foods

Review
Functionality of Ingredients and Additives in Plant-Based
Meat Analogues
Konstantina Kyriakopoulou * , Julia K. Keppler and Atze Jan van der Goot

Food Process Engineering, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands;
[email protected] (J.K.K.); [email protected] (A.J.v.d.G.)
* Correspondence: [email protected]

Abstract: Meat analogue research and development focuses on the production of sustainable products
that recreate conventional meat in its physical sensations (texture, appearance, taste, etc.) and
nutritional aspects. Minced products, like burger patties and nuggets, muscle-type products, like
chicken or steak-like cuts, and emulsion products, like Frankfurter and Mortadella type sausages, are
the major categories of meat analogues. In this review, we discuss key ingredients for the production
of these novel products, with special focus on protein sources, and underline the importance of
ingredient functionality. Our observation is that structuring processes are optimized based on
ingredients that were not originally designed for meat analogues applications. Therefore, mixing and
blending different plant materials to obtain superior functionality is for now the common practice. We
observed though that an alternative approach towards the use of ingredients such as flours, is gaining
more interest. The emphasis, in this case, is on functionality towards use in meat analogues, rather
than classical functionality such as purity and solubility. Another trend is the exploration of novel



protein sources such as seaweed, algae and proteins produced via fermentation (cellular agriculture).



Citation: Kyriakopoulou, K.; Keywords: plant protein; meat analogues; vegetarian sausage; vegetarian burger; vegetarian steak;
Keppler, J.K.; van der Goot, A.J. binders; flavours; colourants
Functionality of Ingredients and
Additives in Plant-Based Meat
Analogues. Foods 2021, 10, 600.
https://doi.org/10.3390/ 1. Introduction
foods10030600
The consumption of plant-based protein foods as a replacement for meat in western
countries seems to encounter several barriers, despite the consumers’ awareness over
Academic Editor: Frédéric Leroy
environmental issues [1]. Among the barriers is the unwillingness of consumers to make
this dietary change, due to the enjoyment of eating conventional meat, the nutritional
Received: 6 February 2021
Accepted: 9 March 2021
and sensory appeal, as well as, the convenience that meat offers [2,3]. The development
Published: 12 March 2021
of protein-rich plant-based products with the potential to replace meat in the nutritional
sense is already explored traditionally with the production of tofu, tempeh, seitan, etc.
Publisher’s Note: MDPI stays neutral
Recent meat analogue research and development focuses on the production of sustainable
with regard to jurisdictional claims in
products that recreate conventional meat, not only nutritionally, but also in all of its physical
published maps and institutional affil- sensations including texture, appearance, smell and taste [4]. Respective products that are
iations. available on the market are strips, chunks, patties and burgers, chicken-like blocks, ground
beef-like products, nuggets, steaks, sausages, etc.
Currently, technologies such as extrusion, shearing, spinning, and freeze alignment are
employed or proposed for texturizing vegetable proteins from oilseeds, pulses and grains,
Copyright: © 2021 by the authors.
forming a variety of structures, while fermentation has been used for many years now for
Licensee MDPI, Basel, Switzerland.
the growth of mycoproteins (Quorn). Among these technologies, extrusion and mixing
This article is an open access article
are mostly used in industry, with the rest being in a developing phase. Several recent
distributed under the terms and reviews are summarizing the different structuring technologies, ranging from bottom-
conditions of the Creative Commons up-approaches such as preparation of individual fibres that are assembled to meat like
Attribution (CC BY) license (https:// structures (like in the case of mycoproteins), or top-down approaches, where the dough is
creativecommons.org/licenses/by/ formed into structured products (for example through extrusion) [5–7]. Less well summa-
4.0/). rized is the fact that the type of structure achieved is dependent on the functional properties

Foods 2021, 10, 600. https://doi.org/10.3390/foods10030600 https://www.mdpi.com/journal/foods

Foods 2021, 10, 600 2 of 29

of the ingredients used. It is therefore relevant to have a closer look at the ingredients used
in currently available meat analogues.
A typical plant-based meat analogue contains, apart from protein in textured and
non-textured form, a significant amount of water, flavourings, oil or fat, binding agents
and colouring agents. However, most of the ingredients used in those products are highly
refined. That is the reason that meat analogues face more and more criticism for being
artificial products [8]. The use of less refined ingredients is a development that gains
traction in recent years [9–11]. To maintain new innovation in meat analogues, it is impera-
tive to understand the role of each ingredient (refined or not) and their interplay to find
alternatives that are better appreciated by consumers.
In this overview, a first introduction is given to the different types of plant-based meat
analogues produced through top-down technologies. This is followed by a discussion of the
key ingredients of plant-based meat analogues. Special focus is put on the protein sources,
and how origin and extraction methods affect their exerted functionality. We elaborate on
the requirements for the development of different categories of meat analogues and we
report cases and examples where specific ingredients could be used for the development of
new meat analogues.

2. Meat Analogue Products Formulation

Consumer preference studies in Western countries have shown that meat-eaters are
more willing to switch to plant-based meat analogues when the products mimic meat
in texture and sensorial properties and can be incorporated in a meal context that fits
with the consumer’s expectations [12,13]. Focusing on this consumer segment more and
more companies have launched plant-based products on the market resembling meat. The
meat analogue product categories addressed in this study are minced products (burger
patties and nuggets), muscle-type products (chicken or steak-like cuts) and emulsion-
type products (such as sausages). The following sections summarize the characteristics
of these products, as well as their requirements in terms of ingredients and additives.
In Section 3, we provide information on how these requirements can be fulfilled by the
currently available plant-based ingredients.

2.1. Emulsion-Type Products

Plant-based products such as sausages, frankfurters, bologna, mortadella, etc. are
examples of emulsion-type products. Similarly to the animal-based formulation, plant-
based products consist of a substantial amount of water, proteins, fats, carbohydrates
(gums, fibres, starch, etc.), salt and spices. The inspiration for the formulation of many
plant-based emulsion-type products comes from meat and meat extender applications,
where high protein non-meat substances partially replace meat [5].
In animal-based emulsion-type products, finely chopped meat from different sources
(pork, beef, mutton, etc.) is used to form a stable mixture that binds water and traps fat,
giving the product its characteristic texture when cooked [6]. Depending on the types of
meat used, a variety of products is produced ranging from high-quality all-meat sausages
to economy-style sausages, where lower meat quality cuts are augmented with higher fat
levels [7]. Although different parts of the animal are used in sausage production, lean meat
is usually separated firstly and mixed with water and salt to maximize protein extraction.
Afterwards, it is blended with the rest of the fat meat, spices and binders for the final
formulation [14,15].
Lean meat contains soluble myofibrillar proteins with desired water-holding capacity
and emulsifying properties [7]. Based on this, it can be expected that plant-based proteins
with similar functionality as myofibrillar proteins in terms of solubility, water-holding and
emulsification capacity can be a suitable replacement. Similar to their meat counterparts,
plant-based emulsion products can comprise multiple emulsion systems (mostly W/O/W).
However, next to good emulsification capacity, the plant-based ingredients used should
present the ability to form coherent and strong gels.
Foods 2021, 10, 600 3 of 29

Proteins: Multiple plant proteins have the function of binding water, stabilise emul-
sions and gels, among which are soy protein, gluten, pea proteins, potato proteins (see
Section 3.1). To achieve a coarser texture in emulsion-type formulations, there is also
the possibility to add proteins in a texturized form in order [16]. Though, proteins are
often combined with non-protein binders or fillers such as polysaccharides (e.g., fibres
and starches (see Section 3.2)). The addition of those ingredients originates from the fact
that it is known already that the presence of plant proteins often leads to a reduction in
gel formation/elasticity in cooked emulsion products [17]. In recent product formulations
such as “mimic-würstel” and “mimic-mortadella”, the use of less refined ingredients such
as bean protein flour, chickpea flour and wheat flour as well as tofu has been suggested [18].
Amongst others due to the use of these ingredient combinations, the dry matter content of
the products is higher than their meat counterparts. That has effects on juiciness.
Binders: Several soluble binders such as soy protein isolate, methylcellulose, car-
rageenan and modified starches are used in emulsion-type meat analogues. Their role is to
improve the textural properties of the products, providing the desirable gelling and thick-
ening (see Section 3.2). Additionally, they can contribute to emulsion stability, reducing oil
leakage and purge loss.
Fats: Fat is an essential component as it improves juiciness, tenderness, and overall
palatability of the emulsion-type products. The stability of both moisture and fat binding in
the highly hydrated gel protein matrix is important. For meat products, rind emulsions or
fat pre-emulsions are used, aiming at fat stabilization, preventing any fat separation during
cooking and coalescence of the fat on the surface of the product [14]. Similar requirements
are also expected for the plant-based counterparts where plant oils and fats are used (see
Section 3.3). The type of fat (fat with a high or low melting point) seems to be less important
for finely ground sausages applications like frankfurters, whereas fats with higher melting
points are used when manufacturing cooked coarse cutting sausages or emulsion-type
products with fat inclusions like mortadella [15]. Stabilisation of the fat in such plant-based
foods can be achieved with the selection of the plant proteins with good emulsification
capacities or even the use of native oleosomes [19] (see Section 3.1). Next to that, the use of
protein-rich ingredient where oil is still in its native oleosome structure can also form fat
containing gels when heated, which can be suitable for this type of products [20].
Others: Colourants and spices are also added to the product to resemble meat emulsion-
type products. Heat stable colourants colours are mainly used, but those can be of natural
origin (see Section 3.4.1). For example, fermented rice flour and paprika oleoresins were
used in plant-based bologna formulations (Smart Deli® Bologna by Lightlife) to a typical
pink colour. A variety of natural savoury spices and meaty aromas are available [21] and
are selected according to the type of product the meat analogue mimics. Salt remains
an important taste enhancer. However, when in contact with proteins, it affects their
functionalities [22]. Current nutritional trends aim at reducing salt content and especially
sodium, leading to some product formulation challenges, not only for sausage-types of
products but also for comminuted products and whole-cuts.

2.2. Burgers, Patties and Nuggets

Plant-based products resembling ground and bound animal-based meat products,
aim at recreating their distinct bite, chewiness, succulence and firmness. Animal-based
burgers, patties and nuggets, consist mainly of proteins and fats and to a lesser extent
of seasoning, salt and binders (such as wheat crumb, starches and fibres). Although in
smaller quantities, salt changes the structure of proteins and toughens the products [23],
while binders provide water and fat retention, and improve the texture and appearance
of the product [24]. Plant-based comminuted products follow closely the recipes of the
corresponding animal products. The majority of the protein components is first transformed
into a meat-like fibre structure that resembles ground meat, known as textured vegetable
protein (TVP), and then mixed with the rest of the ingredients for the final formulation.
 Foods 2021, 10, x FOR PEER REVIEW 4 of 30

Foods 2021, 10, 600 while binders provide water and fat retention, and improve the texture and appearance 4 of 29
of the product [20]. Plant-based comminuted products follow closely the recipes of the
corresponding animal products. The majority of the protein components is first trans-
formed into a meat-like fibre structure that resembles ground meat, known as textured
vegetable protein (TVP), and then mixed with the rest of the ingredients for the final for-
Proteins: The protein is often texturized using low moisture extrusion cooking (Figure 1A).
mulation.
The most prevalent
Proteins: TVPs
The protein usedtexturized
is often in meat analogues are those
using low moisture based on
extrusion soy, wheat
cooking (Figureor pea protein
and
1A). The most prevalent TVPs used in meat analogues are those based on soy, wheat or sources that
mixtures thereof. Nevertheless, there is an increasing number of protein
can
pea be texturized
protein and have
and mixtures theNevertheless,
thereof. potential to there
be used
is anfor the development
increasing of new plant-based
number of protein
sources that can
burger-type be texturized
products and have3.1).
(see Section the potential
Hydrated to be
TVPused
as for
anthe development
ingredient of a meaty and
gives
new plant-based burger-type products (see Section 3.1). Hydrated TVP as an ingredient
chewy texture to the product and provides desirable juiciness in the final product formulation.
gives a meaty and chewy texture to the product and provides desirable juiciness in the
Research is focused on the structuring potential of new protein sources and their ability to
final product formulation. Research is focused on the structuring potential of new protein
retain
sourceswater during
and their storage
ability to retain and to release
water it uponand
during storage heating andit deformation.
to release However, TVP
upon heating and
alone, similarly to ground meat, cannot form a coherent product,
deformation. However, TVP alone, similarly to ground meat, cannot form a coherent which makes the use of
binders
product, unavoidable.
which makes the use of binders unavoidable.

Figure 1. Technologies for plant protein texturization: (A) low moisture extrusion, (B) high mois-
Figure 1. Technologies for plant protein texturization: (A) low moisture extrusion, (B) high moisture
ture extrusion and (C) shear cell technology. The person in figure has given the consent to use this
extrusion
photo. and (C) shear cell technology. The person in figure has given the consent to use this photo.

Binders:Egg
Binders: Egg protein
protein and and methylcellulose
methylcellulose are the are mainthe main candidates
candidates in commercial in commercial
products, while
products, while wheat
wheat gluten
gluten canplay
can also alsothis
play this
role as itrole as ita network
creates creates when
a network
hydratedwhen hydrated
and helps
and helpsbind
bind thethe
TVPTVPandand
otherother
ingredients together.
ingredients The texture
together. The and mouthfeel
texture andofmouthfeel
the of the
products are
products arefurther
furtherimproved,
improved,with with
the use of texturizers
the that present
use of texturizers high
that water holding
present high water holding
moisture and can make the burger softer and juicier. For the latter ingredient require-
moisture and can make the burger softer and juicier. For the latter ingredient requirements,
ments, protein isolates, protein concentrates and polysaccharides (see Section 3.2) can be
protein
used.
isolates, protein concentrates and polysaccharides (see Section 3.2) can be used.
Fats: The mouthfeel of juiciness
Fats: The mouthfeel of juiciness is alsois also affected
affected by the fatby in the fat in the
the product, product,
which can which can
be
be liquid
liquid ororsolid
solid plant-based
plant-based fat, emulsified
fat, emulsified or free or
(seefree (see3.4).
Section Section
In many3.4).cases,
In many
a cases, a
combination
combination ofof liquid
liquid (such
(such as sunflower
as sunflower and oil)
and canola canola oil) and
and solid solidcoconut
fats (like fats (like
or coconut or
palm oils)
palm oils)isisused
used to to
achieve the right
achieve balance
the right (see for(see
balance example the Beyond
for example Burger
the Beyond® and
Burger® and
the Impossible™ Burger). Preferably the fats in the burger are solid at
the Impossible™ Burger). Preferably the fats in the burger are solid at room temperature room temperature
and turn liquid when the product is heated. This gives the product a pleasant mouthfeel,
and turn liquid when the product is heated. This gives the product a pleasant mouthfeel,
similar to corresponding meat products.
similar to corresponding
Others: Moreover, “bleeding”meatvegetarian
products.burgers attempt to create the feeling of juic-
Others: Moreover, “bleeding”
iness by using beetroot juice and at the same vegetarian
time givingburgers
the productattempt to create
a characteristic meatthe feeling of
juiciness by using beetroot juice and at the same time giving the product a characteristic
meat colour (see Section 3.4). Research and development on plant-based burgers are
furthermore focused on achieving even better juiciness and improving the appearance and
taste of these products by developing new colour changing compounds, flavourings and
aroma precursors.

2.3. Chicken-Like and Steak-Like Products

Another category of meat analogue products aims at mimicking whole-cut meats, like
chicken meat, pork and beef steak, that are characterized by the presence of long fibres or
layered structure. Plant-based products mimicking this fibrous or layered structure are
produced via extrusion mainly. The products are processed further by freezing, curing,
marinating and cooking to achieve the final structure, colour, tenderness, aroma and
Foods 2021, 10, 600 5 of 29

flavour change. So far, extrusion can be used to make small pieces of those products, while
the shear cell technology (which is still in development) has the potential to make large
pieces of fibrous plant-based products [25] (Figure 1B,C). The advantage of these products
is that the desired structure of the final product is already there, so there is no need for
reconstitution as it is required for the burger-type products. This can significantly reduce
the ingredient list since binders and other texturizing agents can be omitted. However,
this means that the structuring step is the key step in the formation of both a fibrous and a
juicy product.
Proteins: Soy protein is the most commonly used ingredient. Not only isolated but
especially less refined forms of soy protein such concentrates are used in extrusion ap-
plications [26,27]. With the use of an isolate, in several cases, additional components
such as wheat gluten or carbohydrate fibres, are used for the formation of a multi-phase
blend [28,29]. The mechanism of fibre formation is then based on the alignment of those
phases [25]. Finally, solidification of the structure is achieved usually by cooling. The
products take their shape based on the equipment used.
Binders: On the contrary to the previous meat analogues categories, binders are not
necessary for whole-cut-type meat analogues due to the structuring technology used.
Fats: Fat is added to a limited extend during the structuring step, while the texturized
products can be enriched with fat in a later stage (through marination). Liquid oils seem
to be the preference of the industry for these types of products (see for example the
vegan NoChicken chunks by The Vegetarian Butcher). However, there is still room for
improvement, especially when the products are mimicking raw steak-like pieces where
marbling effects maybe be desirable (see Section 3.3). Technologies to texturize vegetable
fat are currently explored for this purpose [30].
Others: Colouring agents and flavours (including salt) can be either added during the
structuring procedure or applied in the form of a marinade afterwards (for more details
see Section 3.4), depending on whether the product mimics raw or cooked meat.
Regarding the flavour addition, most applications are based on marination afterwards,
since the conditions used inside an extruder are detrimental for flavour compounds. In a
patent by Ojah, it is reported that the marination process through infusion is more successful
if the wet extrudate product is first frozen and then thawed prior to diffusion [31]. This
means that cooling and freezing steps after the extrusion process can be beneficial.

3. Plant-Based Ingredients for Meat Analogues

In the following sections, different ingredients are described that are used or suggested
for the production of the aforementioned categories of meat analogue products. We look
into the role of bulk ingredients, such as proteins, oils and fat, and ingredients used in
smaller quantities, such as binding agents, flavouring and colourants, on the desired
texture and appearance of meat analogues. The required functionalities of the different
types of meat analogues dictate which ingredients can be successful for each specific
product development. Ingredients can have multiple properties and functionalities. Within
this review, we discuss their purpose and end with suggestions for a route to create more
natural plant-based products.

3.1. Plant Proteins

Although the selection of plant protein ingredients is the starting point for product
development, the actual choice is often dictated by the protein availability, the yield of the
crops and the protein extraction potential. A common characteristic observed among the
most frequently used plant-based ingredients is that they are originally by-products of the
food industry (primarily oil or starch production). For example, soy meals collected after oil
extraction were formerly used as animal feed. However, the high protein content, balanced
amino acid composition, wide availability, low price and the specific protein functionalities
of soy (such as good gelling properties and water holding capacity) [4], made way for the
production of protein-rich food ingredients that are used in meat analogue applications.
Foods 2021, 10, 600 6 of 29

The popularity of soy however decreased in recent times in western countries [32] because
of criticism on crop production (i.e., deforestation of the rainforest) [33] and potential
negative health effects associated with the presence of specific antinutritional factors [34].
Protein from wheat, which contains a high amount of gluten, is another frequently used
ingredient. Gluten has unique film-forming properties that result in small fibres when
applied in meat analogues. Besides, it is cost-effective because the starch present in wheat
flour is used industrially as well. The main drawback is that part of the population is
intolerant to gluten.
Apart from soy protein and gluten, other protein-rich oilseeds and leftovers from oil
production are also considered as an ingredient for meat analogues, for example, sunflower
and rapeseed meals. In addition to that, more and more crops are explored for their protein
content, for example, rice, other cereal and bean flours. Derivatives of these crops such as
meals, concentrates and isolates are used in traditional and novel meat analogues [4,35].
However, despite the increased interest in alternative protein crop, just meeting the demand
for nutritional and functional characteristics is not enough; the socio-economic viability
is also important. Lupin is an example of such a crop. Despite the huge potential and
market demand for lupin-based ingredients/products, lupin cultivation in Europe remains
largely insufficient to guarantee a steady supply to the food industry [36]. In the following
sections, we look into currently used and promising protein ingredients for the three meat
analogue categories: emulsion-, burger- and muscle-type products.

3.1.1. Soy protein

Soybeans contain a mixture of water-soluble and insoluble proteins, of which the
whole aqueous extractable proteins can be separated into storage globulin and whey
fractions by acidification to pH 4.5–4.8. The extractable globular proteins are classified into
four protein categories 2S, 7S, 11S and 15S according to their sedimentation coefficients. The
7S (β-conglycinin) and 11S (glycinin) fractions represent more than 80% of the proteins [37].
The protein content (type and ratios between different proteins) [38,39] and the presence of
additional compounds (such as carbohydrates) [40,41] determine the functional properties
of soy ingredients. For example, Tarone et al. (2013) found that gels produced at pH
3 of soy protein fractions rich in 7S exhibited higher stress at rupture and higher water
holding capacity than those of rich in 11S [38]. In another research, the hydrophobic
interactions of the proteins and thus the foamability and surface elastic behaviour of the
soy proteins were affected by the presence of soyasaponin [40]. Moreover, the addition of
external carbohydrates, such as inulin, is found to proportionally improve tofu hardness
and enhance the incorporation of protein into the gel matrix [41].
Soy protein isolates and concentrates are the most commonly used ingredients for
sausage-, burger- and meat muscle-like meat analogues. The production process of those
ingredients determines the composition of the ingredients and their functionality. Both
soy milk and defatted soy flour are used for making protein-rich ingredients. In the case
of soymilk, produced by aqueous extraction of whole soybeans, concentration or spray
drying is used to yield a powder of 45–50% protein content and ~30% fat [42,43]. Defatted
soy flour with a protein content of about 50% [44], is used to make concentrates and isolate
through a process known as fractionation. The concentrates specifically are extracted with
aqueous alcohol or acid solvent resulting in final protein content of 70%. The isolates,
which have a protein content of 90%, are produced using alkaline extraction followed by a
precipitation step in acidic pH and neutralisation [45].
Regarding the protein functionality requirements for the different meat analogues
(see Section 2), these can be achieved both by using highly refined and only enriched soy
protein ingredients. Table 1 provides a summary of the functional quality as well as the
application potential of different soy ingredient, as well as other protein-rich ingredients.
Post-treatment processing, such as toasting or moisture heating [46,47], or even mixing
with other protein or polysaccharides [48] is suggested for tuning the protein properties.
For meat analogue applications, protein purity does not have to be so high. Stronger
Foods 2021, 10, 600 7 of 29

mixtures of soy protein isolate (SPI) and gluten or soy protein concentrates (SPC) have been
used for the production of TVP based patties [4], while less refined ingredients have also
been used for soy emulsions and gels for sausages [49,50], or for structuring muscle-like
products [51–54].

Table 1. Summary of already used protein ingredients for meat analogue applications.

Protein Ingredient Composition (%w/w) Functionality Application in Meat Analogues

Structuring process: Extrusion,
shear cell, spinning, freeze
Soy isolate structuring
Good solubility, gelling and Role: Protein source, texture,
(alkaline/acid precipitation ~90 % protein
emulsification binder, base for fat substitutes,
treatment) emulsifier
Products: Burger patties, minced
meat, sausages
Structuring process: Extrusion,
Soy isolate Decreased solubility, increased shear cell
~90 % protein, denatured due to Role: Protein source, texture,
(additional heat treatment/ water holding capacity, good
heat treatment binder, base for fat substitutes
toasted isolate) gelling Products: Burger patties, minced
meat, sausages
Process: Extrusion, Shear cell
Role: Protein source, texture,
binder
Soy concentrate ~70 % protein Good texturization properties Products: Burger patties, minced
meat, sausages, muscle-type
products
Process: Freeze structuring
Soy milk High solubility, good Role: Emulsifier, texture
>45% protein, ~30 % fat Products: Tofu and yuba
(spray-dried powder) emulsification properties
production
Process: Extrusion
~43–56% protein, ~0.5-9% fat, Role: Texture, Binder
Soy flour/meal (defatted) Water binding capacity and fat Products: Burger patties, minced
~3–7% crude fibre, >30% total retention, native protein
carbohydrate meat, sausages, muscle-type
products
Structuring process: Extrusion,
Binding, Dough forming/ shear cell
75–80% protein, 15–17%
Wheat Gluten isolate Cross-linking capacity via S-S Role: Adhesion, texture
carbohydrates, 5–8% fat Products: Burger patties,
bridges, low solubility
muscle-type products
Process: Extrusion, shear cell,
spinning
Water and fat binding,
Role: Emulsifier, texture, Binder
Pea isolate ~85% protein emulsification, and firm texture Products: Burger patties, minced
after thermal processing meat, sausages, muscle-type
products

3.1.2. Wheat gluten

Wheat gluten is a key ingredient for many analogues. It has an attractive price for
the industry as it is a by-product of bulky wheat starch production. In contrast to soy,
extraction of gluten from wheat is done by washing out the soluble and dispersible com-
ponents with water only, leaving behind the insoluble protein. Apart from binding and
dough forming capacity, wheat gluten has additional desired functionality (including
viscosity, swelling, nutritional quality) [55,56]. Its characteristic functionality allows it to
be used both as a binder and as a structuring agent. The use of gluten in extrusion or
shear cell can transform raw materials into fibrous structures, providing a base for both
whole-cut and minced meat-types of analogues [57,58]. Gluten forms thin protein films
upon simple deformation and elongation, transforming the meat analogue dough into
a fibrous material [53]. This three-dimensional network is a result of disulphide protein
linking [59], which causes also the formation of fibrous structures during high-moisture
extrusion [26,60,61]. Disulphide bonds can be intramolecular or intermolecular, depending
on the protein class. For gliadins, the low/medium molecular weight monomeric proteins,
mostly intramolecular disulphide bonds will be formed, while for glutenins intermolecular
disulphide bonds are more likely [62]. This means that gluten functionality is determined
by the ratio of glutenins:gliadins. Isolation of specific protein subunits [63], modification
of the protein during extraction (for example the use of non-reducing and reducing con-
Foods 2021, 10, 600 8 of 29

ditions [64] or hydrostatic pressure and temperature [65]), and interaction of gluten with
other compounds such as polyphenols [66] and alkali salts [67] can lead to different degrees
of cross-linking, which affect the eventual structure of the gluten in the meat analogues.
Through hydrolysis other properties like solubility, foaming, and emulsifying qualities can
be improved [68], broadening the application field of gluten. Current trends however are
going towards replacing gluten due to its correlations with celiac diseases, although its
unique properties are difficult to replace. Suggestions have been made to replace gluten
with other types of proteins mimicking the properties of gluten after the addition of hy-
drocolloids, fermentation or hydrolysis [69,70]. The use of cross-linking enzymes such as
transglutaminase or phenolic compounds and fibres with cross-linking potential is also an
option for the modification of the alternative proteins [71–73].

3.1.3. Legume proteins

Legume proteins from pea, lentil, lupine, chickpea, faba bean, mung bean and other
types of beans have been examined on their functional properties, such as emulsification,
foam stabilization and gel formation [74–79]. Among those plant proteins, pea protein
has gained a lot of attention since it can be used in meat products and meat analogues in
several forms based on final product formulation, the technology used, and any regulatory
requirements [80]. The properties of the pea ingredients can be affected by the pea cultivar,
the extraction process and the actual protein composition (legumin/vicilin ratio) [79].
The ability of pea ingredients to bind water and fat, and to generate a firm texture after
thermal processing, allows them to act as binders, fillers, and functional improvers [80].
Structuring of pea protein by high moisture extrusion [81,82] and shearing [83] to produce
meat analogue fibres has been successful. However, pea-based structures were found
weaker than their soy-based counterparts [84,85]. This explains why often hydrocolloids
are added in the case of pea protein-containing meat analogues.
Next to pea proteins, chickpea-, lentil-, faba bean-, mung bean- and lupine proteins
also have good emulsion and foam stabilisation capacities [86–91], placing them as good
alternatives to soy for sausage-type meat analogues. Unfortunately, proteins from lentil,
lupine and faba bean present weaker gelling capacities than soy [84,92–94], limiting their
application range. Mung bean and chickpea on the other hand present good gelling prop-
erties and are therefore more promising for meat analogues [75,86,87]. Recent research is
focused on understanding and improving the functionality of ingredients (mostly isolates).
For example, ultrasonication has been employed to improve gelling properties and thermal
stability for legume proteins under optimized treatment conditions [95].
In case the ingredient after fractionation does not possess the desired functionality,
it is possible to apply post-treatment steps to modify the protein composition and func-
tionality. For example, pH shifting, the use of NaCl, calcium or high hydrostatic pressure
during fractionation can change the protein structure, which in turn affects properties like
gelation [93,96]. Thermal post-processing treatments of the ingredients, such as toasting,
were also found a good way to modify legume ingredients, increasing their water hold-
ing capacity and decreasing their solubility [47,97]. An alternative is to use mixtures of
ingredients to obtain the right functionality [98]. Mixing different legume proteins with
polysaccharides or even using less-refined protein-rich ingredients can be a way to improve
the functionality of legume proteins [99–101]. Vogelsang-O’Dwyer et al. (2020) showed that
dry fractionated protein-rich faba bean flour exhibited superior functionality compared
to isolate produced through acid extraction/isoelectric precipitation. The former showed
higher protein solubility (~85%) at pH 7, increased foaming capacity and good gelling
ability [102]. Therefore, many researchers look at the properties of mildly fractionated
legumes, that are produced by milling and air classification [77,103–107].

3.1.4. Rapeseed, sunflower and other seed proteins

In addition to legumes and soy, oilseeds such as rapeseed/canola, sunflower, and
others have also gained interest. Although most seed isolates and concentrates are not
Foods 2021, 10, 600 9 of 29

commercially available yet, they seem to have promising functionality for replacing soy in
plant-based food formulations. The fact that many of these proteins are by-products of the
oil industry, makes their valorisation quite attractive economically. Limitations observed
due to the presence of antinutritional factors or polyphenols that can react with the proteins
and can hinder their application for human nutrition [108] are currently addressed by using
extraction protocols [109,110], fermentation processes [111,112] or crop breeding [113].
Salgado et al. (2012) reported that sunflower protein concentrates have moderate water
holding capacity values, similar to commercial soy protein isolates that are used as a
thickening agent. Moreover, sunflower proteins are known to have good stability for
emulsions and foams, comparable to commercial soybean protein isolates and bovine serum
albumin [114]. Malik et al. (2017) report enhancement of the emulsification and the foaming
capacity of sunflower protein isolates when the pH is kept near the isoelectric point (pH 4–5)
while applying heat [115]. To act as an emulsifying agent, high protein purity was not a
prerequisite according to Karefyllakis et al. (2019). They showed that mildly fractionated
ingredients from sunflower containing three main classes of macromolecules (proteins,
polysaccharides and oil bodies) exhibited satisfactory emulsification performance [116].
Moreover, sunflower protein concentrates also showed a gelation capacity comparable
with commercial proteins used as gelling agents in different applications [114]. Lastly,
the treatment of sunflower isolates with ultrasonication lead to the formation of stronger
gels when applying a temperature of 95 ◦ C. The firmness was further increased as the
temperature decreased to 25 ◦ C [117]. The protein heating causes the unfolding of proteins
and the exposure of its hydrophobic groups, which allows the formation of non-covalent
interaction among the denatured protein molecules reinforcing the colloidal network upon
cooling [117].
Similarly to sunflower proteins, rapeseed proteins, consisting mainly of cruciferin
(11S globulin) and napin (1.7–2S albumin), can gel under high pressure or heat and thus
might promote meat-like textures [118,119]. However, differences in the properties of the
rapeseed protein subunits have been reported; cruciferin forms a strong heat-set gel under
alkaline conditions, while napin in general forms a weaker gel [120]. Ainis et al. (2018)
reported that rapeseed proteins as a whole formed solid gels only at pH 7, while at pH
5 and pH 3 they had viscous-like behaviour after heating [121]. Other researchers report
that gelation of rapeseed and canola proteins typically involves the addition of fixatives
(e.g., transglutaminase), the chemical modification of the proteins (e.g., succinylation
and acetylation), and the use of polysaccharide mixtures [122,123]. Apart from gelation,
rapeseed proteins exhibit good emulsification and foaming properties [124,125], which can
promote their application in sausage-type meat analogues. These properties are further
improved even at alkaline pH by the presence of polysaccharides such as gum Arabic [126].
Quinoa seeds, belonging to the category of pseudo-cereals, have also gained attention
as a protein source. Less refined quinoa ingredients such as whole seeds and flours have
been encountered in meat products as meat extenders in nuggets [127], gelling agents
in mortadella [128], fat replacers in burgers [129], and as binders and means to reducing
nitrate/nitrite additions in sausage-type products [130,131]. Research is focused on un-
derstanding how quinoa protein (rich in 11S globulin and 2S albumin) behaves. Quinoa
isolate presents average solubility (~50%) at neutral pH, however, its water absorption,
emulsifying and foaming properties are similar to that of soy protein [132]. Good gelation
properties are also reported [133,134], together with fibre-like connections in the gel net-
work at low pH and divalent salt addition heat gelation procedures [135]. So far, there
is limited research on the use of quinoa in extrusion processes (apart from low moisture
extruded snacks). This suggests that quinoa protein is more suitable as an ingredient for
sausage-type products and as a binding/thickening agent for TVP based products.
Research on other seed proteins, such as chia and pumpkin seed proteins, is still in
a pioneering stage and thus focused on protein isolation (including alkaline treatment
and dry fractionation) and the resulting functionality [136–139]. For chia protein-rich
ingredients, emulsifying stability was the highest for less refined fractions, while the
Foods 2021, 10, 600 10 of 29

protein-enriched fractions through dry fractionation exhibited better protein solubility,

water absorption, foaming and least gelling capacity [136]. These properties indicate their
potential to be used as an ingredient for emulsion-type products like sausages. Similar to
chia seed protein, the solubility of pumpkin seed proteins is also rather low (<20%) in the
acidic pH region (pH < 5), but it drastically increases at pH above pH 6 [140]. Pumpkin seed
protein with denaturation temperatures above 90 ◦ C seems to be more resistant than other
proteins [139,140], which however might be the reason that these proteins lack important
functionalities thus further modification should be taken into consideration [141].

3.1.5. Other plant proteins (Peanut, Potato, Zein, Hemp)

Protein-rich streams such as peanuts, hemp, potato proteins and corn zein fractions are
also promising sources of protein in food applications. Already in earlier research peanut
flour was texturized using single-screw extrusion and presented functionality comparable
to that of textured soya flour [142]. As potential ingredients for meat analogues, peanut
protein concentrates showed comparable oil binding and foaming capacity to soy protein
isolates. Moreover, they presented higher viscosity and gel formation after heating [143].
Rehrah et al. (2009) formulated a defatted peanut flour meat analogue with a protein
content of 55%. The physicochemical and sensory properties of the product were similar
to soy-based TVP, and the researchers suggested that it can be used for the development
of ground beef like substitutes [144]. Recently, high-moisture extrusion products with a
fibrous structure were also reported [145,146].
Proteins isolated from hemp seed meal (a by-product of the edible hemp oil industry)
were found to exert a range of functionalities, regarding solubility, emulsification, water
and oil holding capacity. These functionalities were affected by the extraction and isolation
process [147–150]. An interesting property of hemp ingredients for meat analogue applica-
tion is the least gelling concentration, which ranges from 12% w/w for the hemp meal to
22% w/w for the isolate [150]. Next to that, evidence was reported on the potential use
of hemp protein concentrate for the substitution of SPI in high moisture extrusion meat
analogues [151].
Potato proteins and zein, either separate or as a blend, can be used as meat ana-
logue ingredients. Potato protein has good emulsification, foaming and gelation proper-
ties [152–154], making it a good texturizer. It consists of the majority from patatin and has
a low thermal denaturation temperature (55–75 ◦ C) at which it forms a gel network [154].
Thermally formed gels from potato protein isolates were obtained at pH 3 and pH 7
with minimal gelation temperatures around 45–50 ◦ C [155], which can be beneficial for
applications where a low temperature is required. However, similar to the rest of the
proteins, potato protein properties can change with the use of different protein isolation
methods [156] or post-treatment modification using enzymes [157].
Zein has been successfully used in stabilizing oil-in-glycerol emulsion-gels fortified
with antioxidants as a healthy substitute for margarine in cake preparation, as a fat ana-
logue in mayonnaise formulation [158], stabilization of foam and emulsions [159], and
in the production of gluten-free bread or dough [160,161]. Glusac et al. (2018) reported
gel-like structure formation of zein-potato protein stabilized used in meat analogues [162].
Recent research on the formulation of zein fibres by Mattice and Marangoni (2020) showed
that individual fibres of the smallest scale can be produced by electrospinning. In addition,
a web-like network can be produced by antisolvent precipitation, while an oriented fibrous
network could be achieved by mechanical elongation. These zein fibres when incorpo-
rated into model meat analogue soy protein isolate gels, could potentially create texture
mimicking chicken meat [163].

3.1.6. Outlook of Plant Protein Usage for Meat Analogues

Soy, pea and gluten are so far the main ingredients encountered in commercial meat
analogues. Those ingredients have in common that they are widely available, and are
by-products of already established food /ingredient production lines, which goes along
Foods 2021, 10, 600 11 of 29

with the low cost. Research so far aimed at determining the most relevant functional prop-
erties for meat analogue applications, among which gelling and emulsification are mostly
investigated. Generally stated the functional properties of readily available ingredients
are not optimised for meat analogue applications, since their fractionation processes were
designed before this application became important. It is therefore interesting to investigate
options to tune the properties of these ingredients towards meat analogue applications,
by blending ingredients (for example proteins with polysaccharides) or by inducing mod-
ification through post-treatments (inducing covalent interactions during processing for
example [164]).
From the overview above we also see that many protein sources are explored and a
broad range of properties are reported. Several promising protein ingredients however
are not commercially available. Probably, the economic feasibility is hampered by the
complexity of the fractionation process and the lack of use of any by-products of those
novel crops. However, new crops can be used to develop fractionation processes that are
aimed at meat analogue applications, for which that inclusion of non-protein components
can be accepted, leading to reduced by-products stream. This means that more options are
available to improve both functionality and resource use (see also Section 4). In addition,
more insights on how the ingredients behave can help to find the right directions to improve
the overall ingredient production process.

3.2. Binding and Texturizing Agents

The formulations of currently available meat analogues include several ingredients
from animal or plant origins, which act as stabilizers, gelling agents, thickeners, emulsifiers.
These ingredients can bind water and/or fat and can provide adhesion for the TVP particles.
For sausage type products, binders are used to improve the smoothness and consistency of
the product, and to maintain a juicy product by retaining the desirable moisture and fat. For
meat emulsion products, phosphates (E450) and NaCl improve the binding properties of
myofibrillar proteins [165,166], however, they are not well perceived by consumers that de-
mand more natural ingredients [167,168]. Recent research on meat products has shown the
potential of using citrus fibres [169] or fibre-rich fractions of cereals as alternatives binders
for bologna-type products [131]. For non-vegan meat analogues, egg white or albumen
can provide the desired functionality (used for example in The vegetarian butcher’s Little
willies). Alternatively, the use of methylcellulose, carrageenan, locust bean gum, calcium
alginate and other ingredients are encountered (see for example the Knacki Vegetale by
HERTA (vegetarian product) and the Garden Gourmet Sensational sausage braadworst
by Nestle (vegan product)). Arora et al. (2017) showed that mushroom-based sausage
analogues containing 5% saturated fat and produced with carrageenan and xanthan gum
exhibited improved textural properties (purge loss and emulsion stability) compared to
those produced with soy protein concentrate and casein [170]. This improvement can be
attributed to the gelling and thickening abilities of polysaccharides [171] and explains their
increasing use as meat analogue binders and extenders. Despite the commercial application
of polysaccharides in vegan products, the fact that E numbers or a long ingredient list with
scientific equivalent names have to be labelled on the package [for exampleE461 (methyl-
cellulose), E407 (carrageenan), E410 (locust bean gum), E412 (guar gum) and E415 (xanthan
gum)] does not appeal to consumers. This has to do with the perception of consumers
regarding their naturalness [172,173] and their impacts on health and wellness [174].
Clearly, the use of egg is not an option for vegan burger products. In those products,
the requirements for binders are slightly different, however. Next to water and fat binding
capacity, the binder helps to improve the texture and appearance of the burger by gluing
the minced particles together. The functionality of hydrocolloids such as methylcellulose,
hydroxypropyl methylcellulose, long fibre cellulose, corn zein and alginates, to bind tex-
turized vegetable proteins, to improve oil encapsulation and to reduce oil absorption, is
well documented [175–178]. Among these ingredients, methylcellulose is the most com-
monly used ingredient and can be found in many signature products of meat analogue
Foods 2021, 10, 600 12 of 29

companies despite its E number. Its binding capacity in combination with properties such
as the unique reversible thermal gelation, the abilities to control ice crystals formation
and reduced cooking loss [179–181], have made it a key ingredient for both comminuted
products and emulsion type products. As a replacement of methylcellulose other hy-
drocolloids or combinations thereof can be considered. Alginate solutions can provide
adhesion for particular materials [6] as in the presence of divalent cations they form a
cold-set gel [182]. Additionally, enzymes like transglutaminase that induce crosslinks
between the protein molecules can be used, improving the binding properties and the
sliceability of finely-textured plant-protein products [6]. However, the use of enzymes is
not perceived well from the consumers’ point of view due to its correlation with celiac
disease [183]. Although enzymes, used as processing aids, may not have to be labelled
when they are inactivated by cooking before final packaging [184], their usage does add
to the costs of the ingredients. Oil can also play a binding role, especially at increased
concentrations. Oil interacts with the proteins and promotes hydrophobic interactions
and aggregation resulting in a uniform gel network [185]. However, despite the possible
alternatives listed above, no clear alternative exists for methylcellulose at this moment that
provides all functionalities needed.
Among the plant-based meat analogues, whole-cut products (like chicken chunks)
have the lowest demand for additives; the recipes are simpler and the products do not need
adhesion. However, some products could benefit from the use of binders when it comes
to water and fat retention, which can improve the mouthfeel and juiciness perception.
Mattice and Marangoni (2020) suggested the addition of gums to improve the water
holding capacity of zein fibres destined for chicken-like meat analogue formulations [163].
Possible additional ingredients for such applications are polysaccharides such as pectin,
guar gum, carrageenan, cellulose, methylcellulose [177,186–191]. Polysaccharides can
be also introduced during the thermomechanical processing, where they improve the
rheological properties of mixtures and the water-binding capacity of meat analogues,
as was observed in the case of soy/pectin based meat analogues produced in the shear
cell [192]. For more natural and clean label products, however, the selection of ingredients
that naturally contain polysaccharides such as protein concentrates or protein-rich flours is
advised for the production of extruded or sheared products.

3.3. Fat, Oil and Oil Substitutes

Fat contributes to the juiciness, tenderness and flavour release, which are important
attributes for meat products [193,194]. Therefore, the addition of fat or oil in meat analogues
can have similar advantages. Emulsion-type meat analogues currently available on the
marked such as sausages have a fat content of up to 25%, which is similar to corresponding
meat products [15]. In these products, fat is pre-emulsified and introduced together with
the protein and the rest of the ingredients in the mixing stage (see Section 2.1 Emulsion-type
products). This differs from burger-type analogues based on TVP, where fat is introduced
through mixing the (pre-processed) ingredients, in the cold mixing step to create the final
product. Muscle cut mimicking meat products could benefit from practices applied to
corresponding meat products, where fats or oils are injected into the muscle to improve
characteristics and consumer acceptability [195–197].
Fat should be properly balanced in the selected formulations as it influences the
structure, rheological properties (fluidity, plasticity, texture) and sensory characteristics of
the products [198]. Pre-emulsification of fat can be achieved by using a variety of plant-
based proteins (see Section 3.1). The extraction and use of native plant oleosomes has also
been suggested as an emulsifier [199]. The formulation of gels using pure oleosomes [200]
or in combination with proteins [201] and carbohydrates [202] have been reported, which
can be interesting for emulsion-type applications or comminuted products. For whole cut
meat analogues, the use of emulsified and crosslinked fat crystal networks [30,203] and
oleogels [204,205] is considered promising when aiming at a marbling structure.
Foods 2021, 10, 600 13 of 29

The fats and oils mostly used in meat analogue applications originate from soy,
sunflower, rapeseed, canola, corn, palm, coconut and sesame oil. Interestingly, the co-
products of many of them: defatted meals, flours and their derivatives (protein concentrates
and isolates), are used or considered as the protein sources of meat analogues. This raises
the question of whether it is necessary to fractionate these oilseeds to proteins and oils.
The oilseed ingredients rich both in fat and proteins could be another way to introduce
lipids into a meat analogue recipe. Ingredients such as full-fat soy flour and (naturally
containing fat) mildly fractionated soy isolates have been used to produce fibrous meat
analogues structures using the shear cell technology [46]. The addition of fat/oil during
processing, however, can affect the structuring potential of the ingredients. When used in
high quantities (>5%) during thermomechanical processing (extrusion and shearing), slip
can is observed which negatively influences the shear forces and thus the fibre formation
process [206]. Additionally, it was reported that extrusion of recipes containing more
than 15 wt% of oil, negatively affected the alignment of the macromolecules and the fibre
formation for the meat analogues, due to excessive material lubrication [207]. Thus, one
can take into consideration the intended use of the material (whether this will be used in
the structuring process or as a binder in the final formulation).
Abundant consumption of fat is associated with adverse health effects, which ex-
plains that part of the research on meat products, focusses on reducing the actual fat
content [208,209]. Research on fat substitutes consisting of water and functional ingredi-
ents is picking up [210–213]. Fat substitutes may also be considered for the formulation
of low-fat meat analogues, where juiciness, succulence and texture need improvement.
Fat substitutes already used in the food industry are protein particles (from milk, eggs, or
plants (i.e., soy protein isolate)), modified lipids, such as synthetic lipids (Olestra® sucrose
polyester); carbohydrates, including modified starches, resistant starches, dietary fibres,
amorphous cellulose fibre (Z-trim® ), etc. [214–218]. Among these, carbohydrates have
gained a lot of attention in meat applications. Amorphous cellulose fibre has been success-
fully used for partial replacement of pork back fat in fermented sausages [219]. Dietary
fibres such as inulin and fructooligosaccharides have been used in low quantities in low-fat
Italian-type salami [220], while alpha-cyclodextrin and wheat fibre have been proposed
as ingredients in chicken frankfurters [221]. Another successful example is konjac gels
combined with other ingredients (starch, carrageenates, gellan gum) which have been used
in the formulation of reduced/low-fat meat products such as frankfurters, bologna, fresh
sausages and pork nuggets [222,223]. The positive aspects of fat substitutes do not stop on
sensorial benefits. The use of rice-starch oleogels in beef burgers diminished cooking loss
and fat absorption as well [205].

3.4. Flavour and Colouring Agents

Meat analogues acceptance is largely determined by their visual appearance and
flavour. After providing the right texture and shape, the focus is on colour or colour
changes during preparation that helps the product to resemble meat. Most commonly used
ingredients for meat analogues, like soy protein and gluten, have a beige or yellow-brown
colour. Thus, the colour of the basic meat analogue structure is far different from the well-
accepted red colour of unprocessed meat or the brown colour of cooked meat. Therefore,
colourants are added to the ingredient mix. The mixing can be done before any structuring
treatment i.e., high-temperature shearing and extrusion (for muscle-type products), or with
the rest of the ingredients in the final product formulation stage (for sausage and burger
types of products). Depending on the application stage and the type of product, colouring
requirements can differ. For example for sausage-type applications, heat-stable red hues
are used, while for raw meat-type analogues browning or decolouring of the original red
hues is desired. The difficulty in the latter case is the determination of proper red colour
hues that present high storage stability at the pH value of the meat analogue but degrade
or brown upon heating.
Foods 2021, 10, 600 14 of 29

3.4.1. Colouring Agents

Heat stable colouring ingredients, such as caramel colours, malt or annatto, turmin,
cumin, erythrosine and carotenoids, such as carotene, canthaxanthin and lycopene [224–229],
can provide the desired hues for products such as sausages or meat analogues mimicking
cooked final products. These colourants are chemically synthesised or naturally derived
components; the latter has gained popularity and come in the form of extracts or dry
powders. Carotenoid-rich extracts like paprika oleoresins and annatto seed extracts, as well
as lycopene-rich extracts from tomatoes, have been used in meat-based products, such as
fermented sausages [229–231]. Red yeast (Monascus purpureus) products made by fermenting
rice, are used traditionally in Chinese cuisine for the colouration of duck meat and has
also been introduced in tofu and other food products [232]. Studies on natural colourants
indicate that those ingredients possess antioxidant properties suggesting that they could act
as nitrite replacers in meat products [229,233]. The combination of colouring agents (paprika
oleoresin, lycopene and red yeast rice) has been successfully applied in “non-meat-based
sausages” based on soybean isolates, gluten, soy oil, egg white, carrageenan, and modified
starch [227]. Due to their stability, these colourants fit well for final product formulations.
Meat analogues marketed as a replacer for “raw” meat products (whole-cut meat
type, burgers or minced meat) require colour changes upon cooking. Therefore, heat-
stable colouring agents, even of natural origin, need to be replaced by or combined with
colourants that allow a colour change similar to that of meat upon cooking or frying. For
meat, it is observed change from red to pink or grey-brown, due to the denaturation of myo-
globin, occurs at a temperature range around 75 ◦ C [234,235]. To mimic this, betanin and
beetroot extracts are proposed as additives attributing a “raw meat” colour [225,236,237]
and undergo colour changes due to thermal degradation [238]. Beetroot extract was
the choice of colourant for the “raw” burger formulation by Beyond Meat™. Impossible
Foods™ created a “bleeding” plant-based burger with the use of soy leghemoglobin, which
is now recombinantly produced. Leghemoglobin gives their product the colour of fresh
meat, while after cooking the desired browning together with meat-like aromas was ob-
served [226,239]. Chemical changes including Maillard reactions influence the colour of the
products not only during the preparation of final by the consumer but also during thermo-
mechanical structuring (extrusion or shearing). This explains why heat-labile colourants
and reducing sugars are used in various combinations depending on the meat analogue
production technology and the characteristics of the final product [225]. Reducing sugars
already reported in meat analogue formulation patents are dextrose, maltose, lactose,
xylose, galactose, mannose and arabinose [225,236].
Despite the available embodiment methodologies and the variety of colourants, the
final products are not always of the highest possible quality. In many applications, mis-
matches among the pH range of the colourant and the pH of the meat analogue are the
cause of the colour problems. Therefore, acidulants, such as citric acid, acetic acid, lactic
acid or their combinations are added to the formulation [240]. However, pH changes are
known to alter the properties of the proteins, affecting the proteins structuring as well
as the taste of the final meat analogues. Besides, proteins have a buffering capacity, thus
require large quantities of additives to induced pH changes. Furthermore, along with the
colouring agents colour retention aids such as maltodextrin and hydrated alginate, are used
to inhibit or control the colour migration from the dyed structured meat analogue [240].

3.4.2. Flavouring Agents

Like with the colouring agents, the selection of the flavour depends on the final
product formulation. Starting from the selection of the plant protein ingredients, it is
known that soy and legume ingredients have unpleasant flavour profiles and intrinsic
off-flavours, which hinders the acceptability of the products [241]. Astringent and bitter
flavours are encountered due to the presence of glycosides, like saponins, and phenols,
such as isoflavones, catechins and phenolic acids [21,241]. Off-flavours such as grassy and
beany flavours are formed due to lipid oxidation [242]. Practises such as defatting, removal
Foods 2021, 10, 600 15 of 29

and deactivation of lipoxygenases and even fermentation of the ingredients are suggested
to remove the undesirable compounds [243].
Meat in itself has a quite neutral taste, while meat analogues mostly mimic processed
meat products (see sections above). Thus, apart from correcting the off-flavours, desired
aroma and taste needs to be introduced depending on whether the final product mimics
marinated meat, burgers or sausages. When measuring commercial meat analogue burger
products, He et al. (2021) examined the effect of cooking on aroma formation and found that
the fatty acid composition and the volatile flavour substances profile are mainly determined
by the flavouring ingredients used [244]. The selection of the right spices can thus increase
consumer acceptance of those products.
For flavouring of meat analogues natural savoury spices, meaty and savoury aromas
are currently used [21]. Besides these, the use of precursors is explored as well [245].
Earlier research was focused on volatile components produced during the cooking of
meat [246–249]. With this knowledge, meat-like flavours have been produced using precur-
sors like reducing sugars (glucose, xylose, fructose, and ribose), amino acids (cysteine, cys-
tine, lysine, methionine, proline, serine, threonine), vitamins (such as thiamine), nucleotides
and iron complexes (e.g., ferrous chlorophyllin or heme-containing proteins) [245,250,251].
Chemical reactions, such as Maillard reactions [252,253], create new characteristic flavour
substances from sugars and amino acids [254,255]. Recently, Chiang et al. (2020) explored
the use of Maillard-reacted beef bone (MRP) hydrolysate to provide meat sensory aspects
in extruded soy and gluten-based products [256]. Even though the added ingredient does
not comply with a vegetarian and/or vegan diet, the produced flavour can increase the
acceptance of meat analogues among consumers that enjoy eating conventional meat [257].
Modification of plant-based protein with the use of enzymatic hydrolysis was also found
to develop desirable chicken- and beef-like aromas [258,259]. Compounds such as furans
and thiophenes containing sulphur are also known to possess strong meat-like aromas
with exceptionally low threshold values [260], thus cysteine/ribose reactions dominated
by sulphur-containing compounds were used as contributors to an overall roasty, meat-
like aroma [254,261]. Such roast aroma or flavour is the most desired, though a risk of
off-flavour formation exists. Therefore, the optimization of the flavour and taste perception
quality is a challenge [262,263]. Depending on the nature of these compounds, complex
chemical reactions may occur, while under high temperature and pressure treatment
volatile components are released from the material leading to flavour perception changes.
The latter is observed during thermomechanical processing, together with moisture loss
when the material leaves the extrusion die [264].
The protein ingredients used during extrusion play an important role as well. Flavour
compounds, such as aldehydes, ketones, and esters, bind with proteins through hydropho-
bic and even through covalent interaction with cysteines [71,265–267]. During extrusion,
changes in the volatile flavour substances are observed, associated with the microstruc-
ture of the meat analogue, the water distribution and the protein conformation [264].
Guo et al. (2020) reported that an increase in the gluten content of extruded SPI: gluten
formulations lead to volatile losses and a decrease in the volatile retention rate [264]. Extru-
sion reduces the available binding sites for wheat gluten, among others due to disulphide
cross- linking [60,264]. Thus, research on understanding how volatile flavours interact with
the protein matrix can be beneficial for developing meat flavours that can be introduced
during the food structuring step.
Together with aromas and precursors, salt also plays an important role in taste percep-
tion. However, the role of salt is not only as a taste enhancer, but it also contributes to the
extension of the shelf life of the product. In some cases, it also improves the product’s tex-
ture. Salt addition to the protein base of the meat analogues, can lead to the solubilisation
and unfolding of the protein affecting its structuring potential (see Section 3.1). It should
be noted that the protein isolates contain already quite some salt due to the fractionation
process [268]. The high level of sodium in the western diet and the increased health risks
that it brings have raised concerns [269]. Looking for inspiration from the meat industry,
Foods 2021, 10, 600 16 of 29

sodium reduction strategies include the use of salt replacers and/or flavour enhancers.
However, the sensory impact of these components is found to be matrix dependent [270].

3.5. Water
Water in meat analogues has multiple functions. It acts as a hydration medium for
the different dried ingredients and as a plasticizer and reaction agent during processing.
In extrusion processing specifically, water determines the viscosity of the melt, partici-
pates in the chemical reactions (starting with the induction of conformational changes in
protein), influences the friction and acts as an energy transfer (thermal and mechanical)
medium [271]. In low moisture extrusion, the moisture content together with the tempera-
ture affects the expansion and porosity of the product as it is observed for starch-containing
extrudates [272]. With increasing moisture content during highly concentrated plant pro-
tein extrusion, an increase in the reaction rates of proteins is reported [273]. The disulphide
bonds, hydrogen bonds and hydrophobic interactions are promoted at higher moisture
levels. This can lead to a high degree of fibrous structure formation [271], while as already
mentioned in the previous section interaction with the flavour components can also occur.
In addition, many of the functional properties of the proteins as swelling, viscosity,
gelation, emulsification, and foaming are affected by the availability of water in the food
system and the degree of interaction with the biopolymers [274,275]. This is important for
specific meat analogue application such as in sausage-type products where water is needed
for the emulsification process. Moreover, the higher water content can be desirable since
sensory properties such as juiciness and mouthfeel of meat analogues are retained longer.
Besides, meat analogues with high water content can also be baked and cooked similarly
to meat. To sustain the desired water content, a variety of binders with water-holding
properties are used (see Section 3.2). Lastly, the inclusion of water in food reduces the
ingredient costs.

4. Steps for the Production of the Future Meat Analogues

Consumer preference studies revealed that key motivations for people to switch
towards a plant-based diet and thus include meat analogue products in their eating routine
are the health benefits and the price of the products [2]. Possible constructs on the other
hand are food neophobia and meat attachment [2]. This means that future research,
development and innovation on meat analogue formulation and production should address
all these aspects. Currently, the main focus has been given on the development of meat
analogues that recreate conventional meat in all of its physical sensations (appearance,
texture, taste, smell, etc.), however, there is an increasing demand by consumers for
sustainable ingredient sourcing, natural, clean-label and nutritious products.

4.1. Structure Formation

To achieve the desired texture and bite in current meat analogues, familiar ingredients
to the consumer, such as soy, gluten and pea protein are used, however from this review
we see that more and different types of plant proteins can be exploited. Mixing, heating
and (low and high moisture) extrusion are still the most established texturization methods
to create meat-like structures, while there is new research on novel structuring methods
such as shear cell, spinning and 3D printing. For each structuring technology and product
applications (emulsion-, burger- and muscle-type meat analogues), product formulations
are optimised based on the protein ingredients used. Many additional ingredients (binders
and texturizing agents) are added to improve the texture of the final product.
What is known so far is that in sausage type formulation, plant proteins should an-
swer the requirements for solubility, emulsification/fat stabilization and water binding
capacities, while they should also form a firm elastic gel [15,17]. For meat analogues
based on extrusion and shearing, protein-crosslinking is considered the main structuring
mechanism. Properties such as water holding capacity and solubility are often regarded as
good indicators of cross-link density and formation during processing, respectively [276].
Foods 2021, 10, 600 17 of 29

However, covalent cross-links are unfavourable before or during thermomechanical pro-

cessing, since they decrease chain mobility, increase viscosity, and preventing material
homogenization [276], while the also affect other sensorial characteristics (Section 3.4.2
Flavouring agents). The identification of these requirements paves the way to the use of
currently underutilised protein sources.

4.2. Exploring Novel Proteinaceous Ingredients

Novel protein sources are constantly explored. Proteins from crops such as rapeseed
and sunflowers (leftovers from oil production) and legumes, can be considered good alter-
natives to soy and are expected to be implemented soon in meat analogue products [118].
The fact that they come in vast quantities and can provide high-quality proteins makes
them really attractive for the food industry, while their functional properties allow their
implementation in a variety of meat analogue products. Researchers aim at exploring other
protein crops as well such as leaves (RubisCo) and aquatic biomasses such as duckweed,
seaweed and algae, however, the technology to extract proteins from those sources is still
in the initial development stage, making them less competitive towards the plant protein
(examined in this review in short term [277]). There are additionally new developments
on protein sources concerning recombinant or cellular agriculture, which use fungi, yeast
and bacteria for the production of animal proteins or for proteins that are natural for the
microorganism. However, also for this downstream processing and determination of their
functional properties are still subjects to recent research [164].
In addition, there is also a trend to use classical protein sources differently. There is
less focus on purity and more on functionality. This trend aligns with traditional techniques
of making plant-based products such astofu, yuba and tempeh, where flours, milk or even
whole beans were used as main ingredients [7]. These products show the potential of
using less refined ingredients and argue even in the direction of designing or modifying
protein-rich ingredients to fit specific requirements for each application. This approach can
potentially reduce the product’s environmental impact [4,278,279], though the nutritional
quality and the functionality of highly refined and less refined ingredients (and their by-
products) should be carefully considered in such comparison [280,281]. This means that
the subject of the research should be understanding when this switch can be made [18] and
what the actual environmental benefit can be.

4.3. Functionality of Protein Sources

The plethora of plant proteins and their properties suggests that the amino acid
sequence of the protein defines the functionality [276]. The technological functionality of
the proteins though can be further influenced by processing creating new possible uses
for the proteins (see Section 3.1). Apart from technological functionality, plant proteins
should also replace meat nutritionally, taking into consideration the amino acid profile,
digestibility and bioavailability of the proteins [282,283]. An indication of composition
differences between animal proteins, traditional and alternative plant-based protein sources
is given in Table 2. Although some plant proteins may be considered inferior to animal
proteins due to their deficiency in the essential amino acid composition and their low
digestibility [284,285], a combination of ingredients to achieve a balanced amino acid
profile and introduction of treatments that improve digestibility could be an option.
However, as discussed in this review, currently available meat analogues contain apart
from proteins and oils/fats (that are encountered in meat), also carbohydrates that play the
role of binding agents and texturizers. This deviation in the composition can be beneficial
for the target customer segment; meat-eating consumers, whose eating patterns are now
characterized by protein overconsumption, due to high intake of animal-based products
(meat and dairy) [286]. In this case, nutritional requirements in terms of protein intake are
less strict, while consumers can benefit from a product with a diverging composition (i.e.,
containing a higher dietary fibre content, vitamins, minerals, antioxidants, etc.).
Foods 2021, 10, 600 18 of 29

Table 2. Composition (%) and essential amino acid contents (in g/100 g) of different plant and animal protein sources.
Soy
Composition Egg Milk Wheat Flour Pea Seeds Lupine Seeds Sunflower Seed Peanut Flour
(% ) Dried Beef ** Dry Whole Isolate Concentrate Flour Kernels Low Fat

83.3 ± 0.7 37.0 ± 1.1

Protein 48.05 21.91 26.32 63.63 9.61 23.12 36.17 20.78 33.8
(N × 5.7) (N × 5.7)
Lipid 43.9 4.62 26.71 - 0.46 21.8 ± 0.4 1.95 3.89 9.74 51.46 21.9
Carbohydrates 1.13 0 38.42 13.3 ± 0.7 25.41 34.5 ± 2.2 74.48 61.63 40.37 20 31.27
Ash 4.13 1.09 6.08 3.4 +0.0 4.7 6.7 ± 0.7 1.53 2.67 3.28 3.02 5.23
Reference [287] [287] [287] [288] [287] [288] [287] [287] [287] [287] [287]

Recommended daily Essential amino acids content in g/100 g of product

Essential amino acids allowances (RDA in mg, for a Defatted soy Sunflower Seed Peanut flour
70-kg man) Egg Beef Milk Soy isolate Soy concentrate Wheat flour Pea seeds Lupine seeds
flour kernels low fat
Histidine 700 1.202 0.699 0.714 2.303 1.578 1.268 1.4 0.586 1.03 0.632 0.854
Isoleucine 1400 2.434 0.997 1.592 4.253 2.942 2.281 2.0 0.983 1.615 1.139 1.188
Leucine 2730 4.15 1.743 2.578 6.783 4.917 3.828 5.0 1.68 2.743 1.659 2.191
Lysine 2.100 3.339 1.852 2.087 5.327 3.929 3.129 1.1 1.771 1.933 0.937 1.213
Methionine 1050 * 1.495 0.571 0.66 1.13 0.814 0.634 0.7 0.195 0.255 0.494 0.415
Phenylalanine 1750 * 2.53 0.865 1.271 4.593 3.278 2.453 3.7 1.151 1.435 1.169 1.752
Tryptophan 280 0.775 0.144 0.371 1.116 0.835 0.683 Not measured 0.159 0.289 0.348 0.328
Threonine 1050 2.129 0.875 1.188 3.137 2.474 2.042 1.8 0.813 1.331 0.928 1.158
Valine 1820 2.991 1.087 1.762 4.098 3.064 2.346 2.3 1.035 1.51 1.315 1.418
Reference [289] [287] [287] [287] [287] [287] [287] [290] [287] [287] [287] [287]

* These values are reported as the sum of cysteine and methionine, and phenylalanine and tyrosine ** Beef, top sirloin, steak, separable lean only, trimmed to 1/8” fat, choice, raw.
Foods 2021, 10, 600 19 of 29

5. Conclusions
Creating meat analogues that mimic meat both nutritionally and in its physical sensa-
tions can increase people’s willingness to substitute (part of) their meat consumption. The
latest research and development activities on meat analogues have shown that it is possible to
produce meat-like texture with the use of plant-based proteins and technologies such as extru-
sion, shearing and mixing. However, to mimic meat in other sensorial characteristics, such as
colour, aroma and mouthfeel, additional non-protein ingredients are used. The diversity in
ingredient functionality requirements among the different plant-based meat analogues types
(sausages, burgers and whole-cuts) does make product development complicated.
Generally stated, the functional properties of readily available protein-rich ingredients
(mostly highly purified from plant material) are not optimal for meat analogue applications.
This can be seen from the necessity to use additives to improve the texture of the products.
The non-optimal ingredient properties can be attributed to the fact that the ingredients used
in meat analogues were designed before meat analogue applications became important.
This means that there might be room for improvement of the ingredients towards meat
analogue production. Novel functionality can be achieved by applying other fractionation
methods, which aim for functionality rather than purity. Next to that proteins from novel
sources can bring novel ingredients for meat analogue applications.

Funding: This research received no external funding.

Acknowledgments: The authors thank Jarno Gieteling (Food Process Engineering, Wageningen
University) for assistance in taking photographs of the extruder and shear cell.
Conflicts of Interest: The authors declare no conflict of interest.

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