LWT - Food Science and Technology 126 (2020) 109314

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LWT - Food Science and Technology

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Sprouting process affects the lactic acid bacteria and yeasts of cereal, T
pseudocereal and legume flours
Giuseppe Perria, Francesco Maria Calabresea, Carlo Giuseppe Rizzelloa, Maria De Angelisa,
Marco Gobbettib, Maria Calassoa,∗
a
Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Via Amendola 165/a, 70126, Bari, Italy
b
Faculty of Science and Technology, Free University of Bozen, Piazza Università 5, 39100, Bolzano, Italy

ARTICLE INFO ABSTRACT

Keywords: The aim of this work was to investigate how sprouting process may affect the indigenous lactic acid bacteria
Sprouting process (LAB) and yeasts of wheat, barley, quinoa, lentil and chickpea flours. LAB and yeasts inhabiting the raw and
Wheat sprouted flours were described by culture-dependent and -independent approach. Based on community-level
Barley catabolic profiles, the sprouting process led to a strong increase of the Shannon's diversity and substrate richness
Quinoa
indices. Compared to raw flours, the highest ability to consume carbohydrates, polymers, amines, carboxylic
Lentil, and chickpea flours
acid and amino acids sources was found for the microbial communities of wheat, chickpea and lentil sprouted
Lactic acid bacteria
Yeasts flours. Except for LAB detected in quinoa, sprouting process caused significant (P < 0.05) changes in the cell
density of LAB and yeasts. Compared to raw flours, all sprouted flours harboured a different microbiome and a
higher number of LAB strains. Positive correlations (r > 0.70; FDR < 0.05) were found between cell density of
LAB and yeasts with the time of sprouting process and the concentrations of free sugars content in flours.
This study demonstrated that the sprouting process of wheat, barley, quinoa, lentil and chickpea grains
modifies the microbial metabolic activities and composition of the lactic acid bacteria and yeasts of the resulting
flours.

1. Introduction
Cereals and legumes are the most important contributors to the
balanced human diet worldwide, providing significant amount of
macro- and micro-nutrients. Besides such grains, pseudocereals (e.g
quinoa, amaranth, buckwheat), which already had an important role in
human nutrition in the past, are gaining popularity thanks to their re-
markable technological and nutritional properties (Alvarez-Jubete,
Wijngaard, Arendt, & Gallagher, 2010).
In the last decade, the raised attention on healthy nutrition and the
large growth of a food customized market for vegetarian, vegan, and
gluten-free diets, pushed towards an increase of “sprouted grains” as
also underlined by the boost of scientific literature. Advisedly, all these
evidences supported the nutritional traits and phytochemical contents
of these cereal products (Benincasa, Falcinelli, Lutts, Stagnari, &
Galieni, 2019). Because of the significant changes in nutritional and
physicochemical properties occurring in grains, subsequently to the
activation of dormant enzymes, germination has been recognized as a
method to enhance the nutritional quality of whole grains (Donkor,
Stojanovskaa, Ginnb, Ashtonb & Vasiljevica, 2012). Additionally, it has
been proved that in several grain types the sprouting process leads to
several changes even in the microbiota structure (Barret et al., 2015;
Landry, Sela, & McLandsborough, 2018).
Lactic acid bacteria (LAB) are mainly responsible for the unique
performances and valuable nutritional, sensory, shelf-life and texture
traits of the sourdough baked goods (Gänzle, 2014; Gobbetti, Minervini,
Pontonio, Di Cagno, & De Angelis, 2016). LAB may originate from flour,
other dough ingredients and bakery environment (Minervini, Lattanzi,
De Angelis, Celano, & Gobbetti, 2015). However, the effect of sprouting
on the LAB and yeasts population of the flours obtained from cereals,
pseudocereals and legumes, has not been systematically studied
(Montemurro, Pontonio, Gobbetti, & Rizzello, 2019). The knowledge
about the effect of sprouting process on the indigenous pro-technolo-
gical microbes (LAB and yeasts) could be useful to optimize the use of
this kind flours in food production processes.
The aim of this work was to investigate how sprouting process may
affect the indigenous LAB and yeasts of flours. For this purpose, wheat,
barley, quinoa, lentil and chickpea grains were used. LAB and yeasts
inhabiting the raw and sprouted flours were described by a culture-
dependent and –independent combined approach.

∗
Corresponding author.
E-mail address: maria.calasso@uniba.it (M. Calasso).

https://doi.org/10.1016/j.lwt.2020.109314
Received 6 December 2019; Received in revised form 20 February 2020; Accepted 21 March 2020
Available online 26 March 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
G. Perri, et al. LWT - Food Science and Technology 126 (2020) 109314

Shannon's diversity (H′)a, substrate richness (S)b and substrate evenness (E)c calculated for the Community Level Catabolic Profiles (CLCPs)d of wheat, barley, chickpea, lentil, and quinoa flours obtained from grains
2. Materials and methods

Sprouted quinoa

17.67 ± 1.4
3.35 ± 0.1

2.71 ± 0.2
2.1. Grains, sprouting process and milling

P = 0.049 Five different grains were considered in this study: two cereals

P = 0.016

P = 0.313
(wheat, Triticum durum var. Simeto and barley, Hordeum vulgare var.
Nure), a pseudocereal (quinoa, Chenopodium quinoa) and two legumes
(chickpea, Cicer arietinum var. Pascià and lentil, Lens culinaris). Cereals
15.30 ± 2.66
3.11 ± 0.02

2.62 ± 0.05
Raw quinoa

and legumes were purchased by Caporal Grani S.a.s. (Gravina di Puglia,

Bari, Italy); quinoa, was supplied by Quinoa Marche Srls (Jesi, Ancona,
Italy). Three batches of each grain were collected. Grains were sprouted
according to the protocol described by Donkor, Stojanovska, Ginn,
H′, substrate utilization pattern, measured as H′ = −∑ pi ln (pi), where pi is the ratio of the activity of a particular substrate to the sums of activities of all substrate activity.

Ashton, and Vasiljevic (2012) with some modifications (Montemurro

et al., 2019). Sprouting parameters were summarized in Table S1.
Sprouted lentil

3.18 ± 0.03

2.53 ± 0.05
18.00 ± 1

2.2. Community level catabolic profiles

P = 0.009

P = 0.011

P = 0.336

Carbon source utilization patterns of microbial communities of raw

and sprouted flours were assessed on each flour by using BIOLOG 96-
well Eco-Microplates (Biolog, Inc., Hayward, CA, USA) according to
2.84 ± 0.09

12.66 ± 1.1

2.51 ± 0.1

Cavallo et al. (2017). Microplates contained 31 different carbon sources

Raw lentil

(carbohydrates, carboxylic acids, polymers, amino acids, amines and

miscellaneous substrates), in triplicate. The microplates were incubated
at 30 °C in the dark and the color development was measured at 590 nm
every 24 h thereafter up to 72 h with a microplate reader (BIOLOG
Microstation).
Sprouted chickpea

S, the number of different substrates used, calculated as the number of wells with a corrected absorbance greater than 0.25.
2.99 ± 0.7

2.43 ± 0.1
15.33 ± 1

2.3. Lactic acid bacteria and yeasts enumeration and isolation

Ten grams of each flour were homogenized with 90 ml of sterile

P = 0.021

P = 0.029

P = 0.411

peptone water (1% [wt/vol] peptone and 0.9% [wt/vol] NaCl) solution
and serially diluted with sterile peptone water. LAB were counted and
isolated by using De Man, Rogosa and Sharpe (MRS) (Oxoid,
2.90 ± 0.009
Raw chickpea

3.32 ± 0.02

Basingstoke, Hampshire, UK), modified MRS (mMRS) (containing 1%

14.01 ± 1

Value are the means from three independent experiments analyzed in triplicate ± standard deviation.

[wt/vol] maltose, 5% [vol/vol] fresh yeast extract, pH 5.6) and sour-

dough bacteria (SDB) agar medium. All these media were supplemented
with cycloheximide (0.1 g/l). Plates were incubated at 30 °C for 48 h
under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid). Presumptive
P values refer raw vs sprouted data. P < 0.05 was considered as statistically significant.

LAB were isolated from each flour by randomly selecting for each
Sprouted barley

medium at least ten colonies from the plates containing the highest
3.24 ± 0.02

2.58 ± 0.02
18.01 ± 0

sample dilutions. Gram-positive, catalase-negative, nonmotile rods and

cocci isolates were cultivated in MRS, mMRS and SDB broth at 30 °C for
E, the equitability of activities across all utilized substrates: E = H′/log S.

24 h and two times restreaked onto the same agar media, until pure
P = 0.009

P = 0.002

P = 0.064

culture was obtained. All isolates considered for further analyses were
able to acidify the culture medium.
2.61 ± 0.045
13.33 ± 0.66

Yeasts were estimated on Sabouraud dextrose (SDA) (Oxoid) agar

2.94 ± 0.1
Raw barley

supplemented with chloramphenicol (0.1 g/l), incubated at 25 °C for

48 h. Randomly selected colonies (ca. 10 for each flour) of yeasts from
the higher plate dilutions were sub-cultured on Sabouraud dextrose
broth and restreaked onto the same agar media, until pure culture was
obtained.
Sprouted wheat

3.42 ± 0.11

2.58 ± 0.08

2.4. Genotypic characterization and identification of lactic acid bacteria

21.00 ± 0a

and yeasts
before (raw) and after sprouting process.

P = 0.037

P = 0.006

P = 0.168

Genomic DNA of LAB was extracted using DNeasy Blood and Tissue
Kit (Qiagen, SA, Courtaboeuf, France), according to the manufacturer's
instructions. Three oligonucleotides, P4 5′-CCGCAGCGTT-3′, P7
14.67 ± 0.57
e
3.14 ± 0.02

3.01 ± 0.55

5′-AGCAGCGTGG-3′, and M13 5′-GAGGGTGGCGGTTCT-3′, with arbi-

Raw wheat

trarily chosen sequences, were used for biotyping lactic acid bacterium
isolates. Reaction mixture and PCR conditions for primers P4, P7 and
M13 were set according to Siragusa et al. (2009). Identification of
strains was performed using LpigF and LpigR primers targeting 16S
rDNA gene of LAB (De Angelis et al., 2006). Primer designed for the ddl
Indices
Table 1

gene were used for the identification of Enterococcus species (Knijff,

’
H

d
b
a
E

Dellaglio, Lombardi, Andrighetto, & Torriani, 2001).

c
S

2
G. Perri, et al.
Fig. 2. Cell numbers (Log cfu/g) of lactic acid bacteria and yeasts in wheat, barley, chickpea, lentil, and quinoa flours obtained from grains before and after sprouting
process. MRS, de Man Rogosa and Sharp medium; mMRS, modified MRS; SDB, sourdough bacteria medium. Values are the means ± standard deviations from three
independent experiments analyzed in triplicate. Different letters (a-d) indicate significant differences (P < 0.05) in microbial count using different agar medium.
DNA from yeasts was extracted using the Wizard Genomic DNA
Purification kit (Promega Corporation, Madison, WI), according to the
manufacturer's instructions. Yeasts were bio-typed through RAPD-PCR
using primers M13m and RP11 (Del Bove et al., 2009). Yeasts were
identified by using NL-1/NL-4 primers (Kurtzman & Robnett, 1998).
2.5. Total DNA extraction from raw and sprouted flours and Illumina
MiSeq analysis
Total DNA was extracted by using the FastDNA® Pro Soil-Direct Kit
(MP Biomedicals, Santa Ana, CA, United States) coupled to the
FastPrep® instrument (MP Biomedicals, Illkrich, France), according to
the manufacturer's instructions.
DNA samples were used as template in PCR using primers
Firm350F/Firm814R, to amplify the region V3-V4 of the 16S rRNA
gene for analysis of diversity inside the phylum of Firmicutes
(Minervini, Celano et al., 2015) and primers MSd_Fun545F/
MSd_Fun1021R for a fragment of the 18S rRNA region for detection of
fungi. PCR were run by Research and Testing Laboratory (RTL; Lub-
bock, TX, United States) based upon RTL (http://www.
3
LWT - Food Science and Technology 126 (2020) 109314
Fig. 1. Types and quantity of carbon and nitrogen
sources used by the microbial communities of wheat,
barley, chickpea, lentil, and quinoa flours obtained
from grains before and after sprouting process.
Values are the means from three independent ex-
periments analyzed in triplicate. Statistical differ-
ences in substrates consumption between the dif-
ferent conditions are reported in terms of P values.
researchandtesting.com). PCR products were sequenced at RTL Geno-
mics, using the Illumina MiSeq 2x300 paired-end MiSeq platform.
Sequenced reads for each sample were processed through denoising
and chimera detection by using RTL Laboratory's in-house pipeline,
described at https://static1.squarespace.com/static/5807c0ce579fb-
39e1dd6addd/t/5813af0fd482e97e5eb4fcb5/1477685010205/Data_
Analysis_Methodology.pdf.
2.6. Chemical characterization of raw and sprouted flours
Protein content (total nitrogen × 5.7) was determined according to
the ISO 1871:2009 method (2009). Starch, fat and ash were determined
according to the standard AACC method 30–10.01, 76–13.01 and
08–01, respectively (AACC, 2010). The total free carbohydrates and
total dietary fibers (DF) contents of the flour samples were determined
using the method of AOAC (2000). Total free carbohydrates were cal-
culated as the difference [100 - (proteins + lipids + ash +
moisture + total dietary fibres + starch)]. Proteins, starch, fat, ash,
total free carbohydrates were expressed as % of dry matter (d.m.). In-
soluble (IDF) and soluble (SDF) dietary fibers were determined
G. Perri, et al. LWT - Food Science and Technology 126 (2020) 109314

Fig. 3. Dendrogram of combined (primers P4, P7, and M13) RAPD profiles of lactic acid bacterium strains isolated from flours obtained by wheat, barley, chickpea,
lentil, and quinoa grains, before and after sprouting process. The first letters of the code of the strain name (W, wheat; SW, sprouted wheat; B, barley; SB, sprouted
barley; C, chickpea, SC, sprouted chickpea; L, lentil, SL, sprouted lentil; Q, quinoa; SQ, sprouted quinoa) indicates the flour of origin whereas the following letter
(separated by one hyphen) of the strain name indicates the medium used for the isolation (A, de Man Rogosa and Sharp medium, MRS; B, modified MRS; C,
sourdough bacteria medium) and the number represents the progressive number of isolation. Lactic acid bacteria isolates correspond to those of Table 2. Cluster
analysis was based on the simple matching coefficient and unweighted-pair group method with arithmetic average. Clusters are indicated by Roman numerals (I–XII).
E., Enterococcus; P., Pediococcus; L., Lactobacillus; Lc., Lactococcus; S., Staphylococcus; W., Weissella; Leuc., Leuconostoc.

according to Goñi, Diaz-Rubio, Pérez-Jiménez, & Saura-Calixto (2009)
and Montemurro et al. (2019). Total phenols were determined as de-
scribed by Slinkard and Singleton (1977) and expressed as gallic acid
equivalent.
2.7. Statistical analyses
Shannon's diversity and substrate richness indices. Microbial commu-
nities of sprouted flours were faster (P < 0.05) to consume carbohy-
drates, polymers, amines, carboxylic acid and amino acids sources
(Fig. 1). Compared to raw flours, the highest ability in consuming
carbohydrates, polymers, amines, carboxylic acid and amino acids
sources was found in wheat, chickpea and lentil sprouted flours.

All the analyses were carried out at least in duplicate for each batch
of grain (total of six analyses for each type of flour). Analysis of var-
iance (ANOVA) was carried out on transformed data, followed by se-
paration of means with Tukey's honestly significant difference (HSD)
test, using the statistical software Statistica 7.0 for Windows (StatSoft,
Vigonza, Italy). Weighted and unweighted UniFrac distance matrices
and OTU tables were used to perform ADONIS and ANOSIM statistical
tests through the compare_category.py script of QIIME to verify the
microbial populations in the different flours obtained by wheat, barley,
chickpea, lentil, and quinoa grains, whole and after sprouting process.
3. Results
3.1. Community level catabolic profiles of raw and sprouted flours
The community-level catabolic profiles (CLCPs), before and after
sprouting process were investigated. In particular, Shannon's diversity,
substrate richness, and substrate evenness indices were calculated
(Table 1). The sprouting process led to a strong increase of the
3.2. Cultivable lactic acid bacteria and yeasts
Overall, compared to raw flours, sprouted flours harboured higher
cell density of yeasts. On the contrary, sprouting differently reflected on
LAB population depending on the flour considered. Sprouted wheat,
barley and chickpea flours showed ca. 2 Log cycles of presumptive LAB
higher than raw flours (Fig. 2). In lentil, the sprouting led to an increase
of LAB only in MRS and mMRS media, while the cell density on SDB
medium was higher in raw than in sprouted flour. Raw flour of quinoa
harboured a higher cell density of LAB than sprouted flour.
Presumptive LAB (295 isolates, ca. 10 from each media) were sub-
jected to RAPD-PCR analysis. A total of 75 LAB strains were identified
(Fig. 3, Table 2). Compared to raw, sprouted flours harboured a higher
number of LAB strains. In details, wheat flour harboured seven species:
Pediococcus pentosaceus (1 strain), Lactobacillus fermentum (1), En-
terococcus casseliflavus (1), Enterococcus faecium/faecalis (2), En-
terococcus termitis (1), Lactococcus lactis (1). L. fermentum, E. termitis and
E. faecium/faecalis W-B-10 were not identified in sprouted wheat flour.
Compared to raw, sprouted wheat flour harboured other 15 strains

4
G. Perri, et al. LWT - Food Science and Technology 126 (2020) 109314

Table 2
Strains of lactic acid bacteria identified from wheat, barley, chickpea, lentil, and quinoa flours obtained from grains before (raw) and after sprouting processa.
Flours Strainsb Clusterc Closest relative GenBank accession no.
(% identity) d

Raw wheat W-A-6, W-A-8, W-A-10 I P. pentosaceus (100) HQ711360.1

W-C-5 II L. fermentum (99) MF424378.1
W-C-3 V E. casseliflavus (99) NR104560.1
W-C-8 VII E. faecium/faecalis (99) NR_113904.1/MF429765.1
W-B-10 XI E. faecium/faecalis (99) NR_113904.1/MF429765.1
W-C-2 UC E. termitis (98) GQ337037.1
W-A-5 UC Lc. lactis (99.7) MF355117.1
Sprouted wheat W-A-6, W-A-8, W-A-10, SW-B-2, SW-A-1, I P. pentosaceus (100) HQ711360.1
SW-A-2, SW-A-8, SW-B-4
SW-C-7, SW-C-8 III E. faecium/faecalis (99) MN148620.1/MN080437.1
W-C-3 V E. casseliflavus (99) NR104560.1
SW-A-5 VI E. casseliflavus NR104560.1
W-C-8 VII E. faecium/faecalis (99) MN148620.1/MN080437.2
SW-A-6, SW-B-7, SW-B-8 X E. faecium/faecalis (99) KY490546.1/MF429765.1
SW-B-5 UC E. casseliflavus (99.9) MF424694.1
W-A-5, SW-C-4 UC Lc. lactis (99.7) MF355117.1
SW-C-6 UC E. faecium/faecalis (99) MN148620.1/MN080437.2
SW-C-10 UC Lc. lactis MF355117.1

Raw barley B-C-4 III E faecium/faecalis (99) MN148620.1/MN080437.1

B-C-5 V E. casseliflavus (99) NR104560.1
B-B-3 UC E. casseliflavus (99.7) MF424694.1
B-B-7 UC E. casseliflavus (99.7) MF424694.1
B-B-9 UC E. casseliflavus (99.7) MF424694.1
B-C-6 UC E. casseliflavus (99.7) MF424694.1
Sprouted barley SB-B-10 II L. fermentum (99) MF424378.1
B-C-4, SB-B-5, SB-C-6 III E. faecium/faecalis (99.9) MN148620.1/MN080437.1
B-C-5, SB-C-5 V E. casseliflavus (99) NR104560.1
SB-B-7, SB-C-2, SB-C-10 VIII E. faecium/faecalis (99.9) MN148620.1/MN080437.2
Raw chickpea C-A-6 II L. fermentum (99) MF424378.1
C-A-1, C-C-4, C-C-10 IV E. mundtii (99.9) CP029066.1
C-A-2 UC S. hominis MG255965.1
Sprouted chickpea SC-B-4 I P. pentosaceus HQ711360.1
C-A-1, C-C-4, C-C-10 IV E. mundtii (99.9) CP029066.1
C-A-2, SC-A-5, SC-B-6
SC-C-5 V E. casseliflavus (99) NR104560.1
SC-B-2 VI E. casseliflavus NR104560.1
SC-A-4 UC E. termitis (99) GQ337037.1
SC-B-10 UC E. mundtii (99.9) CP029066.1
SC-C-3 UC Enterococcus sp. -

Raw lentil L-B-4, L-B-6, L-B-7 I P. pentosaceus (100) HQ711360.1

L-A-4, L-A-5 VI E. casseliflavus NR104560.1
L-A-8, L-A-2, L-A-3 X E. faecium/faecalis (99) KY490546.1/MF429765.1
L-C-1 XI E. faecium/faecalis (99) NR_113904.1/MF429765.1
L-C-7 UC Enterococcus sp. -
Sprouted lentil L-A-4, L-A-5 VI E. casseliflavus NR104560.1
SL-B4 V E. casseliflavus (99) NR104560.1
SL-A-4, SL-A-3, SL-A-6 IX W. confusa (100%) HQ711354.1
SL-B-3, SL-B-9, SL-B7, SL-B-5, XII E. mundtii (99.8) CP029066.1
SL-A-5 UC W. paramesenteroides KF023208.1
(100)
SL-A-9 UC E. casseliflavus (99) NR104560.1
Raw quinoa W-C-3 V E. casseliflavus (99) NR104560.1
Q-B-2 UC E. casseliflavus (99) NR104560.1
Q-B-6 UC L. fermentum MF424378.1
Sprouted quinoa W-C-3, SQ-C-6 V E. casseliflavus (99) NR104560.1
Q-B-2, SQ-B-5 UC E. casseliflavus (99) NR104560.1
SQ-C-5 UC E. casseliflavus (99) NR104560.1
SQ-C-2 UC E. casseliflavus (99.9) MF424694.1
SQ-C-3 UC Lc. lactis (99.7) MF355117.1

a
Identification was carried out by 16S rRNA and ddl gene sequencing.
b
The first letters of the code of the strain name (W, raw wheat; SW, sprouted wheat; B, raw barley; SB, sprouted barley; C, raw chickpea, SC, sprouted chickpea; L,
raw lentil, SL, sprouted lentil; Q, raw quinoa; SQ, sprouted quinoa) indicates the flour of origin whereas the following letter (separated by one hyphen) of the strain
name indicates the medium used for the isolation (A, De Man, Rogosa and Sharpe medium, MRS; B, modified MRS; C, sourdough bacteria medium) and the number
represents the progressive number of isolation.
c
RAPD-PCR cluster. Clusters are numbered with Roman numerals from I to XII; UC, unclustered.
d
Species showing the highest identity (%) to the strain isolated from flour. The percentage of identity was that shown by performing multiple sequence alignments
in BLAST. P., Pediococcus; L., Lactobacillus; E., Enterococcus; Lc., Lactococcus; S., Staphylococcus; W., Weissella.

5
G. Perri, et al.
Fig. 4. Relative abundance (%) of bacterial OTUs classified at the phylum (A) and family (B) level, found in flours obtained by wheat, barley, chickpea, lentil, and
quinoa grains, before and after sprouting process.
mainly belonging to P. pentosaceus, E faecium/faecalis and Lc. lactis.
E. casseliflavus (5 strains) and E faecium/faecalis (1) dominated in
raw barley flour. E. casseliflavus B-B-3, B-B-7, B-B-9, B-C-6 disappeared
in sprouted barley flour. On the contrary, E. casseliflavus SB-C-5 and L.
fermentum were identified only after sprouting. The number of domi-
nant E faecium/faecalis increased in sprouted compared to raw barley
flour.
Raw chickpea flour harboured E. mundtii (3) and L. fermentum (1)
and Staphylococcus hominis (1). The latter two species disappeared after
the sprouting process. Compared to raw flour, sprouted chickpea flour
showed other dominant LAB species (P. pentosaceus, E. casseliflavus, and
Enterococcus sp.).
Raw lentil flour showed four dominant Pediococcus and Enterococcus
species (P. pentosaceus, E. casseliflavus, E. faecium/faecalis, E. faecium/
faecalis, and Enterococcus sp.). Compared to raw, sprouted flour showed
a strong increase of E. casseliflavus strains while P. pentosaceus was not
identified. Strains belonging to Weissella spp. and E. mundtii were de-
tected only in sprouted lentil flour.
Compared to raw, sprouted quinoa flour harboured a higher number
of E. casseliflavus strains. L. fermentum was identified in raw quinoa
flour while Lc. lactis was found only after sprouting.
After preliminary morphological screening, seventeen strains of
yeasts were randomly isolated from raw and/or sprouted flours.
Clavispora lusitaniae was identified in all samples while Debaryomyces
dominated in sprouted chickpea and raw quinoa flours (Table S2). The
number of strains increased in sprouted compared to raw flours.
3.3. Metagenetic profiles of raw and sprouted flours
Total bacterial Operative Taxonomic Units (OTUs) analyzed by the
ggplot package showed that each sample was characterized by a
6
LWT - Food Science and Technology 126 (2020) 109314
specific microbial profile (Fig. S1A). Firmicutes were found at the
lowest level in raw wheat, barley and lentil flours (Fig. 4A). Within
Firmicutes families, Paenibacellaceae (Paenibacillus sp. and Sacchar-
ibacillus sp.) dominated raw and sprouted wheat flours, and especially,
sprouted barley and quinoa flours (Figs. 4B and 5). Bacillus gibsonii and/
or Bacillus sp., decreased during sprouting process. On the contrary,
Lysinibacillus sp., was higher in sprouted than raw samples. Staphylo-
coccaceae (Macrococcus caseolyticus, S. hominis, S. sciuri and Staphylo-
coccus sp.) were the highest in raw chickpea and quinoa flours and
decreased after the sprouting process.
Within LAB group, Streptococcaceae (mainly Lactococcus lactis and
Streptococcus thermophilus species) were found at the highest level in
sprouted wheat and quinoa flours. The relative abundance of Lc. lactis
ranged from 0.09% (sprouted barley flour) to 51.34% in sprouted
wheat flour. Str. thermophilus, found at the highest level in raw chickpea
flour, decreased after sprouting process. Enterococcaceae (E. casseli-
flavus, E. faecalis, E. mundtii, E. termitis and Enterococcus sp.) in wheat,
chickpea and lentil sprouted flours markedly increased, compared to
raw samples. An opposite trend was found for barley and, especially,
quinoa flours. Within Enterococcaceae, E. casseliflavus was the species
found at the highest levels especially in sprouted chickpea flour
(59.66% of relative abundance). Compared to raw flours, E. casseliflavus
strongly decreased in sprouted barley and quinoa. Lactobacillaceae
species (L. brevis, L. fermentum, L. sanfranciscensis, L. spicheri, P. pento-
saceus) were identified at low level (< 1% of the relative abundance) in
all samples. The sprouting process did not led to an increase in the
relative amount of Lactobacillaceae species. Leuconostoccaceae (W.
confusa and W. paramesenteroides) were found at the highest level in
barley flours. Except for lentil, no statistical (P > 0.05) differences
were found between raw and sprouted flours.
Fungal OTUs profiles showed high similarity between chickpea and
G. Perri, et al. LWT - Food Science and Technology 126 (2020) 109314

Fig. 5. Permutation analysis of the relative abundance (%) of bacterial OTUs classified at the species level, found in flour obtained by wheat, barley, chickpea, lentil,
and quinoa grains, before and after sprouting process.

lentils flours (Fig. S1). All flour samples were dominated by Glomer- raw quinoa flour. Except for sprouted barley flour, Fusarium sp., de-
omycota (Glomeraceae) ranging from 80.61% (sprouted barley flour) to creased during sprouting process.
99.90% (lentil flours) of the total OTU (Fig. 6A). Cladosporium sp. de-
creased after sprouting process (Fig. 6B). Compared to raw flours, Al-
3.4. Chemical features of raw and sprouted flours and correlation with
ternaria sp. was found at lower amount in sprouted wheat and quinoa
microbes
flours. Exception was found for barley flours. Debaryomyces hansenii
was detected in raw and sprouted chickpea, quinoa and sprouted barley
The levels of proteins and fats were not significantly (P > 0.05)
flours. Except for chickpea, Clavispora lusitaniae increased in sprouted
affected by the sprouting process (Fig. 7). As expected, a strong increase
samples. Candida intermedia was detected only in sprouted wheat,
of free carbohydrates was observed during the sprouting process. The
barley and chickpea flours. Plectosphaerellaceae was identified only in
only exception was found for lentil grains. The highest levels of free

7
G. Perri, et al.
Fig. 6. Relative abundance (%) of fungal OTUs classified at the phylum (A) and species (B) level, found in flours obtained by wheat, barley, chickpea, lentil, and
quinoa grains, before and after sprouting process.
carbohydrates were observed in wheat and especially in barley and
quinoa flours. These three grains showed the highest decrease of starch
during sprouting process. A decrease of DF and, especially, IDF after
sprouting was also found. Compared to whole grains, sprouted wheat,
barley and chickpea grains showed higher level of SDF. Sprouting
process led to a significant (P < 0.05) increase of total phenols,
especially for wheat and barley grains.
By considering the species-level (Fig. S2) taxonomic assignments
and significant correlations at FDR < 0.05, OTUs co-occurrence was
investigated. The most significant co-occurrences were identified for
Staphylococcaceae (M. caseolyticus and S. hominis) with Lactobacillus
species, P. pentosaceus, and S. thermophilus. W. confusa was positively
correlated with Halospirulina and Bacillus sp. On the contrary, W. con-
fusa showed negative correlations with Lysinibacillus sp., Lc. lactis,
Staphylococcus sp., Carnobacterium sp., E. casseliflavus and E. termitis. Lc.
lactis was negatively correlated with Halospirulina sp., and Bacillus sp.
Positive correlations (r > 0.70; FDR < 0.05) were found between
cell density of presumptive LAB and yeasts with the time of sprouting
process (previously selected for each grain based on the achievement of
a length of rootlets corresponding to ca. 3/4 of the medium seed length,
before the seedling development) and the concentrations of free sugars
content in flours (Fig. S3). On the contrary, negative correlations were
found with soluble dietary fibers and total polyphenols. At species level,
E. casseliflavus, Enterococcus sp., E. termitis, and Carnobacterium sp., were
positively correlated with the time of sprouting process. On the con-
trary, B. gibsonii, Paenibacillus sp., S. hominis, Saccharibacillus, Staphy-
lococcus sp., L. brevis and L. fermentum showed negative correlations.
Other significant correlations were found among specific OTUs and free
sugars, soluble dietary fibers and total polyphenols.
4. Discussion
The conditions used for cereals, pseudocereals and legumes
sprouting in this study were comparable to those used for the same
grains (Montemurro et al., 2019), rye (Katina et al., 2007) and for in-
dustrial malting of barley, and are known to result in enhanced enzyme
activity (Laitila et al., 2006). The sprouting process increased the
8
LWT - Food Science and Technology 126 (2020) 109314
carbon and nitrogen source utilization patterns (carbohydrates, poly-
mers, amines, carboxylic acid and amino acids). The metabolic profiles
of flours are likely to vary according to changes in the microbial po-
pulation during sprouting process. The cell density of LAB was highest
in flours obtained from sprouted grains. Several authors already ob-
served microbial cell densities in grains between 3.0 and 6.0 Log cfu/g
and 2 or 3 log cycles greater in germinated grains than un-sprouted
grains (Yang et al., 2013).
Although with different magnitudes, sprouting caused a different
assembly of the microbiome component of the related-flours. The shifts
had different extents depending on the grains and microbial group in-
vestigated. According to previous studies (Gobbetti et al., 2016; Nachi,
Fhoula, Smida, Ouzari, & Hassoun, 2019; Weckx et al., 2010), P. pen-
tosaceus, Lc. lactis, E. faecium, E. termitis and L. fermentum were found in
wheat flours, highlighting the dominance of coccus-shaped (e.g. En-
terococcus sp.) LAB. This study showed that the sprouting process of
wheat grains modified the dominant species composition in flour (E.
casseliflavus instead of E. termitis) and strongly increased the total
number of strains. Previously, E. casseliflavus was isolated from maize
flour-based traditional Portuguese sourdough (Rocha & Malcata, 2012)
and during the first step of durum wheat dough fermentation (Alfonzo
et al., 2017). Nachi et al. (2019) showed that autochthonous E. casse-
liflavus strains had interesting technological features and thus could be
used in Tunisian sourdough production. However, other studies are
needed to define the role of E. casseliflavus in sourdough biotechnology.
Enterococcus species represent contaminants from the environment and
grains processing. They have been reported to be present in water, soils,
cereal flours and other vegetable materials (De Vuyst & Neysens, 2005).
Compared to un-sprouted grains, Lc. lactis and P. pentosaceus strains
strongly increased in wheat flour produced by sprouted grains. Pre-
viously, P. pentosaceus was described as a dominant species in many
mature sourdoughs (De Angelis, Minervini, Siragusa, Rizzello, &
Gobbetti, 2019; Van Kerrebroeck, Maes, & De Vuyst, 2017). Meta-
transcriptome data confirmed the effects of Lc. lactis in the early stage of
sourdough preparation and the dominance of P. pentosaceus with a key
metabolic role in dough fermentation (Weckx et al., 2010). The use of
wheat flours from sprouted grains with enriched amount of Lc. lactis
G. Perri, et al. LWT - Food Science and Technology 126 (2020) 109314

Fig. 7. Proximate composition in protein (A), fat (B), carbohydrates (C), starch (D), total fiber (E), soluble dietary fiber (F), insoluble dietary fiber (G), ash (H) and
total phenols (I) of flours obtained from wheat, barley, chickpea, lentil, and quinoa grains, before and after sprouting process. Data are the means ± standard
deviations from three independent experiments analyzed in triplicate. Values with different superscript letters (within each panel), differ significantly (P < 0.05).
Values are expressed on dry weight basis. Proteins, starch, fat, ash, total free carbohydrates were expressed as % of dry matter (d.m.).

and P. pentosaceus could differently affect the stability of the mature
sourdough LAB biota compared to un-sprouted grains flour (Gobbetti
et al., 2016). Indeed, the highest diversity of LAB could increase the
enzymatic portfolio of the sourdoughs affecting the metabolic activities
during wheat dough fermentation of leavened baked goods (Gänzle,
2014). Upon culture-independent analysis, wheat flour from sprouted
grains was characterized by a low number of LAB OTUs, among which
Lc. lactis and E. casseliflavus were the most important. Sprouting was
shown to reduce the microbial diversity, indicating selection and
dominance by process-dependant microorganisms (Justé et al., 2011).
According to the most recent literature (De Angelis et al., 2019;
Gobbetti, De Angelis, Di Cagno, Polo, & Rizzello, 2019; Gobbetti et al.,
2016) the succession of dominating and sub-dominating populations of
LAB occurring during sprouting process, lead to the assembly of the
sourdough-like bacterial community.
E. faecium and E. casseliflavus were found in barley flours upon
culture dependent analysis. The sprouting process of barley grains
modified the dominant species composition in flour and L. fermentum
was also detected. According to Booysen, Dicks, Meijering, and
Ackermann (2002) the indigenous microbial community of barley
harbours a wide range of microorganisms and the predominant LAB
species were isolated from malt in the germination vessels. When barley
flour was used for sourdoughs fermentation, doughs were characterized
by the presence of L. fermentum (Harth, Van Kerrebroeck, & De Vuyst,
2016). In flour from un-sprouted barley grains, a predominance of
bacterial OTUs assigned to members of the Enterococcaceae, Strepto-
coccaceae and Leuconostoccaceae families were observed. As previously
reported (Justé et al., 2014), Weissella was found as the most dominant
LAB genus during malting of barley, according to the shifts in microbial
community structure, explained by the changing incubation conditions
during the process, such as temperatures and humidity.
Sprouting process of legume grains modified the dominant species
composition in flour (L. fermentum appeared/disappeared in chickpea
and lentil flours, respectively). Among LAB, relevant increases of E.
casseliflavus and Lc. lactis OTUs were found in chickpea flour from
sprouted grains. Increases of Enterococcus spp. and Lactococcus strains
during sourdoughs fermentation were already observed in kabuli
chickpeas (Sáez, Saavedra, Hebert, & Zárate, 2018). Chickpea and lentil
raw flours harboured the highest OTUs belonging to Staphylococcaceae.
Sprouting process led to a decrease of Staphylococcaceae species (S.
hominis, M. caseolyticus and Staphylococcus sp. species). Compared to the
raw quinoa flour, Lc. lactis increased after sprouting.
In agreement with several authors (O'Sullivan, Walsh, O'Mahony,
Fitzgerald, & van Sinderen, 1999), D. hansenii, C. lusitaniae and C. in-
termedia yeasts were variously detected in sprouted flours.
Compared to flours from un-sprouted grains, germinated flours
contain high amount of phenolic compounds (Katina et al., 2007; Singh,
Rehal, Kaur, & Jyot, 2015), soluble fibers (Benincasa et al., 2019;

9
G. Perri, et al.
Montemurro et al., 2019) and sugars. As expected, these differences,
together with the sprouting process parameters (moisture, temperature,
duration) reflect on the derived flours microbiota. Indeed, specific
correlations were found between sprouting time, concentration of free
sugars in flours, total polyphenols and soluble dietary fibers with both
the relative abundance of some bacterial species and the overall cell
density of LAB and yeasts. This latter, in particular, is positively cor-
related with the sprouting process duration and the concentration of the
free sugars. These findings could have technological/practical im-
plications, since a role of the microbial consortium and its metabolome
on the organoleptic characteristics of the flours can not be excluded and
deserve further investigations. Similarly, the microbiota dynamics in
bread-making processes combining sprouting and sourdough fermen-
tation (Hassani, Procopio, & Becker, 2015) must be taken into account
in future researches.
CRediT authorship contribution statement
Giuseppe Perri: Investigation, Formal analysis. Francesco Maria
Calabrese: Data curation, Formal analysis. Carlo Giuseppe Rizzello:
Methodology, Resources. Maria De Angelis: Writing - review & editing,
Project administration, Funding acquisition. Marco Gobbetti:
Supervision, Conceptualization. Maria Calasso: Conceptualization,
Investigation, Writing - original draft.
Declaration of competing interest
The authors have no competing interests.
Acknowledgements
This study was funded by UE-FSE-FSER, PON RI 2014-2020 Action
I.1—“Innovative doctoral of industrial interest”— XXXII cycle, PhD
Program “Soil and Food Sciences— DOT1302942”—Grant n°1.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lwt.2020.109314.
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