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The Protein Transition: The science behind alternative proteins

LECTURE 3

Plant-based meat: Leveraging plants to create alternative protein products

Instructor: Priera Panescu, Ph.D.

Senior Scientist, Plant-Based Specialist

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Agenda

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Plant-based meat overview

Crop development for plant-based meat

Ingredient optimization for plant-based meat

End product formulation and manufacturing

Looking ahead

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Plant-based meat overview��

Definition of plant-based meat
Opportunities across the value chain

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Plant protein product spectrum

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Beans, lentils, mushrooms, etc.

Tofu, tempeh, seitan, etc.

Bean burgers

Meat mimics

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Plant-based product definitions

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Plant-based meat, eggs, and dairy are produced directly from plants.

Like animal products, they are composed of protein, fat, vitamins, minerals, and water. Next-gen plant-based options look, taste, and cook like conventional meat, �and offer complex carbs and fiber.

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Proteins 101

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Amino acid sequence affects protein physicochemical properties.

Protein molecular weight and configuration impact functionality and texture.

Each species (plant or animal) has a unique composition of protein types.

Plants also contain non-protein components including starch, �fiber, and oil.

Lecture 2 of this course, “What is meat?” goes into detail about protein functionality and structure, but here are some few key points especially important to plant proteins. First, the amino acid sequence of a protein affects its physicochemical properties. For examples, amino acids can be hydrophobic, hydrophilic, ionically charged, or reactive. Which amino acids are present, and how they are sequenced has a significant effect on protein properties, like solubility, water and fat holding capacity, emulsifying ability, and structure. The resulting overall protein molecular weight and configuration also has impacts on its texture and functionality. For example, proteins with high beta-sheet structure content are generally less digestible than those with higher alpha-helical content. Additionally, each species has a unique composition of protein types, so different plant proteins will have different properties, textures, and structures. Lastly, in addition to proteins, plants mostly contain carbohydrates, fiber, oil, and other compounds.

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Technological insight:

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Globular proteins

Fibrous proteins

Storage

Structure

the core goal of plant-based meat is persuading plant proteins to act like animal proteins

Two types of proteins that are especially important when comparing plant and animal meat are globular proteins and fibrous proteins. Globular proteins act as functional and structural proteins, have irregular amino acid sequences, are more water soluble, and have tightly packed, spherical structures that are more sensitive to environmental conditions like pH and temperature changes. Fibrous proteins are typically more structural and protective and form fibrous structures like connective tissue and muscle fiber. Fibrous proteins have repetitive amino acid sequences and are mostly water insoluble. Most plant proteins are globular and most animal meat proteins are fibrous. As a result, the core goal of plant-based meat is persuading globular proteins to develop the function and structures of fibrous proteins. To accomplish this goal, there are many opportunities across the value chain.

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How is plant-based meat made?

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INGREDIENT PROCESSING

Raw materials are isolated and functionalized by mechanical and chemical processes to create optimal ingredients for the end product.

END PRODUCT FORMULATION AND MANUFACTURING

Texturization

The correct mix of ingredients and processes are established to create the desired taste, texture, smell, and structure

SOURCE SELECTION

Characterize new crop sources to diversify the available inputs for plant-based meat.

OPTIMIZATION

The source material is optimized via breeding or engineering.

Crop development

Ingredient optimization

Formulation & manufacturing

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Crop development for plant-based meat��

Plant protein sources
Crop breeding for protein functionality

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While soy and wheat are still dominant,

Brand	Soy	Wheat	Mycoprotein	Pea	Other
Beyond Meat				✓	✓
Boca	✓	✓
Dr. Praeger’s	✓			✓	✓
Field Roast		✓
Gardein	✓	✓
Impossible Foods	✓
Lightlife	✓	✓		✓
Morningstar	✓	✓			✓
Quorn			✓
Tofurky	✓	✓

Source: GFI 2020

many of the top 10 U.S. plant-based meat retail brands have been integrating soy and other proteins

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Crop development focused on plant proteins

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Protein content & quality

Nutrition & claims

Allergenicity, intolerance

Consumer perception

Source (geographic, �commercial)

Functionality

Familiarity with use

Cost

Aroma, flavor, texture, mouthfeel, color

There are many considerations when choosing the optimum plant protein ingredient, including:

Historical use

Certifications

Availability

Safety

Regulatory

Historically, plant proteins used for meat alternatives are derived from commodity crop sidestreams:

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Plant proteins with growth potential

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Potential to be cost effective (byproduct of 3^rd biggest oilseed). Needs scale-up & commercial development.

Scale-up needed for cost improvement. Excellent properties & starch byproduct used for noodles & other foods.

Attractive attributes. Volume can expand until available precursor from starch processing is consumed.

Sustainable, excellent properties. Needs scaling and commercial development to increase volume & decrease cost.

Great potential if byproduct utilization (starch) is improved.

Alternative plant proteins need a competitive value proposition to bring about growth.

To compete directly with wheat and soy a major question is how well they texturize.

This table displays examples of novel proteins that have the potential for use in plant-based product formulations. Sunflowers are the 3rd biggest oilseed globally and have high protein content, so sunflower proteins have a lot of potential. Mung bean protein has gained traction lately as it is in Beyond Meats burger patty (along with pea, brown rice, and faba bean proteins). Potato proteins are in Impossible burgers and while potatoes have low protein content, the proteins have great functionality and digestibility. Duckweed protein is emerging as a sustainable and functional protein alternative. Overall, the theme with these crop sources is that they have proteins with good potential, but the commercial development, like scaling and lowering the cost of production, are necessary. Part of this development includes evaluating sidestream uses. For instance, chickpea, navy bean, and oat have proteins with interesting properties, but protein extraction is only commercially viable if a use for the starch sidestreams is improved.

There is a lot of opportunity for researchers to discover new crop sources, improve their commercial use, and evaluate how well they texturize compared to soy, wheat, and animal proteins.

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Algae, seaweed, and aquatic plants

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Algae, seaweed, and aquatic plants offer a particular portfolio of opportunities as plant protein sources:

High in protein �(e.g. 9-25%)

Omega-3 fatty acid content

Scalable (can be �grown very efficiently �and inexpensively)

Whole-plant harvesting

Minimal land use

Coloration (red seaweeds like dulse turn brown when cooked)

Duck Weed

Dulse

Spirulina

Examples:

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Resource: The Plant Protein Primer

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The Plant Protein Primer is a basic overview of plant proteins, processing, and choice parameters that looks at key metrics and provides a framework for thinking about plant protein sources.

Download at gfi.org/resource/plant-protein-primer

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Protein powders are heterogeneous mixtures

Source: McClements and Grossmann Compr. Rev. Sci. Food Saf. 2021.

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	Protein in fraction (%)	Molecular Weight (kDa)	Isoelectric point (pI)	Melting Temp. (T_m) (ºC)	Comments
Soy
Glycinin (11S)	36 to 51	300 to 380	4.5	93	Trimer, globulin
β-conglycinin (7S)	17 to 24	150 to 200	5	80	Hexamer, globulin
Pea					Multimers
Legumin (11S)	55 to 80	360	4.5	75 to 79	Globulin
Vicilin (7S)		150			Globulin
Convicilin		280			Globulin
Albumin (2S)	18 to 25	50	6.0	110	Albumin
Canola
Globulins	60	14 to 59	4.5	84 to 102	Mainly cruciferin (12S)
Albumins	20				Mainly napin (2S)
Glutelins	15 to 20
Prolamins	2 to 5

Plant proteins are classified based on solubility
Globulins	saline soluble
Albumins	water soluble (neutral)
Prolamins	alcohol soluble
Glutelins	water soluble (acidic or basic)

Protein functionality is dependent on protein composition.

While we often refer to crop proteins as a single entity, crop proteins are actually heterogenous mixtures of proteins. For example, soy protein isolate is mostly made up of glycinin and beta-conglycinin proteins. The protein fraction breakdown is listed here for soy, pea, and canola protein. Generally, plants proteins fall into one of four categories depending on their solubility. Globulins are saline soluble, albumins are soluble in neutral water, prolamins are soluble in water-alcohol mixtures, and glutelins and soluble in acid or basic water. For soy, glycinin and beta-conglycinin are both globulins. Pea protein also contains mostly globulins, but does have a relatively high percentage of albumin proteins. Finally, canola is also mostly globulin, but has relatively high percentages of albumins, glutelins, and prolamins.

The ultimate protein functionality and structure is dependent on the distribution of these protein fractions. For instance, in soy, glycinin has better gelation abilities than beta-conglycinin, so for plant-based meat, high glycinin percentages are preferred.

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Crop genetics and breeding for protein functionality

Source: Ahmad et al. J. Agric. Food Chem. 2021.

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Crop traits of interest for plant-based meat:�

High total protein content
Optimal protein fraction composition
Bland protein flavor
Good protein digestibility
High micronutrient concentration
Minimal anti- and non-nutrient factors
Low toxicity and allergenicity
Suitable protein functionality
Minimal processing required to enrich the protein fraction

Besides the exploration of native crop sources and growth conditions, raw ingredient functionality and composition can be controlled by selecting and breeding crop cultivars. Crops have been traditionally bred for increased yield, but shifting the focus to include plant protein functionality would bolster food science and products. Some agronomic and seed quality traits that are desirable for plant-based meat include (list off). Traits that are observable and quantifiable are referred to as the phenotype of the plant. The phenotype of a plant can be mapped to section of crop genes called quantitative trait loci.

Conventional breeding methods rely on naturally occurring genetic variations, where parent crops are cross-bred and their progeny are selected for further breeding until the trait of interest is at a desired expression level. This type of breeding doesn’t necessarily require genetic information, but can be informed and directed by understanding crop genes.

While conventional crop breeding can be viewed as the 50/50 transfer of genes from two crops into a new crop, genetic engineering is the transfer of selected genes from one organism into a crop. Genetic engineering can incorporate more versatile genes and is more precise, transferring only the desired gene and to a desired location. However, for more complex, multi-gene, naturally-occurring traits, traditional breeding can be less time-intensive, especially in conjunction with speed breeding and other technologies.

Moreover, genome editing technologies, like CRISPR, are becoming more popular and are able to transform plants genes through deleting, inserting, or substituting nonnative gene into plant DNA.

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Ingredient optimization for plant-based meat

Wet and dry crop fractionation
Sidestreams
Post-enrichment protein modifications

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Commercial proteins are complex mixtures

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Enhance

protein gel strength�(less network disruption)
formulation flexibility
protein nutrient density
digestive tolerance

Reduce

extrusion die slip
starch texture
fiber mouthfeel
antinutritional components

Challenges

maintaining functionality
cost
natural perception

isolate

concentrate

flour

protein concentration

0%

100%

oil starch fiber

properties depend on source and process

Proteins are often extracted to improve their properties for a set of applications. This usually focuses on the removal of macromolecular components including oil, starch and fiber. The type of process will determine which components are retained and which are removed. For protein isolation, crops are first made into a flour then enriched further. The primary starting material for the plant-based meat production process is typically plant protein concentrate (comprising 60-70% protein) or isolate (comprising at least 90% protein).

The goals for purification of plant proteins are to reduce impurities which could negatively affect extrusion, mouthfeel, texture, or digestion of the end product. Moreover, increased protein content can improve protein gel strength, formulation flexibility, protein nutritional density, and digestibility.

There are some challenges associated with protein extraction. The enrichment process can intentionally or unintentionally alter the functional and nutrition properties of the proteins in a variety of ways, including denaturation, hydrolysis, chemical modification & cross-linking. Moreover, scalability and cost for commercial use of these enrichment techniques still needs to be improved. Lastly, consumers sometimes view crop fractionation as “overly processed”, but there are some clean label approaches to ingredient optimization.

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Details about enrichment processes

Source: Schutyser et al. Trends in Food & Technology, 2015.

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Crops are fractionated to separate macromolecules.

Protein enrichment can remove small impurities like antinutrients and off-flavors.

Fractionation strategy effects protein:

Protein types recovered
Properties
Yield

These flow charts represent example crop fractionation processes for protein enrichment. These are split between wet or dry fractionation steps. In wet fractionation, solvents are utilized to extract, precipitate, or centrifuge proteins, oil, carbohydrates, and fibers. When focused on protein enrichment, protein solubility in water and other extraction solvents plays an important role in which methods are applied. Generally, wet fractionation begins with milling seeds then preparing a flour suspension for component extraction. For oil-rich sources, flours are typically first defatted with non-polar solvents, like hexane, to remove oil. Water soluble components are then extracted in basic aqueous solution to solubilize protein and remove insoluble carbohydrates. The protein suspension is then precipitated in acidic aqueous solution. The soluble carbohydrates are removed and the remaining solids are dried and mostly contain protein.

Dry fractionation also begins with milling, although more rigorously milling into smaller particles is often required. Flour can then be separated without the use of additional water and solvents through air classification. Less aerodynamic particles, like large starch granules, are separated from more aerodynamic particles, like protein bodies, forming coarse and fine fractions. Dry fractionation is usually not as effective as wet fractionation.

Note that besides carbohydrates, oils, and fibers, efficiently removing small molecular impurities like antinutrients is important. Also, there are many different chemical, physical, and biological protein processing methods besides the ones outlines here. Fractionation strategies are very important because they can affect the types of proteins recovered, their properties, and the overall protein yield.

Whether starch, oil, or protein are the main goal for crop fractionation, there are opportunities for using the whole crop if the overall fractionation process is developed for various applications. Byproducts that are generated from a main process are referred to as side streams.

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Applications of sidestreams

Sidestreams as inputs and outputs during plant-based meat production:

Applying sidestreams lowers production costs and promotes zero-waste economies.

Biomass sidestreams that arise from plant-based meat production have potential utility in cultivated meat and microbial fermentation production.

Source: Kamal et al. Compr. Rev. Sci. Food Saf. 2021.

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Food waste protein source	Crude protein content (g/100 g)	Reference
Buckwheat bran	27.8	Mattila et al. 2018
Flaxseed	20.9
Rapeseed press cake	35.7
Wheat bran	11.05
Hops	21.63
Brewer dry grain	19.96	Haile et al. 2017
Papaya seed	25.9	Filho et al. 2012

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Functionality

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Dispersibility
Solubility
Viscosity
Gelation
Emulsification
Foaming
Water holding
Oil holding

Solvent (e.g. pH, salt, Aw)
Temperature
Time
Pressure
Shear
Concentration

Proteins are often expected to have useful attributes:

These functions may be dependent on:

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Post-enrichment protein modification

Source: Biorender

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Proteins can be further modified via denaturation, hydrolysis, modification, and cross-linking.

Protein modifications are changes to the sequence and/or structure, implemented through chemical, physical, or biological processing. These treatments can optimize and diversify their structural and physicochemical properties, making proteins more water-soluble and improving their techno-functional properties, such as emulsification, water or fat holding, gelation, and foaming abilities. The resulting proteins can be easier to apply in formulations and can impart enhanced end product digestibility and organoleptic properties.

On the lefthand side of this slide, there are a few examples of chemical modifications including phosphorylation, acetylation, glycosylation, and disulfide formation. These modifications can also be achieved using enzymes. Other enzymatic modifications of plant proteins include hydrolysis, glycation, cross-linking, and catalyzing the degradation of impurities. The example on the right shows a common technique used to cross-link proteins together by using the enzyme transglutaminase which catalizes the amide bond formation between glutamine and lysine residues.

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Resource: State of the Industry Report

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The 2020 State of the Industry Report: Plant-Based Meat, Eggs, and Dairy is a snapshot of the major developments of plant-based food industry in 2020 and predictions for trends in 2021.

Download at gfi.org/resource/plant-based-meat-eggs-and-dairy-state-of-the-industry-report/

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Formulation and manufacturing of plant-based meat

Plant-based fats and other ingredients
High-moisture extrusion
Shear cell technology

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Plant-based fat alternatives

Animal adipose tissue is a complex matrix of collagen and fat molecules that is solid at room temperature and gradually melts.

Coconut oil and cocoa butter are native plant fats that are semi-solid at room temperature. Otherwise, plant fats tend to be liquid at ambient temperatures.

Sustainable alternatives are being explored: oleogels, microbially-derived fats, etc.

Animal-free sources of omega-3 fatty acids are also of interest to researchers.

Source: Chapter 6 – Modification of Fats and Oils 1998.

In conventional meat, fats improve juiciness, tenderness, and palatability. Like proteins, animal-based fats are challenging to replicate with plant-based ingredients. This is because animal adipose tissue is a complex matrix of collagen and fat molecules that is solid at room temperature and gradually melts when heated. Generally, plant lipids are liquid oils at room temperature. The graph on the right shows that rapeseed oil, labeled C, has 0% solid content around ambient temperatures while lard and butter, labelled E and F on the graph, have good solid content at gradually decrease solid content at cooking temperatures. Coconut oil, labelled A, has a higher melting point than rapeseed oil, but is still semi-solid at room temperature and melts rapidly. Currently, coconut oil, cocoa butter, and other semi-solid plant oils are used to replicate animal fat, but there’s room for optimization.

Other alternative technologies for fat alternatives include oleogels, microbially-derived fats, and stabilized fat emulsions. Another important challenge for plant-based meats are sourcing and incorporating animal-free omega-3 fatty acids. Omega-3 fatty acids are traditionally found in fatty fish, so they’re particularly important for seafood alternatives.

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Resource: Formulating with animal-free ingredients

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Formulating with animal-free ingredients was published in Food Technology and describes advances and opportunities to use animal-free ingredients to create meat, egg, and dairy alternatives.

Download at gfi.org/resource/formulating-with-animal-free-ingredients/

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Restructured vs. whole muscle type products

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PATTY

LINK

SLICED

SASHIMI

WHOLE MUSCLE

RESTRUCTURED

CUBED

FLAKED

For plant-based meats, plant proteins should be texturized to replicate the fibrous nature of animal proteins. How products are texturized and manufactured depends on the desired end product. Restructured meats are ground, shredded, or flaked products like burger patties, links, or shredded chicken breast. These types of meats are typically easier to replicate since they do not closely resemble the original animal muscle. Whole muscle meat cuts, like sashimi, steak, or other sliced meat, is more difficult to replicate because the hierarchical animal muscle structure is intact. The process of reforming plant proteins to achieve these goals is called texturization.

Products featured (top to bottom, left to right): Impossible burger, Beyond Meat sausage, Good Catch tuna, Quorn chicken pieces, Ocean Hugger’s ahimi, Gardein’s chicken scallopini

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Texturization basics

Texturization of proteins is the process of creating a 3-dimensional structure to match the texture, mouthfeel, and appearance of animal-based meat

Denatures and aligns proteins
Forms fibers that resemble muscle tissue
Produces end products such as granules, shreds, and chunks

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Production technology landscape

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Twin screw high moisture extrusion

Low moisture extrusion

3D printing &

electrospinning

Shear

cell

technology

Enzymatic functionalization

& texturization

Fermentation

Built-for-scale

production

EXISTING

MECHANICAL INNOVATION

BIOLOGICAL INNOVATION

DESIGN & ENGINEERING INNOVATION

Mixing,

molding,

forming

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Extrusion cooking

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Extrusion transforms native ingredient biopolymers (inputs) into a continuous semi-solid (output). To complete this process, a screw system within a barrel conveys mass (a combination of dry ingredients, water, and/or oil) through a die (small opening).

Source: Pietsch et al. ThermoFisher Whitepaper, 2021.

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Low- vs. high-moisture extrusion

Extrusion inlet/process moisture content > 50%
Whole muscle type products; best resembles muscle striata (e.g., fillets and strips)
Extruded in a high-moisture form, then passed through one or more steps including marinating, coating, and cooling (refrigeration/freezing)

HIGH-MOISTURE EXTRUDATE

Extrusion inlet/process moisture content < 35%
Restructured meat products (e.g., patties and links); made by hydrating TVP in water or broth (may also include oils)
Hydrated product is blended with other ingredients to impart the taste, texture, color, and aroma of meat

LOW-MOISTURE EXTRUDATE

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Emerging: shear cell technology

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Source: Birgit L. Dekkers Creation of fibrous plant protein foods. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018). ISBN 978-94-6343-319-8

Shear cell technology allows for independently controlled shear rate, temperature, and residence time, unlike extrusion where these three parameters are coupled. Two device geometries have been developed for shear cell.

The first is cone-in-cone where the bottom cone is rotating while the top cone is stationary, and the material placed in between is sheared. During shearing, material can be heated from both sides, and to prevent moisture evaporation, the shear cells are sealed at the rim. The speed of the rotating bottom cone and the angle between the two cones determines the rate of deformation.

The second geometry is a couette, or cylinder-in-cylinder device which consists of a stationary outer cylinder and a smaller rotating inner cylinder. The two concentric cylinders induce Couette flow in the gap, which is to a large degree similar as the flow inside a cone-in-cone device

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Emerging: shear cell technology

calcium caseinate soy protein isolate-wheat gluten blend soy protein concentrate

Source: Birgit L. Dekkers Creation of fibrous plant protein foods. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018). ISBN 978-94-6343-319-8

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Resource: Plant-based meat manufacturing by extrusion

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The Plant-based meat manufacturing by extrusion guide provides a basic overview of extrusion and process flow diagrams of whole muscle meat and restructured meat products.

Download at gfi.org/resource/plant-based-meat-manufacturing-guide/

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Looking ahead�

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Animal product alternatives will occur along a spectrum

Fully plant-based

Fully cultivated

Hybrid products

Tofu

Plant-based burger with cultivated fat

Synthetic gelatin

Cultivated meat

Impossible burger

100% fermentation-derived

Growth factors from fermentation

Fermentation-derived flavoring ingredients

Enzymes can improve protein functionalization or coagulation

The first of which is looking toward fermentation-derived and cultivated ingredients to form superior hybrid products. Ultimately, in the future we won’t see these 3 alternative protein production platforms as distinct industry segments, but rather as one large industry where we produce animal product alternatives across a spectrum, utilizing ingredients from one or more technology. Products from microbial fermentation are likely to play a role along the entire alternative protein spectrum, making them a critical component of the industry. Examples include:

- Enzymes to improve functionality

Ingredients to improve sensory characteristics. For example, the Impossible Burger, which is plant-based but uses fermentation to produce lehemoglobin, the signature ingredient that gives it that bloody appearance and meaty taste.
Growth factors for cultivated meat
100% fermentation derived ingredients to replace animal-based versions

�

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Data will be central to the next phase of plant-based meat

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Thank you!

Check out GFI’s the science of plant-based meat explainer: https://gfi.org/science/the-science-of-plant-based-meat/

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