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Effect of Lactobacillus brevis - based bioingredient and bran on microbio-logical, physico-chemical and textural quality of yeast leavened bread duringstorage

Valerio Francesca, Di Biase Mariaelena, Caputo Leonardo, Creanza TeresaM., Ancona Nicola, Visconti Angelo, Lavermicocca Paola

PII: S1466-8564(13)00141-0DOI: doi: 10.1016/j.ifset.2013.09.003Reference: INNFOO 1059

To appear in: Innovative Food Science and Emerging Technologies

Received date: 30 April 2013Accepted date: 11 September 2013

Please cite this article as: Francesca, V., Mariaelena, D.B., Leonardo, C., M., C.T.,Nicola, A., Angelo, V. & Paola, L., Effect of Lactobacillus brevis - based bioingredi-ent and bran on microbiological, physico-chemical and textural quality of yeast leav-ened bread during storage, Innovative Food Science and Emerging Technologies (2013), doi:10.1016/j.ifset.2013.09.003

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Effect of Lactobacillus brevis - based bioingredient and bran on microbiological, physico-

chemical and textural quality of yeast leavened bread during storage

Valerio Francescaa, Di Biase Mariaelena

a, Caputo Leonardo

a, Creanza Teresa M.

b, Ancona Nicola

b,

Visconti Angeloa and Lavermicocca Paola

a*,

a Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126

– Bari

b Institute of Intelligent Systems for Automation, National Research Council, Via Amendola 122/D-

I - 70126 – Bari

*Corresponding author:

E-mail: paola.lavermicocca@ispa.cnr.it

Phone: +39 080 5929356; Fax : +39 080 5929374

Mailing address: Istituto di Scienze delle Produzioni Alimentari – CNR, Via Amendola

122/O, 70126 – Bari, Italy

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Abstract

The effects of wheat bran and of a Lactobacillus brevis based-bioingredient (LbBio), obtained after

growth in flour-based medium, on quality of yeast leavened wheat bread (WWB) were investigated.

Bran was used in bread formulation by substituting a part (20 g/100 g) of white wheat flour (WBB),

while LbBio was used instead of the water content (WWB+LbBio and WBB+LbBio). The use of

LbBio in WWB resulted in the biological acidification of the dough due to lactic, phenyllactic and

OH-phenyllactic acid contents determining a high fermentation quotient value and an improved

bread texture and microbiological quality. Conversely, wheat bran reduced the specific volume and

crumb hardness during storage at 25 °C, and affected the antibacterial ability of LbBio during 30°C

storage. Our findings demonstrated that LbBio counteracted the negative effects of bran and

allowed to obtain an enriched fibre bread with specific volume and soft crumb comparable to bread

without bran.

Industrial relevance

Bread is a perishable food with a short microbiological and physico-chemical shelf-life. The main

microbiological alteration occurring into few days after baking is the “rope spoilage” caused by

spore-forming bacteria originating from raw materials. This phenomenon, often misinterpreted as a

sign of unsuccessful dough leavening and not visible from outside, is more common under

industrial production conditions during the hot season causing large economic losses in the warm

climates of Mediterranean countries, Africa and Australia. The use of sourdough often controls this

alteration even if the industrial application of this traditional process is limited by the long

leavening times. In this study an innovative procedure for the preparation of yeast-leavened bread

comprising the addition of a fermentation product from Lactobacillus brevis grown in a flour-

based medium, has been applied. The resulting fementation product (LbBio bioingredient) acts as a

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sourdough acidifying the dough and improving the textural, physico-chemical and microbiological

properties of the resulting bread. The application of bioingredient LbBio could represent an

innovative strategy in industrial bread production to obtain acidified yeast leavened products

thus preventing the ropy spoilage and reducing the negative effects of bran addition.

Keywords: ropy spoilage, firmness, spore-forming bacteria, organic acids, bioingredient; two-ways

ANOVA.

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1. Introduction

Bread is one of the principal components of the human diet, but it generally undergoes staling

process and microbial contamination within few days from its production. In particular, after baking

bread can be spoiled by moulds (mainly Penicillium and Aspergillus species) and heat resistant

spore-forming bacteria, naturally occurring in raw materials and foods of vegetable origin, and

surviving to the cooking process (Rosenkvist & Hansen, 1995). A recurrent microbiological issue

for bakery industries is represented by the ropy spoilage mainly associated to the presence of spores

of Bacillus species in raw materials (Pepe, Blaiotta, Moschetti, Greco & Villani, 2003; Valerio, De

Bellis, Di Biase, Lonigro, Giussani, Visconti, Lavermicocca, & Sisto, 2012). The spores of these

microorganisms survive in the central part of baked bread, where the temperature values reach up to

97-101°C for some minutes. Even though the spore-forming loads are very low in flour (about 2.0

log spores/g), the baking process and the subsequent storage conditions of bread (temperature ≥ 25

°C, water activity≥0.95, pH>5) favour the germination of heat-resistant spores and their increase in

total viable counts up to about 7.0 log CFU/g in bread crumb within two days, causing the loss of

freshness and worsening of the bread quality (Rosenkvist & Hansen, 1995; Viedma, Abriouel,

Omar, López, Gálvez, 2011). As recently demonstrated (Valerio et al., 2012), flour and other raw

materials (brewer yeast, improvers, etc) used to make bread are contaminated by a great variety of

spore-forming bacteria mainly belonging to the genus Bacillus and which include also potential

toxigenic species (Bacillus cereus group). Recently, some authors (De Jonghe, Coorevits, De Block,

Van Coillie, Grijspeerdt, Herman, De Vos & Heyndrickx, 2010) demonstrated the ability of

Bacillus amyloliquefaciens species, a common bread contaminant, to produce heat-labile cytotoxic

substances and a heat-stable cytotoxic component. Generally, microbial contamination levels of

bread higher than 5 log CFU/g are associated to the onset of spoilage process and the elevated risk

of foodborne illness when the causative agent is a toxigenic species (Kramer & Gilbert, 1989).

Spore-forming bacteria are naturally occurring in soil and contaminate wheat and other cereal grain

flours. In particular, bran, arising from the outer part of the grain kernels, contains a higher content

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of different microorganisms than those found in endosperm flours; among these microbes the spore-

forming bacteria could be related to the bread spoilage over its storage (Rosenkvist & Hansen,

1995).

In certain cases, bran has been used to replace part of the formulated flours in order to increase the

dietary fibres (DF) content in the bakery products, since wheat bran typically contains

approximately 45% of dietary fibre, of which about 95% is non-soluble fibre (Pomeranz, 1988;

Cornell & Hoveling, 1998). Nevertheless, health claims related to the ability of wheat bran fibres to

increase faecal bulk and to reduce intestinal transit time were accepted (EFSA, 2010) for labelling

food high in fibres in agreement to the European Regulation (EC) No 1924/2006. Besides DF, other

compounds concentrated in the outer layers of the grains, such as oligosaccharides and

phytochemicals, are gaining more and more importance in improving the nutritional and functional

quality of bread (Chavan & Chavan, 2011).

Furthermore, wheat bran-based flour blend can affect the rheological properties of bread dough

(Katina, Salmenkallio-Marttila, Partanen, Forssell & Autio, 2006a) and final bread quality attributes

that are consistent with reduction in volume, increasing in crumb firmness, and changing in flavour

(Laurikainen, Härkönen, Autio & Poutanen, 1998; Chavan & Chavan , 2011). In bread-making

these drawbacks are usually overcome by adding commercial enzyme mixtures or fermented wheat

bran to the dough during bread-making (Laurikainen et al., 1998; Katina, Laitila, Juvonen,

Liukkonen, Kariluoto, Piironen, Landberg, Åman, & Poutanen, 2007; Damen, Pollet, Dornez,

Broekaert, Van Haesendonck, Trogh, Arnaut, Delcour, Courtin, 2012). Well studied is also the

application of sourdough that, owing to fermentation by Lactobacillus sanfranciscensis,

Lactobacillus brevis and Lactobacillus plantarum, efficiently affects bread crumb properties and

controls moulds and bacterial spoilers (Niku-Paavola, Laitila, Mattila-Sandholm & Hikara, 1999;

Lavermicocca, Valerio, Evidente, Lazzaroni, Corsetti & Gobbetti, 2000; Katina, Sauri, Alakomi &

Mattila-Sandholm, 2002; Ström, Sjögren, Broberg & Schnürer, 2002; Pepe et al., 2003; Sjögren,

Magnusson, Broberg & Schnürer, 2003; Katina, Heiniö, Autio & Poutanen, 2006b; Corsetti &

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Settanni, 2007; Valerio, De Bellis, Lonigro, Visconti & Lavermicocca, 2008; Gerez, Torino,

Obregozo & Font de Valdez, 2010; Coda, Cassone, Rizzello, Nionelli, Cardinali & Gobbetti, 2011;

Wang, Yan, Wang, Zhang & Qi, 2012). Recently, some authors (Komlenić, Ugarčić-Hardi, Jukić,

Planinić, Bucić-Kojić & Strelec, 2010) observed a modification of the rheological properties of

wheat flour dough, and in particular a reduction of bread hardness and an increase in specific

volume after the addition of biological (L. brevis preferment and sourdough) acidifiers. In fact, the

fermentation of dough with LAB enhances the level of organic acids that was reported to be

involved in the reduction of dough mixing time and in a significant desirable weakening of dough

(Delcour & Hoseney, 2010). The resulted bread has greater volume, lower density, softer crumb and

higher slice height (Arendt, Ryan & Dal Bello, 2007). On the other hand, a moderate dough

acidification also enhanced wheat flour proteinase activities that, at the optimal pH values (3.8 -

4.1), influence the extensibility of gluten and the final quality of bread (Thiele, Gänzle & Vogel,

2002; Schober, Dockery & Arendt, 2003). The beneficial effect of LAB observed in sourdough can

be obtained by the addition of LAB-derived acidifiers, even if their effects on physico-chemical,

textural and microbiological quality of yeast-leavened bread formulated with wheat bran, needs to

be further investigated. Thus, the aim of the current work was to apply a L. brevis-based

bioingredient in yeast-leavened wheat bread containing bran to improve the final quality of enriched

fibre bread.

2 Materials and Methods

2.1 Bacterial cultures

Lactobacillus brevis LMG P-25726 was isolated from sourdough and deposited in the Belgian

Coordinated Collections of Microorganisms (BCCM/LGM, Gent, Belgium). For long-term storage,

stock cultures were prepared by mixing 8 mL of a culture with 2 mL of Bacto glycerol (Difco,

Becton Dickinson Co., Sparks, MD, USA) and freezing 1 mL portions of this mixture at -80 °C.

Culture was stored frozen (-80°C) in MRS broth (Oxoid LTD, Basinstoke Hampshire, England)

supplemented with 20% Bacto glycerol (Difco) and subcultured twice before use.

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2.2 LbBio bioingredient preparation

The bioingredient (LbBio) was prepared inoculating 5 mL of an overnight (37°C, 150 rpm) L.

brevis LMG P-25726 culture in a flour-based medium obtained by a mixture of white wheat flour

(100 g), water (500 mL), demineralised whey powder W714 (5 g) (ProfileTM

90, Kerry Ingredients,

Listowel, Ireland) and fructose (5 g) and incubated at 37 °C, 150 rpm for 18 hours. The final

product was combined, instead of water amount, with ingredients of wheat bread according the

formulations reported in Table 1. As a control the flour-based medium (FBM) incubated in the same

conditions (37°C, 150 rpm, 18 hours) but not inoculated with the L. brevis strain LMG P-25726,

was used.

2.3 Bread production

The bread formulation was optimized and standardized within the European Seventh Framework

Programme project DREAM (Design and development of realistic food models with well

characterized micro- and macro-structure and composition,

http://dream.aaeuropae.org/AboutDREAM/tabid/56/Default.aspx). All ingredients were mixed and

cooked in a kneading machine (Princess

Home Breadmaker, type 1936; Princess Household

Appliance BV, Breda, Netherlands). Bread types were prepared according to a standard recipe as

reported in Table 1. Breads containing the FBM instead of the LbBio or water were also made as

controls (WWB+FBM and WBB+FBM). Finally, dough pH and total titratable acidity (TTA) were

determined after the leavening step. TTA was measured according to AOAC Method No.: 981.12

(AOAC, 1990). Loaves were stored for 3 days at 30°C, for microbiological analysis, and at 25°C

for the textural analysis.

2.4 Organic acid quantification in the bioingredient and in the dough

The bioingredient was centrifuged (8422 × g, 10 min) and the supernatant was freeze-dried,

resuspended in HPLC mobile phase (0.005 mol/L H2SO4, Fluka, Deisenhofen, Germany) and

passed through a micro-concentrator (Ultracel-3k, Amicon, Danvers, MA, USA) with a molecular-

mass cut-off of 3000 Da, by centrifugation (7000 × g, 2°C, 1 h). Solutions were loaded onto the

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column. In the case of the dough, 10-gram portions were homogenized with 90 mL of sterile tap

water in a blender for 2 min. Ten millilitre-aliquots were centrifuged (8422 × g, 10 min, 10 °C) and

the supernatant was freeze-dried, resuspended in mobile phase (0.005 mol/L H2SO4) and then

passed through a 3000 Da cut-off micro-concentrator by centrifugation (7000 × g, 2°C, 1 h). The

resulting solutions were loaded onto the column. The analysis of organic acids (lactic, acetic,

propionic, phenyllactic, hydroxyl-phenyllactic, valeric, isovaleric, butyric, isobutyric acids), in the

bioingredient and in the dough, was performed by HPLC (AKTABasic10, P-900 series pump,

Amersham Biosciences AB, Uppsala, Sweden), using a Rezex ROAorganic acid H+ (8%) column

(7.80×300 mm, Phenomenex, Torrance, CA, USA) and a 3-channel UV detector (Amersham

Biosciences 900) set at 210 and 220 nm. The mobile phase was pumped at a flow rate of 0.6

mL/min through the column heated to 70°C. Quantification of the organic acids were performed by

integrating calibration curves obtained from the relevant standards.

2.5 Microbiological quality of raw materials and bread

Wheat flour and bran batches used in bread-making were checked for the presence of natural spore

–forming contaminants as reported in Valerio et al. (2012) with slight modifications. Briefly, 20 g

of each sample was diluted with 180 g of a sterile Maximum Recovery Diluent (MRD, Oxoid) and

homogenized in a stomacher (Seward, London, United Kingdom) for 2 min. The suspension was

filtered through sterile Whatman paper No 4 (Whatman, Maidstone, United Kingdom) to remove

the coarse material and heat treated for 10 min at 80 °C to select spores. Therefore, the suspension

was pour plated (1 mL) and decimally diluted and spread plated (100 μl) on Plate Count Agar

(PCA, Oxoid). In the case of raw materials expected to be contaminated at low levels (<100

spores/g), the suspension was centrifuged (8422 × g, 10 min) after heat treatment and the pellet

resuspended in one-tenth of the original volume.

To enumerate bacterial contaminants of bread samples produced in laboratory and stored at 30°C,

loaves were aseptically sliced after baking and cooling and slices were individually sealed in sterile

polyethylene bags. At each sampling time (T0, T1day and T3days), 20 g of each sample was diluted

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with 180 g of sterile MRD and homogenized in a stomacher for 2 min. The food homogenate was

decimally diluted and suspensions plated on PCA agar.

Plates were incubated for 24 h at 30 °C and the number colony forming units (CFU) was counted.

Detection limit of bacterial count was 1 CFU/g.

Bread slices were also monitored during storage for rope appearance, by evaluating sweet rope

odor, discoloration of the crumb and sticky threads.

2.6 Physicochemical and firmness properties of bread

After cooking the bread loaves not subjected to microbiological analysis were cooled for 2 h and

evaluated for their weight and for volume and specific volume using the rapeseed displacement

approved method 10-05.01 (AACC International 2010). Furthermore, the loaves were sliced to

obtain six transversal slices (25 mm-thickness) that were analyzed for crumb firmness (defined as

the force required to compress the bread slice) by a Zwicki-line uniaxial testing machine (Zwick,

Ulm, Germany) with a 500 N load cell and TestXpert version 6.01 software was used. Briefly, all

slices from each loaf were compressed by 6.25 and 10 mm (25 and 40% deformation, respectively)

with a crosshead speed of 1.7 mm/s to measure the crumb hardness according to the approved

method 74-09, 2000 (AACC International 2000). These strains correspond to the common practice

to squeeze bread between the forefinger and opposed thumb to evaluate the crumb freshness or

between incisor teeth at the first bite (Bourne, 2002). Water activity values were measured on the

crumb of cooked loaves after cooling using a Decagon AquaLab Serie 3 (Steroglass, Perugia, Italy)

and the pH was recorded with a portable 110 pH meter (Oakton Instruments, Vernon Hills, IL,

USA) supplied with Double Pore D electrode (Hamilton, Bonaduz, Switzerland).

2.7 Statistical analyses

Data are presented as mean values ± standard deviation. Comparisons during time were done by

using the one-way Analysis of Variance (ANOVA), while bread types were compared by using the

two-ways ANOVA (Hogg & Ledolter, 1987). The function “anovan” of the software Matlab

R2012a (The MathWorks, Inc.) was used for testing the effects of the two factors (presence of bran

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and/or LbBio) and their interactions on the mean values of the measured variables. To determine

which pairs of levels of the factors were significantly different, the Tukey’s test was adopted

(Hochberg & Tamhane, 1987). Statistical significance was assessed at a level of 5%.

3 Results

3.1 Microbiological quality of bread types during storage and organic acid quantification

Microbiological analysis of flour and bran batches used in this work indicated a low level of

bacterial spores (<103 spores/g of flour or bran) even though, the total bacterial count in baked

control bread types with and without bran (WBB and WWB) reached a concentration level close to

5 log CFU/g after only one day storage at 30°C (Table 2). After baking (T0) some differences in

bacterial counts among bread types were observed and could be related to the different batch used

for each experiment. When bread formulation was modified replacing water with a relevant amount

of the liquid bioingredient LbBio, the total bacterial count as well as the ropy spoilage appearance

were reduced after 3 days only in bread prepared with white wheat flour (WWB+LbBio), while the

efficacy of LbBio was loweredd when bread was prepared with bran (WBB+LbBio) (Table 2).

When a flour-based medium (FBM) was used as a control instead of LbBio, no inhibition of

bacterial count was observed.

The production of lactic, acetic, phenyllactic (PLA) and OH-phenyllactic (OH-PLA) acids was

evaluated in LbBio, after growing L. brevis LMG P-25726 for 18 h, in FBM and in the resulting

dough samples (Table 3). No trace of propionic, valeric, isovaleric, butyric, isobutyric acids was

found both in the LbBio, FBM and in dough samples. The concentration of lactic acid, acetic acid,

PLA and OH-PLA produced by L. brevis LMG P-25726 in the bioingredient was found to be

significantly higher (p>0.05) than in the flour based medium. Dough samples with bioingredient

contained all the acids and showed the higher molar lactic/acetic acid ratio (fermentation quotient,

FQ). Conversely, dough samples not prepared with LbBio contained only acetic acid (except for

WWB) and showed FQ values lower than 1 (Table 3).

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In order to assess the influence of each factor (bran and/or LbBio) and their interaction on bread

quality, a two ways ANOVA test was performed (Table 4). The analysis demonstrated the ability of

LbBio to significantly affect the dough content of lactic acid, PLA and OH-PLA regardless of the

presence of bran and this effect determined the reduction of bacterial count after three days storage.

Whereas, significant effect of bran and interaction between the two factors (LbBio and bran) were

observed for the acetic acid in dough and for bread contamination (three day-storage), although in

these bread formulations (WBB and WBB+LbBio) bacterial counts were not modified (Table 2 and

Table 4). These observations were explained by the multiple comparison Tukey’s test performed to

establish which pairs of levels of the factors were significantly different for each variable (Fig. 1).

The analysis confirmed that a significant inhibitory effect on bread contamination was exerted by

the LbBio (Table 2; Fig. 1, comparison 2), but this ability was influenced by the presence of bran.

In fact, the addition of LbBio to dough together with bran did not produce any significant changes

on bread contamination (Table 2; Fig. 1, comparison 5). The addition of bran to the dough without

LbBio did not modify its contamination level (Table 2; Fig. 1, comparison 1), whereas bran added

to dough with LbBio (WBB+LbBio) led to an increase in the bacterial count of baked bread with

respect to the formulation lacking bran (WWB+LbBio) (Table 2; Fig. 1, comparison 6). At the same

time LbBio significantly reduced the effect of bran on the acetic acid content in dough (Table 3 and

Fig. 1, comparisons 1, 6).

3.2 Physico-chemical and textural properties of bread

The effect of bran and/or LbBio on some physico-chemical and textural properties of bread is

reported in Tables 5 and 6, Figures 1 and 2. Results indicated that white wheat bread (WWB) was

significantly different from bread containing wheat bran (WBB) in all properties except for water

activity, bread pH and hardness at 25% deformation (Tables 5 and 6, Fig. 1, comparison 1). The use

of LbBio significantly affected dough and bread pH values in WWB which resulted most acidified

while this effect was limited by the presence of bran (Table 5, Fig. 1, comparisons 2 and 5). At the

same time, a significant increase in the TTA values was observed in both dough types after LbBio

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application (WWB+LbBio and WBB+LbBio). The two ways ANOVA confirmed that the addition

of LbBio significantly affected dough and bread pH and dough TTA values (Table 5) and that this

effect was significantly influenced by bran addition (see the interaction between factors

Bran*LbBio in Table 5).

Discussion

Results of this work indicated an effective role of Lactobacillus brevis LMG P-25726 in the LbBio

technological and antimicrobial efficacy and confirmed the applicability of LAB-based

fermentation products as bioingredients in yeast-leavened bread.

The microbiological quality of breads during storage resulted improved and the rope spoilage was

delayed or hampered, in the presence of LbBio, confirming the synergistic role of organic acids as

previously observed (Valerio et al., 2008; Mantzourani, Plessas, Saxami, Alexopoulos, Galanis &

Bezirtzoglou, 2014). Although a high acetic acid content and FQ down to 1 can be associated to

enhanced inhibitory efficacy against rope producing bacteria (Röcken, 1996), the presence of only

acetic acid in breads not prepared with LbBio did not warranty bacterial inhibition. Additionally,

other authors demonstrated that acetic acid can determine a shorter and harder gluten and that the

production of lactic acid is advisable to increase elasticity of dough (Gobbetti, Corsetti & Rossi,

1995). In fact, the high molar lactic/acetic acid ratio (FQ>1) generally obtained in sourdough is

associated to pleasant odour, sensory properties and improved shelf-life. In our yeast-leavened

bread types, the presence of the LbBio allowed to obtain optimal FQ, TTA and pH values

comparable to those previously reported for sourdough bread (FQ>1, TTA: 4.8-6.2; pH: 4.8-5.1)

(Katina et al., 2002; Mantzourani et al., 2014).

Additionally to the influence on microbiological quality, the application of LAB bioingredients in

bread-making could have positive effects on some physico-chemical and textural properties of

bread as widely reported (Komlenić et al., 2010; Pepe, Ventorino, Cavella, Fagnano & Brugno,

2013). Acids produced during fermentation can influence the mixing behaviour of dough, since the

gluten protein solubility was proved to cause an increase in softness and elasticity of gluten

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(Wehrle, Grau& Arendt, 1997; Takeda, Matsumura & Shimizu, 2001). In addition, the dough

acidification could enhance enzymatic activity of wheat proteases, causing an increase in free amino

acid content and improved bread flavour (Thiele et al., 2002; Clarke, Schober, Dockery, O’Sullivan

& Arendt, 2004; Rizzello, Coda, Mazzacane, Minervini & Gobbetti, 2012). In our study, a

significant increase in TTA values was observed in both dough types containing LbBio and values

were comparable to those obtained in L. brevis sourdough (Komlenić et al., 2010). Furthermore, the

addition of bran significantly influenced the effect of LbBio on pH and TTA values (see the

interaction between factors Bran*LbBio in Table 6). This result is consistent with the acidity

increase of dough when sourdough was prepared with bran fractions, than confirming an interaction

occurring between LAB and dietary fibres (Rizzello et al., 2012). Recently, Pepe et al. (2013)

demonstrated the contribute of LAB producing exopolysaccharides (EPS) in combination with

immature wheat grain flour containing fructo-olgosaccharides (FOS), in improving the nutritional

and technological characteristics of breads. The ability of LAB to produce EPS is frequently

exploited in bread technology to reduce the amount of additives since these metabolites influence

the viscoelastic characteristics and stabilize the rheological properties of dough (Palomba, Cavella,

Torrieri, Piccolo, Mazzei, Blaiotta, Ventorino & Pepe, 2012). Moreover, the use of rich-FOS

prebiotics can stimulate the microbial metabolism leading to an increase in TTA (Pepe et al., 2013).

4 Conclusions

The bioingredient Lactobacillus brevis–based LbBio, acting as a sourdough, counteract the negative

effects of bran allowing to obtain an enriched-fibre bread with overall quality comparable to that of

the reference white wheat bread. The study also indicated that bread formulation can modulate

bacterial behaviour during storage and consequently the bread spoilage. Further investigations are

needed to better evaluate the positive and negative interactions on bread quality occurring when

different levels of dietary fibers are used in the presence of the bioingredient LbBio.

Acknowledgments

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This research has received funding from the European Community's Seventh Framework

Programme (FP7/ 2007-2013) under the grant agreement no FP7-222 654-DREAM.

N.A. and T.M.C were supported by Project FIRB CAROMICS RBAP11B2SX.

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Figure 1. Multiple comparison test on each pair of factor levels.

Figure 2. Slices of bread loaves produced with A) white wheat flour (WWB), B) white wheat flour

and wheat bran (20% w/w of wheat flour) (WBB), C) white wheat flour and LbBio (WWB+LbBio),

and D) white wheat flour, wheat bran (20% w/w of wheat flour) and LbBio (WBB+LbBio).

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Table 1. Bread formulations containing or not wheat bran and/or Lactobacillus brevis-based

bioingredient (LbBio) and/or the flour-based medium (FBM) as control.

Bread typea

WWB

WBB WWB+LbBio WBB+LbBio WWB+ FBM WBB+ FBM

Ingredient Mass (g)

White wheat flour 350 280 350 280 350 280

Wheat bran - 70 - 70 - 70

Salt 6.3 6.3 6.3 6.3 6.3 6.3

Margarine 10.5 10.5 10.5 10.5 10.5 10.5

Dry yeast 5.25 5.25 5.25 5.25 5.25 5.25

Tap water 210 245 - 35 - 35

LbBio - - 210 210 - -

FBM - - - - 210 210

a White wheat bread (WWB), White bran bread (WBB), White wheat bread+LbBio (WWB+LbBio),

Wheat bran bread+LbBio (WBB+LbBio), White wheat bread+ FBM (WWB+ FBM), Wheat bran

bread+ FBM (WBB+ FBM).

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Table 2. Rope spoilage and total viable count (log CFU/g) of bacterial contaminants naturally

occurring in white wheat bread (WWB) and wheat bran bread (WBB) containing or not the

bioingredient (WWB+LbBio or WBB+LbBio) or the flour based medium (WWB+FBM or

WBB+FBM) and stored at 30°C for different times.

Bread type T0 T1day T3day Rope (T3day)

WWB 2.96±1.10aa

4.61±1.10a 6.93±1.03b ++b

WWB+LbBio 2.67±1.32a 2.74±0.90ab 4.52±1.01b -

WWB+FBM 2.58±0.26a 2.61±0.09a 6.10±0.43b +

WBB 1.15±0.21a 4.80±1.17b 7.34±0.89c ++

WBB+LbBio 2.62±2.13a 3.08±1.53ab 6.63±0.98b +

WBB+FBM 1.38±0.12a 3.52±0.26a 7.17±0.54b ++

a Means ± standard deviation of three replicates; means with different letters in the same row

indicate statistically significant differences between samples by the Tukey test (p < 0.05).

b Rope occurrence in bread crumb: -: no ropey alteration; +: typical ropey smell; ++: ropey slime.

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Table 3. Organic acid content in the bioingredient LbBio, in the flour-based medium (FBM) and in

the dough samples prepared with white wheat flour (WWB) or a mixture of white wheat flour and

wheat bran (WBB) and containing or not the LbBio or the FBM.

Lactic acid Acetic acid PLA OH-PLA FQa

Ingredient mmol/L

LbBio 30.43 ± 5.23 ba 4.37 ± 1.01a 0.05 ± 0.02a 0.02 ± 0.01a

FBMc

3.90± 0.84b 9.11± 0.06b -d b - b

Dough sample mmol/Kg

WWB <DLe a <DLa <DL a <DLa -

WWB +LbBio 9.31±2.71b 3.43±1.47a 0.02 ± 0.01b 0.01± 0.00b 2.71

WWB+FBM <DLa 2.58±2.40a <DLa <DLa 0.18

WBB <DLa 11.13±1.7b <DLa <DLa -

WBB+LbBio 9.36±5.39b 4.60±2.74ac 0.02±0.01b 0.01±0.01b 2.03

WBB+FBM <DLa 8.32±2.66bc <DLa <DLa 0.07

a Fermentation Quotient: molar ratio between lactic and acetic acids

b Means ± standard deviation of three replicates; means with different letters in the same column

indicate statistically significant differences between samples by the Tukey test (p < 0.05).

c Flour-based medium not inoculated with Lactobacillus brevis LMG P-25726 and used as control.

d not found.

e DL: detection limit. In LbBio and FBM DL were: 3.60 mM (lactic acid), 1.62 mM (acetic acid),

0.038 mM (PLA), 0.015 mM (OH-PLA). In dough DL were: 2.26 mmol/Kg (lactic acid), 1.37

mmol/Kg (acetic acid), 0.0004 mmol/Kg (PLA), 0.0008 mmol/Kg (OH-PLA).

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Table 4. Two-ways Anova test p values to measure the effect of the addition of bran and/or LbBio and their interaction on bacterial contamination

of bread during storage at 30°C for different times and on the organic acids content of dough.

Bacterial

contamination of

bread

Organic acids content in dough

factors T0 T1day T3day Lactic acid Acetic acid PLA OH-PLA

Bran 0.1616 0.7735 0.0008 0.9875 0.0001 0.8048 0.2396

LbBio 0.3673 0.1081 0.0000 0.0009 0.1507 0.0012 0.0003

Bran*LbBio 0.1842 0.9319 0.0195 0.9875 0.0005 0.8048 0.2396

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Table 5. Physico-chemical properties of bread types and two-ways Anova test p values to measure the effect of the addition of bran and/or LbBio

on physicochemical properties of bread and dough.

Bread dough Bread

Bread type Dough pH TTAa Specific volume sample

(cm3/g)

Water activity

(aw)

Bread pH

WWB 5.24± 0.37ba 1.96 ± 0.51 3.20 ± 0.51 0.94 ± 0.03 5.56 ± 0.50b

WWB+LbBio 4.57± 0.14a 4.45 ± 0.07 3.10± 0.00 0.94 ± 0.01 4.82 ± 0.18b

WBB 5.43± 0.15a 3.96 ± 0.06 2.65 ± 0.23 0.96 ± 0.01 5.49 ± 0.14a

WBB+LbBio 5.17± 0.11a 5.70 ± 0.14 2.65 ± 0.35 0.95 ± 0.01 5.20 ± 0.16a

Two-ways Anova

p values

factors

Bran 0.0000 0.0000 0.0341 0.0678 0.0705

LbBio 0.0000 0.0000 0.8036 0.7384 0.000

Bran*LbBio 0.0490 0.0452 0.8229 0.3396 0.0041

avalues represent volume (mL) of NaOH 0.1 N. TTA was determined using the AOAC method N° 981.12

b Means ± standard deviation of three replicates; means with different letters in the same row indicate statistically significant differences between

samples by the Tukey’s test (p < 0.05).

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Table 6. Crumb hardness (N) of bread containing or not bran and/or the bioingredient LbBio during storage at 25°C for three days. Two-ways

Anova test p values to measure the effect of the addition of bran and/or LbBio on crumb hardness subjected to 25% and 40% deformation force.

Crumb hardness (N) at 25% deformation Crumb hardness (N) at 40% deformation

Bread type T0 T1day T3day T0 T1day T3day

WWB 6.49±1.55aa 14.85±1.39b 29.04±1.28c 16.79±2.35 a 22.37±3.34a 35.08±6.57b

WWB+LbBio 8.28±0.98a 11.32±2.70a 23.28±2.72b 14.99±1.92a 21.70±0.92b 29.27±3.43cA

WBB 16.81±0.92a 20.78±2.53a 32.85±4.04b 19.49±8.55a 35.65±4.40b 46.38±6.24c

WBB+LbBio 10.11±1.52a 17.89±3.97b 30.78±2.65c 18.72±1.43a 32.09±1.42b 42.78±1.97c

Two-ways Anova

p values

Bran 0.0000 0.0014 0.0002 0.1884 0.0000 0.0000

LbBio 0.0003 0.0499 0.0057 0.5924 0.0942 0.0248

Bran*LbBio 0.0000 0.8275 0.1565 0.8293 0.2465 0.5825

a Means ± standard deviation of three replicates; means with different letters in the same row (at the same deformation force) indicate statistically

significant differences between sampling times by the Tukey’s test (p < 0.05).

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Highlights

Lactobacillus brevis-based bioingredient (LbBio) improves bread texture and quality

Bread formulation influences bacterial behavior in bread and the consequent spoilage

LbBio counteracts the negative effects of bran