Critical Reviews in Food Science and Nutrition

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Recent insights in flavor-enhancers: Definition,

mechanism of action, taste-enhancing ingredients,
analytical techniques and the potential of
utilization

Athanasia Vasilaki, Eleftheria Panagiotopoulou, Thomas Koupantsis,

Eugenios Katsanidis & Ioannis Mourtzinos

To cite this article: Athanasia Vasilaki, Eleftheria Panagiotopoulou, Thomas Koupantsis, Eugenios
Katsanidis & Ioannis Mourtzinos (2021): Recent insights in flavor-enhancers: Definition, mechanism
of action, taste-enhancing ingredients, analytical techniques and the potential of utilization, Critical
Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2021.1939264

To link to this article: https://doi.org/10.1080/10408398.2021.1939264

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CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
https://doi.org/10.1080/10408398.2021.1939264

REVIEW

Recent insights in flavor-enhancers: Definition, mechanism of action, taste-

enhancing ingredients, analytical techniques and the potential of utilization
Athanasia Vasilakia, Eleftheria Panagiotopouloua, Thomas Koupantsisa, Eugenios Katsanidisb, and Ioannis
Mourtzinosb
a
Research and Development Department, PROVIL S.A, Thessaloniki, Greece; bDepartment of Food Science and Technology, Faculty of
Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece

ABSTRACT KEYWORDS
The consumers’ demand for clean-label food products, lead to the replacement of conventional Umami; kokumi; natural
additives and redesign of the production methods in order to adopt green processes. Many ingredients; taste enhancer;
researchers have focused on the identification and isolation of naturally occurring taste and flavor food ingredients;
sensory evaluation
enhancers. The term “taste enhancer” and “flavor enhancer” refer to umami and kokumi compo-
nents, respectively, and their utilization requires the study of their mechanism of action and the
identification of their natural sources. Plants, fungi and dairy products can provide high amounts
of naturally occurring taste and flavor enhancers. Thermal or enzymatic treatments of the raw
materials intensify taste and flavor properties. Their utilization as taste and flavor enhancers relies
on their identification and isolation. All the above-mentioned issues are discussed in this review,
from the scope of listing the newest trends and up-to-date technological developments.
Additionally, the appropriate sensory analysis protocols of the naturally occurring taste-active com-
ponents are presented. Moreover, future trends in using such ingredients by the food industry can
motivate researchers to study new means for clean-label food production and provide further
knowledge to the food industry, in order to respond to consumers’ demands.

Introduction ingredients familiar and accepted by the consumers, without

any controlled additives and produced with environmen-
Nowadays, due to consumers’ requirements, innovative steps
tally-friendly processes (Asioli et al. 2017). According to
in the food industry have been taken toward the optimiza-
Aschemann-Witzel, Varela, and Peschel (2019), “there isn’t a
tion of the processes used and the production of high-added
commonly accepted definition of a ‘clean label’ product … ,”
value, high-quality and nutritive foods (Probst et al. 2015).
but it can be perceived as a descriptive term of products
These requirements are a result of “food related crises” dur-
ing past decades, consumers’ skepticism regarding food without components that are seen unfavorably by consumers
additives and controversial technologies, such as genetically (Aschemann-Witzel et al. 2019).
modified organisms (B
an
ati 2011; Grunert, Bredahl, and Food additives, firstly mentioned in the 1800s, have been
Scholderer 2003). As an example, a number of food addi- used for extending the shelf-life of food products and
tives have been implicated in causing allergic reactions and improving their sensory characteristics and general quality
lifestyle related diseases (Asioli et al. 2017). (Carocho et al. 2014). In 1920, processed foods became an
The consumers’ demand for natural products often important part of human nutrition and many additives were
weighs more than even the nutritional profile, for the selec- authorized for food use (Carocho et al. 2014). According to
tion of a food product. This observation reinforces the need CBI (2020), the European food additives market was valued
for the replacement of artificial additives by natural alterna- almost 12,5 billion US dollars in 2018, and will show a
tives (Mintel 2020). In addition, consumers often perceive growth rate of 5.1% annually, between 2018–2023. These
natural ingredients as safer and more sustainable solutions economic data show the current and future market size of
for the environment (Mintel 2020). Moreover, “wellbeing” is food additives production, which is the consequence of the
a trend that has been strengthened during the COVID-19 wide range of food additives use by the industry.
pandemic (Mintel 2021). In this context, healthy eating Flavor enhancers are defined as a wide category of ingre-
encompasses broader health concerns such as mental and dients, which belong to food additives, used to enhance the
emotional health aspects (Mintel 2021). sensory characteristics of a food, and especially its taste and
As a response to all the above-mentioned consumer flavor. They were first introduced in mid to late 1960, and
requirements, the food industry begun formulating “clean- the term describes the compounds, that when added in
label” products, i.e., food products containing only small quantities, intensify food flavor (Maga and Yamaguchi

CONTACT Ioannis Mourtzinos, [email protected]

ß 2021 Taylor & Francis Group, LLC
2 A. VASILAKI ET AL.

1983). The term “flavor enhancer” may include many com- (such as thermal or enzymatic treatment) that can enhance
pounds that have the ability to increase the intensity of fla- the taste properties of the F/TE, and, also, in techniques to
vor characteristics or give a more complex flavor profile to identify and isolate such compounds. The aim of this review
the final product (Kuninaka 1978). The way that these com- is to list and summarize the relevant data regarding natural
pounds act and the characteristics that the final product F/TE and, additionally, to clarify the definition of the terms
acquires, are some factors that can be used to categorize used to describe F/TE, with special reference to future per-
them (Kuroda and Miyamura 2015; Maga and spectives of F/TE isolation and use in food products.
Yamaguchi 1983).
Monosodium glutamate (MSG) is widely incorporated in
Umami
foodstuffs to enhance taste and palatability (Harada-
Padermo et al. 2021). Many studies have been conducted Umami is one of the five tastes also described as
focusing on MSG substitution by the addition of naturally “savorness.” In 1909, umami was identified as the fifth basic
occurring glutamic acid and 5-ribonucleotides. When mush- taste, by studying the effect of glutamic acid on food flavor
room extracts and tomato concentrates were added in coming from kombu (Ghirri and Bignetti 2012). This com-
chicken soup, they were found to impart similar umami pound is one of the most used taste enhancers, followed by
intensity to MSG (Wang et al. 2019). When shiitake mush- salt, inosine 50 -monophosphate (IMP) and guanosine 50 -
room extract was incorporated in minced meat product, the monophosphate (GMP), that act synergistically. Several years
umami taste intensity was slightly higher than that of MSG later, the histidine salt of 50 -inosinic acid was identified in
(Dermiki et al. 2013b). bonito tuna, and many studies on naturally occurring sour-
According to data retrieved from market specialists and ces of taste enhancers followed (Maga and
food and beverage supply managers, MSG is a low-cost food Yamaguchi 1983).
ingredient, available in abundance through a consistent Commercial production of MSG and its addition into
worldwide supply flow. At an average net price of 1500$/ foods began in 1960. Utilization of IMP and GMP as taste
MT, MSG has been globally established as a popular taste enhancers soon followed. Large quantities of IMP and GMP
enhancer at an affordable price. MSG’s natural counterparts, were produced in Japan, which had an annual production of
such as yeast extracts or fermentation derived umami ingre- 4680 tons by 1974 (Maga and Yamaguchi 1983).
dients, are considerably higher priced products, often 5 to
25 times the cost of MSG. Taking into account their higher
addition level along with their higher cost, natural taste Mechanism of action of umami compounds
enhancers could affect the cost of clean label products. It is The mechanism of action of umami molecules is still under
therefore essential for the food industry to document the investigation. As described by Winkel et al. (2008), there are
value and communicate the measurable benefits of clean two possible classes of receptors of umami molecules. The
label ingredients to the consumer, in order to compensate first one is the G-protein-coupled receptors (GPCRs), which
for the higher price. are located in the taste buds on the tongue, and are the
Besides umami components that can enhance food taste same with those located in the brain for the detection of
when added in small quantities, kokumi is referred as a glutamate and MSG molecules, having many binding sites.
second category of flavor enhancers and describes a certain They are also known as GPCRs and they function as mem-
taste property, a key factor for palatability and it is distinct brane receptors (Winkel et al. 2008). Binding of umami
from the five basic tastes—sweet, sour, salty, bitter and compounds leads to the activation of different signal trans-
umami (Kuroda and Miyamura 2015). “Kokumi” is a term duction pathways in the receptor cell. The proposed mech-
used to describe the delicious mouthfeel and it can be better anism of action for this type of receptors is that the binding
explained by the use of terms mouthfulness, continuity and of umami molecules to the metabotropic glutamate receptors
complexity (Feng et al. 2016; Kuroda and Miyamura 2015). (mGluR) site activates the membrane hyperpolarization
The interesting point is that, by observing the existing litera- through the presence of calcium (Caþþ) and chlorine (Cl-)
ture on taste enhancers, differences between umami and ions (San Gabriel et al. 2005).
kokumi components are not clearly described due to the The second possible receptor system is the heterodimer
complexity of taste/flavor terms and the chemical similarities T1R1/T1R3. Taste Receptors Type 1 (T1Rs) belong to the
between some of the compounds that belong in these cate- same class of mGluRs and are considered responsible for the
gories. The term “taste enhancer” will be used for umami perception of sweet and amino acid taste compounds
compounds and the term “flavor enhancer” will be referred (Ghirri and Bignetti 2012). Through molecular conformation
to kokumi ingredients. of the ligand binding sites of T1R1 and T1R3, it is believed
Thus, flavor/taste enhancers (F/TE) play a significant role that glutamate (Glu) can be bound to both T1R1 and T1R3
on many commercial food products consumed daily. It is of subunits, stabilizing the closed conformation of T1R1 and
essential interest to study, define and categorize the main the open conformation of T1R3, according to Zhang et al.
classes of F/TE and their mechanism of action. Many studies (2008) and L
opez Cascales et al. (2010). T1Rs receptor’s way
on raw materials or foodstuffs which contain high concen- of action is presented in Figure 1. The study of Li et al.
tration of taste-active components can be found in the lit- (2002), shows that the T1R1/T1R3 heterodimer presents
erature. Alongside, researchers are interested in processes high sensitivity and selectivity for L-glutamate, and to some
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3

Figure 1. Mechanism of umami taste signal transduction. An umami compound (U) binding to T1Rs receptor activates the Ga subunit, which in turn affects the
cAPM concentration. The same binding also stimulates the phospholipase C pathway (PLC), which includes the motivation of calcium cation channel through inosi-
tol triphosphate (IP3) action and the final membrane depolarization (Modified Figure, Kinnamon et al. 2009).

extend to L-aspartate, but it does not respond to sweet taste research studies, it was discovered that aspartic acid has the
compounds or other amino acids. same property and the proportion of these two amino acids
As described by Yamaguchi (1991), who studied the syn- is crucial for the intensity of umami taste perception
ergistic effect of ribonucleotides and glutamic acid, IMP and (Kurihara 2009; Rotzoll, Dunkel, and Hofmann 2006).
GMP taste is weak when alone, but when MSG is present, By studying the structural relationship between taste
glutamic acid detection threshold is noticeably lower. The compounds and especially umami compounds, it seems that
fact that ribonucleotides cannot activate T1Rs receptors but activation of umami receptors can mainly occur by L-amino
amplify the response of the receptors to glutamate can acids with five carbon atoms or purine ribonucleotides with
explain their mechanism of action (Nelson et al. 2002). 6-oxygen groups. Particularly, L-forms of a-amino acids pre-
Many researchers study this mechanism of action, and the sent similar taste to L-glutamic acid, given that they have 4
most prominent theory is that ribonucleotides could bind to to 7 carbons. On the contrary, L-glutamine, which is not an
an adjacent site in order to stabilize the closed conformation acidic amino acid, but possesses the same number of carbon
of the T1R1 domain (Zhang et al. 2008). atoms and the same isomeric structure with L-glutamic acid,
Due to the high specificity of T1Rs receptors and the syn- does not have taste potentiator activity. The differences
ergism between glutamic acid and 50 -ribonucleotides, it was among the isomeric structures of amino acids in taste
suggested that this type of umami receptors is the enhancing activity is another field that has to be studied
most important. thoroughly (Maga and Yamaguchi 1983).
Regarding in vivo studies about umami perception, it was Besides amino acids, 50 -ribonucleotide content was found
found that the differences observed in the perception of to be crucial for umami taste intensity, and especially guany-
late and inosinate, which are found mainly in animal-based
umami taste among human subjects are a result of non-syn-
foodstuffs (Kurihara 2009). Such compounds seem to act
onymous polymorphism at the amino acid sequence of
synergistically to L-amino acids and their mechanism of
T1R1 domain (Ghirri and Bignetti 2012). During studies of
action was studied by Suess, Festring, and Hofmann (2015).
mice taste receptors and their response to umami taste, it
The synergistical effect between amino acids and ribonucleo-
was found that mice lacking T1R3 domain have diminished
tides was reported by Kurihara (2009) by using the example
but not eliminated perception of umami compounds
of kombu. Kombu, which contains high amounts of glu-
(Damak et al. 2003). tamic acid, does not elicit strong umami taste but its umami
In other studies (Chaudhari, Pereira, and Roper 2009; taste can be increased by adding bonito flakes which contain
Yasuo et al. 2008) it was proposed that the two different inosinate. The same effect is presented by adding vegetables
classes of umami receptors, the mGluR and T1Rs receptors, and/or cheese in beef or chicken. This synergism was
contribute to umami perception but they have different reported by Kuninaka (1960) and was further studied by
roles. T1Rs are located at the anterior tongue and contribute Yamaguchi (1967). In order to explain the different activity
to taste perception whereas mGluR are located at the poster- among some forms of nucleotides, researchers used artificial
ior tongue and their role is umami taste discrimination and natural compounds, similar to ribonucleotides. In these
from the (Kinnamon, 2009)other tastes (Yasuo et al. 2008). studies, it was found that 50 -ribonucleotide structures with a
hydroxyl-group in the 3 and 6 position are necessary to gen-
Compounds exhibiting umami taste activity erate taste activity. Among 50 -ribonucleotides, GMP, IMP
and XMP (xanthosine-50 -monophosphate) exhibit taste
Regarding compounds that exhibit umami taste activity, the enhancing activity with varying intensity. This has led to the
first recognized ingredient was L-glutamate, and after many conclusion that compounds with minor differences at the 20 -
4 A. VASILAKI ET AL.

Table 1. Common umami compounds found in plant, fungi and dairy materials.
Umami compound Plant material Main results Reference
1. Amino acid
Mushroom Aspartic acid Zhang et al. 2013, Phat et al. 2016,
Glutamic acid Poojary et al. 2017
Tomato Aspartic acid Zhang et al. 2015, Oruna-Concha et
Glutamic acid al. 2007, Sorrequieta et al. 2010,
Dermiki 2013a, Mounayar,
et al. 2013
Potato Aspartic acid Morris et al. 2007, Zhang et al. 2008,
Gluatamic acid Coulier et al. 2011

Green tea Aspartic acid, Glutamic acid

Soy Aspartic acid, Glutamic acid Kim et al. 2017, Van Ho and Suzuki
2013, Karr-Lilienthal et al. 2004,
Lioe, Takara and Yasuda 2006
Cheddar and Swiss Cheese Glutamic acid Drake et al. 2007
2. Organic acids
Green tea Succinic acid
Tomato Succinic acid, Malic acid, Citric acid Beullens et al. 2008
Cheddar and Swiss Cheese Succinate, Lactate, Propionate Drake et al. 2007

3. Ribonucleotides
Mushrooms AMP, CMP, GMP, IMP, UMP Phat et al. 2016, Zhang et al. 2013,
Poojary et al. 2017
Tomato AMP, CMP, GMP, UMP Oruna-Concha et al. 2007, Chew et al.
2017, Dermiki et al. 2013b
Potato 50 -GMP, 50 -AMP, 50 -UMP, 50 -IMP, 50 - Morris et al. 2007, Coulier et al. 2011
XMP, 50 -CMP
Green tea AMP, GMP

site do not have different taste qualities but different Fungi origin
enhancing intensities (Maga and Yamaguchi 1983). Mushrooms. The first report about components that contrib-
The third chemical class that can elicit or intensify ute to umami taste in mushrooms was published in 1961
umami taste of food, is low molecular weight organic acids. and the umami taste was attributed to guanylate (Nakajima
A research study examining the reconstitution of morel et al. 1961). The majority of umami substances and their
mushroom taste by the identification of the different frac- accumulation in mushrooms are affected by many factors
tions of taste, suggested that the compounds that mainly such as the species, the maturity stage, the part of mush-
affect the final taste properties were glutamic, aspartic and room and also the storage time (Zhang et al. 2013). MSG-
succinic acid (Rotzoll, Dunkel, and Hofmann 2006). like compounds are located mainly at the mycelia in certain
Reconstitution experiments are described in the sensory species like Phellinus linteus, however, in Hypsizygus mar-
evaluation part of this article. One year later, Ninomiya et moreus L-glutamate, L-aspartate and ribonucleotides can be
al. (2002) suggested that umami taste of Emmenthaler found in high amounts at the fruit body (Liang et al. 2010;
cheese is synergistically created by the action of acetic, pro- Lee, Jian, and Mau 2009). In Cordyceps militaris and
pionic, lactic, succinic and glutamic acid, while Drake et al. Antrodia camphorate species, the highest ribonucleotide con-
(2007) came to the same conclusion for Cheddar cheese. tents were detected in mycelia forms. Regarding maturity
Also, mat-cha tea and tomato include high amounts of stage, researchers found that stage 5 of maturity can provide
organic acids, crucial for umami taste activity in the final the highest accumulation of aspartic acid, glutamic acid,
product (Kaneko et al. Karr-Lilienthal et al., 2004; Beullens adenosine-50 -monophosphate (AMP), GMP and XMP in
et al. 2008). Umami compounds that have been identified so Volvariella volvacea (Chang et al. 2001).
far and their origin are presented in Table 1. In many cases, mushrooms undergo several processes
that increase their flavor and the amounts of umami-taste
compounds (Svuecs 1950). Dehydration was used to
Natural materials with umami taste properties
reinforce certain taste characteristics and dehydrated edible
Existing literature focused on the methodology of synthesiz- mushrooms can be categorized into three flavor types; the
ing and studying the mechanism of action of umami taste light colored, which present strong umami taste, the tan col-
ingredients (Villard et al. 2003; Cairoli et al. 2008; Morelli, ored with meaty, buttery and savory taste and the dark col-
Manitto, and Speranza 2011). This review is focused on ored type with beefy, meaty and chocolate-like flavor
umami compounds naturally occurring in raw materials, (Holtz 1998).
and especially in plants, fungi and dairy products. In recent A common process used to increase the amount of
years, many researchers contributed to the identification of umami compounds in some widely cultivated plants is the
plant-, fungi- and dairy- derived umami ingredients, either solid-state fermentation through exploitation of mushroom
as such or enhanced through thermal processes, fermenta- strains as inoculums. Inonotus obliquus strains were inocu-
tion or enzymatic hydrolysis. lated into cooked rice to improve and enhance the umami
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 5

taste of rice and similar results were given by adlay and showed that pyroglutamic acid was found in higher concen-
buckwheat fermentation with Pleurotus strains (Lin et al. trations (2.6 times) than glutamic acid, but in the raw sam-
2012; Chen et al. 2012). ples glutamic acid concentration was higher (Zhang and
Besides amino acids and ribonucleotides, umami taste Peterson 2018).
can be provided by high concentration of organic acids. An
excellent example of such a fermentation, targeted in organic Soy. The main product of soybeans with umami taste is soy
acids increase, is the production of kimchi by complex lactic sauce. Soy sauce is a liquid condiment and a main seasoning
acid fermentation (Zhang et al. 2013). in Asian cuisine. Its production process varies in different
countries. The common production process includes the two
steps of fermentation, called koji (the initial solid-state fer-
Plant origin
mentation) and moromi (the brine fermentation). The dif-
Although umami is a taste firstly detected in animal-based
ferent processes used in different Asian countries originated
foods, many plants possess high concentrations of umami
taste compounds. from the differences in the proportion of the raw materials
used in the formulas. The Chinese traditional recipe includes
predominantly the use of soybeans and less wheat, however
Tomato. Tomato has recently been studied as a taste enhan-
in Japan equal amounts of these two ingredients are mixed
cer. Oruna-Concha et al. (2007) found that differences in
(Diez-Simon et al. 2020). The difference on wheat amount
taste perception depended on the part of the fruit that is
affects the sugar availability in the fermentation process,
consumed. The differences have been explained through
thus the final products differ in their flavor characteristics
amino acid and ribonucleotide analysis, combined with sen-
sory evaluation of flesh and pulp. The glutamic acid concen- and components (Devanthi and Gkatzionis 2019). The con-
tration in pulp was almost 4 times higher than in the flesh centration of glutamate in soy sauce is considered as a qual-
and AMP concentration in flesh and pulp was 80 and ity indicator (Van Ho and Suzuki 2013). The fermentation
295 mg/kg, respectively. Tommonaro et al. (2021) pointed process leads to the cleavage of proteins into peptides and
out that the differentiation among tomato varieties and the free amino acids, mainly attributed to the action of
accumulation of taste-active components at the different Aspergillus proteases (Van Ho and Suzuki 2013). The trad-
parts of the fruit is a key-factor of industrial use. itional fermentation process takes place in non-sterile envir-
In order to exploit industry by-products, Zhang et al. onment by the action of the indigenous microflora. At
(2015) studied the production of glutamic acid through industrial scale, the process begins with the soybeans and
enzymatic hydrolysis of tomato seeds. The obtained product, wheat fermentation by Aspergillus oryzae or Aspergillus sojae.
after processing with papain (activity of 18.22%, pH 3 at Brine is added at the initial mixture and the process can last
40  C for 6 hours) contained 727,6 lg/mL glutamic acid and from a few months up to 4 years (Devanthi and Gkatzionis
149.9 lg/mL aspartic acid. 2019). Many studies have been conducted in order to eluci-
Several studies have been published in the field of glu- date and optimize the conditions of the fermentation pro-
tamate level (Sorrequieta et al. 2010; Kisaka and Kida 2003) cess and their effect on the quality and the flavor of the
or AMP accumulation (Yamaguchi and Ninomiya 2000) in obtained product (Harada et al. 2017; Devanthi and
tomatoes. In 2017, Chew et al. worked on gene overexpres- Gkatzionis 2019). In order to study the efficiency of germi-
sion in tomato in order to increase umami-taste compo- nated soybeans and the resulting product of their fermenta-
nents. The results were not unequivocal, as an increase in tion, (Choi et al. 2011; Coulier et al., 2011) used germinated
deaminase can lead to the reduction of glutamate amounts, soybeans to produce Korean soy sauce. The fermentation
an observation that can lead to a more extended investiga- process was conducted under different illumination condi-
tion of glutamate production mechanism. tions and researchers concluded that the sensory characteris-
tics of the different products, and especially their umami
Potato. Ribonucleotide content in potatoes varies depending taste, did not present statistically significant differences.
on the thermal processing conditions during cooking. However, the savory taste seems to be higher in soy sauce
Morris et al. (2007), studied the nucleotide concentration in from non-germinated soybeans (Choi et al. 2011). Kim et al.
raw, boiled and steamed potatoes. During steaming for (2017), in an attempt to clarify the taste-active components
8–10 minutes, 50 -GMP and 50 -AMP reached a maximum of Korean soy sauce, followed a reconstitution experiment.
plateau, a fact that confirms the opinion that steaming is the They found that sweetness, saltiness and umami taste inten-
best thermal process for preserving the taste characteristics sities were similar between the control sample and the
of potato. reconstituted Korean soy sauce. The fraction responsible for
In their effort to quantify the umami compounds con- umami taste was detected through an omission test (Kim et
tained in potatoes, researchers identified another compound al. 2017). Many studies have focused on the effect of brine
with taste enhancing properties. This compound was pyro- on fermentation and the chemical and sensory characteris-
glutamic acid, a compound similar to glutamic acid, that is tics of the final product. Van Ho and Suzuki (2013), sug-
found to accumulate in higher levels in fried potatoes than gested that the addition of 9% sodium chloride (NaCl) brine
in raw. Thermal processes like frying could lead to the for- inhibits the glutaminase activity and, thus, inhibits the glu-
mation of substances with desirable taste characteristics. The tamine transformation to glutamate. In order to prevent this
saliva analysis during sensory evaluation of fried potatoes, inhibition, they added bacterial c-glutamyl transpeptidase in
6 A. VASILAKI ET AL.

the moromi stage of fermentation and the glutamic acid ethanol:water mixture 1:1 and stirring for 6 hours and
concentration increased greatly, compared to the control Morris et al. (2007) extracted the taste-active components by
sample (Van Ho and Suzuki 2013). using a buffer solution consisting of methanol/water/acetic
acid during an extraction in a blood rotator. Buffer solutions
Green tea. Mat-cha tea is a daily consumed infusion in were also used to extract amino acids from defatted tomato
Japan, widely studied for its bioactive ingredients. seeds and a liquid-liquid extraction was applied for the iso-
Researchers have also focused on the sensory characteristics lation of amino acids from mat-cha tea infusion, by using
and especially on umami compounds. Among several stud- dichloromethane and ethyl acetate (Zhang et al. 2015;
ies, the study of Suzuki et al. (2002) suggests that the great Kaneko et al). On the contrary Oruna-Concha et al. (2007)
umami taste presented in mat-cha infusions depends on the- used aqueous HCl 0,01 N solution to extract amino acids
anine (N5-ethyl-L-glutamine) content, which is the main from tomato flesh and pulp. As Zhao et al. (2019) presented
free amino acid present in the tea. Theanine is present on in their study, in many modern techniques, such as high
an average of 1.5–2% per dry weight and is synthesized by temperature and high-pressure treatment, microwave and
theanine synthetase in the roots of the tree. It is synthesized ultrasound assisted extraction, the use of organic or other
by the reaction of glutamic acid and ethylamine and its syn- toxic solvents is avoided. The selectivity of the process can
thesis was studied by the use of bacterial c-glutamyltrans- be increased, and the sensory characteristics are maintained
peptidase (Suzuki et al. 2002). due to the purification of the target compounds by the use
Besides L-theanine, Kaneko et al. identified AMP and of microporous resin adsorption or membrane separation
glutamic acid as key taste contributors and during the frac- techniques. Initially, fat and other high-molecular weight
tionation conducted by the same researchers, they revealed molecules should be removed using extraction, precipitation
that theogallin, L-theanine and succinic acid act synergistic- and centrifugation techniques in the initial mixture.
ally to glutamic acid, to create the final umami taste inten- Macromolecules could hinder the next steps of fractionation,
sity of the infusion. isolation and identification of the umami compounds. In
many cases, preparative chromatography is used for both
Legumes and cereals. These two classes of plants include fractionation and separation, based on different eluent con-
many species and grains studied as a matrix of protein ditions. More often, a gradient elution of taste active com-
hydrolysis application. Hydrolyzed plant proteins are widely ponents is used, and the main solvents are solutions
used in order to increase the umami and glutamate-like taste consisting of ethanol, methanol and water, followed by the
of foodstuffs. They can be produced by two different techni- addition of organic acids. In many cases, a derivatization
ques. The first one is the acidic hydrolysis which results in step is preceding that is necessary for the detection of the
the production of many co-products and high concentration chemical compounds (Morris et al. 2007; Zhang and
of salt. The second one includes the use of enzymes under Peterson 2018). Common identification methods are mass
mild conditions, that result in bitter-tasting products (Wang spectrometry or the usage of amino acid analyzer kits
et al. 2016; Aaslyng et al. 1998). Among the researchers that (Zhang et al. 2015). Effective methodologies are compiled
studied the enzymatic hydrolysis of wheat, Wang et al. and presented in Table 2 and Figure 4.
(2016), tried to mask the bitter taste of the final products by Chemical analysis is the most crucial step in identifying
the use of b-cyclodextrin as a carrier of the hydrolyzed plant the chemical compounds contained in a specific food
protein. Besides wheat, rice (Selamassakul et al. 2020) has matrix. The combination of chemical analysis with sensory
already been studied as a possible source of protein hydrol- evaluation could support the identification of the compo-
ysate with taste and flavor enhancement properties. As it nents that are responsible for the overall taste of the
comes to legumes, soy (Aaslyng et al. 1998) and pea (Zhou food products.
et al. 2021), are some of the already studied materials in the
field of taste/flavor enhancers in the form of Hydrolyzed
Sensory evaluation of umami fractions/compounds
Vegetable Proteins (HVP). HVP, produced by enzymatic or
acid hydrolysis, have been studied extensively, with special Sensory evaluation is a fundamental methodology for the
interest in consumer acceptance (Jeon et al. 2020; Kaneko study of taste-active components. Every sensory evaluation
et al., 2006). procedure of such compounds, fractions or final products is
comprised of two parts; the training of the panelists and the
main sensory evaluation sessions.
Extraction, fractionation and identification of
In many cases, the training sessions are based on triangle
umami substances
tests of a standard solution of glutamate salts (Ottinger and
Umami substances can be isolated using extraction and frac- Hofmann 2003; Lioe, Takara, and Yasuda 2006). In a tri-
tionation processes. Different methodologies have been pro- angle test, assessors are asked to distinguish the different
posed, depending on the chemical and physicochemical solution among three samples, two of which contain water
characteristics of the targeted components. The first step of and one contains MSG. In some other cases, assessors are
the process usually includes blending of the raw or proc- asked to take part at taste detection threshold tests (3-
essed material and maceration in an extraction solvent. In Alternative Forced Choice, 3-AFC test), by tasting a specific
the case of potato, Zhang and Peterson (2018) used number of solutions contain MSG and other synergistically
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 7

Table 2. Examples of extraction, fractionation, separation and identification of umami compounds.

Method/Conditions/Solvents Material Reference
Extraction 50% EtOH (aq) Potato Morris et al. 2007
MeOH/Water/Acetic Acid Potato Zhang and Peterson 2018
Dichloromethane/Ethyl Acetate Mat-cha tea
0,01 N HCl Tomato Oruna-Concha et al. 2007
Microwave Mushroom Yang et al. 2019
Fractionation Preparative HPLC Potato Zhang and Peterson 2018
Column Chromatography Mat-cha Kaneko et al.
Separation/Identification Capillary Electrophoresis Tomato Oruna-Concha et al. 2007
GC-MS tomato Oruna-Concha et al. 2007
HPLC Potato Morris et al. 2007
Identification NMR Mat-cha
NMR Potato Zhang and Peterson 2018
LC/MS-ToF Potato Zhang and Peterson 2018

acting compounds (Dermiki et al. 2013a; Ottinger and 1986). This kind of testing is still used with some modifica-
Hofmann 2003). tions. Zhang and Peterson (2018) and Filho et al. (2020)
In 2007, Oruna-Concha et al. suggested that because studied the influence of glutamate components on the taste
MSG contains sodium, there is a need to investigate the sen- of potato chips and tomato sauce, respectively. The taste-
sitivity of the panel to MSG and correlate it to sensitivity to active components were applied on the sample and assessors
sodium chloride. The MSG taste threshold was assessed by were asked to evaluate saltiness and umami taste at 5–45 s
providing them with samples containing specific (Poojary et or 5–60 s by using a 10-point linear scale for the rating of
al., 2017) of MSG and comparing them against a reference the samples.
sample. The assessors were subsequently divided into three In any case, the choice of the sensory evaluation test
groups; assessors who had a higher sensitivity threshold to depends on the purpose of the study, the samples given and
MSG (glutamate tasters), assessors with same sensitivity to the experience of the panelists on sensory evalu-
MSG and NaCl (hypotasters) and assessors who had higher ation techniques.
sensitivity to NaCl (nontasters).
Regarding the main part of sensory evaluation, many
Kokumi
approaches have been studied and the sensory evaluation
test choice depends on the case of the study. For example, if The term kokumi is used to describe the continuity, thick-
a study is conducted in order to determine the fraction of a ness, mouthfulness and, also, the complexity of foodstuffs
material responsible for the umami taste, a taste profile ana- and is thought to be a key factor for palatability (Feng et al.
lysis of the sample against the recombinates is chosen. The 2016; Shibata et al. 2017). The word kokumi comes from
term “recombination/reconstitution experiments” is used to the word “koku,” which is used to describe food palatability.
describe the process, after fractionation of the start material, “Koku” is the whole sensation that it is caused by the sum
in which researchers combine the individual isolated frac- of food properties, while kokumi is used specially for the
tions. Many of these studies used the technique of omission taste and flavor properties (Nishimura 2019; Toelstede,
test by reconstituting the sample and omitting or excluding Dunkel, and Hofmann 2009).
one fraction at a time (Ottinger and Hofmann 2003; Kim et Actually, it was suggested that “koku” is derived from the
al. 2017). In many cases, a study is conducted in order to combination of three sensations, that act as the three basic
determine and quantify the taste characteristics of a final elements of kokumi properties, as it is presented in Figure
product or an isolated fraction. The protocol usually fol- 2; Continuity/Lingeringness, mouthfulness and complexity
lowed is quantitative assessment (QDA-Quantitative (Nishimura 2019). In sensory tests, many different words
Descriptive Analysis) on a point scale or an unstructured can be chosen to evaluate these attributes such as mildness/
scale (Selamassakul et al. 2020; Wang, Zhou, and Liu 2020; balance/smoothness or richness/depth and aftertaste, while
Dermiki et al. 2013a; Kim et al. 2017). To apply such kind the three basic elements have a specific meaning.
of test, a vocabulary of necessary terms and attributes that Continuity/Lingeringness is used to describe the long-lasting
can characterize the given samples is developed (Oruna- effect and the aftertaste, while mouthfulness is used by
Concha et al. 2007). researchers in order to define the spread of the taste sensa-
Some other approaches suggest the application of isoin- tion in the whole mouth. The term “complexity” is used to
tensity test, a comparative duo test between the components describe the thickness of the taste sensation and more pre-
under study and a dilution series of aqueous solutions of cisely, it is formed by the combination of many different
MSG. Taste dilution factors for umami is another test taste, aroma and texture compounds in the food matrix (Li,
applied, in order to determine the maximum dilution of the Zhang, and Lametsch 2020; Nishimura 2019).
sample under study in which an umami taste could
be detected.
Mechanism of action of kokumi compounds
Time intensity tests have been applied in order to deter-
mine the long-lasting effect and the change at the intensity Many studies have been conducted in order to clarify the
of umami taste during mastication (O’Mahony and Ishii interaction between tastants and taste receptors. It has been
8 A. VASILAKI ET AL.

Figure 2. “Kokumi” consists of three basic elements; complexity, continuity and mouthfulness when seen through a sensory “prism.”

suggested that kokumi substances can interact with the the object of extended study (Feng et al. 2016; Eric et al.
extracellular calcium receptor which belongs to the family of 2014; Kuroda and Miyamura 2015; Shibata et al. 2018). For
G-protein receptors and they can be affected by the binding instance, alliin is a sulfur-containing amino acid that has
of some peptides with specific chemical characteristics been studied by Ueda et al. (1990) as a component that can
(Ohsu et al. 2010; Maruyama et al. 2012; Kuroda and increase continuity, mouthfulness and complexity of
Miyamura 2015; Amino et al. 2018). The binding process umami solutions.
includes the interaction between the receptor and the With reference to substances formed during thermal
amino- or carboxyl- group of amino acid residues of the processing, alapyridaine has been proposed as a molecule
peptides. The binding process and the signals created during of prominent importance in beef broth (Dunkel, K€ oster,
the interaction of receptors and kokumi substances have and Hofmann 2007; Ottinger and Hofmann 2003).
been proposed in the study of Kinnamon (2012), and espe- Maillard reaction products have been widely investigated
cially the action of T1R1/T1R3 receptors. for their kokumi inducing properties, while Eric et al.
Among the compounds that have been studied for their (2014) combined sulfur-containing amino acids and sugars
kokumi flavor properties, c-glutamyl-tripeptides had to produce Maillard products with mouthfulness and
attracted researchers’ interest. Kuroda and Miyamura (2015) meat-like flavor properties (Nishimura 2019; Yamamoto
studied tripeptides mechanism of action and their ability to 2019; Kurobayashi et al. 2019; Ogasawara, Katsumata, and
increase kokumi characteristics. In order to determine the Egi 2006; Song et al. 2013).
chemical characteristics responsible for kokumi properties At this point, it is important to stress out the differences
and flavor-enhancing properties, researchers used many dif- among the terms “umami,” “flavor-enhancing” and
ferent c-glutamyl-tripeptides and glutathione as reference “kokumi.” Many researchers who studied the differences
between these properties, the components that these taste/
substances. The tripeptide which presents the higher
flavor characteristics are derived from and their mechanisms
increase for kokumi properties, besides glutathione, seem to
of action, support that one tastant has two distinctive ways
be c-Glutamyl-Valyl-Glycinyl peptide (c-Glu-Val-Gly) and
of action; the first one is to induce a particular taste, and
the receptor is activated by the presence of 3 mM of this tri-
the second is to affect the other flavorants by means of syn-
peptide (Maruyama et al. 2012).
ergism, antagonism, masking, etc. (Yamamoto 2019;
As stated above, kokumi substances interact with other
Yasumatsu et al. 2015). Umami is a unique taste. Umami
taste molecules such as umami, sweet and salty ingredients
ingredients can act as taste enhancers and synergistically
and there is high positive correlation between calcium-sens-
they can increase the intensity of other flavors. Kokumi is
ing receptor (CaSR) activity and taste activity of these ingre-
not a single unique taste, but the term describes specific fla-
dients (Ohsu et al. 2010). It has been reported that 6.5% of vor properties, while kokumi components act like flavor-
human taste cells react to kokumi substances, 28% to bitter enhancer, eliciting only slight taste or no taste at all when
tastants and 5% to umami ingredients (Maruyama et they are added in water.
al. 2012). Additionally, there are extended studies about interac-
The mechanism of action of other substances mentioned tions between kokumi and umami ingredients and the syn-
above that elicit kokumi flavor properties is still unclear. ergism modifications in taste and flavor derived from their
combination in different concentrations (Yamamoto 2019;
Compounds exhibiting kokumi flavor properties Feng et al. 2016; Ueda et al. 1990). Umami solutions con-
taining MSG and IMP can be positively affected by the add-
Kokumi compounds belong to a wide range of chemical ition of low amounts of garlic extract (Ueda et al. 1990),
classes and their presence has been widely studied in both while glutathione enhances more the taste of ribonucleotide
fresh and processed food. Many researchers have already solutions than monopotassium glutamate (MPG) solution
studied the kokumi flavor properties of c-glutamyl-peptides, (Yamamoto 2019). The interaction between umami and
as it was mentioned previously (Yang et al. 2021; Feng et al. kokumi compounds is presented in Figure 3. Specifically, in
2016; Amino et al. 2018; Nishimura 2019; Yamamoto 2019; the left part of the figure, the taste response of an umami
Kuroda and Miyamura 2019). solution presents lower mouthfulness intensity and lower
Additionally, sulfur-containing amino acids and their aftertaste, while the “spread of the taste” is lower too, with-
reaction products after thermal processing have also been out harmonious sensory response. By the addition of
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 9

kokumi ingredients, the mouthfulness and continuity are Food products with kokumi flavor properties
greater and the flavor response is richer and smoother
As described above, there are many categories of kokumi
(right part).
Many studies have been done in the field of “dose- compounds and they can be found in many different food-
response effect” of kokumi components. As “dose-response stuffs, from raw materials to processed products. They can
effect” is called the ability of some substances to enhance be created in processed products by hydrolysis of proteins,
specific taste properties of foodstuffs when added in sub- thermal processing or fermentation, which usually include
threshold concentrations, when, in super-threshold concen- enzymatic hydrolysis, as well. Many studies have focused on
trations, they elicit different taste/flavor characteristics. For the identification of the sensory and chemical characteristics
instance, bitter tasting compounds contained in yeast extract of these compounds. A short review of the plant and yeast
elicit flavor-enhancing effects when present in subthreshold materials with high concentrations of kokumi compounds
concentrations (Liu et al. 2015). The same effects have been and the processes used to increase the concentration of these
observed in cheese and in the way that oxylipins, derived by ingredients are cited below.
processed avocados, act (Toelstede, Dunkel, and Hofmann
2009; Degenhardt and Hofmann 2010). Furthermore, there Yeast origin
are scientific data claiming that kokumi taste can be per- Yeast extract. Except from the plant-origin food products
ceived only in the presence of two or more basic tastes cited, yeast extract is the most widely studied and used
(Yang et al. 2019). kokumi ingredient by the food industry. Many studies have
been conducted in order to identify the components which
are responsible for its special flavor properties and explain
their action in a food matrix (Liu et al. 2015; Wang et al.
2019; Pereira, Ugucione, and Pereira-Filho 2017 Phat et al.,
2016). Sensory evaluation of the interaction between yeast
extract and mushroom concentrate has also been studied
and compared to MSG addition in chicken soup (Wang et
al. 2019). Yeast extract has been used successfully in order
to mask the negative sensory properties of sodium chloride
replacers in salted meat products (Vidal et al. 2020) and
dairy products (Silva et al. 2018). The worldwide market
size of yeast extract was valued at about 1.3 billion USD,
and its growth rate until 2027 is expected to be 5.9%, annu-
Figure 3. The “umami-kokumi interaction” diagram; Left part: the taste ally (Grand View Research 2020). According to the same
response of an umami solution presents lower mouthlness and continuity, with report, 38% of the annual production of yeast extract is con-
no complexity and smoothness. Right part: After the addition of kokumi ingre-
dients, mouthfulness and continuity increase, while the overall taste can be
sumed by the food and beverage industry. While many stud-
described as smoother, richer and more complex (Modified figure, ies have been done in the field of yeast extract, and this
Nishimura 2019). yeast origin material is widely used during the last decades,

Figure 4. Umami and kokumi compound determination and analysis flow diagram.
10 A. VASILAKI ET AL.

this review will not include more information, as it is more description of certain fractions implied that they possessed
focused on alternative flavor enhancers. kokumi properties (Su et al. 2011).
To elucidate and determine the substances that are
Plant origin. Allium species. Allium species, including onion, responsible for these flavor properties, Su et al. (2012) used
garlic and leek, belong to a widely used plant family of food the A. oryzae crude protease extract and after hydrolysis, a
ingredients. They impart a characteristic taste and aroma in fractionation was used to separate and isolate the peptides
raw or cooked foodstuffs. Garlic was firstly studied as produced. The peanut hydrolysate exhibited umami enhanc-
kokumi flavor enhancer in 1990 by Ueda et al. who reported ing activity when added in umami solution and two novel
that water extracts of garlic could elicit a slight sweet taste peptides were recognized as umami enhancing peptides (Su
and weak aroma properties. However, when added in et al. 2012).
umami dishes, these extracts presented kokumi flavor char-
acteristics, such as continuity, mouthfulness and thickness. Beans. White beans (Phaseolus vulgaris L.) belong to the
The same trend was observed by the addition of onion family of legumes and they are widely used as a food ingre-
extracts into umami dishes (Ueda, Tsubuku, and dient and as a part of traditional cooked dishes. There are
Miyajima 1994). many studies about legume fermentation processes, focusing
With regard to kokumi flavor ingredients in garlic, on the high concentration of sulfur-containing compounds
researchers suggest that the kokumi properties are induced and peptides at the final products. The flavor activity of
mainly by sulfur containing compounds, like alliin, c-glu- these compounds, however, has not been evaluated thor-
tamyl-allyl-cysteine (GAC), c-glutamyl-allyl-cysteine-sulfox-  c et al. 2012).
oughly (Zili

ide (GACSO) and methyl-cysteine-sulfoxide (MeCSO). One of the few studies focused on white beans’ flavor
When the taste and flavor of aqueous solutions of GAC and impact, is that of Dunkel, K€ oster, and Hofmann (2007).
GACSO were analyzed by sensory evaluation, it was found Aqueous extracts of cooked or uncooked beans were pre-
that they exhibited slight garlic-like flavor, while MeCSO pared and their sensory profile was evaluated both in aque-
presented a more leek-like flavor. When added in subthres- ous solutions and in umami chicken broth. It was proposed
hold concentrations in umami model soup they enhanced that white beans contain c-glutamyl peptides, which impart
the final flavor of the product, exhibiting a more rounded mouthfulness, complexity and richness by enhancing the fla-
and complex flavor (Ueda, Tsubuku, and Miyajima 1994). vor properties of umami chicken broth. The taste evaluation
This study was the first that reported GACSO as a flavor of the same products in aqueous solutions revealed that the
enhancing compound and it was suggested that it resulted taste in this medium was weaker (Dunkel, K€ oster, and
from GAC conversion during processing (Ueda, Tsubuku, Hofmann 2007).
and Miyajima 1994).
Onion contains similar kokumi flavor compounds to gar- Soybeans. Soybeans are also widely used legumes, both for
lic, as its aqueous extracts contain high amounts of sulfur- oil and fermented condiment production. Although soy-
containing and c-glutamyl-peptides. It is reported to exhibit beans are well-known for their fermentation products and
characteristic and intense flavor when consumed uncooked, their umami compounds produced during this process, they
while cooking imparts a sweeter flavor and the sulfur-con- contain high amounts of c-glutamyl peptides which impart
taining compounds are dramatically changed (Ueda, kokumi flavor activity (Shibata et al. 2017; Su et al. 2011; Li
Tsubuku, and Miyajima 1994). Alliin, which is abundant et al. 2013). After protein hydrolysis of soybean meal, in
also in garlic, is a sulfoxide that determines the kokumi many cases a fractionation process was conducted, in order
properties of onion. Likewise, propenyl-cysteine-sulfoxide to identify the fractions responsible for kokumi impact. The
(PeCSO) and c-glutamyl-propenyl-cysteine-sulfoxide (c-Glu- c-glutamyl-peptide fraction was tested in a control solution
PeCSO) are two other ingredients of onion responsible for containing MSG, IMP and salt, and it was found that it can
its kokumi properties (Ueda, Tsubuku, and Miyajima 1994). enhance the umami properties and affect the flavor by
increasing the mouthfulness and complexity (Shibata et al.
Peanut. Peanut (Arachis hypogea L.) is a widely used crop, 2017; Zhang and Peterson 2018). Besides that, researchers
with high amounts of fat. After the extraction of oil, peanut have studied the chemical and flavor characteristics of
meal is produced. The exploitation of the industrial waste Maillard Reaction Products (MRPs) derived from reactions
has been extensively studied. Defatted peanut meal has only among soybean peptides, xylose and cysteine. The crucial
been used as animal feed or fertilizer. The enzymatic factor in these cases was the processing temperature and the
hydrolysis of peanut protein by alcalase, ProtamexV R and presence of cysteine, as it was shown that MRPs formed at
papain can produce peptides with a wide range of chemical 100  C without the presence of cysteine, elicit flavor enhanc-
and biological properties. Su et al. (2011), utilized defatted ing properties, while formation at 140  C in the presence of
peanut meal through hydrolysis by Aspergillus oryzae, the cysteine reveal “meaty” flavors (Zhang and Peterson 2018).
microorganism responsible for soy fermentation. The result Addition of cysteine could also reduce the formation of bit-
of this fermentation and hydrolysis process was the produc- ter-tasting compounds, and it is positively correlated to sul-
tion of flavor enhancing fractions, which were evaluated as furous compound formation (Zhang and Peterson 2018). At
umami, salty and full-bodied. Although the resulting prod- the same study it was reported that during heating of pepti-
ucts were not reported as kokumi ingredients, the sensory des in the presence of xylose without cysteine, the final
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 11

Maillard reaction products (MRPs) presented umami and chemical and physicochemical characteristics of the targeted
salty taste enhancement, while heating at 140  C in the pres- compounds. As it is presented in Figure 4, first step of ana-
ence of cysteine imparted a more meat-like flavor enhance- lysis includes extraction, using solvents with chemical affin-
ment and lower bitterness (Zhang and Peterson 2018). ity to the targeted substances. In many cases aqueous
Other researchers suggested that heating at 120  C the pepti- extraction is used (Dunkel, K€ oster, and Hofmann 2007;
des produced by soy hydrolysis, in the presence of cysteine Charve, Manganiello, and Glabasnia 2018; Eric et al. 2013;
and xylose, can maximize kokumi ingredients formation Ueda et al. 1990) and pH adjustment in order to exclude
(Habinshuti et al. 2019). Oligosaccharides, and especially sta- the isolation of unwanted products (Eric et al. 2013). In
chyose and raffinose, is another chemical class of ingredients some cases, the usage of ethanol as extraction solvent is pre-
that has been reported to act synergistically to c-glutamyl- ferred (Ueda et al. 1990; Ueda, Tsubuku, and
peptides, which can enhance the intensity of kokumi proper- Miyajima 1994).
ties imparted by peptides (Shibata et al. 2017). Regarding the fractionation processes, ultrafiltration using
cellulose membranes with different molecular weight cutoffs
Corn. Corn sauce is a fermented product, produced by is used, targeting the fractions of <1 kDa and 1–5 kDa,
enzymatic hydrolysis of corn starch. Initially, glutamic acid and which contain the kokumi flavor-active ingredients (Charve,
its salts and acetic acid were considered to be the main con- Manganiello, and Glabasnia 2018; Su et al. 2011; Su et al.
tributors for umami and kokumi taste/flavor properties of corn 2012; Liu et al. 2015).
sauce. During reconstitution experiments and sensory evalu- When needed, subfractionation is used at the fractions
ation of the reconstitutes, however, it was revealed that the considered to elicit flavor enhancing properties. The more
afore-mentioned components were not the only compounds widely-used technique is gel filtration or gel permeation
responsible for umami and kokumi taste/flavor properties. chromatography. Charve, Manganiello, and Glabasnia (2018)
Alanine, g-glutamyl-peptides and Amadori products generated used a mobile phase consisting of formic acid aqueous solu-
from the reaction between fructose and glutamic acid contrib- tion and acetonitrile, in a gradient elution system, while
ute to corn sauce’s special taste and flavor characteristics, when Habinshuti et al. (2019) used an isocratic elution process
they are present in subthreshold concentration. Although in with methanol and acetonitrile. The detector used in most
this study kokumi sensation was not evaluated though sensory studies was Ultra-Violet (UV) or Diode Array Detector
evaluation, researchers used this term to describe a specific (DAD). Gel filtration chromatography was also applied by
chemical compound class that was identified in the corn sauce Su et al. (2012) and Eric et al. (2013), while the latter per-
samples. In addition, the study included the term “taste modu- formed gradient elution using acetonitrile/water and tri-
lating ingredients” that can be a wider class, including kokumi fluoroacetic acid.
components like glutamic acid derivatives (Charve, When sensory evaluation is needed for specific fractions
Manganiello, and Glabasnia 2018). Habinshuti et al. (2019), or subfractions, preparative High Performance Liquid
compared the corn MRPs to the MRPs generated from soy- Chromatography (HPLC) is widely preferred (Ueda et al.
bean Maillard products, and suggest that the former elicit 1990; Ueda, Tsubuku, and Miyajima 1994; Liu et al. 2015),
more umami, meaty and salty taste with mouthful perception and the chemical characteristics of the targeted substances
due to their higher amount of cysteine. are taken into account for the selection of the eluent system.
For example, cycloalliin was isolated by isocratic elution
Sunflower. Many studies focus on defatted plant meals, as a assisted by potassium monophosphate buffer and acetonitrile
means for utilizing industrial waste. Besides soybeans and as mobile phase (Ueda et al. 1990). For the determination of
peanut, sunflower seed defatted meal is another good yeast extract flavor ingredients, aqueous formic acid solution
example of this approach. Sunflower seed defatted meal was was used as mobile phase A and methanol as mobile phase
the subject of Eric et al. (2013) extended research. They pro- B, through gradient elution (Liu et al. 2015). The choice of
posed and optimized the conditions for producing taste- the column type of the HPLC system is another crucial fac-
active compounds by using a combination of reducing sug- tor that affects the elution of targeted ingredients and their
ars, cysteine and peptides produced by the hydrolysis of separation from other compounds. Flavor-active components
sunflower seed defatted meal. These compounds were found from onion were purified by elution through Hþ column,
to enhance the flavor of an umami broth by adding a more while components from garlic had chemical affinity to OH-
complex and, in some cases meat-like, flavor. Their findings column (Ueda et al. 1990; Ueda, Tsubuku, and
were in agreement to these of Ogasawara, Katsumata, and Miyajima 1994).
Egi (2006), who concluded that Maillard peptides between In the case of c-glutamyl peptides, identification is an
specific molecular weight limits (1–5 kDa) can affect posi- essential step to find the exact amino acid sequence of these
tively the taste of umami solution. peptides, and provide a deeper comprehension of the com-
pounds responsible for the kokumi flavor. In most studies,
mass spectrometry and NMR spectrometry play important
Extraction, fractionation and identification of
role. Ueda et al. (1990) used both of these techniques in
kokumi substances
order to identify onion ingredients, while Ueda, Tsubuku,
The processes chosen for the extraction, fractionation and and Miyajima (1994) used Filter Dynamic Measurement
identification of flavor-active compounds depend on the System (FDMS), Proton-Nuclear Magnetic Resonance (H1-
12 A. VASILAKI ET AL.

NMR) and Carbon-Nuclear Magnetic Resonance (C13-NMR) 2011). The scale used also depends on the objectives of the
spectrometry. The same spectrometry techniques were study and typically vary between a 5- and 10-point scale
applied by Charve, Manganiello, and Glabasnia (2018), but (Eric et al. 2017; Liu et al. 2015; Su et al. 2011).
in combination to HPLC/MS profiling. In recent years, A usual technique to evaluate kokumi flavor is the time
more complex techniques and more modern equipment pro- intensity rating. As Shibata et al. (2017) suggest, panelists
vide easier identification of targeted compounds. Some were requested to rate the flavor intensity of the given sam-
examples of analytical equipment used are systems such as ples by scoring at a 0–10 non-graduated scale at specified
Matrix Assisted Laser Desorption/Ionization-Time-Of- times after tasting. Thickness was rated after 5 sec of tasting,
Flight/Time-Of-Flight-Mass-Spectrometry (MALDI-TOF/ continuity after 20 sec of tasting and mouthfulness as the
TOF-MS) and Liquid-Chromatography-Quadrupole (LC-Q- intensity of reinforcement throughout the mouth.
TOF-MS/MS), which are based on physicochemical charac- When a study is based on sensory evaluation of a widely
teristics of targeted compounds in order to separate and used kokumi tastant, such as yeast extract, it contains many
identify them, by the use of more than one detector to pro- stages of sensory evaluation procedures to cover a wider
vide positive identification (Liu et al. 2015; Su et al. 2012). range of flavor properties. Taste recognition threshold and
taste dilution factor are usually determined through triangle
Sensory evaluation of kokumi fractions/compounds tests and sequential dilutions in umami soup or broth (Liu
et al. 2015).
Sensory evaluation is one of the most important steps for More recent studies combine the sensory evaluation of
flavor compounds identification and it is used either as a kokumi compounds by a panel of trained assessors and ana-
basic and starting point of analysis or as the final step of the lytical methods like e-tongue and e-nose analysis, in order
procedure. The taste evaluation processes for the character- to give a more comprehensive insight (Yang et al. 2021;
ization of kokumi compounds are similar to those of umami Zhang and Peterson 2018).
tastants, with modifications at the testing medium and
examined attributes of the products.
The processes typically include the evaluation of aqueous Current and future trends
extracts of plant materials, without fractionation or isolation The use of MSG can enable the reduction of salt in food-
of the key flavor compounds. Deodorization is often used as stuffs, because it enhances the perception of saltiness (Reis
a first step, to avoid odor interference (Ueda et al. 1990; Rocha et al. 2020). Natural substitutes of MSG can also be
Ueda, Tsubuku, and Miyajima 1994). The lyophilized plant
used to help reduce salt, while, at the same time, maintain-
extract is usually evaluated alone dissolved in water and
ing food palatability and acceptance. Likewise, natural
compared to umami soup or chicken broth containing the
umami components could help reduce the use of sweeteners
tested extract (Ueda et al. 1990; Charve, Manganiello, and
or other additives, enabling the production of clean label
Glabasnia 2018).
food products.
In order to specify the contribution of each taste com-
Kokumi taste ingredients are promising substitutes for
pound at the final product’s flavor, re-engineering tests and
fat, MSG and flavor components such as caramel artificial
taste recombination experiments are chosen. In this type of
flavorings and meat-like flavorings. The complexity and
procedure, flavor compounds are added in an aqueous
length of the taste produced by fat can also be created by
medium at their naturally occurring concentration and the
difference between the natural flavor and re-engineered sol- c-glutamyl peptides and their interaction with other food
utions are compared and contrasted (Charve, Manganiello, flavorants (Kuroda and Miyamura 2015). Umami-enhancing
and Glabasnia 2018; Su et al. 2012). properties of c-glutamyl peptides can reduce the MSG
In many cases, isolated kokumi compounds or fractions required for improved palatability. Sulfur-containing com-
are evaluated dissolved in water and their attributes are pounds can improve the meat-like flavor of vegan and vege-
compared to those elicited by their addition in umami solu- tarian meat mimetic products and make them more
tions or model broths (Ueda et al. 1990; Charve, acceptable to consumers. Caramel-like artificial flavorings
Manganiello, and Glabasnia 2018; Eric et al. 2013). Su et al. can be reduced by the use of Maillard reaction products
(2012), used another approach, in which they perform a sen- (Eric et al. 2014). Additionally, the consumer demand for
sory evaluation by comparing the flavor-active fractions of low-sodium content in processed foods has led researchers
peanut extract and the whole crude peanut extract without to investigate the use of kokumi ingredients for masking the
fractionation. In order to evaluate flavor substances gener- negative sensory properties of sodium chloride replacers by
ated by thermal treatment, such as Maillard reaction prod- using kokumi ingredients (e.g., yeast extract) (Vidal et al.
ucts, these are added into umami solutions after thermal 2020; Silva et al. 2018).
processing (Eric et al. 2013). In the field of sensory evaluation practices, there is an
The attributes tested in each study are different and the increasing demand for the development of objective evalu-
score scale used depend on the aim of the study. Usually, ation techniques, using biomimicking methods. Apart from
the terms of scoring include umami, umami-enhancing, the use of electronic tongue, 3D-bio-printing and microflui-
mouthfulness, complexity, long-lasting flavor, bitterness, dics technology can increase the selectivity and detection
saltiness and caramel-like (Habinshuti et al. 2019; Su et al. threshold of umami molecules (Wang, Zhou, and Liu 2020).
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 13

Another trend is the exploitation of kokumi and umami usually incorporated in umami broths to augment the flavor.
components, such as glutamic acid, aspartic acid and 50 - To elicit specific flavor properties, kokumi components need
ribonucleotides from shiitake mushroom by-products the presence of umami compounds in the food medium/
(Lentinula edodes) that could be present in industrial waste matrix. The taste of these compounds can be distinctive in
or agricultural by-products (Harada-Padermo et al. 2020). the presence of umami-tasting compounds, and the sensory
Targeted compounds could be isolated by applying green evaluation of kokumi ingredients is usually conducted using
extraction protocols, using environmentally friendly solvents chicken or meat broth as the tasting medium. (Eric et al.
such as water, thereby, reducing the environmental impact. 2013; Liu et al. 2015; Ueda et al. 1990).
Usually, the thermal treatment and enzymatic hydrolysis In this review, an extended presentation of scientific data
that precede the extraction process, liberate umami and in the field of natural taste and flavor enhancers is included.
kokumi components and also cause chemical modifications The clarification of the specific sensory properties of taste
that reduce or eliminate off-flavors. A typical example is the and flavor enhancers and their mechanism of action could
hydrolysis of proteins (Zhu and Damodaran 2018). Another help the food industry to utilize them extensively and avoid
benefit of aqueous extraction is that the extracts are rich in the addition of conventional tastants. Plant, fungi and dairy
polar compounds, such as polar taste enhancers, while non- products can act as sources of natural taste-active ingre-
polar ingredients with potentially unwanted taste properties dients. Through the appropriate thermal and enzymatic
are usually excluded. treatment of these components their properties can be inten-
The final extract can be characterized chemically by sified. Furthermore, the analytical techniques of separation
many different analytical techniques, from fast-track meth- and identification are of special interest for the food indus-
ods to scan the resultant extract, to more sensitive and com- try, regarding the production of more green and natural
plicated processes to identify the active taste/flavor food ingredients. All the above-mentioned steps can lead to
ingredients. However, sensory evaluation of the final F/TE successful and effective use of naturally occurring com-
ingredients and their incorporation in the food matrix can pounds, promoting consumers health. However, further
provide useful information about the synergistic action of research on the functionality of these compounds is needed
naturally occurring and/or derived F/TEs with other to promote the incorporation of F/TE in daily consumed
food components. foods. Recent consumer demands make a thorough study of
A natural flavor enhancer can replace an artificial one, these components imperative, and the concept of multifunc-
and in some cases alter its technological properties, through tional food ingredients should be considered, because of the
the addition of functional components. For example, advantages of their use in different scientific fields, including
Maillard type products can also be used as a natural food food science, environment and health.
coloring material and peptides can be studied for their
foaming ability, oil and water retention capacity.
As it was mentioned in the introduction section, natural Disclosure statement
food additives could lead to increased production costs for The authors of the present paper declare no conflicts of interest.
food industry. But, is adopting “green” processes and using
plant, fungi and dairy foods to produce F/TE, really cost
raising practices? “Green process,” without petroleum or References
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