were prepared by mixing 2 mL phosphate buffer (0.005 M, 0.2 M NaCl, pH 7) with 1 mL canola oil and adding biomass powder in the following concentrations 8, 16, 32, 64 and 128 mg powder/mL oil. The emulsions were thereafter mixed in a high-shear homogeniser (Ystral, D-79282, Ballrechten-Dottingen, Germany) for 1 min at 24,000 rpm. After mixing, the emulsions Were incubated at 4°C for 1 h to stabilize.

Emulsions were first evaluated in a light microscope (Olympus BX50 fluorescence microscope, Tokyo, Japan) which can be seen in Figure 5. Oil droplets of emulsionscreated with a) 64 mg/mL freeze-dried fungal biomass powders, b) 128 mg/mL freeze- dried fungal biomass powders. The white scale bars correspond to 50 um.

Figure 5 shows that it was possible to form emulsion droplets with freeze-dried fungal biomass. After incubation of emulsions, particle size distribution was measured by static laser light (Mastersizer 2000, Malvern Instruments, Worcestershire, UK). Emulsions were added to an obscuration rate of between 10-20% and refractive indexes were set to 1.33 and 1.46 for the serum and oil phase, respectively. The particle size distributions can be seen in Figure 6 and further emulsion data is presented in Figure Figure 6 i||ustrates the particle size distributions of emulsions created with freeze-dried fungal biomass powders at different concentrations. The particle size distribution of emulsions made with freeze dried fungal biomass shows an overall decrease in droplet size with an increase of powder concentration. However, with increasing concentration there is also an increase in larger particles (> 100 um) which is likely to be powder aggregates. ln-order to try and minimise these aggregates and create even smaller emulsion droplets, a high-pressure homogeniser was used. Emulsions with 1 mL canola oil and 99 mL distilled water and 64 or 128 mg freeze dried fungal biomass/mL oil samples were mixed. First, the samples were vortexed for 5 min before they were put in a high-pressure homogeniser (Niro Soavi Lab Homogenizer PandaPLUS 2000, GEA, Germany) circle run for 3 min at 200 bar.

Figure 7 shows a graph illustrating oil droplet size of fungal biomass stabilised emulsions and shows that emulsion droplet sizes overall tend to decrease with increased concentration of fungal biomass added. From Figure 7 it is also clear that the high- pressure homogeniser treatment reduces the droplet sizes compared to the high shear emulsion mixer.

Example 4. Extraction of fungi protein into a liquid media Methods to break cells g of frozen biomass was defrosted in 250 mL distilled water for 30 min at ambient temperature. After thawing, pH was adjusted to 10 with NaOH (1 M) and mixed for 5 min at 25,000 rpm (Ultra Turrax T25, Staufen im Breisgau, Germany). Samples were then feed into a high-pressure homogeniser (Niro Soavi Lab Homogenizer PandaPLUS 2000,GEA, Germany) and passed through two passages at 900 bar.

Adjustment of pH for ce/I disruption After the high-pressure homogeniser, different extraction pH was tried. pH was adjusted to between 2-12 using NaOH or HCI, both 1 M. Samples were thereafter centrifuged (5250 x g, 90 min) and the supernatant was collected.

Protein content was measured both according to the Dumas method explained above and using BCA assay kit. For the BCA analysis, 500 uL sample were diluted in 4.5 mL distilled water. From this, 100 uL was added to 2 mL BCA working reagent and gently mixed. The samples were incubated for 30 min at 37°C and thereafter cooled to ambient temperature before the absorbance was measured at 562 nm. The absorbance was compared to a standard curve to calculate protein concentration. The protein concentrations from the Dumas and BCA methods had a Pearson correlation coefficient of 0.99. The result of the protein concentration in the supernatants are presented in Figure 8. This figure shows that after pH 6, the protein concentration will increase with pH, and the maximum concentration was found in the highest pH measured, pH 12. The minimum protein concentration was found around pH 4 and it is therefore assumed that the isoelectric point is close to pH Example 5. Use of extracted protein powder for beverage formulations Protein precipitation and concentration The supernatant from Example 4 extracted at pH 12 was pH adjusted to 4, which was believed to be the isoelectric point, to precipitate the proteins into a protein isolate. From this, the supernatant was removed, and the precipitated protein isolate was thereafter re- adjusted back to pH 7 before drying using freeze-drying or spray-drying. The samples were freeze dried for 6 days in a laboratory freeze dried (Hetosicc, Freeze dryer CD 12, Birkerod, Denmark) and spray-drying was performed in a Büchi Mini Spray Dryer B-290 (Büchi Labortechnik AG, Flawil, Switzerland) with an inlet temperature of 150°C, outlet temperature of 80°C, aspiration 90-100% and pump at 35%.

The protein content of the final isolate powders was determined, using Dumas method explained above, and was 77.2% of the dry matter for both isolates, compared to 54.1% of the dry matter in freeze-dried fungal biomass. The moisture content in all powders were less than 2%.

To evaluate the solubility of the isolate powders and the freeze-dried fungal biomass in Example 3, the absorbed protein concentration in an emulsion was determined. First, powders were dissolved in phosphate buffer (see Example 3) and centrifuged at 5000 x g for 30 min. The protein concentration in the supernatant was thereafter determined using BCA assay kit explained above. The percentage of soluble proteins were determined by the following formula: protein content in supernatant Soluble protein (%) = . _ protein content in powder Soluble protein at different concentration of emulsifier is presented in Figure After the first centrifugation, the powders were used to make an emulsion (see Example 3) which was also centrifuged at 5000 x g for 30 min whereafter the oil phase was discarded. The serum phase was thereafter centrifuged again at 5000 x g for 30 min and the protein concentration in the supernatant was determined using BCA assay kit. The absorbed protein concentration (Cp) was determined as the difference in proteins dissolved in the phosphate buffer and in the emulsion serum phase.

FDB stands for freeze-dried biomass, HPH stands for high-pressure homogeniser, FDPI stands for freeze-dried protein isolate and SDPI stands for spray-dried protein isolate.

Figure 9 shows a decrease in soluble proteins with an increased concentration of the different powders. The freeze-dried fungal biomass has the smallest content of soluble proteins in the phosphate buffer, which could indicate that too much processing can affect the properties of the proteins.

Creating oil-in-water emulsions using iso/ated extracted protein powder Emulsions with protein isolate powders were made and evaluated as explained in Example 3. The droplet sizes are presented in Figure 10, with the oil droplet sizes of the freeze-dried samples illustrated at the left and oil droplet sizes of the spray dried samples illustrated at the right.

The particle size of emulsions made with protein isolates shows a decrease in droplet size with an increase of powder concentration. The freeze-dried powders have a better emulsifying capacity as the oil droplets are smaller. Similar to Experiment 3, it is possibleto reduce the droplet size further by using high-pressure homogenisation. Size distributions of emulsions with 128 mg powder/mL oil are compared in Figure Figure 11 illustrates the particle size distributions of emulsions with 128 mg powder/mL oil. The left image shows an emulsion mixed in a high shear mixer and the right image shows an emulsion mixed with a high-pressure homogeniser. FDB stands for freeze-dried biomass, HPH stands for high-pressure homogeniser, FDPI stands for freeze-dried protein isolate and SDPI stands for spray-dried protein isolate.

Figure 11 shows that the spray-dried isolate powder has less aggregates compared to the other two powders. However, the distributions of the emulsions from the high-shear mixer are quite similar and might be limited in size by the mixer. From the size distributions of the emulsions mixed with the high-pressure homogeniser it is clearer that the isolates can create smaller emulsions than the freeze-dried biomass as they have more particles with a size smaller than 1 um.

From the measured droplet sizes, the emulsification capacity (EC) can be calculated using the following equation: 6<ß EC = Gp * dm (my/mz) where, Cp is the calculated absorbed protein concentration, ds; is the surface weighted mean, and cl) is the dispersed phase volume fraction. Emulsifying capacity for the different powders and concentrations can be seen in Figure 12. FDB stands for freeze-dried biomass, FDPI stands for freeze-dried protein isolate and SDPI stands for spray-dried protein isolate.

The emulsifying capacity was better for the protein isolates compared to the freeze-dried fungal biomass. For most concentrations, the freeze-dried protein isolate powder showed the highest emulsifying capacity.

Foaming Properties Foaming properties were determined using a modified version of the method described in Lonchamp et al. 2019. 15 mL solution of 1% w/v spray-dried protein isolate, freeze-dried protein isolate, and freeze-dried fungal biomass were prepared in 50 mL glass beakers, respectively, and stirred for 1 h. The diameter of the glass beakers was 33 mm. Thesolutions were then frothed for 3 min using handheld whisk-type frother (Ikea, Sweden) at constant speed provided by the whisker. The height of the resulting foam was measured immediately after whisking and every 10 min until the foam collapsed. The foaming ability was expressed as the initial height of the foam while the foam stability was measured as the time required for the foam to fully collapse. All samples were measured duplicate. Figure 13 illustrates the foaming properties of fungi biomass powder vs protein isolate. FDB; freeze-dried biomass, FDPI; freeze-dried protein isolate, SDPI; spray-dried protein isolate.

From Figure 13, it is shown that using freeze-dried fungal biomass to create a foam is not as beneficial as using freeze-dried or spray-dried isolates. Fungal biomass powder is therefore more suited in beverages where foam should be avoided, such as sport nutrition or infant formula.

Example 6. Use of protein extracted to liquid phase for formulation of beverages Extraction of proteins to liquid media Fungal biomass was grinded twice in a meat grinder using a disk with pore size 3 mm and thereafter diluted in water ratio 1:4. The pH of the biomass mixture was adjusted to 10 or 12 and the solution was thereafter filtered through a 40 um filter where the supernatant was collected. The supernatant was either left at the extraction pH or the pH was adjusted back to 7. Additionally, a control sample at extraction pH 7 was also performed.

Dry matter content of both supernatant and retentate was carried out like the method explained above and was measured in duplicates. Protein content was determined using BCA kit as explained above, but with an incubation for 2 h at ambient temperature instead of 30 min at 37°C and was measure in triplicates. Protein content in supernatant and retentates from different extraction methods is presented in Figure ln Figure 14 it is obvious that a higher extraction pH will lead to a higher protein content in the supernatant which agrees with Figure 8. Adjusting the pH after extraction will lead to a slightly lower protein content due to dilution, but it will not result in major changes.

Emu/sification index 22.5 mL of biomass protein liquid or deionised water with 1 % emulsifier was mixed with 7.5 mL canola oil to make 1:4 oil-in-water emulsions. The emulsions were mixed for 1 min using a high shear homogeniser at 22,000 rpm.After mixing the emulsions, 10 mL was left in a measuring tube in ambient temperature so that phase separation between emulsion and serum could be measured. This was done by leaving the emulsions overnight and visually evaluate different phases using the scale on the tube. The phases were recorded in mL and emulsification index was calculated according to the following equation: Vemulsíon *total volume Emulsification index (%) = Emulsification index for different fungal biomass stabilised emulsions was recorded in duplicates and results are shown in Figure Figure 15 shows that the emulsification index for emulsions created with fungal biomass was between 28% and 50%. The highest emulsification index was obtained with freeze dried fungal biomass, followed by the supernatants where pH was re-adjusted to 7. The results indicate that the fibers in the biomass will contribute to an emulsion creation. By adjusting the pH back to neutral after extraction, the results suggest that emulsification properties are enhanced.

The highest and lowest emulsification index for fungal biomass were compared to commercially available protein powders and emulsifiers, which are presented in Figure Figure 16 shows that using fungal biomass as an emulsifier, either as powder or as extracted protein liquid, results in comparable emulsification indexes as other commercially available alternatives.

Foaming Properties Protein liquids were used to measure foaming ability and stability according to Lonchamp et al. 2019. 15 g of different protein solutions was measured in a 50 mL beaker and the hight of liquid was measured. The solution was thereafter frothed using a handheld milk frother (Coline), and hight was recorded every 10 min until the foam was gone, or until 270 min had passed. The initial foam height was determined as the foaming ability and the stability was determined as time before the foam was gone. The height of foam, expressed in % of initial liquid height, over time for the different solutions can be seen in FigureFigure 17 shows that foaming ability is mostly influenced by pH, where higher values are seen for extraction pH 12 compared to 10 and 7. The foaming stability, however, seems to benefit from re-adjusting the pH to 7 after protein extraction.

The fungal biomass with the highest and lowest foaming stabilities were compared in terms of foaming properties to commercially available protein powders and emulsifiers, which is presented in Figure ln Figure 18 it is shown that foaming ability and stability of fungal biomass protein liquids are comparable with commercially available protein powders and emulsifiers.

Example 7. Creation of fungi-based beverages with beneficial environmental impact Ca/culations of Environmental Impact The environmental impact of pure mycoprotein has either been describes in literature or calculated from a detailed study of a mycoprotein production plant. For calculations of the environmental impact of a mycoprotein drink, the impact of producing 1kg of mycoprotein with 15 g /100 g of protein has been considered to be 1,14 kg CO2e of GHG emissions, 0.69 m2 of used land, and 524 L of water consumption. Based on this, the production from fungal biomass has been assumed to be of a similar kind as of an oat drink production, assuming an extraction of 46% of the fungal proteins from fresh biomass into water. For this, the following equation was used to obtain a drink using 1% protein content (similar content to existing oat drinks in the market), where Ithe impact value to be calculated (GHG, land usage or water usage).

Imycopnbíomass 1,, - = + (1 - + 1 - + 1 ycopr.,dr1nk - oatproductwn oatpackagzng oatflfransport Proteznmycopn >< 0,_ Ioatfarmíng) The results are plotted in comparison with milk and popular plant-based drinks in Figure 20, in which all values except from mycoprotein were taken from Smedman, A. et al. 2010.

Claims (17)

Claims

1.A liquid dairy replacement product intended for human consumption, containing fungi biomass or protein derived from fungi biomass, with a fungal protein content between 0.5 and 13 g / 100mL.

2.The liquid dairy replacement product according to c|aim 1, in which the phytic acid content is between 0 and 0.5 g/100 mL.

3.The liquid dairy replacement product according to any one of the preceding claims, in which said liquid dairy replacement product is a solution or suspension of fungi biomass particles with a concentration of biomass bet\Neen 1% and

4.The liquid dairy replacement product according to c|aim 1 or 2, in which said product is an oil in water emulsion comprising oil droplets and said fungi biomass being in an aqueous solution or suspension, the oil in water emulsion having a fat content within the range of from 1 to 30 mL/ 100mL.

5.The liquid dairy replacement product according to c|aim 4, wherein the oil droplets has droplet sizes within the range of from 1 to 100 um.

6.The liquid dairy replacement product according to any one of claims 1 to 5, with wherein the liquid dairy replacement product has a foaming capacity within the range of from 50% to 250% height of foam volume compared to height of initial liquid, as measured according to the foaming capacity test disclosed herein.

7.The liquid dairy replacement product according to any one of claims 1 to 6, with a foaming stability of at least 250 min before collapse of foam.

8.The liquid dairy replacement product according to any one of claims 4 to 7 with an emulsification capacity of from >0 to 1 m2/mg of fungal biomass powder, preferably within the range of from 0.1 to 0.6 m2/mg powder.

9.The liquid dairy replacement product according to any one of claims 4 to 8, with an emulsification index between 20% and 70%, preferably between 25% and 50%.

10.A method for preparing a liquid dairy replacement product according to claim 1, comprising the steps of a. cultivating fungi in a liquid fermentation in an aerated bioreactor, to obtain a fungi biomass; b. drying the fungi biomass obtained in step a) either by controlled low vacuum dehydration or by freeze drying; and c. suspending the dried fungi biomass obtained in step b) in an aqueous solution to form a suspension containing between 1 and 20 g/L of dry biomass content and to obtain the liquid dairy replacement product.

11.The method according to claim 10, wherein the dried fungi biomass suspension is mixed, such as in a high-shear homogeniser, with a vegetable oil to obtain an oil in water emulsion.

12.The method according to claim 11, wherein the fungi biomass suspension is mixed with the vegetable oil at least 1 min at 20,000 rpm or more.

13.A method for producing a liquid dairy replacement product according to claim 1, comprising the steps of; a. cultivating fungi in a liquid fermentation in an aerated bioreactor, to obtain a fungi biomass; b. optionally drying or freezing the fungi biomass obtained in step a) c. providing the fungi biomass obtained in step a) or b), i.e. in fresh, frozen or powdered form, in an aqueous solution to form a suspension containing between 1 and 20 g/L of dry biomass content; d. adjusting the pH of said suspension to a value within the range of from 10 and 13; e. subjecting the suspension from step d) to a high-shear mixing step, optionally with a subsequent high pressure homogenization step, while keeping the pH within the range of from 10 to 13, thereby extracting protein from the fungi mycelium; and 25 30 f. adjusting the pH to to a value within the range of from 5.5 to 7.5 and thereby obtaining the liquid dairy replacement product.

14.A method for producing a liquid dairy replacement product according to claim 1, comprising the steps of; a. cultivating fungi in a liquid fermentation in an aerated bioreactor, to obtain a fungi biomass; optionally drying or freezing the fungi biomass obtained in step a) providing the fungi biomass obtained in step a) or b), i.e. in fresh, frozen or powdered form, in an aqueous solution to form a suspension containing between 1 and 20 g/L of dry biomass content; adjusting the pH of said suspension to a value within the range of from 10 and 13; subjecting the suspension from step d) to a high-shear mixing step, optionally with a subsequent high pressure homogenization step, while keeping the pH within the range of from 10 to 13, thereby extracting protein from the fungi mycelium; and adjusting the pH to a value within the range of from 3.5 to 4.5 to promote protein precipitation; collecting and drying the precipitates, optionally with freeze drying or spray drying; and suspending the dried precipitates obtained in step g) in an aqueous solution to obtain the liquid dairy replacement product.

15.The method according to claim 13 or 14, wherein the suspension comprises the product obtained in step f) from the method according to claim 13 or in step h) from the method according to claim 14, such as in a high-shear homogeniser, is mixed with a vegetable oil to obtain an oil in water emulsion.

16.The method according to claim 15, wherein the suspension is mixed with the vegetable oil at least 1 min at 20,000 rpm or more.

17. The method according to any one of claims 10 to 16, wherein the viscosity of the final preparation is adjusted to within the range of from 2 mPa-s to 400 mPa-s, optionally with addition of xanthan gum.