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Biotechnological production of non-volatile flavor compounds

Bruno Nicolau Paulino, Adones Sales, Lorena de Oliveira Felipe,

Glaucia Maria Pastore, Gustavo Molina, Juliano Lemos Bicas

PII: S2214-7993(21)00025-4
DOI: https://doi.org/10.1016/j.cofs.2021.02.003
Reference: COFS 689

To appear in: Current Opinion in Food Science

Please cite this article as: Paulino BN, Sales A, de Oliveira Felipe L, Pastore GM, Molina G,
Bicas JL, Biotechnological production of non-volatile flavor compounds, Current Opinion in
Food Science (2021), doi: https://doi.org/10.1016/j.cofs.2021.02.003

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 Biotechnological production of non-volatile flavor compounds

Bruno Nicolau Paulinoa; Adones Salesb; Lorena de Oliveira Felipec; Glaucia Maria
Pastored; Gustavo Molinae; Juliano Lemos Bicasf*
a. Faculty of Pharmaceutical Sciences, Federal University of Amazonas (UFAM).
Manaus – AM, Brazil. ORCID: 0000-0002-3714-7466. brunofcf@ufam.edu.br
b. Serviço Nacional de Aprendizagem Industrial - Unidade Portão – Rua Padre
Leonardo Nunes, 180, Portão, 80330-320, Curitiba – PR, Brazil. ORCID: 0000-
0002-2103-9639. adonessales@gmail.com
c. Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki, 305-0006, Japan. ORCID: 0000-0002-3384-4395.

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lorenaob@gmail.com
d. Department of Food Science, School of Food Engineering, University of Campinas.

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Campinas – SP, Brazil.
ORCID: 0000-0001-9631-0158. glaupast@unicamp.br
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e. Institute of Science and Technology, UFVJM. Diamantina – MG, Brazil. ORCID:
0000-0002-1409-6846. gustavomolinagm@gmail.com
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f. Department of Food Science, School of Food Engineering, University of Campinas.
Campinas – SP, Brazil.
ORCID: 0000-0002-5252-4004. jlbicas@gmail.com
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*Corresponding author. Address: Rua Monteiro Lobato, 80. Departamento de Ciência de

Alimentos, Faculdade de Engenharia de Alimentos – Unicamp. Campinas, SP, 13083-862, Brazil.
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Phone: +55 19 3521-0065; E-mail: jlbicas@gmail.com

Highlights
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 Recent advances in bioproduction of non-volatile flavor compounds are

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presented.
 Umami- and kokumi-tasting substances can be produced through
biotechnological approaches
 Enzymatic conversion is the most employed strategy for production of emerging
low-caloric and high-intensity sweeteners

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Abstract
The market for food additives has continuously grown and been modulated to meet
consumer demands, mainly those related to sensory quality, sustainability and health concerns.
In this context, compounds used to impart or improve food flavor characteristics are particularly
important, and the methods employed to produce such substances are associated with their
acceptance by both industry and consumers. In this review, we present the most recent advances
in the biotechnological production of non-volatile flavor compounds, particularly the microbial
and enzymatic strategies to produce umami and kokumi-related substances, emerging low-
caloric and high-intensity sweeteners.
Keywords: food additives; biotechnological products; umami; kokumi; sweeteners.

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1. Introduction
Flavor is usually referred to as a complex multifactorial sensation that stems from the
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interaction between olfaction (aroma), gustation (taste), and touch sensations. Moreover, it is
known that other parameters influence flavor perception, such as appearance, temperature, and
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sound. While olfaction reflects the perception of volatiles that activate odor receptors in the nasal
cavity and touch sensations from the stimulation of trigeminal nerve endings (pungency,
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coolness) and texture (crunchiness), for instance, the taste is usually attributed to non-volatile
soluble substances that interact with taste buds located in the tongue (and throughout the
digestive tract). Although sweet, sour, salty, bitter and umami have been referred to as the five
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basic tastes in the gustation process, other taste candidates (metallic, fatty acid, “starchy”) have
also been proposed [1,2].
Different strategies, including chemical syntheses, microbial and enzymatic processes,
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have been developed for producing taste compounds, mainly nucleotides or other umami-taste
substances, and sweeteners [3–5]. Following consumer demands for natural, healthier,
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socioeconomically responsible and eco-friendly foodstuffs [6,7], biotechnological processes

represent an emerging strategy to supply flavor compounds for the food industry. Besides being
possibly labeled as natural (EC No 1334/2008, Code of Federal Regulations Title 21 / FDA) [8],
such “biotech” flavor compounds may be produced from renewable, abundant, and inexpensive
starting materials (e.g. lignocellulose wastes) [9]. Thus, this is an attractive and challenging field
in the biotechnology, increasing the number of studies on enzymatic and microbial production of
these added-value compounds [10,11].

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 Many non-volatile flavor compounds produced by microbial or enzymatic processes
(e.g., monosodium glutamate - MSG, acidulants, syrups) have a well-established market, while
others are in developmental stages or have just started to be marketed. As an example, Cargill in
collaboration with Evolva has been marketing since 2017 the steviol glycosides EverSweet
(mainly the rebaudiosides D and M) produced using yeast [8]. Besides, the technological
development in this field can illustrated by the recent patents related to the main compounds
mentioned in this review.

Table 1

Thus, this mini-review will focus on the most recent advances regarding the

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biotechnological production of sweeteners, umami- and kokumi-tasting compounds, but not
MSG, acidulants, syrups, and polyols, which have been reported in other reviews [12–14].

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2. Umami- and kokumi-tasting substances
Amino acids, nucleotides and peptides are examples of non-volatile flavor compounds
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used as flavor enhancers and for providing umami and kokumi tastes, besides being considered
to partially replace salt in low sodium foods [15–17]. Although MSG is the most traditional and
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studied flavor enhancer, this section will focus on nucleotides and peptides.
The most economically important nucleotides (nucleosides 5’-monophosphate) for the
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food industry are inosine-5’-monophosphate (5’-IMP) and guanosine-5’-monophosphate (5’-

GMP), which are umami-tasting compounds used as flavor enhancers in different food products
[18]. The global market for nucleotides as food ingredients was US$403.1 million in 2014 and is
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expected to reach US$809.3 million by 2022 [19]. On an industrial scale, these compounds can
be produced by chemical and biological (microbial and enzymatic) methods, or by a combination
of both (chemoenzymatic methods). However, a growing interest for enzymatic methods has
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been evidenced, since they are considered safer, eco-friendly, and more aligned to actual market
demands. In the last years different enzymes have been reported for enzymatic synthesis of
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nucleotides, including nucleoside kinases, acid phosphotransferases, phosphoribosyl transferases,

synthetases, adenylate deaminases, 5’-phosphodiesterases, and phosphoribosyl transferases [20].
The process for producing nucleotides may start from isolated (e.g., pyrimidine or
purine nucleobases, and pentose sugar) or complex substrates (e.g., yeast RNA). In the first case,
the regioselective phosphorylation of nucleosides and phosphate donors may be achieved by
using nonspecific acid phosphatases [18]. The source of nucleosides for this process can be
obtained by microbial fermentation [21]. For example, the purine biosynthetic pathway of

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Ashbya gossypii was designed (disruption of two key genes), resulting in a redirected metabolic
flow to excrete 200 times more (2.2 g/L) inosine (the precursor of 5′-IMP) in relation to the wild-
type [21]. Similarly, a 41% increase (to reach 19 g/L) in the production of guanosine (precursor
of 5′-GMP) could be achieved when the purine operon from the purine biosynthetic pathway was
deregulated and the respiratory chain energy generation was optimized in Bacillus
amyloliquefaciens XH7 [22].
However, industrial processes for large-scale production of 5’-nucleotides is mostly
based on the enzymatic degradation of yeast RNA using phosphodiesterase (nuclease P1) to form
the four types of 5’-NMP (GMP, AMP, CMP and UMP), which are further separated by ionic
chromatography(e.g hyper-cross-linked resins) [23]. Among these 5’-NMP, only 5’-GMP
exhibits umami taste, but 5’-AMP can be converted into another umami-tasting nucleotide, 5’-

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IMP, by the action of 5’-adenylic acid deaminase (AMP deaminase) [24]. The recent advances in
the enzymatic production of nucleotides comprise the prospection of novel enzymes with better

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catalytic properties as well as the improvement of free or immobilized enzyme systems [25,26],
as illustrated next. Figure 1 presents the biotechnological production of umami-taste nucleotides
(5’-GMP and 5’-IMP).
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Fig.1
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The production of AMP deaminase by new strains has been reported in the last years
[27]. For example, an Aspergillus niger strain isolated from soil was used to produce this enzyme
by solid-state fermentation [24]. In this study, yeast powder was employed as a substrate for the
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5’-IMP production and different amounts of AMP deaminase (0.6-18 mL) at 40°C and pH 6.0
were tested. The results showed an increase in the conversion ratio for higher amounts of AMP
deaminase and conversion ratios around 85% were obtained when 6 to 18 mL of enzyme were
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used, yielding a production of 2 mg/g of IMP.

The enzymatic synthesis of 5’-NMP from purine bases by a non-immobilized
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recombinant hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermus

thermophilus expressed in E. coli BL21 was reported by [20]. The best conditions were achieved
by using 10 mM of guanine or hypoxanthine, which resulted in product yields of approximately
50% of 5’-GMP and 45% of 5’-IMP after 20 min or 10 min at 60°C and pH 8.5-10.5,
respectively. This enzyme was further immobilized onto magnetic glutaraldehyde-activated
microbeads (named MTtHGXPRT3) and could yield up to 7.5 mM of 5’-IMP or 3.8 mM of 5’-
GMP starting from 40mM or 10 mM of 5-phospho-α-D-ribosyl-1-pyrophosphate, 80mM of

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hypoxanthine or 20 mM of guanine, 48 mM or 12 mM of MgCl2, and 30 µg of MTtHGXPRT3,
respectively [28]. These reports were important to demonstrate that this enzyme (free or
immobilized) was highly active at relatively high pH values and temperatures required to
solubilize high concentrations of low water-soluble bases.
Specific nucleases have also been applied for the 5’-NMP production from yeasts. As an
example, the heterologous expression of Penicillium citrinum nuclease P1 gene nucP1 in A.
niger resulted in a nuclease P1, which could yield up to 49.90 mg of 5’-NMP per g of dry yeast
hydrolysate (substrate) [29].
Other enzymatic approaches for the production of 5’-NMP are also available, such as a
two-step enzymatic system using phospholipase D (PLD) from Streptomyces netropsis and

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phospholipase C (PLC) from Bacillus cereus [30]. In the first step, PLD was able to transfer a
phosphatidyl residue to the 5’-hydroxyl of nucleosides and form a phosphorylated product, while
PLC was used for a selective hydrolysis producing nucleoside 5’-monophosphates. An overall

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yield of 63% in the preparative synthesis of 5’-AMP could be achieved.
Besides nucleotides, some peptides are also responsible for umami taste. In the past 50
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years, 52 umami-tasting peptides have been reported [31]. One of the most prominent examples
is LGAGGSLA, known as a beefy meaty peptide (BMP) or “delicious peptide”, which has been
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used as a flavor enhancer since the 1970s. More recently, the production of this octopeptide was
reported in an engineered Escherichia coli. However, the production of a single gram of the
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desired product required 94 g of the fusion protein and, therefore, 6700 g of wet E. coli (at 15
g/L) or 448-L of fermentation broth. Despite the relatively low yield, this study was important to
confirm the umami taste of this peptide [32]. Moreover, the tripeptide glutathione (GSH) is
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known to improve some tastes, and acts on the calcium receptors of the tongue. It interacts with
MSG and IMP, also yielding umami and kokumi sensations [33]. The production of GSH by
genetically modified microorganisms has also been investigated and the expression of the
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mutated Cys4p, Yap1p, and Gly1p genes was important to reach better yields [34].
The influence of microbiological metabolites on kokumi taste has also been explored.
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This sensation is given by the perception of mouthfulness, complexity, and continuity, in

addition to reinforcing the sweet, salty, and umami tastes, and is usually associated with fish
sauce, soy sauce, shrimp paste, cheese, and beer, for example [35]. The microbiota analysis of 10
samples of “Pla-ra”, a Thai fermented freshwater fish, revealed the presence of 12 bacteria
genera, including Tetragenococcus spp., Staphylococcus spp., and Lactobacillus spp. The
analysis of the 18 and detection of 17 glutamyl peptides revealed that the tripeptide γ-Glu-Val-
Gly was the most potent kokumi substance [36]. In another study, the presence of umami- and

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kokumi-tasting (γ-L-glutamyl-L-valine) molecules were detected in a Lactobacillus plantarum
fermentation product that was further applied in a bakery product as a flavor enhancer, providing
a 50% reduction in NaCl without significantly modifying its acceptance [37].
Enzymatic processes for producing kokumi compounds have also been reported, mostly
using γ-glutamyl transpeptidase (GGT) and glutaminase to yield γ-glutamyl compounds from
amino acids or protein hydrolysates [38,39]. For instance, the synthesis of kokumi-imparting
dipeptide γ-Glu-Leu using GGT from Bacillus licheniformis achieved the maximum conversion
yield (around 52%) when 200 mM of both L-Gln and L-Leu were reacted at 50°C and pH 9.0
after 5 h of incubation with 2.0 U/mL of GGT [40]. Additionally, the glutaminase from B.
amyloliquefaciens catalyzed a γ-glutamyl transpeptidation to produce γ-[Glu](n=1,2,3,4)-Val and
γ-[Glu](n=1,2,3,4)-Met peptides [41]. After 14 h of reaction, the yields of γ-[Glu](n=1,2,3,4)-Val

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peptides ranged between 5.33 to 35.03% with the most desirable molar ratio of Gln:Val of 600
mM:400 mM, while the yields of γ-[Glu](n=1,2,3,4)-Met peptides ranged between 6.75 to

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21.06% with the most desirable Gln:Met molar ratio of 800 mM: 400 mM. Similarly, γ-
[Glu](n=1,2,3,4,5)-Phe peptides were produced by using the same enzyme with yields varying
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between 0.13% to 49.64% and an optimal molar ratio of Glu:Phe of 300 mM:100 mM [42].
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3. Sweeteners
Sweeteners can be divided into caloric (starch-based sweeteners and sweetening syrups),
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low-caloric (polyols and emerging sweeteners or rare monosaccharides), and high-intensity

sweeteners (e.g. acesulfame potassium, advantame, aspartame, neotame, saccharin, Siraitia
grosvenorii extracts, and steviol glycosides) [3]. The main advances in biotechnological
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production of emerging and high-intensity sweeteners will be discussed next.

3.1 Emerging low-caloric sweeteners (rare monosaccharides)

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This section will be dedicated to discussing the enzymatic production of rare

monosaccharides (Figure 2), such as D-tagatose, D-allulose, and D-allose, whose interest has
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increased in the last decades due to their health (e.g. anti-hyperglycemic, antioxidant, anti-cancer,
anti-inflammatory, and others) and technological benefits (e.g. modulation of rheological
properties, enhanced textural properties, increased oxidative stability and foaming capacity,
inhibition of crystallization) [43–49]. In this context, sugar syrups and other food products
containing rare monosaccharides can be considered promising due to their functional label in
addition to their improved technological and sensory properties [50,51].

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 Fig.2

D-tagatose, a C-4 epimer of D-fructose, is a value-added compound that can be

efficiently produced by enzymatic methods. This rare monosaccharide is considered GRAS, has
a low glycemic index, and exhibits 92% of sucrose sweetness [52]. Its production can be carried
out via D-galactose isomerization by L-arabinose isomerase (LAI). Recent advances on this
subject include the use of alternative and complex substrates (in this case yielding D-tagatose-
monosaccharides mixtures) and the characterization of the enzymatic reactions involving new
LAI isolated from different microorganisms. These approaches are reported next.
Cheese whey has been considered as a starting material to produce sweetening syrup
containing D-tagatose and D-fructose. In this case, a multienzymatic system comprising β-

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galactosidase, LAI, and D-xylose isomerase co-immobilized on acrylic epoxy activated supports
has been employed to hydrolyze lactose and further isomerize the resulting monosaccharides

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(galactose and glucose) into the desirable products. This procedure yielded a complete lactolysis,
and the levels of D-tagatose and D-fructose in the final syrup reached, respectively, close to 45%
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and between 48-53%, at temperatures between 50°C and 60 °C [53].
Whey permeate is another complex substrate for D-tagatose production. A two-step
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process, for instance, employed a purified β-galactosidase from E. coli for hydrolyzing lactose
and LAI-producing Lactobacillus plantarum (untreated and permeabilized) immobilized in
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alginate for converting D-galactose into D-tagatose. Starting from whey permeate with 300 g/L
of D-galactose, this procedure yielded D-tagatose productivity of 2.54-2.75 g/L/h after 48 h of
reaction at 25°C, resulting in a final concentration in a range of 122-132 g/L [44].
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In recent years, researches have been investigating other LAI sources to produce D-
tagatose. A new LAI isolated from L. brevis was able to convert D-galactose (at 50mM) into D-
tagatose, yielding a conversion rate of 43% after 23 h at 65°C. This enzyme exhibited optimum
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pH around 7 and required divalent cations, such as Mn2+, Co2+, and Mg2+, while Cu2+ and Ca2+
inhibited this enzyme [54]. The LAI produced by E. coli BL21 expressing the gene araA from
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Clostridium hylemonae resulted in a similar conversion rate (45.9% for 10 mM D-galactose) at a

similar temperature (60°C), but the reaction time was much shorter (2 h of incubation). This LAI
also exhibited a better enzymatic activity at pH 7-7.5 and required Mg2+ as a cofactor, but the
optimum temperature was lower (around 50°C) [52].
D-allulose, also known as D-psicose, is a low-calorie C-3 epimer of D-fructose with
GRAS status and 70% of sucrose sweetness [3,49]. This rare sugar can be produced by the

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epimerization of D-fructose using D-tagatose-3-epimerase (T3E) or from D-glucose involving
the combination of D-xylose isomerase and D-allulose-3-epimerase (A3E) [3].
The use of recombinant enzymes has been widely employed in such processes. T3E
from Cabaelleronia fortuita expressed in E. coli, for instance, could convert fructose (at two
different initial concentrations, 200 and 500 g/L), into D-allulose, reaching 63.8 and 147 g/L,
respectively. The same enzyme could also convert D-tagatose (concentrations of 200 and 500
g/L) into 138.4 and 314.2 g/L of D-sorbose, respectively [55]. This is one of the few studies
reporting D-sorbose production, that could be used as an alternative sweetener with different
healthy benefits [46,56]. Besides using fructose solutions, D-allulose production was also
investigated using more complex raw materials, such as the following examples.

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The production of a high-fructose syrup (HFS) containing D-allulose in a one-pot/two-
enzyme system using inulin-rich substrate (Jerusalem artichoke tubers) has been reported [57]. In
this study, a new exo-inulinase from Bacillus velezensis and A3E from Ruminococcus sp. were

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used, resulting in 1.47 g/L of D-allulose production at 50°C. The ratio of D-glucose:D-
fructose:D-allulose in the final HFS was 1:3:1. Another procedure for the D-allulose production
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involved sucrose (present in cane molasses) hydrolysis by thermostable invertase to yield high-
fructose cane molasses, which was further biotransformed by immobilized Corynebacterium
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glutamicum expressing A3E from Paenibacillus senegalensis. This one-pot two-step reaction
system could produce 61.2 g/L of D-allulose from cane molasses [58].
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An alternative process for converting D-glucose into D-allulose involved the use of an
engineered E. coli co-expressing both D-glucose isomerase (DXI) from Acidothermus
cellulolyticus and A3E from Dorea sp. Starting from 500 g/L of D-glucose, the final D-allulose
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and D-fructose concentrations were 89.1 g/L and 204.4 g/L, respectively, representing a D-
glucose:D-fructose:D-allulose ratio of 6.5:7:3 [59].
D-allose is a GRAS monosaccharide that can be produced by the reversible enzymatic
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isomerization of D-allulose, which is usually carried out under mild pH conditions (7.0-9.0) and
temperatures in a range of 60-85°C using different isomerases, such as L-rhamnose isomerase
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(LRI), D-ribose-5-phosphate isomerase (RPI), D-galactose 6-phosphate isomerase and glucose 6-

phosphate isomerase [3,60]. As already mentioned for the D-allulose production, most of the
biocatalysts used in the process are recombinant enzymes. LRI from Clostridium stercorarium,
for instance, was used to produce 199 g/L of D-allose at 70°C, pH 7.7, 1mM Mn2+, and 600 g/L
of D-allulose, which represents a conversion rate of 33% [61].
However, the use of allulose solutions as starting materials is not an economical
approach, therefore other substrates and enzymes have been investigated. For instance, fructose

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can be first converted into D-allulose by T3E or A3E, which is further isomerized into D-allose
using one the isomerases mentioned above. In this sense, a one-pot reaction using two
isomerases, including A3E from Flavonifractor plautii and RPI from Clostridium thermocellum,
achieved optimal conditions at pH 7.5, 60°C and 600 g/L fructose, yielding 79 g/L of allose
(conversion rate of 13%). This study also found that both enzymes required cobalt ions (1 mM
Co2+) [62]. Another one-pot enzymatic process, carried out at 65°C, pH 8.5 for 5 h, involved the
use of fructose and immobilized enzymes (A3E from Ruminococcus sp. and LRI from Bacillus
subtilis) produced by recombinant E. coli and resulted in a final product with a D-fructose:D-
psicose:D-allose mass ratio of 6.6:2.4:1.0 [63].

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3.2 High-intensity sweeteners
Steviol glycosides are zero-calorie diterpenes sweeteners obtained from extracts of
Stevia rebaudiana Bertoni leaves, with the most important being stevioside (5-10%),

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rebaudioside A (2-5%), and rebaudioside C (1%). The bitter aftertaste associated with stevioside
is particularly important because it limits the use of the Stevia extract in many food formulations
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[64]. Therefore, enzymatic modifications, mainly transglycosylation, have been considered a
solution to improve the sweet taste of steviol glycosides [65] (Figure 3).
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Stevioside glycosylation catalyzed by β-glucosidase, glucansucrase, and other enzymes
has been one of the strategies to transform stevioside into better tasting steviol glycosides,
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improving the sweet taste of Stevia sweeteners [64, 66]. In this sense, computational analyses
have been useful to predict structure modifications associated with the elimination of the bitter
aftertaste of stevioside [67].
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Fig.3
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The transglycosylation reaction catalyzed by dextransucrase from Leuconostoc

mesenteroides can be considered an interesting procedure to transfer glucose moiety from
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sucrose to different positions in stevioside, resulting in steviol glucosides. A high conversion

yield (98%) has been described applying 4 U/mL of enzyme, 800 mM of sucrose, 50 mg/mL of
stevioside at 28°C for 6 h [66]. The enzymatic modification of stevioside with trans-α-
glucosylation by a recombinant glucansucrase from Lactobacillus reuteri 180 has also been
reported. In this case, a mathematical model indicated that the best reaction conditions could be
achieved with 31mM of stevioside, 525 mM of sucrose, 10U/mL of Gtf180-ΔN-Q110E mutant
enzyme at 37°C. Under these conditions, the conversion yield reached 95%, and 28 mM of

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steviol α-glucoside was produced [68]. The same enzyme was also applied in the α-glucosylation
of rebaudioside A (RebA) using sucrose as a glucose donor for synthesizing glucosylated
derivatives with improved taste properties. Higher conversions (95%) and 115 g/L of α-
glucosylated derivatives (mostly mono-α-glucosylated RebA) could be achieved after 3 h of
incubation at 37°C/pH 4.7/1 mM CaCl2 with 5 U/mL of enzyme. Even higher concentrations of
α-glucosilated products (270 g/L) could be obtained with a fed-batch system [69].
The transglycosylation of RebA applying a recombinant dextransucrase of Leuconostoc
lactis EG001 produced in E. coli BL21 has also been reported aiming to produce glycosylated
derivatives with increased storage stability (4-80°C for 24-48 h) in beverages (soft drink and
orange juice). The reaction mixture consisted of 80 mM of RebA, 0.4 M of sucrose, 13 mM of

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MgCl2, and 2.1 U/mL of enzyme at pH 5.2. After 6 h of incubation at 30°C, the conversion of
RebA to O-α-D-glucosyl-(1′→6′)-RebA reached 86.5% [70].
The biotechnological production of other isoprenoid sweeteners usually found in plants,

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such as mogrosides and cucurbitacins from Cucurbitaceae and glycyrrhizin from Glycyrrhiza
glabra, have also been reported [71]. Mogrosides are 300 times sweeter than sucrose. Its
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biosynthesis, similarly to other triterpenes, starts from squalene. Therefore, the microbial
production of such sweeteners requires the overexpression of the enzymes associated with the
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squalene biosynthesis. The squalene synthase enzyme from Siraitia grosvenorii was cloned and
characterized. This enzyme was then expressed in Escherichia coli showing optimal conversion
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of farnesyl pyrophosphate to squalene at 37 °C and pH 7.5 [72].

A different approach to produce biotechnological sweeteners involved the evaluation of
the ability of several microbes to perform selective hydrolysis of glycosidic bonds in mogroside
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V to convert it into siamenoside I, which is sweeter and has better tasting than other mogrosides.
The most promising results were associated with the beer yeast Dekkera bruxellensis, which
selectively hydrolyzed the mogrol glycosides and promoted the conversion of the mogroside V
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natural sweetener into siamenoside I [73].

Besides the aforementioned isoprenoid sweeteners, a new candidate of low-calorie
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natural sweetener is 5-ketofructose (5-KF). This compound could be produced by Gluconobacter

oxydans overexpressing a G. japonicus fructose dehydrogenase and using fructose as a substrate
[74]. In a 2-L reactor, concentrations, product yields and time-space yields up to, respectively,
489 g5-KF/L, 0.98 g5-KF/gfructose and 8.2 g5-KF/L.h could be achieved, which are quite outstanding
results for a bioprocess. Moreover, 5-KF showed an identical sweet taste quality as compared to
fructose, and a similar intrinsic threshold concentration (16.4 mmol/L).

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 4. Concluding remarks
Considering the recent advances in the biotechnological production of non-volatile
flavor compounds discussed in this review, this strategy may sustainably supply healthier food
additives, being aligned with the modern market demands. Industrial processes are becoming
viable by employing robust biocatalysts, optimized processes conditions and alternatives and/or
low-cost substrates (e.g. yeast biomass from brewing process for umami nucleotide synthesis;
by-products from the dairy industry for rare monosaccharide production), among others.
Moreover, the use of engineered microorganisms involved in the direct biosynthesis or for
specific modifications in flavor additives has also increased in the last years. Therefore, the
adoption of these processes in the food industry tends to be increased in the future.

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 5. CRediT author statement
Bruno Nicolau Paulino, Adones Sales, Lorena de Oliveira Felipe, Gustavo Molina,
Glaucia Maria Pastore, Juliano Lemos Bicas: Conceptualization, Writing - Original Draft,
Writing - Review & Editing. Glaucia Maria Pastore, Juliano Lemos Bicas: Funding
acquisition, Supervision.

6. Conflicts of interest statement

Nothing declared.

7. Acknowledgments

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LO Felipe is grateful to the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT - Monbukagakusho) for the Doctoral Grant (recipient nº 177307) provided

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by the Japanese Government. BN Paulino, G Molina, GM Pastore and JL Bicas acknowledge the
Coordination for the Improvement of Higher Education Personnel (CAPES) – Finance Code 001.
-p
G Molina thanks the Minas Gerais Research Funding Foundation (Fapemig) (grant number
APQ-01056-17). GM Pastore and JL Bicas acknowledge the National Council of Technological
and Scientific Development (CNPq) (grant number 400411/2016-4) and the São Paulo Research
re
Foundation (FAPESP) (grant numbers 2015/503331, 2016/21619-7, 2020/05536-0, and
2020/04941-8) for the financial support.
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 References and recommended Reading
Papers of particular interest, published within the period of review, have been
highlighted as:
• of special interest
•• of outstanding interest

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Tables and figures

Captions:

Figure 1. Three ways to biotechnologically produce umami-taste nucleotides (5’-GMP

and 5’-IMP): 1) production of nucleosides by fermentation, followed by phosphorylation to 5’-
nucleosides monophosphate (5’-NMP); 2) production of 5’-NMP through enzymatic hydrolysis
of microbial RNA by phosphodiesterase (PDE); 3) direct production of NMP by fermentation.
5’-AMP produced from different ways may be deaminated to 5’-IMP using 5’-adenylic acid
deaminase (AMP deaminase). Continuous arrow describes microbial processes, while dashed
arrow describes enzymatic strategies.
Figure 2. Enzymatic production of rare monosaccharides (structures highlights) from

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carbohydrates present in dairy by-products, sugarcane molasses, and inulin-rich products. LAI:
L-arabinose isomerase; T3E: D-tagatose-3-epimerase; DXI: D-xylose isomerase (glucose

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isomerase); A3E: D-allulose-3-epimerase; LRI: L-rhamnose isomerase; RPI: D-ribose-5-
phosphate isomerase; GalPI: D-galactose-6-phosphate isomerase; GluPI: glucose-6-phosphate
isomerase; T3E: D-tagatose 3-epimerase. -p
Figure 3. Enzymatic conversion of the main steviol glycosides (steviosides,
rebaudioside A and C). GD: glucose donor; GSase: glucansucrase; 1,3Gase: β-1,3-glucanase;
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UGTase: UDP-glucosyltransferase; SSase: sucrose synthase; DSase: dextransucrase.
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Table 1. Recent patents involving some of the non-volatile flavor compounds mentioned in this
text.
Target Patent details (title, year, patent
number)
Allulose Carvalho, 2019, US20190376101A1
Kokumi Method for preparing natural kokumi
flavor, 2017, AU2014293958B2
Mogroside Methods of producing mogrosides and
compositions comprising same and uses
Brief description
Allulose production by enzymatic
process using saccharide substrate
Two-step fermentation using fungi and
bacteria for kokumi production, and
more
Method of synthesizing a mogrol or
mogrol precursor product from a

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thereof, 2017, US20170283844A1 mogrol precursor substrate
Mogroside Methods and materials for enzymatic In vitro method for producing a

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synthesis of mogroside compounds, 2018, mogroside compound, and more
US9920349B2
Nucleic Method for producing nucleic acid Nucleic acid-based seasoning
acid seasoning, 2019, US20190223480A1
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production comprising a
ribonucleotide-containing material
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with a nucleosidase activity
Steviol Process for the enzymatic preparation of Stevioside cleaving by an enzyme
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steviol from stevioside, 2018, mixture from Aspergillus niger for

KR101814220B1 steviol preparation
Steviol Microbial production of steviol Production of steviol glycosylation
glycosides glycosides, 2019, US10463062B2 products (e.g. rebaudioside M or D)
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using engineered enzymes and

engineered host cells, and more
Stevioside Method for preparing glucose-based Glucose-based stevioside production
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stevioside in enzymatic variable by enzymatic temperature-changing

temperature and high throughput, 2019, process, and more
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WO2019136916A1
Tagatose Enzymatic synthesis of D-tagatose, 2019, Enzymatic conversion of sugars to
KR20190054187A tagatose

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 5’-NMP
Nucleosides
Phosphatase
Phosphate donnor

(G)
5’-GMP
PDE

1
(A) 5’-AMP
PDE AMP 5’-IMP
2 deaminase
RNA
(C)
3 PDE Intermediates
5’-CMP for synthesis of
pyrimidine-

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(U) based drugs in
PDE pharmaceutical
industry

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5’-UMP

FIGURE 1 -p
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 Dairy
by-products
LAI T3E
Lactose D-galactose D-tagatose D-sorbose
β-galactosidase

Low glycemic index

Sugarcane Low caloric value
molasses D-glucose Health and technological benefits

Invertase DXI
Sucrose

LRI
A3E RPI
Inulin-rich T3E GalPI

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products exo- GluPI
inulinase D-fructose D-allulose D-allose
Inulin

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HFS

FIGURE 2
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 Stevia rebaudiana extract

GSase

Stevioside
GD
1,3Gase
UGTase O-α-D-glucosyl-(1”6’) stevioside
+ SSase

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GSase
DSase

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Rebaudioside A

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O-α-D-glucosyl-(1”6’) rebaudioside A
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Rebaudioside C

FIGURE 3
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