Methods for forming texturized, cell-based meat analogues

Abstract

Provided herein are texturization methods that allow for replication of the fibrous texture of animal flesh using a large amount of cells while using a high throughput methodology at temperatures below typical extrusion temperatures. The texturization methods incorporate Plantago seed-derived polysaccharide, including for example, psyllium. Suitable sources of cells may be used in such texturization, including animal-derived cells, finfish-derived cells, and yeast cells. The resulting texturized cell mass has visual fibrosity, and may be used to produce one or more downstream products, including food products.

Description

32676-20001.40 METHODS FOR FORMING TEXTURIZED, CELL-BASED MEAT ANALOGUES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 678,944, filed August 2, 2024, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates generally to texturization methods, and more specifically to methods to introduce fibrous texture into a cell mass using Plantago (Plantaginaceae) seed-derived materials. BACKGROUND

[0003] Typically, textured proteins are made by extrusion, but the actual texture comes from alignment and crosslinking of proteins that happens in the die of the extruder. Other methods for creating texture include electrospinning, extrusion with alginate (wet spinning), freeze structuring, as well as mixing proteins and polysaccharides. Currently, existing extrusion texturization methods typically require higher temperatures, e.g., 90°C or 100°C or higher, and conditions that can cause protein unfolding and impact alignment. Existing extrusion technologies are also typically challenged by fat addition that can result in destructuring and decreased strength.

[0004] Thus, what is needed in the art are alternative methods that replicate fibrous texture of animal flesh that can be performed on a commercially viable scale. BRIEF SUMMARY

[0005] In some aspects, the texturization methods provided herein allow for replication of the fibrous texture of animal flesh. In some embodiments, the texturization methods provided herein allow low temperature texturization of cells while using a high composition of cells (e.g., greater than about 50%, or even greater than about 90%) in a high throughput methodology.

[0006] In some aspects, provided is a method using Plantago (Plantaginaceae) seed- derived materials, including for example psyllium, to obtain fibrous texture. In some embodiments, the method comprises mixing a cell mass with Plantago (Plantaginaceae) seed- 1MOFO-35800624532676-20001.40 derived materials at temperature that is lower than typical extrusion temperatures to produce a texturized cell mass. In some variations, the cell mass comprises animal-derived cells, yeast cells, or plant cells. In one variation, the cell mass comprises finfish-derived cells. In some variations, the Plantago (Plantaginaceae) seed-derived material is psyllium.

[0007] In some embodiments of the foregoing, the mixing involves extrusion (e.g., the texturization method uses an extruder), or high-speed sheer mixing (e.g., the texturization method uses a food processor or blender, such as a Thermomix), or a combination thereof. In some variations, the temperature at which the cell mass and psyllium are mixed is less than 100°C, less than 90°C, or between 40°C and 85°C.

[0008] In another aspect, provided is a texturized cell mass produced according to any of the methods described herein. DESCRIPTION OF THE FIGURES

[0009] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0010] FIGS. 1A and 1B depict graphs that show the effects of visual fibrousness and hardness, respectively, for psyllium only as compared to psyllium and cells over various psyllium concentrations. (FIG. 1A n=5 technical replicates; FIG. 1B n=3 technical replicates).

[0011] FIG. 2 depicts a graph showing the temperature impact on firmness post- extrusion.

[0012] FIGS. 3A and 3B depict graphs that show effects of visual fibrousness and hardness, respectively, with changing psyllium concentrations. (FIG. 3A n=5 technical replicates; FIG. 3B n=3 technical replicates).

[0013] FIGS. 4A and 4B depict graphs that show effects of visual fibrousness and hardness, respectively, with changing solids inclusion. (FIG. 4A n=5 technical replicates; FIG. 4B n=3 technical replicates). 2MOFO-35800624532676-20001.40 DETAILED DESCRIPTION

[0014] The following description sets forth exemplary compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0015] In some aspects, the methods provided herein provide benefits of texturizing with Plantago (Plantaginaceae) seed-derived polysaccharides, such as psyllium. In certain embodiments, the texturization methods can generate a fibrous texture in a high throughput setting at lower temperatures (e.g., relative to the traditional texturization methods). Fiber formation was unexpectedly observed during low temperature extrusion trials upon inclusion of plant-derived fibers to cells. In some embodiments, the methods provided herein also do not use ions to crosslink polysaccharides present in the texturized cell mass. Plantago Seed-Derived Materials

[0016] The methods provided herein use Plantago-derived seed materials to introduce fibrosity into a cell mass. Plantago (Plantaginaceae) is an annual or perennial, stemmed, or stemless herb, which has more than 200 species, including for example Plantago ovata, Plantago asiatica, Plantago major, and Plantago depressa. In some variations, the methods use Plantago-seed derived polysaccharides. In some variations, the methods use materials (or polysaccharides) derived from a plant in the genus Plantago. In certain embodiments, Plantago-seed is Plantago ovata-seed.

[0017] In one embodiment, the methods provided herein use psyllium. In some variations, the psyllium comprises arabinoxylan. In certain variations, the psyllium comprises highly branched arabinoxylan. In some variations of the foregoing, the psyllium further comprises xylose, uronic acid, galactose, rhamnose, glucose and / or mannose.

[0018] In some variations of the foregoing, the Plantago-derived seed material is provided in powder form. In other variations, the Plantago-derived seed material is provided in hydrated form. Cell Mass

[0019] In some embodiments, the cell mass is provided as a cell slurry. 3MOFO-35800624532676-20001.40

[0020] In some embodiments, the cell mass comprises animal-derived cells. In certain embodiments, the cell mass comprises finfish-derived cells. In other embodiments, the cell mass comprises yeast cells.

[0021] The cell mass provided may be determined by dry weight vs. wet weight. Further, it is generally well understood in the art how to calculate wet basis from dry basis.

[0022] In other variations, the cell mass has a solids content, by dry mass, between about 5% and 25%. In other variations, the cell mass has a solids-to-moisture ratio between about 0.01 and about 0.35. The moisture present in the cell mass may vary depending on various factors, including other components that may be present, including fat content. Mixing

[0023] In some embodiments, the mixing involves extrusion. In some variations, the texturization method provided here uses an extruder. In other embodiments, the mixing involves high-speed sheer mixing. In some variations, the texturization method uses a mixing device that has a heating element, a motor for fast or slow blending and stirring, and a weighing scale, such as a Thermomix. In other embodiments, any combination of the mixing methods described herein may be used. Any suitable equipment that is commercially available and any suitable techniques known in the art may be employed to perform the mixing.

[0024] In some variations, the temperature at which the cell mass and the Plantago seed- derived materials (including, e.g., psyllium) are mixed is less than 100°C, less than 95°C, less than 90°C, or less than 85°C; or between 40°C and 95°C, between 40°C and 90°C, or between 40°C and 85°C.

[0025] In one aspect, provided is a method for producing a texturized cell mass, comprising: mixing and / or shearing a cell mass provided in the form of a slurry (e.g., a cell slurry) with Plantago seed-derived materials (including, e.g., psyllium) a suitable temperature as described herein to form a mixture. In some embodiments, the method further comprises extruding the mixture at a second temperature, wherein the second temperature is lower than the first temperature, to yield a texturized cell mass that has fibrosity. In some embodiments, the method comprises providing a cell slurry feed to a heated extruder barrel; providing Plantago seed-derived materials (including, e.g., psyllium) to the heated extruder barrel; 4MOFO-35800624532676-20001.40 mixing and / or shearing the cell slurry feed and Plantago seed-derived materials (including, e.g., psyllium) in the heated extruder barrel at a first temperature below 100°C, or between 40°C and 85°C to form a mixture; squeezing the mixture through a cooling die. In some variations of the foregoing, the Plantago seed-derived materials (including, e.g., psyllium) is provided to the heated extruder barrel after the cell slurry feed is provided to the heated extruder barrel. In other variations, the Plantago seed-derived materials (including, e.g., psyllium) is added to the heated extruder barrel containing the cell slurry feed for uniform distribution, and wherein the mixture is uniformly dispersed within the heated extruder barrel and continuously heated.

[0026] In some embodiments of the foregoing, one or more additional components are mixed with the cell mass and the Plantago seed-derived materials. In some variations, such additional components may include, for example, fat or oil, hydrocolloid, coloring agent, or plant protein (e.g., solid plant protein), or any combination thereof. Texturized Cell Mass

[0027] The methods provided herein introduce fibrosity into the cell mass, resulting in a texturized cell mass. In one aspect, provided is a texturized cell mass produced according to any of the methods described herein.

[0028] The texturized cell mass described herein, including produced according to the methods herein, may be characterized using various methods and techniques well known in the art. The texturized cell mass may be characterized based on, for example, fibrosity, fiber alignment, moisture content, and firmness.

[0029] Fibrosity may be characterized by any suitable methods and techniques known in the art. In some embodiments, the texturized cell mass (e.g., produced according to the methods provided herein) has a visual fibrosity based on a visual fibrousness sore. In some variations, the texturized cell mass has a visual fibrousness score of at least 10, at least 15, at least 20, at least 30, or at least 45; or between 10 and 50, or between 14 and 46, when evaluated by sensory analysis.

[0030] In some embodiments, the texturized cell mass (e.g., produced according to the methods provided herein) has substantially aligned fibers. For example, in one variation where the production method employs extrusion, the fibers of the texturized cell mass are 5MOFO-35800624532676-20001.40 substantially aligned in the direction of flow from the cooling die. In another variation where the production method employs extrusion, the fibers of the texturized cell mass are anisotropic. In other variations where the production method uses a food processor or blender (e.g., Thermomix), the fibers of the texturized cell mass are substantially aligned in the direction of flow. Any suitable techniques known in the art may be used to assess fiber alignment, including the use of microscopy.

[0031] In some embodiments, the texturized cell mass (e.g., produced according to the methods provided herein) has a moisture content between 60% and 95%, between 70% and 99%, between 60% and 90%, or between 70% and 90%.

[0032] In other embodiments, the texturized cell mass (e.g., produced according to the methods provided herein) has a firmness of greater than 100 g, or between 100 g and 2500 g. Downstream Uses

[0033] The texturized cell mass described herein, including produced according to any of the methods described herein, may be incorporated into an edible product, such as a food product. For example, in one aspect, the texturized cell mass described herein may be used as a component that mimics muscle in forming a cell-cultured fish composition. In some embodiments, the cell-cultured fish composition is a cell-cultured tuna composition.

[0034] In one aspect, provided is a cell-cultured tuna composition, comprising: a fat mimetic fraction comprising any of the texturized cell mass described herein (including produced according to the methods herein); and a fat mimetic fraction comprising fat and gelling agents. In some embodiments, wherein the fat mimetic fraction is injected into the fat mimetic fraction. In other embodiments, muscle mimetic fraction and the fat mimetic fraction are marbled. In some embodiments, the cell-cultured tuna composition further comprises gelling agent dispersed within the composition to hold the composition together. EXAMPLES

[0035] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation. 6MOFO-35800624532676-20001.40 Materials and Methods Materials

[0036] All raw materials other than the bluefin tuna cells used in the examples below were procured from commercially available sources.

[0037] Bluefin tuna cell slurry was produced as described by the expansion and concentration of suspension bluefin tuna cells. Stable cell lines of Bluefin tuna cells were expanded under growth conditions. The cells proliferated in dilute conditions under controlled pH and temperature conditions. The cell slurry was concentrated by centrifugation. These slurries were viscous solutions akin to a suspension that may flow like a liquid, with a water content of about 80-95%. The cell slurry was frozen then thawed for use in a subsequent formulation. Manufacturing By Extrusion

[0038] The following setups were used for extrusion processing. The first setup used a twin-screw extruder equipped with micro-plunger feeder that was capable of handling less than 100g of material per batch. The dimensionless aspect ratio of the cooling die was 75 and the process temperature tried were 90 ℃ and 120 ℃.

[0039] The second setup used a twin-screw extruder with a cooling die of L / T of 40. Multiple gravimetric and volumetric feeders were used to feed powders and liquids at different zones and different temperature conditions. The process temperature varied between 40 ℃ and 120 ℃. The screw profile was preset by the manufacturer for conveying and kneading based on their knowledge with an output rate at approximately 8 kg / hr. Manufacturing Using Thermomix

[0040] A Thermomix was used to process materials to replicate the shear and unidirectional alignment effect (as observed in extrusion) of various trials to evaluate the level of fibrousness in the final product. Once all ingredients were poured into the Thermomix, temperature was set at the desired temperature between 50 to 85 ℃ and shear was set at 10. The mixture was mixed for 2 minutes once the display temperature reaches the set temperature. All trials were performed in batches of 500g each. 7MOFO-35800624532676-20001.40 Texture Analysis

[0041] A Texture Analyzer was used to perform the texture profile analysis of the product at room temperature. The following test settings were used: 1.00 mm / sec Pre-Test Speed, 1.00 mm / sec Test Speed, 1.00 mm / sec Pos-Test Speed, 50% Strain, Auto (Force) Trigger. Data was analyzed to generate texture parameters such as hardness (g), resilience (%), cohesion, springiness (%), and chewiness. Sensory evaluation

[0042] The product was evaluated for different visual and perceptible sensory parameters using internally trained sensory panelists (n = 5). The product was evaluated first for visual fibrousness by asking panelist to tear apart the product using both the hands and inspect freshly exposed cross section to observe macroscopic fibers running in one direction. Next, panelists were asked to taste (chew) the product and evaluate for four different parameters specifically related to fibrous perception – rate of fiber breakdown, length of fiber during breakdown, fibrousness between teeth, and toothpull. All the samples were evaluated on a scale of 0 to 100 using pre-identified references for individual parameter. Example 1: Texturization of Hydrated Cells in the Presence of Psyllium Fiber

[0043] Bluefin tuna cells with approximate solid content of 15% were evaluated independently for the ability to texturize via extrusion at 90 ℃. This test utilized the extruder as described above. The cells exited through the cooling die as a frothy slurry without any texture formation.

[0044] In a subsequent test, the cells were combined with 6% psyllium fiber and processed at 90 ℃. The extruded slab was both cohesive and had an internal structure showing fibrous appearance with fibers running parallelly in the direction of flow.

[0045] When bluefin tuna cells with approximate solid content of 10% were combined with psyllium fiber at 5% concentration in Thermomix at 85℃, similar results were obtained where the resultant mass had visual fibrousness (30 ± 16 visual fibrousness score) and measurable hardness of 223 ± 44 g. In contrast, no visual fibrousness was observed in either control without cells or without psyllium. 8MOFO-35800624532676-20001.40

[0046] These studies demonstrate the unexpected finding that when cell biomass is processed under the appropriate conditions (temperature and shear) and in the presence of psyllium, they are able to form a solid, texturized product containing observed fibers. Example 2: Concentration Dependence of Psyllium Fiber Texturization

[0047] To understand the psyllium fiber concentration effect on the fibrousness, in the first set of experimentation, the psyllium fiber was added at 2%, 5%, or 10% with water and mixed in the Thermomix at 85℃. In another set, an additional 15% dry yeast cells were added keeping psyllium fiber concentration same at 2%, 5%, and 10%, respectively to understand the effect of solids addition on the texture formation. The concentration and results are shown in Table 1 and FIGS. 1A and 1B. The psyllium fiber at a certain minimum concentration (5%) resulted in measurable hardness and visual fibrousness. Similar results were obtained when the additional solids were added as dry yeast cells, but an increase in both visual fibrousness and hardness resulted from the combination of cells and psyllium. Table 1. Summary of results for visual fibrousness and hardness as a function of psyllium fiber concentration with / without dry yeast cells concentrationExample 3: Temperature Dependence of Psyllium Fiber Texturization

[0048] Processing temperature had a distinct effect on the firmness of the product coming out of the extruder when combined at the same concentration and same processing conditions except temperature (Table 2 and FIG. 2). For example, when psyllium fiber (10%), dry yeast cells (10%), and water (80%) were combined and processed at the indicated temperatures, textural attributes altered with increasing temperature. A change in the process temperature alone altered the final product texture, which ranged from soft chewy to firm rubbery. 9MOFO-35800624532676-20001.40 Notably, a substantial increase in firmness resulted for the highest temperature tested in this study. Table 2. Summary of product outcomes as a function of temperatureExample 4: Dependence of Texturization on Fiber and Cell Concentrations

[0049] Based on the data generated on minimum psyllium fiber concentration needed for visual fibrousness in Example 2, dry yeast cells were combined at 5%, 10%, 15%, or 20% concentration with 5% psyllium fiber in the Thermomix at 85℃ to understand the effect of solids at variable concentrations. Results showed that the hardness values were similar for all trials, repeating the result seen in Example 2 for the same concentration of Psyllium. The visual fibrousness score showed a dose dependent effect on yeast cell concentration and was highest for the trial with 10% dry yeast cells (FIGS. 3A and 3B). This result demonstrates an unexpected non-linear impact of cell concentration on hardness. Example 5: Comparison of Plant Fiber Source on Texturization

[0050] In this example, the psyllium fiber contributed to the formation of visually perceptible texture as compared to other plant fibers like pea fiber, bamboo fiber, oat fiber, and konjac gum when combined at a certain concentration with dry yeast cells and water and mixed in Thermomix at 85℃. Only psyllium fiber formed a cohesive solid mass that could be used for instrumental textural analysis. While others formed a pasty mixture or thick slurry. While psyllium fiber, a complex polysaccharide, is chemically distinct from pea and bamboo fibers (cellulosic mixtures) and konjac gum (glucomannan), it is similar to oat fiber, another complex polysaccharide that contains arabinoxylan. This testing demonstrates the unique attributes of psyllium fiber when compared to other similar materials. Results are summarized in Table 3 below. 10MOFO-35800624532676-20001.40 Table 3. Results of firmness and visual evaluation on products produced with cells and plant derived fibers.Example 6: Comparison of Solids Source on Texturization

[0051] To understand the effect of addition of other non-fiber types of solids sources on the visual fibrousness when combined with psyllium fiber, plant proteins such as rice protein and soy protein isolates and sunflower oil were tested. Each of these solids was added at 10% concentration and psyllium fiber was used at 5% concentration then mixed using Thermomix at 85℃. Data are shown in Table 4 and FIGS. 4A and 4B. Irrespective of the solids source, formulations including psyllium fiber showed visual fibrousness, with improvements for cells and sunflower oil + lecithin over no solids control. The majority of samples with solids plus psyllium resulted in similar hardness, with a slight increase in the presence of soy protein isolate. Notably, the temperatures used in this study are below that used in plant protein extrusion, indicating a different mechanism of action to result in enhanced texture in the presence of psyllium. A control was prepared for each tested solid without psyllium fiber and those formulations resulted in liquid mixtures without fibrousness or measurable hardness (data not shown). 11MOFO-35800624532676-20001.40 Table 4. Summary of results as a function of type of solids with constant psyllium fiber concentration

[0052] The results of this study show maintenance of fiber formation in the presence of the 10% oil concentration. This demonstrates an unexpected characteristic of the psyllium mediated texturization in the presence of high oil content. 12MOFO-358006245

Claims

32676-20001.40 CLAIMS What is claimed is:

1. A texturization method, comprising: combining a cell mass with a Plantago seed- derived polysaccharide to introduce fibrous texture into the cell mass.

2. A method for producing a texturized cell mass, comprising: mixing and / or shearing a slurry of a cell mass with a Plantago seed-derived polysaccharide at a temperature between 40°C and 90°C to produce the texturized cell mass.

3. The method of claim 1 or 2, wherein the method comprises mixing and shearing.

4. The method of any one of claims 1 to 3, wherein the method involves extrusion.

5. The method of any one of claims 1 to 4, wherein the method uses a food processor or blender.

6. The method of any one of claims 1 to 5, wherein the Plantago seed-derived polysaccharide comprises Plantago ovata seed-derived polysaccharide.

7. The method of any one of claims 1 to 5, wherein the Plantago seed-derived polysaccharide comprises psyllium.

8. The method of any one of claims 1 to 7, wherein the Plantago seed-derived polysaccharide is provided in powder form.

9. The method of any one of claims 1 to 7, wherein the Plantago seed-derived polysaccharide is provided in hydrated form.

10. The method of any one of claims 1 to 9, wherein the cell mass comprises animal- derived cells.

11. The method of any one of claims 1 to 9, wherein the cell mass comprises finfish- derived cells.

12. The method of any one of claims 1 to 9, wherein the cell mass comprises yeast cells.

13. The method of any one of claims 2 to 12, wherein the texturized cell mass has visual fibrosity. 13MOFO-35800624532676-20001.40 14. The method of claim 13, wherein the texturized cell mass has a visual fibrousness score of at least 10 when evaluated by sensory analysis.

15. The method of any one of claims 2 to 14, wherein the texturized cell mass has: (i) substantially aligned fibers; or (ii) a moisture content between 60% and 95%; or (iii) a firmness of greater than 100 g, or any combination of (i)-(iii).

16. A texturized cell mass produced according to the method of any one of claims 1 to 15.

17. A texturized cell mass, comprising: cells and Plantago seed-derived polysaccharide, wherein the texturized cell mass has visual fibrosity, and one or more of the following properties: (i) substantially aligned fibers; or (ii) a moisture content between 60% and 95%; or (iii) a firmness of greater than 100 g, or any combination of (i)-(iii).

18. The texturized cell mass of claim 17, wherein the texturized cell mass has a visual fibrousness score of at least 10 when evaluated by sensory analysis.

19. The texturized cell mass of claim 17 or 18, further comprising: plant protein, fat, or coloring agent, or any combination thereof.

20. A muscle mimetic fraction, comprising a texturized cell mass of any one of claims 16 to 19.

21. A cell-cultured tuna composition, comprising: a muscle mimetic fraction comprising a texturized cell mass of any one of claims 16 to 19; and a fat mimetic fraction comprising fat and gelling agents.

22. The composition of claim 21, wherein the white fraction is injected into the red fraction. 14MOFO-35800624532676-20001.40 23. The composition of claim 21, wherein the red fraction and the white fraction create marbling.

24. The composition of any one of claims 21 to 23, further comprising gelling agent dispersed within the composition to hold the composition together. 15MOFO-358006245

Images