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Probiotics in the Sourdough Bread Fermentation: Current Status

Article in Fermentation · January 2023

DOI: 10.3390/fermentation9020090

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 fermentation

Review
Probiotics in the Sourdough Bread Fermentation: Current Status
Ingrid Teixeira Akamine, Felipe R. P. Mansoldo and Alane Beatriz Vermelho *

Bioinovar, Institute of Microbiology Paulo de Góes, General Microbiology, Federal University of Rio de Janeiro—UFRJ,
Rio de Janeiro 21941-902, Brazil
* Correspondence: abvermelho@micro.ufrj.br

Abstract: Sourdough fermentation is an ancient technique to ferment cereal flour that improves bread
quality, bringing nutritional and health benefits. The fermented dough has a complex microbiome
composed mainly of lactic acid bacteria and yeasts. During fermentation, the production of metabo-
lites and chemical reactions occur, giving the product unique characteristics and a high sensory
quality. Mastery of fermentation allows adjustment of gluten levels, delaying starch digestibility,
and increasing the bio-accessibility of vitamins and minerals. This review focuses on the main steps
of sourdough fermentation, the microorganisms involved, and advances in bread production with
functional properties. The impact of probiotics on human health, the metabolites produced, and the
main microbial enzymes used in the bakery industry are also discussed.

Keywords: sourdough bread; microorganism; enzymes; probiotics; fermentation; microbiome

1. Introduction
Sourdough fermentation is a technique that can use several types of flour, such as
wheat, rye, or other cereals, and water. The oldest process of sourdough preparation
is spontaneous fermentation and acidification due to the local microbiota in a complex
interaction, mainly lactic acid bacteria (LAB) and yeasts [1–3]. It is a traditional fermentation
process used for various foods, especially baked goods, improving nutritional and health
Citation: Akamine, I.T.; Mansoldo, benefits [4,5]. The best-known example is bread; however, other applications of this type of
F.R.P.; Vermelho, A.B. Probiotics in fermentation include biscuits, panettone, pasta, and beverages [6–9]. Several varieties of
the Sourdough Bread Fermentation: this traditional sourdough bread recipe have appeared, bringing innovations to the primary
Current Status. Fermentation 2023, 9, process to attend to the bakery industry and the consumer, which appreciates nutritious,
90. https://doi.org/10.3390/ healthy, and tasty food [10]. Sourdough can be used by the bakery industry in several
fermentation9020090 applications through liquid, pasty, or dry formats with live microorganisms. Moreover,
Academic Editors: Farhad Garavand
there are “ready-to-use” sourdoughs with microorganisms inactivated to promote shelf-
and Eoin Byrne
stable products [11].
When sourdough is used, rather than Saccharomyces cerevisiae, a typical baker’s yeast,
Received: 21 November 2022 for the bread dough fermentation, superior properties in bread quality and technological
Revised: 9 January 2023 value are achieved [12].
Accepted: 18 January 2023
Probiotics are safe for human consumption and can produce metabolites that posi-
Published: 20 January 2023
tively influence gastrointestinal [13] and bone health diseases [14]. Beneficial effects in
eczema [15], allergies [16], respiratory tract infection [17], obesity [18], and cognitive and
mental health are also described [19]. Probiotics act as gut microbiota, stimulating a com-
Copyright: © 2023 by the authors.
plex communication with the body that is mediated by the metabolites generated [20].
Licensee MDPI, Basel, Switzerland.
Some LAB and yeasts present in sourdough fermentation are considered and presumed
This article is an open access article to be probiotics [21]. However, a complete analysis, such as a whole-genome analysis, is
distributed under the terms and proposed for better probiotic characterization [22–26].
conditions of the Creative Commons This study aims to review improvements in sourdough fermentation, its enzymes, metabo-
Attribution (CC BY) license (https:// lites, and technological advances to produce sourdough bread with desirable properties.
creativecommons.org/licenses/by/
4.0/).

Fermentation 2023, 9, 90. https://doi.org/10.3390/fermentation9020090 https://www.mdpi.com/journal/fermentation

Fermentation 2023, 9, 90 2 of 33

2. Sourdough Fermentation Types: Inoculum and Technology Processes

Sourdough fermentation can be classified according to the inoculum (type I, II, and
III) and the technology process (type 0, I, II, and III). According to inoculum, type I uses
the microorganisms present in the dough, which results in spontaneous fermentation and
utilizes back-slopping to propagate the sourdough microbiota. This process is common in
artisan bakeries and homes. Control parameters include temperature, number, and time of
back-slopping in artisan bakeries. Type II inoculums are frequently utilized in industrial
processes, where a starter culture is added to sourdough fermentation according to the
objectives and results desirable in the final process. Type III is a hybrid and combines type
II by using a starter culture with type I for propagating the sourdough with back-slopping.
Type III is common in artisan bakeries and industrial ones [27].
According to the production and process, sourdough fermentation can also be classi-
fied into four major types. The simple type 0 fermentation starts with a flour–water mixture
and is allowed for a limited time. LAB species that are naturally present in the flour,
grow faster are more prolific than yeast. Aplevicz et al. 2013 [28] compared the growth of
two Lactobacillus paracasei strains with two Saccharomyces cerevisiae strains. LAB had the
highest counts over time and microbial growth after 10 h of fermentation. The highest
value for LAB strains was 8.91 log CFU/g, and then 8.66 log CFU/g. One S. cerevisiae strain
reached 8.03 log CFU/g, and the other, 0.21 log CFU/g. At 6 h of fermentation, LABs were
8.66 log CFU/g and 8.33 log CFU/g. Yeasts showed 7.18 log CFU/g and 7.08 log CFU/g.
The growth kinetics of six strains of LAB: Fructilactobacillus sanfranciscensis, were
compared with five strains of yeast: Kazachstania humilis, from sourdough, by Altilia et al.
2021 [29]. Three predictive models were used to evaluate the behavior of co-cultivated
microorganisms: The zwitwering model based on Gompertz’s equation, Baranyil and
Roberts’ function, and Schiraldi’s function. The results showed that F. sanfranciscensis
strains significantly steer the growth kinetics affecting the ratio of bacterial to yeast cells,
where the yeast strains of K. humilis adapt to the bacterial strains. The authors discuss
the possibility of metabolic interactions for stabilizing the sourdough consortium through
communication, such as quorum sensing, to control population density, among other
functionalities. In another co-culture article with LAB and yeast, the yeast population
size decreased in the presence of LAB regardless of the strain, while the LAB’s population
size was rarely influenced by the presence of yeast [30]. In type 0 fermentation, bioactive
molecules and organic acids (lactic and acetic acids) are produced lowering the pH (pH~4),
but it is controversial if this type of fermentation is a true sourdough. The time limit is
insufficient to produce other characteristic products of sourdoughs and is more known as
sponge dough [27].
Type I is a traditional method for preparing sourdough and consists of spontaneous
fermentation in a flour–water mixture at an ambient temperature of less than 30 ◦ C for 24 h
or less and back-slopping frequently. This fermentation increases the quality of the final
baked good [27,31].
Type II is when a starter culture initiates the fermentation process and is used to
develop desirable characteristics for the baking industry. It is a sourdough in liquid form
with controlled parameters [31,32].
Type III can be dried or lyophilized. It is preferable for use in an industrial bakery
because it has a higher stability quality than fresh sourdough [31,33]. The fermentation
temperature used is above 30 ◦ C and in a single time stage between 24–72 h [34].
The fermentation process will change depending on the added materials, such as
the addition of naturally rich products in microorganisms: fruits, yogurt, or another
material [35]. It is observed that it is challenging to classify sourdough fermentation due
to some nuances in the process production [34]. Figure 1 summarizes the major types of
production and process of and their characteristics.
Fermentation 2023, 9, x FOR PEER REVIEW 3 of 35

Fermentation 2023, 9, 90 3 of 33
nuances in the process production [34]. Figure 1 summarizes the major types of
production and process of and their characteristics.

Figure 1. Sourdough production of the four types of processes. Type II and III can be scaled to an
Figure 1. Sourdough production of the four types of processes. Type II and III can be scaled to an
industrial level.
industrial level.
3. Sourdough
3. Sourdough Fermentation:
Fermentation: Major Major Pathways
Pathways
The sourdough
The sourdough fermentation
fermentation process
process generates
generates mainly
mainly acids,
acids, alcohols,
alcohols, aldehydes,
aldehydes,
esters, and ketones; it is the primary route of volatile organic compounds (VOCs) [36].
esters, and ketones; it is the primary route of volatile organic compounds (VOCs) [36].
Sourdough bread
Sourdough bread hashas aacomplex
complexprofile,
profile,strongly
strongly influenced
influenced bybythethe
compounds
compounds generated
gener-
during
ated fermentation
during fermentation by the bydiverse arrayarray
the diverse of microorganisms
of microorganisms present, mainly
present, yeasts
mainly and
yeasts
LAB.LAB.
and
Wheat flour
Wheat flour isis composed
composed of of starch
starch (70–75%),
(70–75%), waterwater (14%),
(14%), proteins (10–12%), non- non‐
starch polysaccharides
starch polysaccharides (2–3%), (2–3%), lipids (2%),
(2%), and soluble
soluble carbohydrates
carbohydrates such as maltose, maltose,
sucrose,and
sucrose, andglucose
glucose(1.55–1.85%)
(1.55–1.85%) TheThe starch
starch is degraded
is degraded intointo glucose
glucose and maltose
and maltose by flourby
flour amylase and glucoamylase by some sourdough LABs.
amylase and glucoamylase by some sourdough LABs. Maltose is degraded into glucose Maltose is degraded into
glucose
by by the maltose
the enzyme enzyme phosphorylase
maltose phosphorylase
from LAB from
andLAB and by alpha‐D‐glucosidase,
by alpha-D-glucosidase, a maltasea
maltase
from from Saccharomyces
Saccharomyces yeasts. In yeasts. In the the
the dough, dough, the available
available carbonis source
carbon source is thus
thus maltose,
maltose, by
followed followed
sucrose, byglucose,
sucrose,andglucose,
fructose, andwith
fructose, with some trisaccharides
some trisaccharides such as
such as maltotriose
maltotriose
and raffinoseand raffinose [37].
[37].
The
Theglucose
glucoseconcentration
concentration increases
increases during fermentation
during because
fermentation other complex
because carbo-
other complex
hydrates are metabolized
carbohydrates are metabolizedby LAB and
by LAByeasts. andTheyeasts.
disaccharide lactose can belactose
The disaccharide fermented
can bybe
microbial
fermentedenzymes liberating
by microbial enzymesglucose and galactose
liberating glucose after cleavage.after
and galactose Starting from glucose,
cleavage. Starting
homofermentative LAB produces lactic
from glucose, homofermentative LABacid throughlactic
produces glycolysis, while heterofermentative
acid through glycolysis, while
LAB generates, besides
heterofermentative LABlactic acid, CO
generates, 2 , acetic
besides acid,
lactic and/or
acid, CO2,ethanol [38]. and/or
acetic acid, Two major fer-
ethanol
mentation
[38]. Two routes are found: lactic
major fermentation fermentation
routes are found:(LF) andfermentation
lactic alcoholic fermentation (AF). In
(LF) and alcoholic
lactic fermentation,
fermentation (AF).the In pyruvate molecules formed
lactic fermentation, by glucose
the pyruvate oxidationformed
molecules from the byEmbden–
glucose
Meyerhof–Parnas or glycolytic pathway are reduced to lactic acid
oxidation from the Embden–Meyerhof–Parnas or glycolytic pathway are reduced to lactic (homolactic fermentation).
Streptococcus, Lactobacillus, and Enterococcus use this route (Figure 2A1). Another possibility
is the pyruvate originating from a mixture of lactate, ethanol and/or acetic acid, through
 Fermentation 2023, 9, x FOR PEER REVIEW 4 of 35

Fermentation 2023, 9, 90 acid (homolactic fermentation). Streptococcus, Lactobacillus, and Enterococcus use this4 route
of 33
(Figure 2A1). Another possibility is the pyruvate originating from a mixture of lactate,
ethanol and/or acetic acid, through the oxidation of the coenzymes NADH + and H+ by
theoxidation
the lactate dehydrogenase, andNADH
of the coenzymes CO2 from a glucose
+ and molecule
H+ by the lactate until ribulose 5 phosphate
dehydrogenase, and CO2
(heterolactic fermentation).
from a glucose molecule until ribulose 5 phosphate (heterolactic fermentation).

Figure 2. Major sourdough fermentation pathways. (A) Lactic fermentation. (B) Alcoholic fermentation.
(A1), homolactic
Figure 2. Majorfermentation; (A2), heterolactic
sourdough fermentation fermentation.
pathways. (B3),
(A) Lactic alcoholic fermentation
fermentation. fromfermen‐
(B) Alcoholic yeast.
tation.
The (A.1), homolactic
formation fermentation;
of ethanol and (A.2),
CO2 from the heterolactic
reduction fermentation.
of pyruvate (B.3),alcoholic
characterizes alcoholicfermentation.
fermentation
from yeast. The formation of ethanol and CO2 from the reduction of pyruvate characterizes alcoholic
This fermentation is used by genera such as Leuconostoc, Bifidobacterium, Weissella,
fermentation.
and some Lactobacillus. Some glycolytic enzymes are missing in these bacteria, and they
use the pentose-P
This pathway
fermentation to degrade
is used the such
by genera glucose (Figure 2A2).
as Leuconostoc, ClassificationWeissella,
Bifidobacterium, accord-
ing
andtosome
the type of lactic fermentation
Lactobacillus. defines
Some glycolytic threeare
enzymes major Lactobacillus
missing groups: group
in these bacteria, and theyI:
obligate
use the homofermentative
pentose‐P pathwaylactobacilli. Hexoses
to degrade the are(Figure
glucose exclusively
2A2).fermented to lactic
Classification acid
according
via Embden–Meyerhof–Parnas
to the type pathway.
of lactic fermentation defines threeThe bacteria
major have the
Lactobacillus enzyme
groups: groupfructose-1,6-
I: obligate
bisphosphate aldolase, but phosphoketolase is absent. Due to this, pentoses and
homofermentative lactobacilli. Hexoses are exclusively fermented to lactic acid via the gluconate
are not fermented. This group includes the species L. acidophilus, L. delbrueckii, and L.
salivarius; group II: facultative heterofermentative lactobacilli. Hexoses are fermented to
lactic acid almost exclusively via the Embden–Meyerhof–Parnas pathway. The bacteria
Fermentation 2023, 9, 90 5 of 33

possess both aldolase and phosphoketolase enzymes, and ferment not only hexoses but
also pentoses. In the presence of glucose, the enzymes of the phosphogluconate pathway
are inhibited. This group includes L. casei, L. paracasei, L. plantarum, and L. curvatus. Organic
acids, CO2 , alcohol, and H2 O2 could be produced; group III: composed by the obligate
heterofermentative Lactobacilli. They have the enzyme phosphoketolase but not aldolase
and ferment sugars in a heterofermentative mode. Hexoses are fermented via the phospho-
gluconate pathway, producing lactate, ethanol (acetic acid), and CO2 in equimolar amounts.
Pentoses enter this pathway and can also be fermented [39–42].
After the enzymatic hydrolysis of pentosans and other complex carbohydrates, pen-
toses such as d-xylose, ribose, and l-arabinose are liberated in rye and wheat flour. Het-
erofermentative strains can ferment pentoses through part of the 6-phosphogluconate
pathway [43]. Pentoses and hexoses are simultaneously rather than successively fermented
by sourdough Lactobacilli. Lactobacilli are important during the fermentation of sour-
doughs, and they can ferment pentose carbohydrates without producing CO2 because they
have a constitutive phosphoketolase. Facultatively heterofermentative lactic acid bacteria
produce the phosphoketolase enzyme in response to the presence of pentoses [42,44]. The
contribution of LAB to the flavor of sourdough bread is associated with the production of
lactic acid (fresh acidity) and acetic acid (sharp acidity). The conversion of amino acids such
as phenylalanine (sweet), isoleucine (acidic), glycine, serine, and alanine (vinegar/sour) to
aldehydes and ketones can form additional flavor compounds [36].
Alcoholic fermentation (AF) is characterized by the formation of ethanol and CO2 from
pyruvate reduction (Figure 2B). AF is a metabolic pathway of yeasts. In this fermentation,
yeast produces gas that promotes dough conditioning and increased volume and imparts
desirable aromas and flavors in baked goods. A synergy occurs between the dough and
the yeast, and in this context, the rate of CO2 production is determined by the activities
of the glycolytic yeast enzymes. The retention of CO2 produced by yeast is a function of
the wheat or cereal flour. It is essential to point out that glucose formed by LAB amylases
from amide is a significant source of this substrate for yeasts. Other compounds are volatile
organic compounds (VOCs), which arise from this interaction between yeasts and LABs.
Diverse aldehydes, alcohols, esters, ketones, lactones, sulfur compounds, furan derivatives,
and hydrocarbons are found, conferring flavor and other characteristics to sourdough
bread [32]. Some VOCs are due to yeast fermentation, particularly of S. cerevisiae in mixed
sourdough starters, including ethanol, 2,3-butanedione, 2-methyl-1-propanol, 3-methyl-1-
butanol, and phenyl ethyl alcohol [45]. Other negatively correlated compounds have been
associated with the fermentation of homofermentative and facultative heterofermentative
LAB, including 2,3-butanedione and acetaldehyde [46].

4. Probiotics and Postbiotics in Sourdough and the Impact on Human Health

Probiotics are live microorganisms that confer a health benefit when administered in
adequate amounts. In addition, new terms have been used to name microbial metabolites
from non-viable cells, including paraprobiotics, parapsychobiotics, ghost probiotics, matabi-
otics, tyndallized probiotics, bacterial lysates, and postbiotics. However, the last term is
being mightily used for the vital concept of promoting health. Postbiotics means non-living
microorganisms and is a preparation with inanimate microbial cells or their components
that promote host health. Metabolites and cellular structures from microorganisms from
sourdough are potential postbiotics [47,48].
Sourdough microbiota are involved in a complex interaction that engenders beneficial
compounds for human consumption. With the current molecular methods, a better charac-
terization of sourdough microbiota has been described [49,50]. Moreover, whole-genome
sequencing has permitted more accuracy in confirming the requirements for characterizing
probiotic microorganisms [25].
The probiotics and postbiotics of sourdough provide health benefits in addition to
the essential nutritional components of the bread itself. A fermented food passes through
a process with microorganisms and enzymes converting macronutrients into bioactive
Fermentation 2023, 9, 90 6 of 33

products. LAB is one major microorganism group with several probiotic strains applicable
to cereal fermentation. LAB are classified as Gram-positive bacteria which include low
guanine + cytosine (G + C) content as well as being acid-tolerant, non-motile, non-spore-
forming, and are rod- or cocci-shaped. The main function of LAB is to produce lactic
acid, that is, the acidification [40]. The most common genera found in sourdough were
Lactobacillus, Leuconostoc, Weissella, and Pediococcus. However, the genera Streptococcus,
Enterococcus, and Lactococcus are found, although less frequently [51,52]. The second
group of microorganisms are the yeast, and more than 20 species dominated by the genera
Saccharomyces and Candida are found. LABs and yeasts are naturally found in the microbiota
of the cereal flour used or can be added as a starter. Some native microorganisms found in
sourdough are listed in Table 1.
No strong relation between the microbiota and regional origin has been proven until
now [34,53]. However, in Brazil, Lactobacillus farciminis was the prevalent species rather than
Lactobacillus sanfranciscensis. Other bacteria could be present. At a temperature of 30 ± 1 ◦ C,
unusual bacterial groups such as Pseudomonas and Enterobacteriaceae can proliferate. At a
temperature of 21 ± 1 ◦ C, there may be a prevalence of LAB, which are safer for human
consumption [54].
Other factors that have been identified that could influence the microbiota. Insects
could be a reservoir of Lactobacillus sanfranciscensis [55], commonly found in type I sour-
dough. Lactobacillus delbrueckii and Lactobacillus reuteri groups from vertebrate–host interac-
tions are typical occurrences in type II sourdough [33]. In Italy, bakery LAB species such as
Weissela spp. and Streptococcus spp. are in the local environment [50]. In addition, the water
used in sourdough type I could influence its microbiota [56]. It has been demonstrated
that Lactobacillus, Streptococcus, Enterococcus, and Lactococcus are endophytic components
of wheat and, as such, a source of sourdough microbiota [52]. The sourdough microbiota
changes with decreasing pH, whereas at the beginning, there is abundant Proteobacteria
phylum in mature sourdoughs, which at the end is replaced by Firmicutes [54,57]. De An-
gelis et al. [58] demonstrated that the extraction rate of wheat flour from cultivars Triticum
aestivum and Triticum durum modified the microbiota community in sourdough type I. The
study showed that during fermentations, both controlled and uncontrolled pH conditions
up to 4.0 yield similar LAB and yeast species [59].
A meta-analysis with 583 sourdough-related literature articles in the period 1999–2017
was done by Kerrebroeck et al. 2017 [34] based on the study of species diversity (LAB and
yeasts) and process condition (type I and II). The authors concluded that the species number
of yeasts is lower than LABs and the microbial diversity of type I sourdoughs is lower
than type II. Through the principal component (PC) analysis, three clusters were detected.
The first group had a high relative abundance of Pichia kudriavzevii and/or Torulaspora
delbrueckii. In the second group, a high relative abundance of Wickerhamomyces anomalus
and/or Candida glabrata was established, and the third Saccharomyces cerevisiae and/or C.
humilis species were detected.
A systematic analysis from 1990 to 2020 using “sourdough” as a keyword showed the
following number of articles by geographic location: North America (13), South America
(6), Africa (31), Europe (175), Asia (54), China (20), and Oceania (1—New Zealand). In Latin
America and America, very few studies have been published. Lactobacillus plantarium and
Saccharomyces cerevisiae appeared in several sourdough microbiota characterizations [1]. It
also showed that some bacteria not belonging to the LAB group could significantly function
in sourdough and need to be explored more. Some species are listed in Table 1. On the
other hand, in a study that characterized 500 samples from sourdough microbiota, almost
all from the United States of America (429 samples) showed evidence of different bacterial
groups of the Rhodospirillales order despite the Lactobacillales being the dominant order.
Lactobacillus sanfranciscensis was the prevalent species. It was demonstrated that the acetic
acid bacteria group substantially impacted aroma and leavening properties in sourdough
fermentation [53].
Fermentation 2023, 9, 90 7 of 33

A sourdough starter is in essence, a culture of probiotic yeast and lactic bacteria to

be added to the fermentation process, besides the native microbiome of the cereals and
external contaminations.
Starters also can be used to propagate the sourdough as a live fermentation. In this
context, it is interesting to explore the technological and biological properties of the selected
starter associations of bacteria/yeast to improve the quality of fermentation and bread
properties. Acidification and proteolysis analysis (including free amino acids), and volatile
compound profiles are examples. In a recent review of Landis et al. [53], working with
500 sourdough starters from four continents, it was suggested that geographical location
has little influence on the microbial diversity of bacteria and yeast species. This result
contrasts sharply with widespread assumptions relative to the biogeographic distribution
of starters from sourdough [34]. In Brazil, for instance, Lactobacillus farciminis was the
prevalent species rather than Lactobacillus sanfranciscensis. Table 2 lists some starters used in
sourdough and produced compounds.

Table 1. Native microbiota found in sourdough.

Microbiota Reference
Acetobacter lovaniensis spp.
Acetobacter malorum ssp.
Acetobacter pasteurianus/papaya
Acetobacter tropicalis
Enterecoccus durans
Enterobacter hormaechei/cloacae
Enterococcus faecalis
Enterococcus faecium
Enterococcus gilvus
Enterococcus hirae
Gluconobacter frateurii spp.
Gluconobacter sphaericus spp.
Komagataeibacter cluster 4
Lactobacillus brevis
Lactobacillus coryniformis
Lactobacillus curvatus
Lactobacillus diolivorans
Lactobacillus farciminis
Lactobacillus fermentum
Bacteria [1,34,53,54,60,61]
Lactobacillus gallinarum
Lactobacillus kimchii
Lactobacillus otakiensis
Lactobacillus parabrevis
Lactobacillus paracasei
Lactobacillus paralimentarius
Lactobacillus plantarium
Lactobacillus sakei
Lactobacillus sanfranciscensis
Lactobacillus xiangfangensis
Lactococcus lactis
Leuconostoc
Leuconostoc citreum
Pediococcus
Pediococcus parvulus
Pediococcus pentosaceus
Psychrobacter
Streptococcus
Weissella
Fermentation 2023, 9, 90 8 of 33

Table 1. Cont.

Microbiota Reference
Cida glabrata
Cida humilis
Hanseniaspora uvarum
Kazachstania humilis (synonym Cida humilis)
Kazachstania servazzii
Kazachstania unispora
Kluyveromyces aestuarii
Kluyveromyces lactis
Yeasts Kluyveromyces marxianus [1,34,53,61–63]
Pichia fermentans (synonym Cida lambica)
Pichia kudriazevii
Saccharomyces cerevisiae
Saccharomyces uvarum
Saccharomycestales sp.
Torulaspora delbrueckii
Wickerhamomyces anomalus
Yarrowia keelungensis

Table 2. Starters used in sourdough bread production with identified compounds.

Inoculum Compound Property Reference

Lactobacillus sanfranciscensis and Significantly influences wheat
Glutaminase activity [64]
Lactobacillus reuteri bread flavor
Lactobacillus diolivorans and
Propionate Increase antifungal property on bread [65]
Lactobacillus buchneri
Improved volume, crumb softness,
Weissella cibaria MG1 Dextrans [66]
and shelf-life
Antifungal property improving the
Lactobacillus hammessi Monohydroxy fatty acid [67]
bread shelf-life
Lactobacillus paracasei RN5, Lactobacillus
plantarum X2, Lactobacillus brevis LBRZ7,
Lactobacillus fermentum LBRH10,
Antimicrobial Prevent bacterial and mold spoilage [68]
Lactobacillus buchneri LBRZ6, and
Propionibacterium frendenreichii ssp.
Shermanii NBIMCC 327
Lactobacillus curvatus 750(13), Pediococcus
acidilactici EKO26, Pediococcus pentosaceus
1850(3), Lactobacillus coryniformis pA,
Weissella cibaria EKO31, Pediococcus Reducing allergenic proteins and
pentosaceus EKO23, Lactobacillus Proteolytic activity improving the quality of [69]
plantarum KKp 593/p, Lactobacillus bakery products
helveticus 10, Lactobacillus plantarum
W37/54, Lactobacillus sakei 750(20), and
Lactobacillus rhamnosus Lr (23)
Lactobacillus curvatus MA2, Pediococcus
Phytase and Improve textural and sensory
pentosaceus OA2, and Pediococcus [70]
antioxidant activities features of bread
acidilactici O1A1
Weissella cibaria PON10030, Weissella
cibaria PON10032, Lactobacillus citreum
Volatile organic compounds Improve the taste of bread [71]
PON 10079, and Lactobacillus
citreum PON10080
Saccharomyces bayanus Aromatic compounds Improve the sensory profile of bread [72]
Potential improvement in nutritional
Propionibacterium freudenreichii B12 Vitamin [73]
and health value of bread
Torulaspora delbrueckii Aromatic compounds Improve the sensory profile of bread [72]
Lactobacillus reuteri LTH5448 and
γ-glutamyl dipeptides Influence in the salty taste [74]
Lactobacillus reuteri 100-23
Fermentation 2023, 9, 90 9 of 33

Table 2. Cont.

Inoculum Compound Property Reference

Isodipeptide bonds, ketones,
Lactobacillus sanfranciscensis, Candida Positive effects on bread rheological
medium-chain fat acids, [75]
milleri, and transglutaminase features, shelf-life, and aroma profile
and alcohols
Lactobacillus acidophilus ATCC20552 and
Antimicrobial Inhibit rope-forming B. subtilus [76]
Bifidobacterium lactis Bb 12
Reduction of fructans, consequently
Kluyveromyces marxianus and
Inulinase FODMAPs in dough prepared with [77]
Saccharomyces cerevisiae
whole wheat flour
Reduction of FODMAPs
Kluyveromyces marxianus Inulinase, phytase [62,78]
Highest porosity and lowest hardness
Saccharomyces cerevisiae and
Phytase activity Phytic acid decrease [79]
Pediococcus pentosaceus
Positive aroma profile of
Gluconobacter oxydans IMDO A845 Higher amount of lactic acid [80]
sourdough bread
Leuconostoc citreum FDR241 glycosyltransferase dextran concentration in sourdough [81]
Enterococcus mundtii QAUSD01 Proteolytic activity Gluten-degrading [60]
Wickerhamomyces anomalus QAUWA03 Proteolytic activity Gluten-degrading [60]
Phenolic acid esterase,
decarboxylases, reductase, Its influence in bread quality
Lactobacillus plantarum [33]
and wide range of needs study
glycosil hydrolases
Increased proteolysis in faba
Weissella cibaria VTT E-153485 Peptidase [82]
bean dough
Increased viscosity in faba
Weissella confusa VTT E-143403 Dextran [82]
bean dough
Pediococcus pentosaceus VTT E-153483 and Reduction of phytic acid in faba
Phytase [82]
Leuconostoc kimchi VTT E-153484 bean dough
Low-salt bread with desirable
Lactobacillus amylovorus DSM19280 6% Organic acid and
shelf-life, and high sensory quality [83]
and Weisella cibaria MG1 18% exopolysaccharide
(volume and crumb texture)
Lactobacillus brevis and Lactobacillus Improve texture and aromatic
Volatile compounds [84]
plantarum at 35 ◦ C properties of sourdough bread
Organic acid, saturated fatty Anti-aflatoxigenic capability and
Lactobacillus reuteri [85]
acid, hydroxy fatty acid antifungal activity
Lactobacillus plantarum 29DAN and L.
Polyphenol Antioxidant activity [86]
plantarum 98A
Lactobacillus plantarum NOS7315,
Lactobacillus rossiae NOS7307, Lactobacillus Improved bread
Synergistic fermentation [87]
brevis NOS7311, and Saccharomyces sensory characteristic
cerevisiae PS7314
Exopolysaccharides (EPS)
Bacillus licheniformis Immunomodulatory potential [88]
Organic acid, higher the Decrease spoilage, increase shelf-life,
Lactobacillus paracasei K5 complexity of volatile and improve sensory properties in [89]
compounds sourdough bread
Enterococcus mundtii QAUSD01 and Toxic gliadin degraded in the
Proteolytic activity [90]
Wickerhamomyces anomalus QAUWA03 sourdough fermentation
Do not interfere in the sensory quality
Lactobacillus plantarum CH1 Antifungal compounds [91]
of bread
Bread with lowly digestible starch
Streptococcus thermophilus Glucosyltransferase B [92]
and textural improvements
Pediococcus pentosaceus SP2 Organic acid content Reduce mold and rope spoilage [93]
High phenolic and
Enterococcus faecium and
antioxidant capacity, Improve bread quality [62]
Kluyveromyces aestuarii
respectively
Inhibition of growth of rope-forming
Lactobacillus reuteri TMW1.656 Reutericyclin [94]
bacilli in bread
Fermentation 2023, 9, 90 10 of 33

Table 2. Cont.

Inoculum Compound Property Reference

Decrease phytate and increase
Wickerhamomyces anomalus P4 Phytase mineral solubilization in [95]
sourdough bread
Levilactobacillus brevis TMW 1.211,
O2-substituted
Pediococcus claussenii TMW 2.340 Improving water binding capacity [96]
(1,3)-β-D-glucan
from breweries
Technological properties of dough
and bread, such as water absorption,
Weissella confusa/cibaria 3MI3 rheology, stability in cold storage,
Dextran [97]
from sourdough bread staling, and syneresis of starch
gels/avoided the resistant
starch formation
Pediococcus lolii B72 and Lactiplantibacillus
Volatiles compounds Improving sensorial acceptability [98]
plantarum E75 from mature sourdough

The microbial production of bioactive peptides, enzymes, organic acids, exopolysac-

charides (EPS), and vitamins are considered the primary metabolites responsible for antiox-
idant, antimicrobial, and probiotic activities [99]. Relative to fermentative processes, many
bioproducts are present in sourdough fermentation. Koistinen et al. [100] demonstrated
118 bioactive compounds in sourdough fermentation. Some of these health benefits and
others are summarized in Table 3.

Table 3. Microorganisms and the compounds they produce, and the health benefits.

Microorganisms Compound Benefit Reference

Probiotics Streptococcus thermophilus,
Lactobacillus plantarum, L. acidophilus, L.
Alfa-gliadin degradation, reduced
casei, L. delbrueckii spp. bulgaricus, Peptidase [101]
wheat allergenic
Bifidobacterium breve, B. longum, and
B. infantis
Gamma-aminobutyric
LAB from sourdough ACE-inhibitory activity [102]
acid (GABA)
Low-glycemic index in the white
LAB from sourdough Multifactors [103]
wheat bread
Antiadhesive properties, inhibition
Lactobacillus reuteri Exopolysaccharide [104]
enterotoxigenic Escherichia coli
Lactobacillus brevis with S. cerevisiae var. Higher total phenolic and a
Chevalieri; L. Fermentum; L. Fermentum lower molar ratio of lactic to Reduce glycemic index [105]
with phytase acetic acid
L. curvatus SAL33 and L. Brevis AM7 Peptide lunasin Cancer preventive [106]
Bifidobacterium strains Phytase Increase iron bioavailability in bread [107]
Weissella ciabaria MG1; L. reuteri VIP, L. Improved nutritional quality of
Oligosaccharides [108]
reuteri Y2 sorghum bread
Decrease phytate levels, improve
L. brevis Phytase [109]
mineral bio-accessibility
L. Sakei KTU05-6 Organic acids, bacteriocins Bio-preservative [110]
Dextransucrase Alfa-glucans/ oligosaccharides or
Weissella confusa LBAE C39-2 [111]
(glycoside hydrolase) glycoconjugates
Vitamin B12, folate,
L. rossiae DSM 15814 from sourdough Nutritional improvement [112]
and riboflavin
Lactobacillus amylovorus DSM 19280 and
Glutamate accumulation NaCl reduction in bread [83]
Weisella cibaria MG1
Nutritional improvement protects
Essential and non-essential
Traditional sourdough LAB against oxidative stress and
amino acids, flavonoids, [113]
starter culture degenerative disease through
antioxidant peptides
phenolic compounds
Fermentation 2023, 9, 90 11 of 33

Table 3. Cont.

Microorganisms Compound Benefit Reference

Fructose metabolized/
LAB from traditional
antifungal and Decrease FODMAPS/ control molds [114]
Austrian sourdoughs
anti-bacillus properties
In mice, decreased: the profile,
Managing gut microbiota, decreasing
insulin resistance,
Lactobacillus plantarum ZJUFT17 from pathogenic and pro-inflammatory
lipopolysaccharide, cytokines [115]
Chinese sourdough microbes, and stimulating
interleukin (IL)-1β, tumor
anti-obesity ones
necrosis factor (TNF)-α
Levilactobacillus brevis TMW 1.211,
O2-substituted Prebiotic effect in bread, improving
Pediococcus claussenii TMW 2.340 [96]
(1,3)-β-D-glucan water binding capacity
from breweries
Lactobacillus plantarum ZJUFB2 from Probiotic effect on gut Prevent insulin resistance and
[116]
Chinese sourdough microbiota modulate gut microbiota.
Dietary fiber, short acid fat
Levilactobacillus brevis TMW 1.2112, Healthy environment in the
chain SCFA, butyrate, [117]
Pediococcus claussenii TMW 2.340 colon, chemopreventive
propionate,β-glucan
Pediococcus pentosaceus F01,
Bread from surplus bread with high
Levilactobacillus brevis MRS4, Γ-aminobutyric acid (GABA) [118]
nutritional value
Lactiplantibacillus plantarum H64, and C48
Weissella cibaria PDER21 α-D-glucan Antioxidant properties [119]

One important point is that most probiotics die when the bread is baked at high
temperatures. However, most health benefits continue, mostly, not with probiotics at
intestinal epithelial cell colonization but in sourdough dough. It is essential to note that
active biomolecules could be present in the baked bread. Different conditions, varying
temperatures, baking time, and fermentation parameters could influence the heat resistance
of some bioproducts, and they could be present as nutrients and fibers such as β-glucan
and resistant starch [120]. Increasingly, the literature discusses this important point. If the
baking time is shortened by increasing the baking temperature or reducing the bread size,
higher residual viability of LAB species may be obtained after baking [121]. Studies with L.
plantarum at three different baking temperatures showed a reduction from 8.8 log CFU/g
to 4–5 log CFU/G [122]. Concerning yeast, the count decreased from an initial value of
9 log CFU/g to 4 log CFU/g with baking at 200 ◦ C for 13 min [121].
During the baking process, cell lysis of microorganisms delivers cellular debris that
may also have beneficial properties functioning as postbiotics. Examples of postbiotic
compounds that are present in sourdough are short-chain fatty acids (SCFAs), secreted
proteins/peptides, bacteriocins, secreted biosurfactants, amino acids, flavonoids, EPS,
vitamins, organic acids, and other molecules discussed in this review. Remains of microbial
cell structures such as peptidoglycan from bacterial cell walls, pili, fimbriae, flagella, cell-
surface-associated proteins, cell-wall-bound biosurfactants, and cell supernatants are also
postbiotic components that have potential health benefits in the host [47,123].
There is both a growing demand from consumers for additive-free, safe, and nutritious
foods and for bread with a longer shelf-life and less staling due to microbial spoilage, and
sourdough offers some advantages [124].
Several microorganisms, mainly LAB, have antimicrobial properties, which favors their
use as probiotics and as a bioprotective culture in fermented products. Koistinen et al. [125]
suggest that metabolites with this effect produced by LAB may synergistically modulate
the local microbial ecology, such as in the gut. Some strains of LAB can produce a variety
of other antimicrobial compounds, such as organic acids (lactic, acetic, formic, propionic,
and butyric acids). Organic acids have been described with antifungal actions against
Aspergillus, Fusarium, Monilia, and Penicillium [126]. Ethanol, fatty acids, enzymes, ace-
toin, hydrogen peroxide, diacetyl, antifungal compounds (propionate, phenyl-lactate,
hydroxyphenyl-lactate, cyclic dipeptides, and 3-hydroxy fatty acids), bacteriocins (nisin,
Fermentation 2023, 9, 90 12 of 33

reuterin, reutericyclin, pediocin, lacticin, enterocin, and others), and bacteriocin-like in-
hibitory substances (BLIS) are also described in the literature [127].
Bacteriocins are a class of antimicrobial proteins or peptides considered safe for the
body and do not induce resistance. This antimicrobial can be applied against harmful
food pathogens and used as a food preservative (preserving foods such as dairy prod-
ucts, canned foods, and meats) and a natural antiseptic with bactericidal or bacteriostatic
effects. Lactococcus lactis is the microorganism producing nisin, the first fully identified
bacteriocin [128]. Among lactic acid bacteria, one of the most versatile and adaptable is
Lactiplantibacillus plantarum, with a long history of use in foods and claims of benefits as
a probiotic. This species has shown promising results in protecting post-harvest fruits
and controlling mold in foods such as beverages and bread [129]. Lactiplantibacillus plan-
tarum strain ITM21B has been used to prolong bread shelf-life due to the production of
antimicrobial substances, such as lactic, acetic, phenyl lactic (PLA), and hydroxy-phenyl
lactic (OH-PLA) acids [130]. Varsha et al. 2014 [131] demonstrated that the antifungal
compounds produced during fermentation resist sterilization temperatures and remain
active adding value for the baking industry.
The antimicrobial effect reduces the salt content in bread. Helping reduce sodium
consumption to the recommended level of 5–6 g/day will benefit health, particularly
regarding blood pressure and cardiovascular disease. Taking into consideration that bread
and cereals represent the major sources of salt in the human diet, microbial bioprocessing
has been proposed to reduce salt content while keeping the flavor [132].
In addition, the hydrolysis of flour proteins leads to the production of amino acids,
organic acids, and other metabolites, improving the nutritional and functional values of
sourdough bread [133].
Dietary sourdough bread increases short-chain fatty acid, isovaleric and 2-methyl bu-
tyric acids, and γ-aminobutyric acid (GABA) content, among other bioactive molecules [134].
The γ-aminobutyric acid GABA is a non-protein amino acid synthesized from L-glutamate
by the glutamate decarboxylase [135]. It is a mammalian neurotransmitter involved in
critical regulatory functions, hypotensive properties, anti-depressive effects, diuretic, and
antioxidant effects [136]. It is present in many medicines and supplements [137].
Several strains of LAB can carry out activities that promote human health, such as
modulating the immune response, preventing cancer, reducing chronic intestinal inflam-
mation, and cholesterol levels. Other functions include improving the intestinal barrier,
inhibiting pathogenic organisms, and beneficial interactions with the endogenous intestinal
microbiota [138]. These bacteria are present in sourdough and liberate bioproducts in bread
dough, such as vitamin B12, folate, and riboflavin [112].
Although probiotics from sourdough are not present in the intestine environment, they
provide gastrointestinal benefits, including the pre-digestion of non-nutritional molecules.
Sourdough bacteria and yeast decrease the phytic acid present in wheat. The phytate
(myo-inositol hexaphosphate) content of whole wheat products is a concern because it
inhibits the absorption of minerals, has a negative effect on the nutritional properties, and
causes gastrointestinal disorders, digestive discomfort, and flatulence. On the other hand,
removing wheat bran and germ reduces the nutritional value of bread. The acidification
during sourdough fermentation stimulates endogenous grain phytase, but LAB and yeast
phytase activity significantly reduces the phytate content of the bread [124]. Another
advantage is the reduction of fermentable oligo-, di-, and monosaccharides and polyols
such as sorbitol and mannitol (FODMAPS) and amylase–trypsin inhibitors (ATIs) from
wheat. Both are related to trigger intestinal symptoms in irritable bowel syndrome and
non-celiac wheat sensitivity. A study by Boakye et al. 2022 [139], described that the
longer sourdough fermentation time (12 h) caused up to 69%, 69%, and 41% reductions
in fructans, raffinose, and ATIs, respectively. Low FODMAPs may be valuable for people
with gastrointestinal disorders.
The reduction of gluten content is important for people with gluten-related disor-
ders. The long fermentation process of sourdough also helps break down gluten proteins
Fermentation 2023, 9, 90 13 of 33

in wheat. Moreover, certain bacteria produce peptidases. Some Lactobacillus strains, in-
cluding L. sanfranciscensis, L.rossiae, L. plantarum, L. brevis, L. pentosus, L. alimentarius, L.
fermentum, L. paracasei, L. casei subsp. casei, and P. pentosaceus degrade gluten. Other gluten-
degrading bacteria have been isolated form sourdough, such as Bacillus spp. [140], and
Enterococcus mundtii and the yeast Wickerhamomyces anomaly from locally fermented sour-
doughs (Khamir) [60]. Gluten has baking properties, but its protein components, gliadin,
and glutenins, could trigger undesirable immune responses, causing inflammation and
damage [141].
Antioxidant and anti-inflammatory activities of peptides from cooked sourdough
breads were described [142], where longer fermentation (72–96 h) increases the production
of aromatic compounds with antioxidant activity [143].
LAB and yeast strains were isolated and molecularly identified from traditional Ira-
nian sourdough. Based on total phenol production and antioxidant capacity assessments,
all the identified traditional sourdough microorganisms significantly produced pheno-
lic compounds. They showed significant antioxidant capacity improving bread’s health
benefits and quality [62].
EPS such as β-glucan, dextran, and inulin, are metabolites from LAB identified in
sourdough fermentation. The β-glucan, for instance, is a prebiotic homopolysaccharide
formed by glucose, presenting substantial health benefits, such as stabilized cholesterol
levels, anti-inflammatory effects, and benefits for probiotic microorganisms [96]. The
conclusions reached by Schlörmann et al. [117] provide significant insights into the gen-
eral chemopreventive and prebiotic effects of LAB-generated sourdoughs and bread. In
addition, exopolysaccharides are produced by LAB in sourdough, favoring great water
retention. β-glucans also contribute to the viscoelastic properties of dough [96]. In the case
of thermal treatment at 60 ◦ C and 80 ◦ C for up to 60 min, the molecular mass of the EPS
was not significantly affected [144].
The glycemic index (GI) is the speed at which sugar enters our body’s bloodstream
and this parameter is affected by probiotics from sourdough. It is reported that sourdough
bread has a lower GI than yeast bread. Bread with carbohydrates and starches, which are
rapidly assimilated, increase the GI. An explanation of the decrease in the glycemic index
is the higher level of lactic acid produced under these fermentation conditions. Lactic acid
effectively diminishes postprandial glycemic and insulinemic responses, whereas acetic
acid brings out a delayed gastric emptying rate. Moreover, the starch availability is reduced
under baking heat, thus allowing the breakdown of sucrose to form EPS such as glucans
which contribute to the rise in dietary fiber content [145,146]. Mutlu et al. [147] discussed
that LAB could contribute to lowering the glycemic index by limiting the digestion rate of
starch and converting glucose from digested starch into several bioproducts such as organic
acids, fatty acids, sugar alcohols, bioactive peptides, and indigestible exopolysaccharides.
Similar results were found by Demirkesen-Bicak et al. [148], where the authors demon-
strated results supporting the consumption of wheat and whole wheat bread produced by
type 2 fermentation due to higher resistant starch and slowly digestible starch, and lower
rapidly digestible starch and glycemic index values showing beneficial effects. However,
some controversy exists in the literature. A systematic review selected 18 studies published
up to June 2021 in the EMBASE, MEDLINE, Scopus, and Web of Science databases. The
authors concluded that the consumption of sourdough bread has a lower influence on
blood glucose compared to that of industrial bread or glucose [149].
In addition to the health effects, sourdough brings flavor, aroma, and better texture
of bread due to LAB enzymatic hydrolysis processes. Flavor from volatile compounds in
sourdough are due to peptidases conversion of glutamine released from cereal proteins
to glutamate, and conversion of arginine to ornithine by LAB during baking. Microbial
and enzymatic reactions, such as lipid oxidation by cereal enzymes, improve flavor and
texture. LAB and yeast in sourdough produce aroma precursors such as free amino acids,
which lead to the generation of aldehydes or corresponding alcohols [126,150]. Table 3
summarizes the functionality of several microorganisms studied in this fermentation. It
Fermentation 2023, 9, 90 14 of 33

was demonstrated that sourdough bread fermentation conditions considerably influence

bioactive compound viability [100]. Furthermore, many studies have described unique
characteristics and molecules formed from the selective use of inoculum as a starter in
bread making.
Using a starter culture and varying the temperature and fermentation time changes
the flavor and texture in sourdough bread [84]. Moreover, selected LAB and manipulated
temperature of sourdough fermentation could increase the β-glucan yield [96]. Methods
for maintaining viable starter cells for use in the baking industry have been studied widely,
such as immobilization techniques [93]. Studies showed that using the spray drying
technique to preserve the viability of lactic acid bacteria and yeasts in the dry mass was
much more effective than other drying techniques [5]. However, this area needs further
studies since they can vary with the cell type or microbial consortia [151]. Studies have
been realized to develop better kinetic control in the spray drying process of LAB, which
is fundamental for their growth and viability [152]. Understanding the stress response
mechanisms used for LAB can help industries develop better processes to produce LAB at
a large scale [153]. Encapsulation of LAB has been demonstrated as a method to improve
the viability of LAB during drying processes [154]. For instance, microencapsulation
of cells by spray drying with cellulose acetate phthalate was more efficient than with
maltodextrin [155]. Using probiotics in bread is a challenge due to the higher temperature
of baking. However, a recent study showed that the strain of Lactobacillus plantarum P8
survived baking and increased the number of its cells during storage [122].

5. Enzymes in Sourdough
Enzymes are responsible for several biochemical events in sourdough fermentation.
Although some enzymes are present in the cereal, most of them are produced by microor-
ganisms. From this perspective, the selection of native yeasts in sourdough for use in
the baking industry has increased, and they are better adapted to improve their stabil-
ity [156,157]. In the same way, bacteria, especially lactic acid bacteria, are widely selected
for use in industrial bread making [71].
Many valuable properties observed in the select starters of bacteria and yeasts are
related to their enzyme activities or involvement in the enzyme activities in the sourdough
system. The wheat grain has enzymes that can be activated or inhibited by the low pH
of the sourdough environment, and the microbiota present in sourdough contributes to
its enzymes in the sourdough metabolic process [3]. The mechanisms of LAB and yeast
enzymatic action during sourdough fermentation are becoming well-known [54]. The hy-
drolases demonstrate an essential role in sourdough bread. However, other enzyme classes,
including transferases and oxidoreductase, are involved in the properties of sourdough
fermentation [81,158]. Figure 3 shows the sourdough fermentation process and the major
enzymes involved.

5.1. Transferases
The more expressive metabolic activities of microbial communities during fermen-
tation are acidification, leavening, and flavor formation, all related to the metabolism of
carbohydrates [159]. Transferases are enzymes that catalyze the transfer of specific func-
tional groups from one molecule to another. Glycosyltransferases (EC 2.4) are involved in
the biosynthesis of exopolysaccharides in sourdough. It is an enzymatic group produced
by the LAB from sourdough microbiota, such as Lactobacillus, Leuconostoc spp., Leuconostoc
citreum, and Weissella species. Glucansucrases are glycosyltransferases from LAB that
split sucrose. The resulting glucose builds EPS-type α-glucan polymers such as dextran,
mutan, alternan, and reuteran. Fructansucrase, found in Gram-negative and Gram-positive
bacteria, is another glycosyltransferase that synthesizes levan (levansucrase) or inulin
(inulosucrase). The inulosucrase is observed only in LAB [160,161].
 the sourdough environment, and the microbiota present in sourdough contributes to its
enzymes in the sourdough metabolic process [3]. The mechanisms of LAB and yeast en‐
zymatic action during sourdough fermentation are becoming well‐known [54]. The hy‐
drolases demonstrate an essential role in sourdough bread. However, other enzyme clas‐
Fermentation 2023, 9, 90 ses, including transferases and oxidoreductase, are involved in the properties of sour‐
15 of 33
dough fermentation [81,158]. Figure 3 shows the sourdough fermentation process and the
major enzymes involved.

Figure3.3. General
Figure General scheme
schemeofofbread sourdough
bread fermentation
sourdough and the
fermentation andmajor enzymes
the major involved.
enzymes EPS:
involved.
Exopolysaccharides.
EPS: Exopolysaccharides.
5.1.EPS
Transferases
formed during sourdough fermentation influences the dough’s viscoelastic prop-
The improves
erties and more expressive metabolic
the texture activities In
and shelf-life. of this
microbial
context,communities duringhydrocolloids
EPS can replace fermenta‐
tionasare
used acidification,
bread improversleavening, and flavordemands
and meet consumer formation,for all related
reduced usetoofthe metabolism
food of
additives [162].
carbohydrates [159]. Transferases are
Adding a glucosyltransferase enzymes
B, made that catalyze the
by Streptococcus transfer ofwith
thermophilus specific
the func‐
activity
oftional groups from one molecule
4,6-α-glucanotransferase in theto another. Glycosyltransferases
preparation of wheat flour dough, (EC 2.4) are involved
results in
in bread with
the biosynthesis of exopolysaccharides in sourdough. It is an enzymatic group
better health benefits. This enzyme increased the number of short branches and percentage produced
ofby the
(α1 →LAB from sourdough
6) linkages in starch,microbiota,
resulting insuch
slowasstarch
Lactobacillus, Leuconostoc
digestibility and lowspp.,retrogradation
Leuconostoc
properties. It also reduced hardness, gumminess, and chewiness, indicating improvements
in texture [92].
Transglutaminase (TGase, EC 2.3.2.13) catalyzes the cross-linking of proteins between
the ε-amino group of a lysine residue and the γ-carboxamide (acyl) group of a glutamine
residue. Which results in an intra- or intermolecular bridge highly resistant to proteolysis,
which leads to an increase in the structure and texture of protein substrates [163,164]. It
is an important enzyme for the food industry, where studies report that its application
improves dough handling properties, increases bread stability, and volume. The protein
polymers resulting from the action of TGase can modify the rheological properties of gluten,
transforming a weak gluten into a strong one [75].
Fermentation 2023, 9, 90 16 of 33

5.2. Oxidoreductases
Oxidative enzymes are applied to treat wheat flour to restore the gluten network.
Those enzymes can be divided into exogenous oxidative enzymes (laccase, glucose oxi-
dase, hexose oxidase) and endogenous oxidative enzymes (tyrosinase, peroxidase, catalase,
sulfhydryl oxidase, lipoxygenase, protein disulfide isomerase, NAD(P)H-dependent dehy-
drogenase, thioredoxin reductase, and glutathione reductase) [165].
Oxidoreductase (EC 1) catalyzes reactions of electron transfer. Glutathione reductase
(EC 1.8.1.7) is the most known enzyme in this group related to sourdough bread properties.
It acts on a sulfur group of donors, with NAD+ or NADP+ as the acceptor [166].
Gluten protein presents intra- and intermolecular chains of disulfide bonds between
amino acids [167]. The glutathione reductase activity, identified in the microbiota of natural
fermentation, interferes with these thiol bonds [158]. The glutathione reductase deficient
mutant strain (Lactobacillus sanfranciscensis) produced bread with a softer texture and
higher specific volume than bread made with traditional biological yeast and non-mutant
Lactobacillus sanfranciscensis activity [167].
Glucose oxidase (EC 1.1.3.4) and pyranose oxidase (EC 1.1.3.10) are oxidoreductases
produced by several fungi, mostly from Aspergillus niger in the case of glucose oxidase.
One molecule of glucose oxidase catalyzes the conversion of β-D- glucose into hydrogen
peroxide (H2 O2 ) and gluconic acid [168]. According to Xu et al. [169] the resulting H2 O2 can
further oxidize the free sulfhydryl units in the gluten protein and promote the formation of
disulfide bonds in the gluten network, thereby strengthening the gluten network. In addi-
tion, glucose oxidase has antimicrobial activity against fungi and foodborne bacteria [170].
These effects enhance the rheological properties, volume and height, and stability of the
bread [169,171,172].
Laccases (benzenediol: oxidoreductase oxygen, EC 1.10.3.2) are multi-copper enzymes
and they belong to a group of so-called polyphenol oxidases. They catalyze the oxidation
of a wide range of substrates while simultaneously reducing molecular oxygen to wa-
ter [173–175]. Laccase activity has been found to enhance the viscoelastic dough qualities
and in recent years it has been used as a dough and bread improver, primarily utilized in
commercial preparations [175,176]. Pentosans are polymerized by laccase and favor the
aggregation of glutenins, which lowers the protein content of glutenin macropolymers.
While not all researchers agree, some authors report that laccases are also important in
baking because they can cross-link the arabinoxylans found in whole flour, improving
the gluten index and inducing hetero-crosslinking between arabinoxylans and the gluten
matrix [165].

5.3. Lyases
Lyases (EC 4) are a group of enzymes that cleave carbon bonds with oxygen, carbon,
nitrogen, and others and eventually form double bonds or a new ring [177].
The glutamate decarboxylase (EC 4.1.1.15) is commonly found in the sourdough envi-
ronment and acts in L-glutamate’s decarboxylation to form γ-aminobutyric acid (GABA), a
non-protein amino acid with various health benefits.
Amino acid catabolism by LAB is related to sourdough properties such as taste and
nutrition. However, these reactions can accumulate toxic biogenic amines, especially
tyramine, cadaverine, and putrescine. It is well-known that a high GABA content in foods is
highly desirable as it is a non-protein amino acid with many health benefits, and some LAB,
such as Levilactobacillus brevis, are excellent producers [178,179]. LAB and yeast are the most
important GABA producers because they are commercially valuable as starters in fermented
foods. GABA has several physiological functions, such as inhibiting neurotransmission
in the central nervous system. Other related functions include relaxation, sleeplessness,
enhanced immunity under stress, increasing the concentration of growth hormone in
plasma, preventing diabetes, inhibiting the invasion and metastasis of various types of
cancer, anti-inflammatory effects, blood pressure regulation, and antioxidant effects. It is
also a hormonal regulator (for review, see Diana et al., 2014 [178] and Polak et al. 2021 [179].
Fermentation 2023, 9, 90 17 of 33

Indeed, fermented foods represent an excellent source of dietary GABA. In a recent

study, Lactobacillus brevis A7 and Lactobacillus farciminis A11, demonstrated high GABA-
producing capability when used to prepare bread containing 20% amaranth flour (amaranth
has a high concentration of essential amino acids) [180].
Calabrese et al. [181] analyzed spontaneous sourdough metagenomes and transcrip-
tomes to identify microorganisms and their role in the fermentation process. Based on
meta-omics data collected and their identified microbiota, a reconstructed synthetic micro-
bial community was established. The results revealed a complex network with dominant,
subdominant, and satellite communities engaged in different functional pathways. Aspar-
tate ammonia-lyase (EC 4.3.1.1), the enzyme that converts aspartate into fumarate, was
uniquely transcribed by Lactiplantibacillus plantarum. This approach was effective and
significant for studying these complex metabolic frameworks bringing new knowledge
into this complex fermentation.

5.4. Hydrolases
Hydrolases (EC 3) are enzymes that use water to break various chemical bond types.
Amylase is the most popular hydrolase in baked good processes. However, peptidases
have received more attention recently due to investigations into their role with gluten.
Other hydrolases have essential roles in the baking processes, such as cellulase, xylanase,
and pentosanase [182]. Table 4 presents the main enzymes used in the bakery and the
microorganisms that produce them, according to Dahiya et al. [183].

Table 4. List of microorganisms that produce the main enzymes used in the bakery.

Enzyme Microorganisms Reference

Xylanase Sporotrichum thermophile BJAMDU5 [184]
Pichia pastoris [185]
Bacillus subtilis [186]
Myceliophthora thermophila BJTLRMDU3 [187]
Phytase Lactobacillus casei [188]
Enterobacter sp. ACSS [189]
Sporotrichum thermophile [190]
Aspergillus niger NCIM 563 [191]
Amylase Rhizopus oryzae [192]
Bacillus subtilis US586 [193]
Bacillus subtilis M13 [194]
Streptomyces badiun DB-1 [195]
Glucose Oxidase Aspergillus niger [196]
Penicillium notatum [197]
Aspergillus niger [198]
Aspergillus niger [199]
Peptidase Rhizopus oryzae [192]
Bacillus subtilis PF1 [200]
Bacillus subtilis [201]
Bacillus pumilus SG2 [202]
Lipase Aspergillus niger MTCC 872 [203]
Pseudomonas fluorescens (NRLL B-2641) [204]
Bacillus subtilis I-4 [205]
Bacillus sp. MPTK 912 [206]
Cellulase Sporotrichum thermophile BJAMDU5 [203]
Streptomyces strain C188 [207]
Cellulomonas uda [208]
Trichoderma reesei NCIM 1186 [209]

5.4.1. Amylase, Inulinase, and Their Impact on Bread Structure and Properties
Several types of amylases act on starch, mainly liberating glucose for the fermentation
and improvement of the bread’s color, flavor, and shelf-life [210]. The main groups are
endoamylases, exoamylases, and debranching amylases. Among the endoamylases, α-
Fermentation 2023, 9, 90 18 of 33

amylase (EC 3.2.1.1) is the main one, acting on α-1,4 bonds in the amylose and amylopectin
chains. The group of exoamylases includes glucoamylase (EC 3.2.1.3), α-glucosidase (EC
3.2.1.20), and β-amylase (EC 3.2.1.2) promoting the hydrolysis of terminal glycosidic units.
Debranching amylases act on α-1,6 branch bonds, and pullulanase (EC 3.2.1.41) is the
leading representative [211].
Starch/amide is present in around 70–75% of wheat flour. It comprises a linear
chain of α-1,4 glycopyranoside (amylose) and a linear chain of α-1,4 glycopyranoside
with α-1,6 branch points (amylopectin). During the baking process, the bread undergoes
several changes in starch retrogradation relative to the crystalline structure of amylose
and amylopectin. These changes occur not only in baking but also during the cooling and
storage processes [212]. Starch retrogradation is a process in which disaggregated amylose
and amylopectin chains in a gelatinized starch paste reassociate to form a more ordered
structure [213]. It is a pivotal issue in bread aging and is the cause of bread hardening.
The amylases produced by LAB strains reduce the aging process during bread stor-
age [214]. Amylase-producing microorganisms in the sourdough environment are es-
sential in converting starch into fermentable carbohydrates such as maltodextrins, mal-
tose, sucrose, and glucose. LAB are sources of maltose phosphorylase, which generate
D-glucose, β-D-glucose, 1-phosphate, and 1,6-α-glucosidase, which hydrolyzes α-(1–6)-
glucooligosaccharides [3]. In addition, wheat has α-amylase, β-amylase, and glucoamylase
activity, but at pH < 4.5, only glucoamylase maintains its activity. Microorganisms in the
sourdough microbiota produce inulinase (β-2, 1-D-fructan-fructan-hydrolase—EC 3.2.1.7)—
a glycosylase that hydrolyzes the β-2.1 bonds of fructose of the fructose polymer, inulin.
Inulinase can reduce the number of oligosaccharides, disaccharides, monosaccharides, and
fermentable polyols (FODMAPs) in bread [78]. FODMAPs are associated with irritable
bowel syndrome. However, it is not appropriate to eliminate these polyols but only to
reduce them to an adequate level, as these compounds are considered prebiotics to maintain
the healthy intestinal microbiota [215]. Kluyveromyces marxianus yeast strains have reduced
the concentration of FODMAPs in whole wheat bread [78].

5.4.2. Cellulase, Phytase, and Xylanase for Mineral Bio-Accessibility Improvement in Bread
Cellulases are classified into endoglucanases (act in the middle of cellulose) and
exoglucanases (cleave the extremities of the polymer). The endoglucanase is the endo-β-
1,4-glucanase (EC 3.2.1.4), cleaving intramolecular bonds of β-1,4-glycosidic. The exoglu-
canases are the β-glucosidase (EC 3.2.1.21) and exo-β-1,4-glucan cellobiohydrolase (EC
3.2.1.91), which cleave ends of the cellulose and glycosidic terminals (liberating Cellobiose),
respectively [216].
Endocellulase acts on cellulose and β-glucan substrates, removing insoluble arabi-
noxylans, contributing to gluten network formation [217]. Subsequently, there is a decrease
in the hardness of the bread and, consequently, an improvement in the sensory evaluations.
Cellulase action may also provide an anti-staling effect, which may be related to alterations
in water distribution between the starch–protein matrix [182]. In the study carried out by
Li et al. (2014) [218], the authors added β-glucanase to a mixture of barley flour (30%) and
wheat flour (70%) with approximately 1.5% glucan as the starting material. As a result,
they noticed an improvement in the maneuverability of the dough, an increase in softness
and elasticity, an increase in the specific volume of the bread, and a reduction in hardness.
The enzyme phytase or myoinositol-hexaphosphate phosphohydrolase is present
in plants, bacteria, yeasts, and filamentous fungi. It hydrolyzes phytate molecules and
releases inorganic phosphate, dephosphorylation-dependent inositol esters, and minerals
linked in the phytate structure [219]. Two classes of phytase are categorized depending on
where the hydrolysis of phytate begins, -3-phytase (EC 3.1.3.8) or 6-phytase (EC 3.1.3.26);
the 3-phytase being the one that removes the orthophosphate group from the position
C3 characteristic of microorganisms. Two classes of phytase are found depending on the
position where the hydrolysis of phytase -3-phytase (EC 3.1.3.8) or -6-phytase (EC 3.1.3.26).
Fermentation 2023, 9, 90 19 of 33

The 3-phytase removes the orthophosphate group from the position C3 characteristic of
microorganisms [220].
Xylan is the main hemicellulosic component of hardwoods and accounts for approxi-
mately 30% of the woody cell wall. Xylanases (EC 3.2.1.8) are enzymes that hydrolyze this
polysaccharide. They are of two types: β-1,4-endoxylanase cleaving internal glycosidic
linkages, and α-D-xylosidase which cleaves xylo-oligosaccharides, and xylobiose forming
xylose, a disaccharide of two xylose molecules [211]. It is important to highlight that
xylanase from various sources has different mechanisms. Two mechanisms can summa-
rize the improvement in bread quality due to the addition of xylanase: (i) the removal of
arabinoxylans from gluten alters the distribution of water between gluten proteins and
arabinoxylans; (ii) pentosan destruction and viscosity reduction effect [182].
Aslam et al. [221] reviewed that wheat is a reservoir of minerals, mainly in the aleurone
tissue between the endosperm and the seed coat. However, these minerals are complexed
with phytate molecules that reduce their bio-accessibility. Xylanase and cellulase showed
satisfactory results to facilitate access into the aleurone tissue for the action of phytase,
improving iron bio-accessibility [222].
Although some aspects of mineral assimilation by the human body need to be consid-
ered, such as diet, blood flow, gut epithelium, and intestinal microbiota [223], some LAB
and yeasts may have phytase activity that improves mineral bio-accessibility in sourdough
bread [79,224–226]. Bifidobacterium strains have demonstrated the ability to increase the
accessibility of iron through its phytase activity during bread fermentation [107].
Pentosanase is a hemicellulase that acts on pentosans, particularly hydrolyzing water-
unextractable arabinoxylan (WUA) into water-extractable arabinoxylan (WEA). The WUA
represents 85% of the total arabinoxylan in whole wheat flour. The WEA affects the bread
making process by reducing viscosity and making the dough more malleable. However,
WUA promotes lower bread quality for interfering with the gluten network because it
competes with gluten for water [169]. Adding pentosanase has shown a good outcome
for bread making, with higher specific volume and crumb texture improvement [227].
Moreover, Martínez-Anaya et al. [228] showed that sourdough with starter modified the
results of pentosanase used in the bread making process, with the fraction of soluble
pentosan increased considerably, and a lower xylose/arabinose ratio.

5.4.3. Lipase and the Baking Technology

Lipase and esterase (EC 3.1) catalyze the reaction that forms ester bonds. The sub-
class triacylglycerol acyl hydrolase (EC 3.1.1.3) acts on triglycerides releasing fatty acids
and glycerol.
Both phospholipase A1 (EC 3.1.1.32) and phospholipase A2 (EC 3.1.1.4) liberated
fatty acids on phospholipids and other lipophilic compounds. These groups are the most
important in the bread making process [210,211,229].
In baking, using lipase to replace emulsifiers, such as monoglycerides, proved to be
an efficient alternative to replace chemical additives [230]. Triacylglycerol acyl hydrolase
can break down triglycerides, which interferes negatively with the gas retention gluten
properties [210]. Sourdough fermentation inhibits the lipase activity due to decreased pH
in the system, which is an efficient alternative for neutralizing the lipase activity in wheat
germ [231]. Wheat germ, rich in vitamins, amino acids, fiber, and minerals, is nutritionally
more important in wheat. However, it causes oxidation in wheat flour during storage
because this needs to be removed before producing wheat flour [232]. A study showed that
wheat germ could produce bread with a superior nutritional, textural, and sensory quality
after the sourdough process to obtain sourdough-fermented wheat germ free of oxidation
potential [233].

5.4.4. Peptidase and Implications for the Gluten Network

Peptidases hydrolyze peptide bonds of proteins and peptides. They are still known as
proteases, proteinases, and proteolytic enzymes [234].
Fermentation 2023, 9, 90 20 of 33

The International Union of Biochemistry and Molecular Biology (IUBMB) classifies

peptidases with criteria based on reaction and catalytic type. Endopeptidases (EC 3.4.21-99)
hydrolyze internal peptide bonds, while exopeptidases (EC 3.4.11-19) remove amino acids
from the ends of peptide chains. These enzymes have a complex classification involving
the reaction and catalytic type and the evolutive relations. The MEROPS platform is a
complete site about peptidases, including their complete classification [234].
Gluten is a complex protein responsible for wheat bread’s most peculiar characteristics,
such as expandability and retention of gas produced in fermentation. Its quality is more
important than quantity [235]. Gluten structure varies with the cultivar and cultivation
conditions [236]. It is composed of gliadin (α, β, γ, ω), glutenin, and polypeptide fractions
that combine with gliadin or glutenin [237]. Figure 4 seeks to demonstrate the complexity
of gluten formation and the water effect. Gluten forms a network retaining carbon dioxide
during baking, making dough expand. It is responsible for dough’s rheological characteris-
tics due to gliadin, which ensures the dough’s viscosity, and glutenin, with the property of
elasticity. During sourdough fermentation, the partial hydrolysis of gliadin and glutenin
proteins occurs because of the acidification and activation of cereal peptidases. In addition,
endogenous flour peptidases became activated at a low pH reducing the gluten disulfide
bonds [38]. Cysteine proteinases in wheat grain are capable of hydrolyzing both gliadins
and glutenin. The hydrolysis of glutenins during sourdough fermentation results in depoly-
merization and subsequent solubilization [238]. Endopeptidases may be applied to improve
the rheological properties, and one example is the endo- and exopeptidases from A. oryzae.
The fungi peptidases are employed to modify wheat gluten by limited proteolysis resulting
in better rheology and shorter dough mixing for a higher loaf [239]. Lactic acid bacteria also
exhibit proteolytic activity, decreasing the content of native gluten [231,240,241]. Several
studies have focused on LAB peptidases and demonstrated that in different ways, that
LAB in sourdough fermentation has a proteolytic activity for gluten hydrolysis [60]. Celiac
disease or gluten enteropathy is an autoimmune disease caused by gluten-containing food
and results in those with it to adhere to a strict gluten-free diet. One characteristic of
gluten is that it is resistant to proteolysis by human digestive peptidases [242]. There is
considerable interest in producing gluten-free bread that is specific for celiac consumers.
The gliadin fraction, especially α2-gliadin, has been considered the most allergenic part
of gluten [243]. Another possible treatment is enzyme therapy, in which peptidases are
ingested with food to increase the hydrolysis of resistant immunogenic peptides containing
proline and glutamine amino acids [244]. The hydrolysis of some parts of gluten could
favor the appearance of allergen terminals and cause more harm than benefit to a celiac
person [243]. On the other hand, hydrolyzing gluten to reduce the allergenic components
potentially benefits non-celiac people [69]. A group of LAB hydrolyzed gluten, but not
the most allergenic part in the central region of alpha-gliadin. However, other allergenic
proteins were degraded, improving the wheat digestibility [245]. A bioprospection analysis
of gliadin-cleaving proteolytic activity among 20 Lactobacillus strains showed that the most
active strain, L. casei, was able to hydrolyze the 33-mer immunogenic peptide of α-gliadin
by 82% in 8 h and completely in 12 h [246]. Bread digestibility might be positively affected
by long sourdough fermentation [148]. L. rhamnosus, Pediococcus pentosaceus, and Lactobacil-
lus curvatus used gluten as the only source of nitrogen, significantly reducing the allergenic
composition of wheat and improving digestibility [69]. Strains of Lactobacillus plantarum
ES137 and Pediococcus acidilactici ES22, isolated from sourdough fermentation, showed high
proteolytic capacity [247]. Lactobacilli in sourdough have proteolytic capacity to produce
γ-glutamyl dipeptides, other peptides, and amino acids as flavor precursor compounds in
the bread produced [248]. Other bacteria, such as Bacillus spp. isolated from sourdough,
are capable of gluten hydrolysis [140].
Fermentation 2023,
Fermentation 9, x90FOR PEER REVIEW
2023, 9, 2321ofof35
33

Figure 4. Some possible interactions of wheat proteins. (A)‐low water content. (B)‐baking mixture
Figure 4. Some possible interactions of wheat proteins. (A)-low water content. (B)-baking mixture
with optimal water content, based on the work of Feng et al. and Schopf and Scherf [249,250].
with optimal water content, based on the work of Feng et al. and Schopf and Scherf [249,250].

6.
6.General
GeneralRegulation
Regulationfor forMicrobes
MicrobesUsed Usedin in Sourdough
Sourdough Bread Bread
Searching
Searchingfor forrules
rules that
that maintain
maintainfood foodsafety
safetyisisaa critical
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growing
use
useofof microorganisms
microorganismsas as foods
foods oror probiotics.
probiotics. At Atthis
thismoment,
moment,there thereisisananeffort
effortatatCodex
Codex
Alimentarius to harmonize rules for probiotics and the use of
Alimentarius to harmonize rules for probiotics and the use of microorganisms in food food microorganisms in [251].
[251].In the United States, there are rules defined by the Food and Drug Administration
(FDA)In for
the the
United States,
use of there are rules
microorganisms defined
in food by the Food
production. It isand Drug Administration
necessary to qualify as a
(FDA) for Recognized
Generally the use of microorganisms
as Safe for Use (GRAS)in foodmicroorganism
production. It [252].
is necessary
In Brazil,tothe
qualify as a
biological
Generally
fermenting Recognized
mixtures and as Safe
theirformicrobial
Use (GRAS) microorganism
content are classified [252].
as aInsupporting
Brazil, the andbiologi‐
pro-
cal fermenting
cessing agent and mixtures and their
are exempted frommicrobial content
registration are classified
by ANVISA [253].asHowever,
a supporting and
food with
nutritionalagent
processing allegations
and areor exempted
probiotic use fromneeds to be registered
registration by ANVISA [253],[253].
and microorganisms
However, food
isolated
with from national
nutritional raw materials,
allegations or probiotic or related
use needs territory, are recognized
to be registered [253],asand national genetic
microorgan‐
patrimony
isms isolated [254],
fromand they need
national raw to be registered
materials, too. territory, are recognized as national
or related
geneticThe requirements
patrimony [254],foranda they
microorganism to be considered
need to be registered too. GRAS include taxonomic
identification,
The requirementsabsence forof virulence, enterotoxins,
a microorganism to behemolytic
considered activity,
GRAS and transferable
include taxonomic an-
tibiotic resistance genes. A probiotic microorganism still needs
identification, absence of virulence, enterotoxins, hemolytic activity, and transferable an‐to prove stress tolerance
(low pH,
tibiotic gastric enzymes,
resistance genes. Abile salts) up
probiotic to levels foundstill
microorganism in the intestine,
needs to prove as well
stressastolerance
adhesion
capacity and antipathogenic activity, and to be approved in clinical
(low pH, gastric enzymes, bile salts) up to levels found in the intestine, as well as adhesion trials [255]. Moreover,
FAO/WHO
capacity and advises in its guidelines
antipathogenic activity,that
and the
to beevaluation
approvedofinprobiotics should
clinical trials be Moreover,
[255]. done using
molecular information
FAO/WHO advises in its from strains, because
guidelines the probiotic
that the evaluation ofproperties
probiotics are related
should to thisusing
be done level
of identification [256].
molecular information from strains, because the probiotic properties are related to this
level In France, since 1993,
of identification [256].a law specifies the characteristics of sourdough bread, such as
pH 4.3, acetic acid 900
In France, since 1993, ppm, at least,
a law with the
specifies sourdough consisting
characteristics of lactic acid
of sourdough bacteria
bread, suchand as
It is possible to add Saccharomyces cerevisiae
pH 4.3, acetic acid 900 ppm, at least, with sourdough consisting of lactic acid bacteriaflour
yeasts. to a maximum of 0.2% on the and
weight.ItIfissourdough
yeasts. possible toisadd dry,Saccharomyces
it needs at least one billion
cerevisiae to a live food bacteria
maximum of 0.2%and on one
the to ten
flour
million If
weight. live yeasts perisgram
sourdough dry, [257].
it needs at least one billion live food bacteria and one to ten
In Europe, since 1997,
million live yeasts per gram [257]. with the novel food regulation, all food that started to be
produced after 15 May
In Europe, since 1997, with 1997 is considered
the novel food a novel food and
regulation, has specific
all food rulestotobebepro‐
that started fol-
lowed [258]. For microorganisms usually found in sourdough and used as a starter, it is
duced after 15 May 1997 is considered a novel food and has specific rules to be followed
Fermentation 2023, 9, 90 22 of 33

challenging to define it as a novel food because sourdough predates 1997 [259]. Therefore,
if the starter used in the sourdough fermentation is unusual or has some additional novel
properties, it could be considered a novel food. For instance, UV-treated baker’s yeast
(Saccharomyces cerevisiae) has been considered a novel food since 2017 because it is alleged
to increase vitamin D2 [260]. Moreover, the EU1151/2012 recognizes that baker products
could have a geographical denomination [11].
Regarding microorganism patents, the United States, Japan, Europe, India, and par-
ticipants of The Trade-related Aspects of Intellectual Property Rights (TRIPS) agreement
consider that microorganisms should be patented [261]. However, Brazil, for instance,
allows patenting only for genetically modified microorganisms [262].

7. Conclusions
The role of microorganisms in sourdough bread has been extensively demonstrated in
the literature. Increasing information is arising about the fermentation process and other
metabolic routes promoting improvements in quality and adding interesting nutritional,
health, and sensory characteristics. Currently, several benefits of probiotic and postbiotics
elements are well established in terms of the bread’s properties, such as rheological and
organoleptic properties, as well as human health and dough quality. However, there are
challenges to overcome to produce a microbial starter or consortium with high technological
properties on an industrial scale. Better digestibility, satiety, and antioxidants, among
other properties, could be obtained. The sourdough ecosystem needs to be explored
more concerning the dynamic of the interaction among the microorganisms, including the
quorum sensing process and the metabolic regulation of sourdough. Molecular and omics
tools are bringing more information about the sourdough microbiome and the complex
metabolic network, allowing the complete identification of the non-cultivable microbial
population and establishing probiotic strains. A better characterization of the postbiotics
present in the dough is also essential. Another promising area of study is relative to the
addition of other microbial products that can improve the final quality of bread. Sourdough
bread and the microorganisms involved in its production are an area for increased research
activity. The breaking and sharing of bread is of biblical and global importance. Further
studies will elucidate the mechanisms of action of these bioproducts, metabolic routes, and
their effects on bread, and the production of safe food leading to technological advances.

Author Contributions: Conceptualization and writing, I.T.A.; writing, formal analysis, original draft
preparation, F.R.P.M.; conceptualization, writing, review, editing, project administration, and funding
acquisition, A.B.V. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded in part by the postgraduate program of the Paulo de Góes
Institute of Microbiology, Federal University of Rio de Janeiro (UFRJ), through the Coordenação de
Aperfeiçoamento Pessoal de Nível Superior (CAPES) [grant number 001], Conselho Nacional de
Desenvolvimento Científico e Tecnológico (MCTI-CNPq) grant code [309461/2019-7], and Fundação
de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), grant code [“CIENTISTA DO NOSSO
ESTADO” 26/202.630/2019 247088].
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Andrew Macrae for the English review and Slide-
Model.com for the diagram in Figure 1. The images in the graphical abstract are from TogoTV (©2016
DBCLS TogoTV) and Servier Medical Art (smart.servier.com).
Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Fermentation 2023, 9, 90 23 of 33

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