International Journal of Food Microbiology 266 (2018) 173–182

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Use of hop extract as antifungal ingredient for bread making and selection of T
autochthonous resistant starters for sourdough fermentation
⁎
Luana Nionellia, Erica Pontonioa, Marco Gobbettib, Carlo Giuseppe Rizzelloa,
a
Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy
b
Facoltà di Scienze e Tecnologie, Free University of Bozen-Bolzano, 39100 Bolzano, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: Aiming at meeting the consumers' demand in terms of bio-preservation, the potential of the combination of the
Hop extract lactic acid bacteria fermentation and the addition of hop extract as natural preservative in breadmaking, was
Antifungal activity exploited. The antifungal properties of a hop (Humulus lupulus) extract were investigated, showing a significant
Sourdough inhibition of the hyphal growth of Aspergillus parasiticus, Penicillium carneum, Penicillium polonicum, Penicillium
Lactic acid bacteria
paneum, Penicillium chermesinum, Aspergillus niger, Penicillium roqueforti. Lactic acid bacteria belonging to species
of Enterococcus feacium, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus curvatus,
Pediococcus pentosaceus, and Pediococcus acidilactici were isolated from hop and subjected to selection based on
kinetics of growth and acidification. The sourdough (hS) enriched with hop extract (hE), started with three
selected strains, had phenols concentration and antioxidant activity higher than those obtained in the same
condition but without the hE. Hop-sourdough used in breadmaking delayed the fungal growth (14 days), giving
a bread characterized by free aminoacids concentration, antioxidant and phytase activities higher than bread
started only with baker's yeast, with or without the addition of hE. Specific volume and cell-total area of the
bread containing hE improved, and its sensory profile was characterized by typical sourdough attributes, and a
moderate bitter/herbaceous perception.

1. Introduction
Current consumer trends regarding the demand for fresh and
minimally processed food products without additional chemical pre-
servatives have promoted the research for new preservation strategy
(Kramer et al., 2015). Naturally occurring compounds that show anti-
microbial activity against spoilage and pathogenic microorganisms
could potentially be applied for food preservation to extend the shelf-
life and to improve the safety of foods and beverages at the same time
(Kramer et al., 2015). Several recent studies were focused on natural
preservatives from plant matrices, such as proteins and peptides or
essential oils, to be used as food ingredients (Kramer et al., 2015;
Rizzello et al., 2017).
The hop plant Humulus lupulus belongs to the family of the
Cannabinaceae has been used for beer brewing since ancient times.
Besides giving beer its typical bitter taste, hop compounds possess
distinctive antimicrobial and antioxidant properties (Kramer et al.,
2015).
The antimicrobial activity of bitter acids, namely α- (humulones)
and β-acids (lupulones), and their isomers, was largely demonstrated
mainly against Gram-positive bacteria (Kramer et al., 2015). Different
fungi are also inhibited by hop acids and prenylchalcones (Mizobuchi
and Sato, 1985). Recently additional antimicrobial compounds were
isolated, including xanthohumol, a broad spectrum anti-infective agent
working towards many bacteria, viruses, fungi and protozoa (Natarajan
et al., 2008), and humulinic acids, a non-bitter derivatives of iso-α-
acids (Schurr et al., 2015).
Besides the recognized potential to reduce the beer spoilage, the
applicability of hop compounds as possible food preservatives has not
been extensively studied, although few studies reported the inhibition
of foodborne pathogens in cheeses (Larson et al., 1996) and meat
products (Kramer et al., 2015).
Overall, the use of antifungal compounds from plants was proposed
for extending the baked goods shelf-life (Coda et al., 2008; Rizzello

Abbreviations: hE, hop extract; WFH, wheat flour hydrolyzate; WFH-he, hop extract wheat flour hydrolyzate; WFH-cp, calcium propionate wheat flour hydrolyzate; WSE, water/salt-
soluble extracts; TTA, Total titratable acidity; DY, dough yield; MIC, minimal inhibitory concentration; ME, methanolic extract; FQ, fermentation quotient; hS, hop sourdough; S, wheat
sourdough; WB, wheat bread; WBcp, calcium propionate wheat bread; WBhE, hop extract wheat bread; SWBhE, hop extract sourdough wheat bread; TFAA, Total free amino acids; aw,
water activity
⁎
Corresponding author.
E-mail address: carlogiuseppe.rizzello@uniba.it (C.G. Rizzello).

https://doi.org/10.1016/j.ijfoodmicro.2017.12.002
Received 23 May 2017; Received in revised form 20 November 2017; Accepted 1 December 2017
Available online 05 December 2017
0168-1605/ © 2017 Elsevier B.V. All rights reserved.
L. Nionelli et al.
et al., 2015). Indeed, fungal contamination is the most common and
costly spoilage for bakeries and, in many cases, it is the factor governing
shelf-life (Rizzello et al., 2017).
It was already demonstrated that the water-soluble extract from
Phaseolus vulgaris cv Pinto contained phaseolin alpha-type precursor,
phaseolin erythroagglutinating and phytohemagglutinin inhibited a
large spectrum of fungal species isolated from bakeries (Coda et al.,
2008). Moreover, the prolonging of the shelf-life through the inhibition
of the mold contamination was recently reported by using pea, lentil
and faba bean flour hydrolysates as ingredients for bread making
(Rizzello et al., 2015, 2017). In these cases, the antifungal activity was
attributed to native proteins (nsLTP, ubiquitin, lectin alpha-1 chain,
wound-induced basic protein, defensin-1, defensin-2) and mixture of
peptides, which were released during hydrolysis from legume vicilins,
lectins and chitinases (Rizzello et al., 2015, 2017). Also an extract of
amaranth seeds was used as an ingredient for the manufacture of
gluten-free and wheat flour breads, and inhibitory activity, due to ag-
glutinin peptides, was achieved during long-term storage under pilot
plant conditions (Rizzello et al., 2009). The same advantage was ob-
served for breads enriched with sourdough-fermented wheat germ, able
to delay fungal growth for at least 28 days (Rizzello et al., 2011).
Besides plant compounds, metabolites produced by sourdough lactic
acid bacteria and non-Saccharomyces yeasts during fermentation have
been screened for their antimicrobial properties (Coda et al., 2008,
2011b, 2013; Rizzello et al., 2011). In particular, lactic acid bacteria are
considered as efficient bio-preservative organisms because of their
acidification capacity through the lactic acid fermentation, but also for
the ability to synthesize or release various antimicrobial and antifungal
molecules, like bacteriocins, organic acids, bioactive peptides (Crowley
et al., 2013; Gerez et al., 2009; Schnürer and Magnusson, 2005).
Despite many promising results recently collected by the scientific
community, the problem of fungal contamination in the baked goods
sector remains still unsolved and much awaited by consumers and
manufactures, especially due to the lacking of a large activity spectrum
when antifungal plant matrices and starters were used singly (Rizzello
et al., 2017).
In this work, a hop extract was characterized for the antifungal
activity and used as ingredient aiming to prolong the shelf-life of bread.
Sourdough fermentation with lactic acid bacteria, isolated from hop,
characterized and selected for pro-technological characteristics and hop
resistance, was employed for bread making. First, the effects of the hop
extract on the selected lactic acid bacteria performances and on the
sourdough characteristics were evaluated. Then, the effects of the ad-
dition of the hop extract, together with the sourdough fermentation, on
the shelf-life, as well as on rheological and sensory features of wheat
bread, were also assessed.
2. Materials and methods
2.1. Hop and hop extract
Commercial Amarillo hop cones (Pinta, Disegna Group, Marostica,
Italy) were used to obtain the hop extract. The proximal composition of
the hop was: moisture, 11.0%; protein, 15.2%; fat, 3.4%; dietary fibers,
46.2%; total soluble carbohydrates, 2.0%; polyphenols and tannins,
4.7%; α-acids 11.1%, β-acids, 6.5%. To obtain the hop extract (hE), 1 g
of milled hop was resuspended in 100 ml of distilled water, homo-
genized, and boiled for 1 h aiming at the isomerization of the hop acids
(Jaskula et al., 2008). After cooling at room temperature, the extract
was centrifuged (10,000 × g) for 10 min to remove eventual suspended
materials and the supernatant was filtered using a 0.22 μm filter (Mil-
lipore Corporation, Bedford, MA 01730). Analysis of the total iso-α-
acids in hE was carried out by HPLC, with an ÄKTA Purifier system (GE
Healthcare, Buckinghmshire, UK) equipped with a Kinetex 2.6 column
(Phenomenex, Torrance, CA, USA) and an UV detector operating at
174
International Journal of Food Microbiology 266 (2018) 173–182
270 nm. The analysis was carried out at 1 ml/min flow, 40 °C, using an
eluent constituted by 85% (vol/vol) methanol, 0.05% phosphoric acid
(85%) and 0.05 mM EDTA (isocratic).
2.2. Antifungal activity of the hop extract
Seventeen indicator microorganisms were used for antifungal assays
since previously identified as the most common baked goods spoilage
molds (Coda et al., 2008; Rizzello et al., 2009). Penicillium polonicum
CBS 112490, Penicillium chrysogenum CBS 111214, Penicillium paneum
CBS 101032, Penicillium albocoremium CBS109582, Penicillium cherme-
sinum CBS117279, Penicillium carneum CBS112297, Eurotium herbar-
iorum CBS117336, Eurotium rubrum CBS150.92, Aspergillus parasiticus
CBS971.97, Aspergillus versicolor CBS117286, P. bialowiezense
CBS110102 and Penicillium brevicompactum CBS28997 were from the
Culture Collection of Centraalbureau voor Schimmelcultures (Utrecht,
Holland); Penicillium roqueforti DPPMAF1, Penicillium aethiopicum
DPPMAF2, Aspergillus niger DPPMAF3, Aspergillus penicilloides F1, and
Wallemia sebi F2 were from the Culture Collection of the Department of
Soil, Plant, and Food Sciences (Bari, Italy). Fungi were grown in Potato
Dextrose Agar (pH 5.6) (PDA, Oxoid) at 25 °C for 24–72 h, with the
exception of W. sebi F2, that was cultivated in Dichloran-Glycerol Agar
Base (DG18, Oxoid). The inhibitory activity of hE was assayed based on
hyphal radial growth rate of fungi (Coda et al., 2013). The hE was
added (25%, vol/vol, final concentration) to sterilized PDA or DG18.
After mixing, aliquots of 20 ml were poured into Petri plates (90 mm
diameter). Control plates contained PDA or DG18 supplemented with
25% (vol/vol) of sterile water. The assay was carried out by placing a 3-
mm diameter plug of growing mycelia onto the center of Petri dishes
containing the culture medium. Plates were incubated aerobically at
25 °C. Three replicates were run simultaneously. The radial growth of
mycelia (colony diameter, mm) in all plates was measured after 6 days
of incubation. Each datum point is the mean of at least four measure-
ments of a growing colony. The percentage of growth inhibition was
calculated from mean values as follows: percentage of inhibition =
[(mycelial growth under control conditions − mycelial growth in the
presence of hE)/mycelial growth under control conditions] × 100.
The effect of hE on the germination of conidia was determined
(Coda et al., 2008, 2013) on the indicator P. roqueforti. Wheat flour
hydrolysate (WFH) was chosen as the substrate since representative of
the chemical composition of wheat flour. WFH was obtained as pre-
viously described by Coda et al. (2013). A suspension of 20% wheat
flour (wt/vol) in tap water was incubated at 30 °C for 18 h under stir-
ring conditions (ca. 200 rpm). After incubation, the suspension was
filtered onto a Whatman apparatus (Polycarp 75 SPF, Whatman Inter-
national Maidstone, England) and added of yeast extract (0.3%, wt/
vol), sucrose, glucose and maltose (0.25% total concentration, wt/vol).
The WFH was sterilized by filtration on 0.22 μm membrane filters
(Millipore Corporation, Bedford, MA01730) and stored at 4 °C before
use.
After growth for 7 days on PDA plates, conidia of P. roqueforti
DPPMAF1 were harvested in sterile water, containing 0.05% (vol/vol)
Tween 80. The count of the conidia in the suspension was carried out
using the Petroff-Houser Counting chamber. A fixed number of ca.
106–107 conidia/ml was added to 5 ml of the mixture of WFH con-
taining 25% (vol/vol) hE (WFH-hE). The mixtures were incubated in
60 mm Petri dishes for 24 h at 25 °C under stirring conditions. WFH
alone and WFH added of 0.3% (wt/vol) calcium propionate (WFH-cp)
were used as controls. To determine the percentage of germinated
conidia (length/width ratio ≥ 2) at 16 and 24 h of incubation, aliquots
of the suspension were examined with a Zeiss (Weimar, Germany) op-
tical microscope (400 × magnification). Three separate replications of
at least 100 conidia were used for each assay. All assays for antifungal
activity were carried out at least in triplicate.
L. Nionelli et al.
2.3. Stability to proteolysis and pH
hE was treated with trypsin (EC 3.4.21.4, Sigma Aldrich Co.) as
described by Atanassova (2003). After treatments, the residual activity
was determined by agar diffusion assay on the indicator P. roqueforti
DPPMAF1. The dependence of the antifungal activity from the values of
pH was investigated by measuring the hyphal radial growth rate of the
indicator P. roqueforti DPPMAF1 in PDA adjusted at pH 4.0 to 8.0 (0.5-
intervals).
2.4. Microbiological analysis, isolation of lactic acid bacteria and genotypic
characterization by Randomly Amplified Polymorphic DNA-Polymerase
Chain Reaction (RAPD-PCR) analysis
For the microbiological analysis, 10 g of hop cones were finely
grounded by the laboratory mill Ika-Werke M20 (GMBH, and Co. KG,
Staufen, Germany) and homogenized with 90 ml of sterile peptone
water (1% [wt/vol] of peptone and 0.9% [wt/vol] of NaCl) solution.
Lactic acid bacteria were enumerated using MRS (Oxoid, Basingstoke,
Hampshire, UK) agar medium, supplemented with cycloheximide
(0.1 g/l). Plates were incubated, under anaerobiosis (AnaeroGen and
AnaeroJar, Oxoid), at 30 °C for 48 h. Cell densities of yeasts and molds
were estimated on Yeast extract Peptone Dextrose Agar (YPD) (Oxoid)
medium (pour and spread plate enumeration, respectively), supple-
mented with chloramphenicol (0.15 g/l), at 30 °C for 72 h. The attri-
bution was confirmed by microscope observation. Total mesophilic
aerobic bacteria were determined on Plate Count Agar (PCA, Oxoid) at
30 °C for 48 h, and total enterobacteria were determined on Violet Red
Bile Glucose Agar (VRBGA, Oxoid) at 37 °C for 24 h.
At least ten colonies of presumptive lactic acid bacteria were ran-
domly selected from the MRS plates containing the two highest sample
dilutions. Gram-positive, catalase-negative, non-motile rods and cocci
isolates were cultivated into MRS at 30 °C for 24 h and re-streaked onto
the same agar medium. All isolates considered for further analyses were
able to acidify the culture medium. Genomic DNA from pure cultures of
bacterial strains was extracted using a DNeasy blood and tissue kit
(Qiagen, SA, Courtaboeuf, France), according to the manufacturer's
instructions (Ahmed et al., 2009). Three oligonucleotides, P4 5′-CCGC
AGCGTT-3′, P7 5′-AGCAGCGTGG-3′ (Corsetti et al., 2003) and M13
5′-GAGGGTGGCGGTTCT-3′ (Stendid et al., 1994), with arbitrarily
chosen sequences, were used for bio-typing of lactic acid bacteria iso-
lates. Reaction mixture and PCR conditions for primers P4 and P7 were
those described by Corsetti et al. (2003), whereas those reported by
Zapparoli et al. (1998) were used for primer M13. RAPD-PCR profiles
were acquired by the MCE-202 MultiNA microchip electrophoresis
system (Shimadzu Italia s.r.l., Milan, Italy), using the DNA-2500 re-
agent kit (100 to 2500 bp) and the 2-logDNAladder (0.1 to 10.0 kb)
(Promega s.r.l., Padova, Italy) according to the manufacturer's in-
structions. Determining the Dice coefficients of similarity and using the
UPGMA algorithm evaluated the similarity of the electrophoretic pro-
files.
2.5. Genotypic identification of lactic acid bacteria
To identify presumptive lactic acid bacteria, two primer pairs
(Invitrogen Life Technologies, Milan, Italy), LacbF/LacbR and LpCoF/
LpCoR, were used for amplifying the 16S rDNA (De Angelis et al.,
2006). The expected amplicons of ca. 1400 and 1000 bp were eluted
from the gel and purified by the Nucleospin gel and PCR clean-up kit
(Macherey–Nagel, Düren, Germany). Primers designed for the recA
gene were also used to distinguish Lactobacillus plantarum, Lactobacillus
pentosus, and Lactobacillus paraplantarum species (Torriani et al., 2001).
PCR products were separated by electrophoresis, purified as described
above, and subjected to Sanger sequencing (Sanger et al., 1977).
Taxonomic strain identification was performed by comparing the se-
quences of each isolate with those reported in the NCBI Reference
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International Journal of Food Microbiology 266 (2018) 173–182
Sequence (RefSeq) database (Altschul et al., 1997). Strains showing
homology of at least 97% were considered to belong to the same species
(Goebel and Stackebrandt, 1994).
2.6. Characterization and selection of the autochthonous lactic acid
bacteria
The acidification capacity of the 13 autochthonous lactic acid bac-
teria strains was evaluated by singly inoculating wheat flour dough
containing 25% (vol/wt) of hE. In details, the lactic acid bacteria strains
were cultivated into MRS broth at 30 °C for 24 h. Cells were harvested
by centrifugation (10,000 × g, 10 min, 4 °C), washed twice in 50 mM
sterile potassium phosphate buffer (pH 7.0) and re-suspended in tap
water at the cell density of ca. 8.0 log cfu/ml. Sixty-two grams of wheat
flour, 25 ml of hE, and 12.5 ml of tap water, containing the cell sus-
pension of each lactic acid bacterium (cell density in the dough of ca.
107 cfu/g), were used to prepare 100 g of dough. Dough yield (DY,
dough weight × 100/flour weight), was 160. Fermentation was carried
out at 30 °C for 16 h.
The characteristics of the flour (Triticum aestivum, cv Appulo) used
were the following: moisture, 14.2%; protein (N × 5.70), 11.5% of dry
matter (d.m.); fat, 1.6% of d.m.; ash, 0.6% of d.m.; and total soluble
carbohydrates, 1.5% of d.m.
Mixing was done manually for 5 min. Wheat doughs were fermented
at 30 °C for 16 h. Kinetics of growth and acidification were determined
and modelled in agreement with the Gompertz equation, as modified by
Zwietering et al. (1990): y = k + A exp.{−exp.[(μmax or Vmax e/A)
(λ − t) + 1]}; where y is the growth expressed as log cfu/g/h or the
acidification rate expressed as dpH/dt (units of pH/h) at the time t; k is
the initial level of the dependent variable to be modelled (log cfu/g or
pH units); A is the cell density or pH (units) variation (between in-
oculation and the stationary phase); μmax or Vmax is the maximum
growth rate expressed as Δlog cfu/g/h or the maximum acidification
rate expressed as dpH/h, respectively; λ is the length of the lag phase
measured in hours. Fermentations were carried out in triplicate. The
experimental data were modelled through the nonlinear regression
procedure of the statistic package Statistica per Windows (StatSoft Inc.,
Tulsa, USA).
2.7. Minimum inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) of the hE towards the
selected strains Lb. plantarum/s2, Pediococcus pentosaceus/s2, and
Lactobacillus brevis/s1, was determined by adding hE to MRS broth at
concentration ranging 0 to 75%. To avoid the effect of the dilution of
the nutrients on bacterial growth due to the hE addition, a 4× stock
solution of sterilized MRS was used to prepare the substrate mixtures
(diluted with sterilized water when necessary), keeping concentration
constant at all the percentages of hE tested. The inoculum was of ca.
5 log cfu/ml. Growth of the lactic acid bacteria strains at 30 °C was
monitored over 16 h by measuring the optical density of the cultures at
620 nm using a microplate reader (Biolog Eco-Microplates, Biolog, Inc.,
Hayward, CA, USA). The MIC was defined as the lowest concentration
of hE required to completely inhibit the growth of the bacterium.
2.8. Sourdough fermentation and characterization
A pool of autochthonous lactic acid bacteria, consisting of Lb.
plantarum/s2, P. pentosaceus/s2, and Lb. brevis/s1 was used to obtain a
wheat flour sourdough containing 25% of hE (hS). Cell suspensions
were prepared as described elsewhere, the DY was 160 and the initial
cell density of lactic acid bacteria was 7.0 log cfu/g. Fermentation was
at 30 °C for 16 h. S was produced without the addition of hE (that was
substituted by tap water), fermented in the same conditions, and used
as control.
Total titratable acidity (TTA) was determined after homogenization
L. Nionelli et al.
of 10 g of dough with 90 ml of distilled water, and expressed as the
amount (ml) of 0.1 M NaOH needed to reach the value of pH of 8.3.
The water/salt-soluble extracts (WSE) of sourdoughs, which were
prepared according to Weiss et al. (1993), was used to analyze organic
acids, and total free amino acids (TFAA). Organic acids were de-
termined by High Performance Liquid Chromatography (HPLC), using
an ÄKTA Purifier system (GE Healthcare, Buckinghmshire, UK)
equipped with an Aminex HPX-87H column (ion exclusion, Biorad,
Richmond, CA), and an UV detector operating at 210 nm. Elution was at
60 °C, with a flow rate of 0.6 ml/min, using 10 mM H2SO4 as mobile
phase (Coda et al., 2011a). Fermentation quotient (FQ) was determined
as the molar ratio between lactic and acetic acids. TFAA were analyzed
by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cam-
bridge Science Park, England) with a Na-cation-exchange column (20
by 0.46 cm internal diameter), as described by Rizzello et al. (2010a,
2010b).
2.9. Total phenols and antioxidant activity
Total phenols concentration and antioxidant activity were de-
termined in sourdoughs and doughs before baking. The 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging activity was determined on
the methanolic extract (ME) of the dough. Five grams of each dough
were mixed with 50 ml of 80% methanol to get ME. The mixture was
purged with nitrogen stream for 30 min, under stirring condition, and
centrifuged at 4600 × g for 20 min. ME were transferred into test tubes,
purged with nitrogen stream and stored at ca. 4 °C before analysis. The
concentration of total phenols was determined as described by Slinkard
and Singleton (1997), and expressed as gallic acid equivalent. The free
radical scavenging capacity was determined as reported by Yu et al.
(2003) and expressed as follows: DPPH scavenging activity (%) =
[(blank absorbance − sample absorbance) / blank absorbance] × 100.
The value of absorbance was compared with 75 ppm butylated hydro-
xytoluene (BHT), which was used as the antioxidant reference.
2.10. Phytase activity
Phytase activity was determined on the water salt/soluble extract
(WSE) of sourdoughs and doughs before baking, by monitoring the rate
of hydrolysis of p-nitrophenyl phosphate (p-NPP) (Sigma, 104-0). The
assay mixture contained 200 μl of 1.5 mM p-NPP (final concentration)
in 0.2 M Na-acetate, pH 5.2, and 400 μl of WSE. The mixture was in-
cubated at 45 °C and the reaction was stopped by adding 600 μl of
0.1 M NaOH. The p-nitrophenol released was determined by measuring
the absorbance at 405 nm (De Angelis et al., 2003). One unit (U) of
activity was defined as the amount of enzyme required to liberate
1 μmol/min of p-nitrophenol under the assay conditions.
2.11. Baking test and bread characterization
To confirm the antifungal activity of hE, four types of wheat flour
bread (DY, 160) were manufactured at the pilot plant of the Department
of Soil, Plant and Food Sciences (Bari, Italy): WB, wheat flour bread
started with baker's yeast; WBcp, wheat flour bread started with baker's
yeast, added of 0.3% calcium propionate (wt/wt on flour weight basis);
WBhE, bread started with baker's yeast, containing 25% (vol/wt) hE;
SWBhE, sourdough bread, containing hS (25%, wt/wt) and hE (25%,
vol/wt).
WB and WBcp were manufactured according to the protocol routi-
nely used for typical Italian bread (Coda et al., 2013), and were used as
controls. Formulas for bread making are described in Table 1.
Doughs, having DY of 160 (corresponding to 62.5% of flour and
37.5% of water or water mixed with hE, respectively), were mixed at
60 ×g for 5 min with a IM 58 high-speed mixer (Mecnosud, Flumeri,
Italy). When hS was added at 25% (wt/wt) to the dough for making
SWBhe, the amount of wheat flour, hE and water were taken into
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International Journal of Food Microbiology 266 (2018) 173–182
Table 1
Formulas used for bread making. The dough yield of all breads was 160 and fermentation
with baker's yeast was allowed at 30 °C for 1.5 h. WB, wheat flour bread started with
baker's yeast; WBcp, wheat flour bread started with baker's yeast and added of 0.3%
calcium propionate (wt/wt on flour weight basis); WBhE, bread started with baker's yeast
and containing 25% (vol/wt) hE; SWBhe, sourdough bread (also including baker's yeast),
containing hop-sourdough (25%, wt/wt) and hop extract (25%, vol/wt).
Ingredients WB WBcp WBhE SWBhE
Wheat flour (g) 187.5 187.5 187.5 140.6
Tap water (ml) 112.5 112.5 37.5 28.1
Hop extract (ml) – – 75 56.3
Hop-sourdougha (g) – – – 75
Calcium propionate (g) – 0.6 – –
Baker's yeast (g) 6 6 6 6
a
Sourdough (hS) was obtained with 62.5% of wheat flour, 12.5% of water and 25% of
hop extract (DY 160). It was inoculated with a pool including Lactobacillus plantarum/s2,
Lactobacillus brevis/s1 and Pediococcus pentosaceus/s2 (ca. 7.0 log cfu/g) and fermented at
30 °C for 16 h.
account in the final calculation of DY and hE, aiming at a correct
comparison to the other samples (Table 1). Baker's yeast was always
added (2% wt/wt, corresponding to a final cell density of yeasts of ca.
9 log cfu/g). Fermentation was at 30 °C for 1.5 h. Before baking, doughs
were characterized as described before. All breads were baked at 190 °C
for 30 min using a Combo 3 oven (Zucchelli, Verona, Italy). Fermen-
tations were carried out in triplicate and breads were analyzed twice.
For each bread, 3 slices were cut. The size of the slice was 12 cm,
height, and 1.5 cm, width. Slices were inoculated by nebulization with
a suspension of 102 conidia/ml of P. roqueforti DPPMAF1 and packed in
polyethylene bags to maintain constant moisture and incubated at room
temperature for 21 days. The contamination was determined by visual
observation of the slices and approximately quantified with a scale from
“−” (no fungal growth observed) to “++++” (surface completely
covered by the mycelia of the indicator mold). Moisture was de-
termined according to the standard AACC method (AACC., 2003).
Water activity (aw) was determined at 25 °C by the Aqualab Dew Point
4TE water activity meter (Decagon Devices Inc., USA).
2.12. Texture, color, and image analyses of breads
Instrumental Texture Profile Analysis was carried out with a TVT-
300XP Texture Analyzer (TexVol Instruments, Viken, Sweden), equipped
with a cylinder probe P-Cy25S and a BVM-test system, using a Texture
Analyzer TVT-XP 3.8.0.5 software (Rizzello et al., 2012). The following
textural parameters were obtained by the texturometer software: hard-
ness (maximum peak force); fracturability (the first significant peak force
during the probe compression of the bread); and resilience (ratio of the
first decompression area to the first compression area).
The chromaticity co-ordinates of the bread crust: L, a, and b, cor-
responding to lightness, chromaticity on a green (−) to red (+) axis,
and chromaticity on a blue (−) to yellow (+) axis, respectively, were
determined by a Minolta CR-10 camera. The color difference, dE, was
calculated as follows: dE = (dL)2 + (da)2 + (db)2 , where dL, da, and
db are the differences for L, a, and b values between sample and re-
ference (a white ceramic plate having L = 93.4, a = −1.8, and b = 4.4
(Ahrné et al., 2007)).
The crumb features of breads were evaluated after 24 h of storage
using the image analysis technology and the UTHSCSA ImageTool
program (Version 2.0, University of Texas Health Science Centre, San
Antonio, Texas, available by anonymous FTP from maxrad6.uthscsa.
edu), as previously described by Rizzello et al. (2012).
2.13. Sensory analysis
Sensory analysis of breads was carried out by 10 panellists (5 male
L. Nionelli et al. International Journal of Food Microbiology 266 (2018) 173–182

and 5 female, mean age: 35 years, range: 18–54 years), according to the
method originally described by Haglund et al. (Haglund et al., 1998;
Rizzello et al., 2010a, 2010b). Elasticity, color of crust and crumb, acid
taste, acid flavour, sweetness, dryness, salty taste, and taste were con-
sidered as sensory attributes using a scale from 0 to10, with 10 the
highest score (Rizzello et al., 2010a, 2010b, 2015). Bitter (taste asso-
ciated with caffeine and quinine) and herbaceous (fresh-tasting notes of
cut grass and herbs) attributes were also included, since identified as hE
descriptors in the preliminary sessions of the panel test.

2.14. Statistical analysis

Data were subjected to one-way ANOVA; pair-comparison of treat-

ment means was achieved by Tukey's procedure at P < 0.05, using the
statistical software Statistica 12.0 (StatSoft).

3. Results

3.1. Antifungal activity of the hop extract

For the production of the hE, a protocol including the boiling aiming
at the isomerization of α- and β-acids was used. It was already reported
that the thermal treatment lead to the isomerization of ca. 30–50% of
the α-acids (Huang et al., 2013; Jaskula et al., 2008), while that of the
β-acids is negligible. As expected, the concentration of total iso-α-acids
in hE was 320 ± 20 mg/ml. The process parameters meets the
common protocols used in the beer industry where hops and hop-de-
rived products are usually boiled for 0.5 to 2.5 h (Verzele and De
Keukeleire, 1991a).
The pH of the hE was 4.90 ± 0.20. The spectrum of activity of the
hE was characterized through the inhibition of the hyphal radial growth
(Fig. 1). Among the 17 species of fungi assessed, the inhibition of A.
parasiticus CBS97197, P. carneum CBS 112297, P. polonicum CBS
112490, P. paneum CBS 101032, P. chermesinum CBS117279, A. niger
DPPMAF3, and P. roqueforti DPPMAF1 was higher than ca. 20%. The
inhibition of P. bialowienzense CBS110102, A. versicolor CBS117286, E.
rubrum CBS150.92, E. herbariorum CBS117336, P. chrysogenum CBS
111214, P. albocorenium CBS109582, and P. aethiopicum DPPMAF2 was
in the range of 8–13% while W. sebi F2, A.s penicilloides F1, and P.
brevicompactum CBS28997 were weakly affected (< 5%) by the pre-
sence of the hE in the media.
The hE was further characterized through the evaluation on the P.
roqueforti conidia germination. After 16 h at 25 °C, spore germination
was the highest for the control WFH alone (Fig. 2). The addition of
Fig. 2. Conidial germination of Penicillium roqueforti DPPMAF1incubated in WFH for 24 h
at 30 °C containing hE (25%, vol/vol). Data are the means for three independent ex-
periments. Bars of standard deviations are also represented.
0.3% (wt/vol) calcium propionate (WFH-cp) or hE (25% vol/vol) sig-
nificantly (P < 0.05) decreased the germination. The same trend was
found after 24 h of incubation in which the presence of hE in broth
decreased the germination of ca. 72 and 55% compared respectively to
WFH and WFH-cp (Fig. 2).
As shown by hyphal radial growth rate of P. roqueforti, trypsin di-
gestion did not affect the antifungal activity of hE, that resulted 35.5%
after the enzymatic treatment. Compared to pH from 4.0 to 5.5, values
ranging from 6.0 to 8.0 caused a decrease of ca. 30% of the inhibitory
activity of the hE towards P. roqueforti.
3.2. Isolation and identification of lactic acid bacteria from hop
Overall, data from the microbiological analysis of the hop showed
low microbial cell densities. Total mesophilic aerobic bacteria and en-
terobacteria were 4.69 ± 0.3 and 2.5 ± 0.2 log cfu/g, respectively.
The number of presumptive lactic acid bacteria was
2.95 ± 0.4 log cfu/g. Yeasts were not found in 10 g of hop, while

Fig. 1. Antifungal activity of the hE obtained from 1 g of

milled hop resuspended in 100 ml of distilled water,
homogenized, boiled, centrifuged, and filtered, as de-
termined by hyphal radial growth inhibition after 6 days of
incubation at 25 °C in PDA containing 25% (vol/vol) of the
extract. PDA containing 25% (vol/vol) of sterile water was
used as control.

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Fig. 3. Dendrogram of combined (primers P4, P7 and M13) RAPD profiles of lactic acid bacteria isolated from hop cones.

molds were 2.34 ± 0.1 log cfu/g.

Gram-positive, catalase-negative, non-motile rods and cocci able to
acidify the MRS broth were subjected to RAPD-PCR analysis. Primers
M13, P4, and P7 generated different patterns (bands ranging from 2500
to 100 bp), and were used for clusters analysis. The reproducibility of
the RAPD fingerprints was assessed by comparing the PCR products
obtained from three separate cultures of the same strain. The dendro-
gram of the 49 isolates is shown in Fig. 3. At the similarity level of 80%,
almost all isolates were grouped into 9 clusters. According to the sta-
tistical elaboration based on the RAPD profiles, three of the 9 clusters
(II, V, VIII) grouped identical isolates (100% similarity). Four strains
that did not group into clusters and 9 strains, as the representatives of
each cluster, were identified by partial sequencing of the 16S rRNA.
The following species were identified: Lb. plantarum (n.2 strains), P.
pentosaceus (n.2) Enterococcus faecium (n.5) Pediococcus acidilactici
(n.1), Lactobacillus helveticus (n.1), Lactobacillus curvatus (n.1), and Lb.
brevis (n.1).

3.3. Selection of lactic acid bacteria
Since the number of isolates was rather elevated, the selection of the
most performing strain was carried out on the 13 strains chosen on the
basis of the RAPD-PCR screening, as previously reported by literature
(Nionelli et al., 2014; Rizzello et al., 2010a, 2010b); acknowledging the
possibility to lose some strains with interesting phenotype features.
The lactic acid bacteria strains were singly used to ferment wheat
flour dough containing 25% (vol/wt) of hE. After fermentation, all
strains reached a cell density ranging from 8.9 ± 0.1 to
9.6 ± 0.2 log cfu/g. The length of the lag phase (λ) of growth varied
from 1.36 ± 0.12 to 3.18 ± 0.09 h. The lowest λ value was observed
for Lb. plantarum/s2 and P. pentosaceus/s2.
The values of Δ pH varied from 1.15 ± 0.04 to 2.45 ± 0.06
(median value of 1.25) (Fig. 4), whereas Vmax varied from 0.27 ± 0.03
Fig. 4. Variation of pH values (ΔpH) of hop sourdough (DY, 160) containing 25% (vol/
wt) of hE and singly inoculated with cluster representative lactic acid bacteria.
Fermentation was carried out at 30 °C for 16 h.
to 0.79 ± 0.02 dpH/h. The highest values of Δ pH were found for Lb.
plantarum/s2, P. pentosaceus/s2, P. pentosaceus/s1, and Lb. brevis/s1
(2.45 ± 0.06, 2.43 ± 0.02, 2.37 ± 0.02, and 2.22 ± 0.03, respec-
tively). Among these strains, P. pentosaceus/s1 showed a latency phase
of growth significantly (P < 0.05) longer than the others
(λ = 3.01 ± 0.10). Based on the shortness of the growth lag phase (λ)
and values of Δ pH, Lb. plantarum/s2, P. pentosaceus/s2, and Lb. brevis/
s1 were selected and used as mixed starter. All the parameters of the
kinetics of growth and acidification of the doughs fermented with such
strains are reported in Table 2. The hE MICs were 55, 52, and 58%,
respectively for Lb. plantarum/s2, P. pentosaceus/s2, and Lb. brevis/s1.

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Table 2
Parameters of kinetics of growth and acidification of sourdoughs (DY 160) containing hop extract (25%, vol/wt) and started with selected lactic acid bacteria strains.

Kinetic of growth Kinetic of acidification

A (log cfu/g) λ (h) μmax (Δlog cfu/g/h) ΔpH (pH units) λ (h) Vmax (Δ pH/h)

Lb. plantarum/s2 1.95 ± 0.08a 1.36 ± 0.12c 0.38 ± 0.03a 2.45 ± 0.06a 2.79 ± 0.10b 0.55 ± 0.03b
Lb. brevis/s1 1.76 ± 0.06b 2.65 ± 0.07a 0.34 ± 0.02b 2.22 ± 0.03b 2.57 ± 0.08c 0.39 ± 0.05c
P. pentosaceus/s2 1.75 ± 0.05b 2.22 ± 0.10b 0.42 ± 0.02a 2.43 ± 0.02a 3.17 ± 0.05a 0.60 ± 0.03a

a–c
Values in the same column with different superscript letters differ significantly (P < 0.05).

Table 3 Table 4
Chemical characteristics of the hop-sourdough (hS, DY 160) containing 25% (v/w) of hE. Fungal contamination of bread slices inoculated by nebulization with a suspension of
hS, started with Lb. plantarum/s2. P. pentosaceus/s2. and Lb. brevis/s1 (initial cell density 102 conidia/ml of Penicillium roqueforti DPPMAF1, during 21 days of incubation at room
of lactic acid bacteria. 7.0 log cfu/g, and fermented at 30 °C for 16 h. A control sourdough temperature. WB, wheat flour bread started with baker's yeast; WBcp, wheat flour bread
(S) was produced without the addition of hE and fermented in the same condition. started with baker's yeast and added of 0.3% calcium propionate (wt/wt on flour weight
basis); WBhE, bread started with baker's yeast and containing hop extract (25%, vol/wt);
S hS SWBhE, sourdough bread (also including baker's yeast), containing hop-sourdough (25%,
wt/wt) and hop extract (25%, vol/wt).
pH 3.82 ± 0.20a 3.85 ± 0.20a
TTA (ml of 0.1 N NaOH/10 g) 11.4 ± 1.0a 12.0 ± 1.1a Days WB WBcp WBhE SWBhE
Lactic acid bacteria cell density (log cfu/g) 9.7 ± 0.2a 9.9 ± 0.3a
Lactic acid (mmol/kg) 151 ± 5a 146 ± 10a 7 ++ − − −
Acetic acid (mmol/kg) 14 ± 2a 12 ± 2a 14 +++ +/− + +/−
FQ 11 ± 2a 12 ± 3a 21 ++++ + ++ +
TFAA (mg/kg) 1603 ± 75b 1850 ± 39a
Total phenols (mmol/kg) 6.17 ± 0.23b 19.87 ± 1.82a The ingredients used for bread making are described in Table 1.
Antioxidant activity1 41.7 ± 2.3b 74.8 ± 2.8a
Phytase activity (U)2 1.83 ± 0.12a 1.76 + 0.15a
contamination of bread slices was observed throughout 21 days
The data are the means of three independent experiments ± standard deviations (Table 4). In all cases, moisture was in the range 28–30%.
(n = 3). Slices of WBcp and SWBhE showed the appearance of fungal myce-
a–b
Values in the same row with different superscript letters differ significantly lium with ca. 7 days of delay with respect to WB. Contamination of
(P < 0.05).
1
SWBhE behaved similarly to WBcp (a moderate growth of the fungal
The antioxidant activity was determined based on the scavenging activity towards
mycelium on the slices surface at 21 days), while a markedly larger
DPPH radical after 10 min of reaction.
2
One unit (U) of phytase activity was defined as the amount of enzyme required to
contaminated surface was observed for WBhE.
release 1 μmol of phosphate per min under the assay conditions.

3.6. Bread characterization

3.4. Sourdough fermentation with selected starters
Autochthonous Lb. plantarum/s2, P. pentosaceus/s2, and Lb. brevis/
s1 were used in mixture to obtain two different sourdoughs; hS, con-
taining the hE, and S, produced without the addition of the hE. The pH
values before fermentation were 5.75 ± 0.10 and 5.95 ± 0.08, for hS
and S, respectively. No significant (P > 0.05) differences were found
for pH and TTA after fermentation (Table 3). Also, the final cell density
of presumptive lactic acid bacteria did not significantly (P > 0.05)
differ among sourdoughs (Table 3).
The kinetics of growth and acidification of the pool of selected starters
obtained in sourdoughs hS and S did not significantly (P > 0.05) differ.
The parameters for growth (mean values) were: A, 1.95 ± 0.08 log cfu/
g; λ, 1.36 ± 0.10 h; μmax, 0.38 ± 0.03 Δlog cfu/g/h, while ΔpH, λ,
and Vmax for acidification were 2.46 ± 0.05 pH units, 1.45 ± 0.09 h,
and 0.80 ± 0.02 ΔpH/h, respectively.
Sourdoughs were characterized by similar concentration of lactic
and acetic acids, and by a FQ ranging from 11 to 12 (Table 3). Also for
phytase activity, no significant (P > 0.05) differences were found.
Contrarily, total phenols were > 2-fold higher in hS compared to S.
Accordingly, the antioxidant activity, determined based on the
scavenging activity towards DPPH radical, was the highest for hS
(Table 3).
3.5. Baking test
According to formulas reported in Table 1, four different breads
were manufactured at the pilot plant. After baking, slices of each bread
were cut and inoculated with a suspension of (102 conidia/ml) of P.
roqueforti DPPMAF1. Slices were packed with polyethylene film to
avoid drying and stored at room temperature. The fungal
Table 5 summarizes the main chemical, textural, and color char-
acteristics of the breads. Overall, the use of calcium propionate (WBcb)
did not significantly (P > 0.05) affected any of the characteristics as-
sessed, when compared to the WB. However, as expected, the addition
of the hE in dough (WBhE) led to a slight but significant (P < 0.05)
decrease of the pH values, which was even more emphasized when also
sourdough was used (SWBhE), reaching the pH value of 4.40 ± 0.10.
An opposite trend was found for TTA, that was the highest for SWBhE, as
the consequence of the lactic fermentation, followed by WBhE. How-
ever, the aw was not affected by the use of hE and hS. The concentration
of total free amino acids (TFAA) found in bread doughs did not differ
between WB and WBcp, however it was significantly higher (P < 005)
than ca. 17 and 41% in WBhE and SWBhE, respectively (Table 5).
The addition of hE markedly increased the concentration of total
phenols in WBhE and SWBhE. In particular, the highest concentration
(> 2-times higher than WB) was found for SWBhE. As expected, anti-
oxidant activity changed according to the total phenols concentration
trend. Indeed, the DPPH scavenging activity of the methanolic extracts
(ME) of WBhE and SWBhE was respectively 23.5 and 32.5% higher than
that found for WB (Table 5).
The phytase activity of WB, WBcp, and WBhE did not significantly
(P > 0.05) differ, but it was significantly (P < 0.05) higher when
sourdough was added to the bread formula.
Among the structural characteristics of breads, specific volume and
hardness (corresponding to the peak force of the first compression of
the product) significantly (P < 0.05) changed as the consequence of
the hS addition. Compared to WB, specific volume of SWBhE sig-
nificantly (P < 0.05) increased, while hardness decreased of the 10%.
Resilience (how well the bread fights to regain its original position) and
fracturability (the first significant peak force during compression of the

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Table 5
Chemical and textural characteristics, and image and color analysis of breads. WB, wheat flour bread started with baker's yeast; WBcp, wheat flour bread started with baker's yeast and
added of 0.3% (wt/wt on flour weight basis) calcium propionate; WBhE, bread started with baker's yeast and containing hop extract (25%, vol/wt); SWBhE, sourdough bread (also
including baker's yeast), containing hop-sourdough (25%, wt/wt) and hop extract (25%, vol/wt).

WB WBcp WBhE SWBhE

Chemical characteristics
pH⁎ 5.58 ± 0.13a 5.48 ± 0.09a 5.09 ± 0.15b 4.40 ± 0.10c
Total titratable acidity⁎ 5.6 ± 0.5c 5.4 ± 0.4c 6.1 ± 0.2b 8.6 ± 0.5a
aw 0.980 ± 0.03 0.975 ± 0.05 0.974 ± 0.08 0.971 ± 0.08
TFAA (mg/kg)⁎ 851 ± 12c 848 ± 21c 998 ± 15b 1198 ± 20a
Total phenols (mmol/kg)⁎ 5.3 ± 0.1c 5.3 ± 0.2c 9.2 ± 0.2b 11.7 ± 0.3a
Antioxidant activity1⁎ 38.5 ± 2.1c 38.7 ± 0.8c 62.0 ± 2.5b 71.0 ± 1.5a
Phytase activity (U)2⁎ 1.51 ± 0.21b 1.48 ± 0.30b 1.52 ± 0.4b 1.75 ± 0.15a
Textural characteristics
Specific volume (cm3/g) 2.71 ± 0.12b 2.66 ± 0.15b 2.69 ± 0.09b 2.92 ± 0.11a
Hardness 2819 ± 15a 2820 ± 12a 2826 ± 15a 2706 ± 15b
Resilience 0.39 ± 0.03 0.37 ± 0.05 0.40 ± 0.02 0.40 ± 0.02
Fracturability (g) 2280 ± 10 2270 ± 15 2300 ± 20 2275 ± 20
Image analysis
Black pixel area (%) 40.2 ± 0.5b 39.8 ± 0.5b 40.6 ± 0.8b 43.5 ± 0.8a
Color analysis
L 55.1 ± 0.2a 56.0 ± 0.2a 51.5 ± 0.2b 41.1 ± 0.2c
a 4.6 ± 0.1b 4.4 ± 0.1b 6.4 ± 0.2a 6.5 ± 0.2a
b 22.2 ± 0.1a 22.1 ± 0.3a 19.8 ± 0.2b 19.7 ± 0.3b
dE 42.7 ± 0.3c 41.8 ± 0.4c 45.4 ± 0.2b 55.2 ± 0.3a

The data are the means of three independent experiments ± standard deviations (n = 3).
a–c
Values in the same row with different superscript letters differ significantly (P < 0.05).
⁎
The determination was carried out before baking.
1
The antioxidant activity was determined based on the scavenging activity towards DPPH radical after 10 min of reaction.
2
One unit (U) of phytase activity was defined as the amount of enzyme required to release 1 μmol of phosphate per min under the assay conditions.

bread), also determined by instrumental Texture Profile Analysis,
ranged from 0.37 ± 0.05 to 0.40 ± 0.02 and from 2270 ± 15 to
2300 ± 20, without significant (P < 0.05) differences among the four
breads.
Digital images were pre-processed to detect crumb cell-total area by
a binary conversion (black/white pixels). The cell-total area percentage
of SWBhE was slightly, but significantly (P < 0.05) higher than those
of the other experimental breads (Table 5).
The addition of hE and hS in bread formula, significantly
(P < 0.05) influenced the color of the crust, leading to a decrease of
lightness (L) and b values and to an increase of the a values (Table 5).
The color difference (dE), which was calculated based on the chroma-
ticity co-ordinates (L, a, and b), did not significantly (P > 0.05) differ
between WB and WBcp, while it significantly (P < 0.05) differed when
hE and hS were used in the bread formula (Table 5).
Few hours after baking, the sensory analysis was carried out (Fig. 5).
No significant (P > 0.05) differences were found for scores attributed
to elasticity. The score for color was similar between WB, WBcp, and
WBhE but it was significantly (P < 0.05) higher for SWBhE. Compared
to WB and WBcp, the breads containing the hE were characterized by
significantly (P < 0.05) higher scores for bitter and herbaceous taste
attributes. Sourdough bread SWBhE was also characterized by the
highest scores for acid taste, acid flavour, salty taste and overall per-
ception of taste (Fig. 5), while a slight but significant (P < 0.05) de-
crease of sweetness was observed (Fig. 5).
4. Discussion
Bitter acids and essential oils are pivotal constituents of hops. The
former, known as α- and β-acids, or humulones and lupulones, re-
spectively, are precursors of beer bittering agents (Verzele and De
Keukeleire, 1991b). Hop varieties contain up to 19% (wt/wt) α-acids
(super-α-hops), which nowadays are, to a great extent, extracted using
liquid or supercritical carbon dioxide (Benitez et al., 1997). As con-
sequence of the most important chemical conversion occurring during
wort boiling, namely, the thermal isomerization of the hop α-acids, six
major iso-α-acids are present in beer, i.e., the trans- and cis-isomers of
isocohumulone, isohumulone, and isoadhumulone. If upon wort
boiling, the isomerization yield of α-acids into iso-α-acids is invariably
low (at most 50–60%) and also subject to variations (Jaskula et al.,
2008), the β-acids are even less soluble and thus their isomerization
during wort boiling is therefore very low (Čulík et al., 2009). In this
study, hop cones containing 11.1% of α- and 6.5% of β-acids were re-
suspended in water and boiled. According to previous study (Huang
et al., 2013; Jaskula et al., 2008), the isomerization of ca. 30% of α-
acids occurred under extraction in the study conditions. The resulting
iso-α-acids are considered the most abundant and effective anti-
microbial hop compounds (Schurr et al., 2015). For centuries it has
been believed that hops protect beer from infection by most organisms,
including pathogens, but it was only in the 20th century that Shimwell
(1937a, 1937b) showed that hop compounds only inhibit growth of
Gram-positive bacteria and not of Gram-negative bacteria. The in-
hibitory mechanism has been already investigated (Simpson and Smith,
1992), and resulted related to the protonophoric action of hop weak
acids (Simpson and Smith, 1992; Simpson, 1993).
To date, the antibacterial activity of hop compounds and the hop
resistance of lactic acid bacteria have been investigated, while the
studies on antifungal activity of hop constituents are limited
(Gerhauser, 2005). However, contamination by fungi is the most
common cause of microbial spoilage and a costly problem for bakeries
(Rizzello et al., 2010a, 2010b). Salts of propionic, sorbic, and benzoic
acids are routinely used as chemical preservatives, but the European
directive on preservatives (Pattison et al., 2004) has decreased the al-
lowable concentrations of sorbate and propionate (0.2 and 0.3%, wt/wt
on flour weight basis, respectively). Prolonged preservation of bread
with a high calcium propionate concentration showed a strong in-
hibitory effect towards many fungi, but it also stimulated resistant
strains of P. roqueforti (Suhr and Nielsen, 2004). Under this study
conditions, hop extract exhibited discrete (> 20%) antifungal activity
against fungi considered the main responsible for bakery goods con-
tamination, A parasiticus, P. carneum, P. polonicum, P. paneum, P. cher-
mesinum, A. niger, and P. roqueforti. The other 10 fungi species were
only weakly inhibited (< 13%) by the presence of hE. A marked de-
crease of the germination of P. roqueforti DPPMAF1 was found in the

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presence of 25% (vol/vol) hE. P. roqueforti was used as the indicator
microorganism for antifungal assays since it corresponds to one of the
most common bread spoilage mold, presenting high resistance to the
preservatives commonly added to baked goods (Suhr and Nielsen,
2004; Coda et al., 2008; Rizzello et al., 2009).
Although the known antimicrobial activity against Gram-positive
bacteria of hop compounds, some strains appear to be able to grow
under such inhospitable conditions. Indeed, in this study, auto-
chthonous lactic acid bacteria were isolated from commercial hop.
Strains belonging to P. pentosaceus (32.6%), E. feacium (24.5%), Lb.
plantarum (20.4%), Lb. helveticus (10.2%), Lb. curvatus (6.1%), Lb. brevis
(4.1%), and P. acidilactici (2%) were identified. Seventy-five percent of
the hop microbiota was dominated by Lactobacillus and Pediococcus
species, which were found as the most hazardous gram positive bacteria
spoiling beer (Sakamoto and Konings, 2003). The representative of each
cluster were characterized for their pro-technological properties and
hop resistance, aiming at selecting strains to be used as starters for
fermentation of wheat doughs containing hop antimicrobial compo-
nents. Based on the kinetics of growth and acidification, three strains
belonging to Lb plantarum, Lb. brevis and P. pentosaceus species, were
found to be suitable for the fermentation of sourdough enriched with
hE. The strains were not inhibited until 52% hop extract in medium.
Hop-sourdough was obtained with one fermentation cycle and without
baker's yeast addition (type II-sourdough). It was characterized and
compared to a control sourdough produced without the addition of hE
and fermented in the same conditions, aiming at evaluating the effects
of the hE on the main biochemical properties and on the starters per-
formances. Under the experimental conditions (25% addition), growth
and acidification rate of the selected strains during sourdough fer-
mentation were not affected by the presence of hE. Nevertheless, as
expected, some sourdough characteristics changed. In particular, total
phenols were significantly higher in hS than the control, thanks to the
abundant hop polyphenols fraction (De Keukeleire, 2000). Conse-
quently, the antioxidant activity of hS followed the same trend. All the
other chemical properties, including organic acids concentration, fer-
mentation quotient, and phytase activity, were strictly influenced by
the lactic acid fermentation (Nionelli et al., 2014; Rizzello et al., 2016)
and thus independent from the addition of hE. In order to evaluate the
effect of the hop extract and sourdough on the technological and
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International Journal of Food Microbiology 266 (2018) 173–182
Fig. 5. Spider web chart of the sensory analysis
data for experimental breads. WB, wheat flour
bread started with baker's yeast; WBcp, wheat
flour bread started with baker's yeast, added of
0.3% (wt/wt on flour weight basis); WBhE, bread
started with baker's yeast, containing hop extract
(25%, vol/wt); SWBhE, sourdough bread, con-
taining hop-sourdough (25%, wt/wt) and hop
extract (25%, vol/wt).
sensory properties as well as the shelf-life of white bread, four bread
types were manufactured at pilot plant conditions and analyzed. As
expected, baker's yeast, used for bread making, was not be affected by
the presence of the hE. It was already found that the ability to increase
the proton-translocating ATPase levels upon exposure to protonophores
contributes considerably to the resistance of S. cerevisiae to hop com-
pounds (Viegas et al., 1998).
In this study, two wheat flour breads leavened with baker's yeast
and produced with or without the addition of calcium propionate, were
used as controls. The addition of hE in the bread formula delayed the
fungal growth at least until 14 days of storage at room temperature and
showed the same activity as the 0.3% (wt/wt) calcium propionate. The
aw value of the bread was not affected by hE addition, thus hypothe-
sizing that the inhibition strictly depends by hop weak acids.
Nevertheless, the combination of hE and sourdough fermentation gave
better results than the incorporation of the sole hE. An abundant lit-
erature already demonstrated the role of the sourdough in the pro-
longation of bread shelf-life (Coda et al., 2008, 2011b, 2013;
Lavermicocca et al., 2000), mainly due to the lactic acid bacteria
acidification during sourdough fermentation, but also to the synthesis
of a wide range of low-molecular-weight compounds (Niku-Paavola
et al., 1999), peptides (Okkers et al., 1999), and proteins (Rizzello et al.,
2011) with antifungal activity. As previously reported for wheat sour-
dough breads fortified with other vegetable ingredients (Nionelli et al.,
2014; Rizzello et al., 2012) the metabolic activities of lactic acid bac-
teria further enhanced the antioxidant and phytase activities and the
free amino acids concentration of SWBhE, that are considered properties
of great interest from the nutritional and functional point of view.
The use of hE and even more the combination with the sourdough
fermentation affected the texture and sensory properties of the bread.
Under the experimental conditions of this study, the color seemed to be
significantly affected by the addition of hE. The sensory acceptability of
the SWBhE was assessed by a panel test, that shown an organoleptic
profile appreciated by the panellists, with high score for the overall
intensity of taste. In particular, SWBhE was characterized by a lower
intensity of dryness and sweetness compared to other breads, and more
pronounced acid and salty taste and flavour, due to the lactic acid
fermentation and the effect of the proteolysis by selected lactic acid
bacteria. Due to the incorporation of the hE, the SWBhE was
L. Nionelli et al.
characterized by a bitter and herbaceous taste compared to the control;
however, it was judged acceptable by the panellists.
In this study, the promising use of hop sourdough as a natural
biopreservative to considerably extend the shelf-life of bread, without
affecting its rheological and sensory properties, was demonstrated.
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