Use of a fungal protein comprising an over expressed native polypeptide to provide a plant-based meat alternative

Abstract

The present invention relates to the use of a fungal protein to create a food product. More particularly, the present invention relates to the use of an overexpressed native polypeptide to create a food product.

Description

[0001]TITLE USE OF FUNGAL PROTEIN TO PROVIDE NON-DAIRY AND NON-MEAT FOOD ALTERNATIVES FIELD OF THE INVENTION The present invention relates to the use of fungal proteins to create a food matrix. More particularly, the present invention relates to the use of a fungal protein which is an overexpressed native protein to create a food matrix. BACKGROUND OF THE INVENTION Consumers increasingly demand alternatives to traditional meat and dairy items such as ground beef, milk and yogurt. Today, there are plant-based meat and dairy alternatives. But plant protein, which is the basis of these alternatives, is expensive to prepare and difficult to use. Plant protein can be obtained from a wide variety of plants, including soy, wheat, oat, and pea. However, extracting the protein from the physical plants can be expensive and typically involves various chemical steps. In addition, the harsh conditions employed in extraction will generally denature the plant proteins. Typically, harsh organic solvents are avoided. However, harsh acidic and basic conditions are required to extract the protein. For example, soy protein is extracted from soybean meal. The soybean meal must first be dehulled and defatted and converted to flour. Soy flour is first extracted with base having a pH over 9.0. The base extract is clarified to remove insoluble material and the supernatant liquid is then acidified to produce a precipitated protein curd which is separated from the whey by centrifugation. The curd in turn is neutralized with alkali to form a proteinate salt. The desired soy globulin proteins must then be separated from contaminating soluble carbohydrates, soy whey protein and salts. This is done by a leaching process involving the use of alcohol / water and further acid and base treatments. Importantly, at the end of the process, the extracted plant protein is denatured (i.e., has lost its original three-dimensional structure). Denatured proteins are difficult to work with in terms of creating plant-based meat and dairy alternatives. Denatured proteins tend to have lower solubility than non-denatured proteins. Also, functional properties like gel, foam and emulsion forming abilities are generally negatively impacted by denaturation. Because typical plant proteins are denatured and have limited solubility, acceptable meat and dairy alternatives composed of such proteins are difficult and expensive to make. For example, plant-based meat alternatives must have a certain hardness or rigidity to seem meat like. To achieve this, high levels of hydrocolloids must be added to the plant-based meat. However, health conscious consumers of alternative meat and dairy products may find the high levels of hydrocolloids in many plant foods to be unacceptable. The poor solubility of plant proteins can sometimes be remedied by using transglutaminase to cross-link the dilute plant proteins in solution. But addition and use of exogenous enzymes can be expensive and time consuming. There is a continuing need for protein sources which do not require extraction (and the resultant denaturation) from plant materials for use in creating meat and dairy alternatives. There is also a continuing need for plant-based meat and / or dairy alternatives that do not need hydrocolloids or the use of expensive and time-consuming exogenous enzymes. SUMMARY OF THE INVENTION In one aspect of the present invention, a food product is presented composed of a beverage or foodstuff and a fungal protein having an overexpressed native polypeptide combined with the beverage or foodstuff. Optionally, the fungal protein is spray dried or freeze dried. Optionally, the fungal protein is derived from Aspergillus, Fusarium, Mucor, Rhizomucor or Trichoderma. Optionally, the fungal protein is derived from Aspergillus. Optionally, the fungal protein is derived from Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus tubigensis. Optionally, the fungal protein is derived from Trichoderma. Optionally, the fungal protein is derived from Trichoderma reesei. Optionally, the fungal protein is derived from Fusarium. Optionally, the fungal protein is derived from Fusarium oxysporum. Optionally, the fungal protein is derived from Rhizomucor. Optionally, the fungal protein is derived from Rhizomucor miehei. Optionally, the fungal protein is the product of fungal fermentation and the titer of the overexpressed native polypeptide is above 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L. Optionally, the food product has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the overexpressed native polypeptide. Optionally, the overexpressed native polypeptide is an enzyme. Optionally, the enzyme is an alpha-glucosidase. Optionally, the alpha-glucosidase is derived from Trichoderma reesei. Optionally, the titer of alpha-glucosidase is at least 100 g / L. Optionally, the enzyme is inactivated. Optionally, the enzyme is inactivated before being combined with the food stuff or beverage. Optionally, the enzyme is inactivated after being combined to the food stuff or beverage. Optionally, the over expressed native polypeptide has superior foaming and / or emulsification and / or gel forming properties compared with a denatured protein. Optionally, the overexpressed native protein contains less than 100 ppm of saponin. Optionally, the overexpressed native protein does not have off flavors. In accordance with an aspect of the present invention, a method for preparing a plant- based meat alternative is presented having the steps of mixing a batter comprising water, plant protein, one or more meat flavors and a fungal protein which is an over expressed native polypeptide; and baking or cooking the batter to provide the plant-based meat. Optionally, the batter further has textured or structured protein derived from plants. Optionally, the plant protein is chickpea, pea, soy, fava, gluten, or oat. More preferably, the plant protein is soy protein or pea protein. Optionally, the batter further includes a transglutaminase. Optionally, the batter further includes one or more hydrocolloids. Optionally, fungal protein is spray dried or freeze dried. Optionally, the fungal protein is derived from Aspergillus, Fusarium, Mucor, Rhizomucor or Trichoderma. More preferably, the fungal protein is derived from Aspergillus. Optionally, the fungal protein is derived from Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus tubigensis. Optionally, the fungal protein is derived from Trichoderma. Optionally, the fungal protein is derived from Trichoderma reesei. Optionally, the fungal protein is derived from Fusarium. Optionally, the fungal protein is derived from Fusarium oxysporum. Optionally, the fungal protein is derived from Rhizomucor. More preferably, fungal protein is derived from Rhizomucor miehei. Optionally, the fungal protein is the product of fungal fermentation and the titer of the overexpressed native polypeptide is above 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L. Optionally, the batter has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the overexpressed native polypeptide. Optionally, the overexpressed native polypeptide is an enzyme. More preferably, the enzyme is an alpha-glucosidase. Optionally, the alpha-glucosidase is derived from Trichoderma reesei. Optionally, the titer of alpha-glucosidase is at least 100 g / L. Optionally, the enzyme is inactivated. Optionally, the enzyme is inactivated before being combined with the batter. It is also an option to inactivate the enzyme after addition to the batter. Optionally, the over expressed native polypeptide has superior foaming and / or emulsification and / or gel forming properties compared with a denatured protein. Optionally, the overexpressed native protein contains less than 100 ppm of saponin. Optionally, the overexpressed native protein does not have off flavors. Optionally, the plant-based meat alternative is a frankfurter style sausage. DESCRIPTION OF FIGURES FIG. 1 shows development in complex shear modulus during a temperature sweep from 40 to 95°C and back to 40°C. Overlayed results for 8% slurries of TrGA protein, Egg White, Soy Protein and pea Protein are provided. FIG. 2 shows liquid drainage curves for foams made with TrGA-protein and egg-white respectively. FIG 3: Particle size distribution curves for emulsions made with A) TrGA-proein, B) egg- white, C) soy and D) pea protein. All emulsions were measured right after preparation (0 days), and after 8 days storage at 5˚C. FIG 4: Hardness of plant-based sausages made without AnGA protein (Reference) or with part of the soy protein isolate substituted with AnGA protein. Error bars represent standard deviation, n = 3. DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nded., John Wiley and Sons, New York (1994), and Hale & Markham, THEHARPERCOLLINSDICTIONARYOFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference. As used herein, the term “glucoamylase (EC 3.2.1.3)” refers to an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, and materials are now described. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a gene” includes a plurality of such candidate agents and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Description of the Preferred Embodiments In one aspect of the present invention, a food product is presented composed of a beverage or foodstuff and a fungal protein having an overexpressed native polypeptide combined with the beverage or foodstuff. Preferably, the fungal protein is spray dried or freeze dried. Preferably, the fungal protein is derived from Aspergillus, Fusarium, Mucor, Rhizomucor or Trichoderma. More preferably, the fungal protein is derived from Aspergillus. Still more preferably, is derived from Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus tubigensis. Preferably, the fungal protein is derived from Trichoderma. More preferably, the fungal protein is derived from Trichoderma reesei. Preferably, the fungal protein is derived from Fusarium. More preferably, the fungal protein is derived from Fusarium oxysporum. Preferably, the fungal protein is derived from Rhizomucor. More preferably, the fungal protein is derived from Rhizomucor miehei. Preferably, the fungal protein is the product of fungal fermentation and the titer of the overexpressed native polypeptide is above 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L. Preferably, the food product has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the overexpressed native polypeptide. Preferably, the overexpressed native polypeptide is an enzyme. More preferably, the enzyme is an alpha-glucosidase. Preferably, the alpha-glucosidase is derived from Trichoderma reesei. Preferably, the titer of alpha-glucosidase is at least 100 g / L. Preferably, the enzyme is inactivated. More preferably, the enzyme is inactivated before being combined with the food stuff or beverage. It is also preferred to inactivate the enzyme after addition to the food stuff or beverage. Preferably, the over expressed native polypeptide has superior foaming and / or emulsification and / or gel forming properties compared with a denatured protein. In other preferred embodiments, the overexpressed native protein contains less than 100 ppm of saponin. In still other preferred embodiments, the overexpressed native protein does not have off flavors. In accordance with another aspect of the present invention, it has been discovered that native fungal proteins can be used to supplement plant proteins to make meat alternatives that do not require hydrocolloids or enzymes to achieve the requisite hardness. Alternatively, use of native fungal proteins in meat alternatives substantially lessons the amount of hydrocolloid that must be added to achieve the requisite hardness and / or the need for the addition of exogenous enzymes to achieve better textual properties of the plant protein. In accordance with an aspect of the present invention, a method for preparing a plant- based meat alternative is presented having the steps of mixing a batter comprising water, plant protein, one or more meat flavors and a fungal protein which is an over expressed native polypeptide; and baking or cooking the batter to provide the plant-based meat. Preferably, the batter further has textured or structured protein derived from plants. Preferably, the plant protein is chickpea, soy, pea, soy, fava, gluten, or oat. More preferably, the plant protein is soy protein or pea protein. Preferably, the batter further includes a transglutaminase. Preferably, the batter further includes one or more hydrocolloids. Preferably, fungal protein is spray dried. Preferably, the fungal protein is derived from Aspergillus, Fusarium, Mucor, Rhizomucor or Trichoderma. More preferably, the fungal protein is derived from Aspergillus. Still more preferably, the fungal protein is derived from Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus tubigensis. In other preferred embodiments, the fungal protein is derived from Trichoderma. More preferably, the fungal protein is derived from Trichoderma reesei. In still other preferred embodiments, the fungal protein is derived from Fusarium. More preferably, the fungal protein is derived from Fusarium oxysporum. In other preferred embodiments, the fungal protein is derived from Rhizomucor. More preferably, fungal protein is derived from Rhizomucor miehei. Preferably, the fungal protein is the product of fungal fermentation and the titer of the overexpressed native polypeptide is above 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L. Preferably, the batter has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the overexpressed native polypeptide. Preferably, the overexpressed native polypeptide is an enzyme. More preferably, the enzyme is an alpha-glucosidase. Preferably, the alpha-glucosidase is derived from Trichoderma reesei. Preferably, the titer of alpha-glucosidase is at least 100 g / L. Preferably, the enzyme is inactivated. More preferably, the enzyme is inactivated before being combined with the batter. It is also preferred to inactivate the enzyme after addition to the batter. Preferably, the over expressed native polypeptide has superior foaming and / or emulsification and / or gel forming properties compared with a denatured protein. In other preferred embodiments, the overexpressed native protein contains less than 100 ppm of saponin. In still other preferred embodiments, the overexpressed native protein does not have off flavors. Preferably, the plant-based meat alternative is a frankfurter style sausage. EXAMPLES Example 1: Preparation of the whole broth samples from GA-producing transformants TrGA producing transformants were initially pre-grown in 250 ml shake flasks containing 30 ml of ProFlo medium. Proflo medium contained: 30 g / L α-lactose, 6.5 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.2 g / L CaCl2·2H2O, 1 ml / L 1000 x trace element salt solution as mentioned above, 2 ml / L 10% Tween 80, 22.5 g / L ProFlo cottonseed flour (Traders protein, Memphis, TN), 0.72 g / L CaCO3. After two days of growth at 28°C and 140 rpm, 10% of the Proflo culture was transferred into a 250 ml shake flask containing 30 ml of Lactose Defined Medium. The composition of the Lactose Defined Medium was as follows: 5 g / L (NH4)2SO4, 33 g / L 1,4-Piperazinebis (propanesulfonic acid) buffer, pH 5.5, 9 g / L casamino acids, 4.5 g / L KH2PO4, 1.0 g / L MgSO4 ·7H2O, 5 ml / L Mazu DF60-P antifoam (Mazur Chemicals, IL), 1ml / L of 1000 x trace element solution. 40 ml / L of 40% (w / v) lactose solution was added to the medium after sterilization. Shake flasks with the Lactose Defined Medium were incubated at 28°C, 140 rpm for 4 – 5 days. Mycelium was removed from the culture samples by centrifugation and the supernatant was analyzed for total protein content (BCA Protein Assay Kit, Pierce Cat. No.23225). The protein profile of the whole broth samples was determined by SDS-PAGE electrophoresis. Samples of the culture supernatant were mixed with an equal volume of 2 x sample loading buffer with reducing agent and separated on NUPAGE® Novex 10% Bis-Tris Gel with MES SDS Running Buffer (Invitrogen, Carlsbad, CA, USA). Polypeptide bands were visualized in the SDS gel with SIMPLYBLUE SafeStain (Invitrogen, Carlsbad, CA, USA). Example 2: Full Scale Production of Fungal Protein via Fungal Fermentation Briefly, spores of a strain were added to 500 mL of medium in a 3 L flask with both side and bottom baffles. The culture was grown in a minimal medium for 48 hours at 34°C in a shaking incubator. After 48 hours, the contents of the flask was added to a 14 L fermenter containing 9.5 L of medium containing 4.7 g / L K2HPO4, 1.0 g / L MgSO47H2O, 4.3 g / L (NH3)2SO4 and 2.5 mL / L of 400X T. reesei trace elements solution (citric Acid (anhydrous), 175 g / L; FeSO4· 7 H2O, 200 g / L, ZnSO4· 7 H2O, 16 g / L, CuSO4· 5 H2O, 3.2 g / L; MnSO4, 1.4 g / L; H3BO3, 0.8 g / L.). These components were heat sterilized together at l2l°C for 30 minutes. A solution of 60% glucose and 0.48% CaCl2 · 2 H2O was separately autoclaved, cooled, and added to the fermenter to a final concentration of 75 g / L glucose and 0.6 g / L CaCl2· 2 H2O. The medium was adjusted to pH 3.5 with 28% NH3and the temperature was maintained at 34°C during the growth period. Once glucose was exhausted, the temperature was dropped to 28°C, and the culture was fed glucose-sophorose. The dry cell weight (DCW), total protein concentration and other parameters were measured, and specific total protein production rate and yield on fed sugars were calculated. Downstream processing consisted of cell-separation and ultrafiltration. Preservatives may be added to avoid protein spoilage. Example 3: Determination of protein concentrations Protein content of samples was determined by the Dumas method. The apparatus used was a Rapid MAX N, Exceed from Elementar (Langenselbold, Germany). Approximately 2g of sample was weighed into iron crucibles designed for the instrument. The crucibles were placed in the autosampler. Protein was determined using a predefined program for analysis). A conversion factor from nitrogen to protein of 6,25 was used. Protein concentrations for the samples used in this study are shown in the Table 1. below: Table 1 Description Protein % Form T GA Di ® TGA E d t t i i 132 Li id Example 4: Comparing rheological properties of protein solutions Diazyme TGA and Sanovo egg-white powder (see Example 3) were compared in this experiment. A solution of Sanovo egg white powder containing 13,2% protein was made in deionized water. Diazyme TGA was used as is. Rheological properties of protein solutions were compared using an Anton Paar Physica MCR 301 rheometer equipped with a bob-cup double gap measuring system. Pretreatment of samples was done by heating in an oven at 40°C before measurement and mixing the samples well before transferring to the rheometer. After loading to the rheometer, the sample surface was covered with silicone oil to prevent drying out at the surface. Oscillation rheology was determined during a temperature sweep from 40 to 95 °C and back to 40 °C, with a temperature gradient of 5 °C / min. The analysis was run in oscillation mode with 1 Hz frequency and a strain of 0.1%. It is seen from the rheology curves in FIG. 1 that a solution of 8% TrGA-protein formed a stable gel upon heating. The complex shear modulus of TrGA-protein, over the temperature sweep, increases from 0.081 Pa to 2894 Pa. A very substantial increase and a clear indication of gel formation. The phase transition temperature or T½-value (the temperature where half of the phase transition has taken place) is around 75 °C. It is seen that TrGA-protein behaves much like egg-white. For egg-white the increase in complex shear modulus is also very substantial (from 6,0 to 5152 Pa). T½for egg-white is 82 °C. Hence, higher than for TrGA-protein, showing that egg-white needs a higher temperature to form a gel than TrGA-protein. Soy and pea protein does not show clear phase shift behavior. Hence, at this concentration gel formation is not observed. Example 5: Comparing foam stability of protein solutions Foam analysis was run as follows: Solutions containing 2% protein were made in deionized water and 10mL was placed in a 100mL glass bottle with wide opening. Funnel with led rings were placed in holders above 25mL measuring cylinders. Foam was produced in the bottles by whipping with a hand-held milk-frotter for 60sec. The bottles with foam, were inverted and placed in the funnel / lead rings. The volume of the liquid which has drained to the measuring cylinder was noted after 1, 2, 3, 4, 5, 10, 15, 20- and 30-min. Foam drainage curves are plotted as liquid volume in the measuring cylinder as a function of time. It is seen from the drainage curves in FIG. 2 that TrGA protein and Egg-white protein behave very similar regarding water drainage from the foam. Egg-white is known to have unique foaming properties (e.g., Cunningham, 1976) Hence the foaming properties shown here for TrGA protein are very interesting from a commercial perspective. Hence, the foaming properties shown here for TrGA protein appear to be commercially significant. Example 6: Comparing emulsion stability of different protein solutions Emulsification analysis was run as follows: Protein solutions, containing 1% protein were made with deionized water. The protein solutions were mixed with rape seed oil in the ratio 80:20, followed by homogenization by Ultra Turrax homogenizer for 30 seconds at 20500 rpm. The emulsions were subsequently cooled down to 5°C as fast as possible. Emulsion droplet size was measured on Malvern Mastersizer 3000 immediately after production and after 8 days. The samples were made homogeneous before measurement by very mild stirring. Between measurements the emulsions were stored at 5°C. It is seen from Figure 3A that TrGA-protein retains small particle sizes over an 8-day period. This shows that TrGA is good at stabilizing emulsions. In comparison, the particle size of an emulsion made with egg-white (Figure 3B) shifts slightly towards the right (larger particles) over the 8-day period, indicating that the egg-white emulsion is slightly less stable than the TrGA-protein emulsion. Already at the onset of the experiment (t = 0 days), the particle size distribution of emulsions made with soy and pea (Figure 3C and 3D respectively) are significantly shifted towards larger particles, compared to TrGA-protein and egg-white emulsions. Larger particle sizes generally give less stable emulsions, but a change in particle size over time is a better way to recognize emulsion stability. Particle size distribution for soy and pea protein emulsions shifts substantially towards larger particles over the 8-day period, showing that these proteins are inferior to TrGA-protein in stabilizing emulsions. Example 7 Use of Aspergillus niger Glucoamylase Protein in plant-based meat alternative For the reference sample, the liquid ingredients used in the recipe are: 275 g tap water, 123.8 g ice and 29.7 g rapeseed oil. Dry ingredients are: 70.7 g soy protein isolate (85,1 % protein), 55g texturized soy protein, 5.6 g table salt (NaCl), 1.65 g onion powder, 0.4 g ground pepper, 0.64 g paprika and 0.85 g glucose. Initially all liquid ingredients were added to a Robot Coupe R602V blender and mixed at high speed for 20 sec. to make an emulsion. All dry ingredients, except texturized protein, were mixed, and blended into the emulsion at medium speed for 20 sec. Texturized protein was added and mixed into the batter by a 10 sec blend at low speed. The final batter was left to hydrate for 1h. After hydration the batter was blended 2 times 1.5 min. In between blends the batter was scraped down from the sides of the container. The batter was further mixed by hand around 2 min. Then portions of the batter were stuffed into 50 mL centrifuge tubes and centrifuged at 4000 rpm for 5min (to remove air bubbles). The tubes were topped up with more batter closed and placed in a 50 ˚C water bath for 1h, then transferred to a 90 ˚C water bath where they incubated for 20 min. After this cook cycle the tubes were placed in plenty of cold water until the temperature had reached room temperature. The resulting plant-based sausages were removed from the tubes and cut in slices of 2,5 cm (the top 2 cm, corresponding to the topping batter, was discarded). Texture profile analysis (TPA) was carried out on the sausages using a TA.XTPlusC Texture Analyser – 650H from Stable Microsystems. A second batch of sausages were made where part of the soy protein isolate was substituted with Aspergillus niger glucoamylase protein (AnGA protein). AnGA protein came in the form of a liquid solution with 18,9% protein (AnGA solution). The protein content was determined as described in Example 3. Specifically, the second batch was produced in the same way as the reference batch, except that the water addition was 228.7 g, soy protein isolate addition was 60.5g and 43.6 g AnGA solution was added. Note that the total amount of protein was kept constant in the two batches. Results on hardness of the sausages are shown in Figure 4. It is seen that the sausage containing AnGA protein was substantially harder than the reference sausage. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. To the extent the content of any citation, including website or accession number may change with time, the version in effect at the filing date of this application is meant. Unless otherwise apparent from the context any step, element, aspect, feature of embodiment can be used in combination with any other.

Claims

CLAIMS What is claimed is:

1. A food product comprising: a beverage or foodstuff and a fungal protein comprising an over expressed native polypeptide combined with the beverage or foodstuff.

2. The food product of claim 1 wherein the fungal protein is spray dried or freeze dried.

3. The food product of either of claims 1 or 2 wherein the fungal protein is derived from Aspergillus, Fusarium, Mucor, Rhizomucor or Trichoderma.

4. The food product of claim 3 wherein the fungal protein is derived from Aspergillus.

5. The food product of claim 4 wherein the fungal protein is derived from Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus tubigensis.

6. The food product of claim 3 wherein the fungal protein is derived from Trichoderma.

7. The food product of claim 6 wherein the fungal protein is derived from Trichoderma reesei.

8. The food product of claim 3 wherein the fungal protein is derived from Fusarium.

9. The food product of claim 8 wherein the fungal protein is derived from Fusarium oxysporum.

10. The food product of claim 3 wherein the fungal protein is derived from Rhizomucor.

11. The food product of claim 10 wherein the fungal protein is derived from Rhizomucor miehei.

12. The food product of any of the preceding claims wherein the fungal protein is the product of fungal fermentation and the titer of the overexpressed native polypeptide is above 10 g / L.

13. The food product of claim 12 wherein the titer of the overexpressed native polypeptide is above 20 g / L.

14. The food product of claim 13 wherein the titer of the overexpressed native polypeptide is above 30 g / L.

15. The food product of claim 14 wherein the titer of the overexpressed native polypeptide is above 40 g / L.

16. The food product of claim 15 wherein the titer of the overexpressed native polypeptide is above 50 g / L.

17. The food product of claim 16 wherein the titer of the overexpressed native polypeptide is above 60 g / L.

18. The food product of claim 17 wherein the titer of the overexpressed native polypeptide is above 70 g / L.

19. The food product of claim 18 wherein the titer of the overexpressed native polypeptide is above 80 g / L.

20. The food product of claim 19 wherein the titer of the overexpressed native polypeptide is above 90 g / L.

21. The food product of claim 20 wherein the titer of the overexpressed native polypeptide is above 100 g / L.

22. The food product of any of the preceding claims comprising at least 1% (w / w) of the overexpressed native polypeptide.

23. The food product of claim 22 comprising at least 2% (w / w) of the overexpressed native polypeptide.

24. The food product of claim 23 comprising at least 3% (w / w) of the overexpressed native polypeptide.

25. The food product of claim 24 comprising at least 4% (w / w) of the overexpressed native polypeptide.

26. The food product of claim 25 comprising at least 5% (w / w) of the overexpressed native polypeptide.

27. The food product of claim 26 comprising at least 6% (w / w) of the overexpressed native polypeptide.

28. The food product of claim 27 comprising at least 7% (w / w) of the overexpressed native polypeptide.

29. The food product of claim 28 comprising at least 8% (w / w) of the overexpressed native polypeptide.

30. The food product of claim 29 comprising at least 9% (w / w) of the overexpressed native polypeptide.

31. The food product of claim 30 comprising at least 10% (w / w) of the overexpressed native polypeptide.

32. The food product of claim 31 comprising at least 15% (w / w) of the overexpressed native polypeptide.

33. The food product of claim 32 comprising at least 20% (w / w) of the overexpressed native polypeptide.

34. The food product of claim 33 comprising at least 25% (w / w) of the overexpressed native polypeptide.

35. The food product of claim 34 comprising at least 30% (w / w) of the overexpressed native polypeptide.

36. The food product of claim 35 comprising at least 35% (w / w) of the overexpressed native polypeptide.

37. The food product of claim 36 comprising at least 40% (w / w) of the overexpressed native polypeptide.

38. The food product of claim 37 comprising at least 45% (w / w) of the overexpressed native polypeptide.

39. The food product of claim 38 comprising at least 50% (w / w) of the overexpressed native polypeptide.

40. The food product of claim 39 comprising at least 55% (w / w) of the overexpressed native polypeptide.

41. The food product of claim 40 comprising at least 60% (w / w) of the overexpressed native polypeptide.

42. The food product of claim 41 comprising at least 65% (w / w) of the overexpressed native polypeptide.

43. The food product of claim 42 comprising at least 70% (w / w) of the overexpressed native polypeptide.

44. The food product of claim 43 comprising at least 75% (w / w) of the overexpressed native polypeptide.

45. The food product of claim 44 comprising at least 80% (w / w) of the overexpressed native polypeptide.

46. The food product of claim 45 comprising at least 85% (w / w) of the overexpressed native polypeptide.

47. The food product of claim 46 comprising at least 90% (w / w) of the overexpressed native polypeptide.

48. The food product of claim 47 comprising at least 95% (w / w) of the overexpressed native polypeptide.

49. The food product of claim 48 comprising at least 99% (w / w) of the overexpressed native polypeptide.

50. The food product of any of the preceding claims wherein the overexpressed native polypeptide is an enzyme.

51. The food product of claim 50 wherein the enzyme is an alpha-glucosidase.

52. The food product of claim 51 wherein the alpha-glucosidase is derived from Trichoderma reesei.

53. The food product of claim 52 wherein the titer of alpha-glucosidase is at least 100 g / L.

54. The food product of any of claims 50 to 53 wherein the enzyme is inactivated.

55. The food product of claim 54 wherein the enzyme is inactivated before addition to the beverage or food stuff.

56. The food product of claim 54 wherein the enzyme is inactivated after addition to the beverage or food stuff.

57. The food product of any of the preceding claims wherein the over expressed native polypeptide has superior foaming and / or emulsification and / or gel forming properties compared with a denatured protein.

58. The food product of any of the preceding claims wherein the overexpressed native protein contains less than 100 ppm of saponin.

59. The food product of any of the preceding claims wherein the overexpressed native protein does not have off flavors.

60. A method for preparing a plant-based meat alternative, comprising: mixing a batter comprising water, plant protein, one or more meat flavors and a fungal protein comprising an over expressed native polypeptide; and baking or cooking the batter to provide the plant-based meat.

61. The method of claim 60 wherein the batter further comprises textured or structured protein derived from plants.

62. The method of claims 60 or 61 wherein the plant protein is chickpea, pea, soy, fava, gluten, or oat.

63. The method of claim 62 wherein the plant protein is soy protein or pea protein.

64. The method of any of claims 60 to 63 wherein the batter further comprises a transglutaminase.

65. The method of any of claims 60 to 64 wherein the batter further comprises one or more hydrocolloids.

66. The method of any of claims 60 to 65 wherein the fungal protein is spray dried freeze dried.

67. The method of any of claims 60 to 66 wherein the fungal protein is derived from Aspergillus, Fusarium, Mucor, Rhizomucor or Trichoderma.

68. The method of claim 67 wherein the fungal protein is derived from Aspergillus.

69. The method of claim 68 wherein the fungal protein is derived from Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, or Aspergillus tubigensis.

70. The method of claim 67 wherein the fungal protein is derived from Trichoderma.

71. The method of claim 70 wherein the fungal protein is derived from Trichoderma reesei.

72. The method of claim 67 wherein the fungal protein is derived from Fusarium.

73. The method of claim 72 wherein the fungal protein is derived from Fusarium oxysporum.

74. The method of claim 67 wherein the fungal protein is derived from Rhizomucor.

75. The method of claim 74 wherein the fungal protein is derived from Rhizomucor miehei.

76. The method of any of claims 60 to 75 wherein the fungal protein is the product of fungal fermentation and the titer of the overexpressed native polypeptide is above 10 g / L.

77. The method of claim 76 wherein the titer of the overexpressed native polypeptide is above 20 g / L.

78. The method of claim 77 wherein the titer of the overexpressed native polypeptide is above 30 g / L.

79. The method of claim 78 wherein the titer of the overexpressed native polypeptide is above 40 g / L.

80. The method of claim 79 wherein the titer of the overexpressed native polypeptide is above 50 g / L.

81. The method of claim 80 wherein the titer of the overexpressed native polypeptide is above 60 g / L.

82. The method of claim 81 wherein the titer of the overexpressed native polypeptide is above 70 g / L.

83. The method of claim 82 wherein the titer of the overexpressed native polypeptide is above 80 g / L.

84. The method of claim 83 wherein the titer of the overexpressed native polypeptide is above 90 g / L.

85. The method of claim 84 wherein the titer of the overexpressed native polypeptide is above 100 g / L.

86. The method of any of claims 60 to 85 wherein the batter comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 % (w / w) of the overexpressed native polypeptide.

87. The method of any of claims 60 to 86 wherein the overexpressed native polypeptide is an enzyme.

88. The method of claim 87 wherein the enzyme is an alpha-glucosidase.

89. The method of claim 88 wherein the alpha-glucosidase is derived from Trichoderma reesei.

90. The method of claim 89 wherein the titer of alpha-glucosidase is at least 100 g / L.

91. The method of any of claims 87 to 90 wherein the enzyme is inactivated.

92. The method of claim 91 wherein the enzyme is inactivated before addition to the batter.

93. The method of claim 91 wherein the enzyme is inactivated after addition to the batter.

94. The method of any of claim 60 to 93 wherein the over expressed native polypeptide has superior foaming and / or emulsification and / or gel forming properties compared with a denatured protein.

95. The method of any of claims 60 to 94 wherein the overexpressed native protein contains less than 100 ppm of saponin.

96. The method of any of claims 60 to 94 wherein the overexpressed native protein does not have off flavors.

97. The method of any of claims 60 to 96 wherein the plant-based meat alternative is a frankfurter style sausage.

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