Innovative Food Science and Emerging Technologies 25 (2014) 2–8

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Innovative Food Science and Emerging Technologies

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Effect of Lactobacillus brevis-based bioingredient and bran onmicrobiological, physico-chemical and textural quality of yeast-leavenedbread during storage

Francesca Valerio a, Mariaelena Di Biase a, Leonardo Caputo a, Teresa M. Creanza b, Nicola Ancona b,Angelo Visconti a, Paola Lavermicocca a,⁎a Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italyb Institute of Intelligent Systems for Automation, National Research Council, Via Amendola 122/D-I, 70126 Bari, Italy

⁎ Corresponding author at: Istituto di Scienze delle PAmendola 122/O, 70126 Bari, Italy. Tel.: +39 080 592935

E-mail address: paola.lavermicocca@ispa.cnr.it (P. Lav

1466-8564/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.ifset.2013.09.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 April 2013Accepted 11 September 2013Available online 20 September 2013

Editor Proof Receive Date 11 October 2013

Keywords:Ropy spoilageFirmnessSpore-forming bacteriaOrganic acidsBioingredientTwo-way ANOVA

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 breadformulation by substituting a part (20 g/100 g) of white wheat flour (WBB), while LbBio was used instead of thewater content (WWB + LbBio andWBB + LbBio). The use of LbBio inWWB resulted in the biological acidificationof the dough due to lactic, phenyllactic andOH-phenyllactic acid contents determining a high fermentation quotientvalue and an improved bread texture andmicrobiological quality. Conversely, wheat bran reduced the specific vol-ume and crumbhardness 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 enrichedfibre bread with specific volume and soft crumb comparable to bread without bran.Industrial relevance:Bread is a perishable foodwith a shortmicrobiological and physico-chemical shelf-life. Themainmicrobiological alteration occurring into few days after baking is the “rope spoilage” caused by spore-forming bac-teria originating from rawmaterials. This phenomenon, often misinterpreted as a sign of unsuccessful dough leav-ening and not visible from outside, is more common under industrial production conditions during the hot seasoncausing large economic losses in the warm climates of Mediterranean countries, Africa and Australia. The use ofsourdough often controls this alteration even if the industrial application of this traditional process is limited bythe long leavening times. In this study, an innovative procedure for the preparation of yeast-leavened bread com-prising the addition of a fermentation product from Lactobacillus brevis grown in aflour-basedmediumhas been ap-plied. The resulting fermentation product (LbBio bioingredient) acts as a sourdough acidifying the dough andimproving the textural, physico-chemical and microbiological properties of the resulting bread. The applicationof bioingredient LbBio could represent an innovative strategy in industrial bread production to obtain acidifiedyeast-leavened products, thus, preventing the ropy spoilage and reducing the negative effects of bran addition.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Bread is one of the principal components of the human diet, but itgenerally undergoes staling process and microbial contamination with-in few days from its production. In particular, after baking, bread can bespoiled by moulds (mainly Penicillium and Aspergillus species) and heatresistant spore-forming bacteria, naturally occurring in raw materialsand foods of vegetable origin, and surviving to the cooking process(Rosenkvist & Hansen, 1995). A recurrent microbiological issue forbakery industries is represented by the ropy spoilage mainly associatedto the presence of spores of Bacillus species in raw materials (Pepe,Blaiotta, Moschetti, Greco, & Villani, 2003; Valerio et al., 2012). The

roduzioni Alimentari, CNR, Via6; fax: +39 080 5929374.ermicocca).

ghts reserved.

spores of these microorganisms survive in the central part of bakedbread, where the temperature values reach up to 97–101 °C for someminutes. Even though the spore-forming loads are very low in flour(about 2.0 log spores/g), the baking process and the subsequentstorage conditions of bread (temperature ≥ 25 °C, water activity ≥0.95, pH N 5) favour the germination of heat-resistant spores and theirincrease in total viable counts up to about 7.0 log CFU/g in breadcrumb within 2 days, causing the loss of freshness and worsening ofthe 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 tomake bread are contaminated by a great variety of spore-forming bacte-ria mainly belonging to the genus Bacillus and which include also poten-tial toxigenic species (Bacillus cereus group). Recently, some authors (DeJonghe et al., 2010) demonstrated the ability of Bacillus amyloliquefaciensspecies, a common bread contaminant, to produce heat-labile cytotoxic

3F. Valerio et al. / Innovative Food Science and Emerging Technologies 25 (2014) 2–8

substances and a heat-stable cytotoxic component. Generally, microbialcontamination levels of bread higher than 5 log CFU/g are associated tothe onset of spoilage process and the elevated risk of foodborne illnesswhen the causative agent is a toxigenic species (Kramer &Gilbert, 1989).

Spore-forming bacteria are naturally occurring in soil and contami-nate wheat and other cereal grain flours. In particular, bran, arisingfrom the outer part of the grain kernels, contains a higher content of dif-ferent microorganisms than those found in endosperm flours; amongthese microbes, the spore-forming bacteria could be related to thebread spoilage over its storage (Rosenkvist & Hansen, 1995).

In certain cases, bran has been used to replace part of the formulatedflours in order to increase the dietary fibres (DF) content in the bakeryproducts, since wheat bran typically contains approximately 45% ofdietary fibre, of which about 95% is non-soluble fibre (Cornell &Hoveling, 1998; Pomeranz, 1988). Nevertheless, health claims relatedto the ability of wheat bran fibres to increase faecal bulk and to reduceintestinal transit time were accepted (EFSA, 2010) for labelling foodhigh in fibres in agreement to the European Regulation (EC) No 1924/2006. Besides DF, other compounds concentrated in the outer layers ofthe grains, such as oligosaccharides and phytochemicals, are gainingmore and more importance in improving the nutritional and functionalquality of bread (Chavan & Chavan, 2011).

Furthermore, wheat bran-based flour blend can affect the rheologi-cal properties of bread dough (Katina, Salmenkallio-Marttila, Partanen,Forssell, & Autio, 2006a) and final bread quality attributes that areconsistent with reduction in volume, increase in crumb firmness, andchange in flavour (Chavan & Chavan, 2011; Laurikainen, Härkönen,Autio, & Poutanen, 1998). In bread-making, these drawbacks are usuallyovercome by adding commercial enzymemixtures or fermented wheatbran to the doughduring bread-making (Damen et al., 2012; Katina et al.,2007; Laurikainen et al., 1998). Well studied is also the application ofsourdough that, owing to fermentation by Lactobacillus sanfranciscensis,Lactobacillus brevis and Lactobacillus plantarum, efficiently affects breadcrumb properties and controls moulds and bacterial spoilers (Codaet al., 2011; Corsetti & Settanni, 2007; Gerez, Torino, Obregozo, & Fontde Valdez, 2010; Katina, Heiniö, Autio, & Poutanen, 2006b; Katina,Sauri, Alakomi, & Mattila-Sandholm, 2002; Lavermicocca et al., 2000;Niku-Paavola, Laitila, Mattila-Sandholm, & Hikara, 1999; Pepe et al.,2003; Sjögren, Magnusson, Broberg, & Schnürer, 2003; Ström, Sjögren,Broberg, & Schnürer, 2002; Valerio, De Bellis, Lonigro, Visconti, &Lavermicocca, 2008; Wang, Yan, Wang, Zhang, & Qi, 2012). Recently,some authors (Komlenić. et al., 2010) observed a modification of therheological properties of wheat flour dough, and in particular, a reduc-tion of bread hardness and an increase in specific volume after the addi-tion of biological (L. brevis preferment and sourdough) acidifiers. In fact,the fermentation of dough with LAB enhances the level of organic acidsthat was reported to be involved in the reduction of dough mixingtime and in a significant desirable weakening of dough (Delcour &Hoseney, 2010). The resulting bread has greater volume, lower density,

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

Bread typea

WWB WBB WWB + LbBio

Ingredient Mass (g)

White wheat flour 350 280 350Wheat bran – 70 –

Salt 6.3 6.3 6.3Margarine 10.5 10.5 10.5Dry yeast 5.25 5.25 5.25Tap water 210 245 –

LbBio – – 210FBM – – –

a White wheat bread (WWB), wheat bran bread (WBB), white wheat bread + LbBio (W(WWB + FBM), and wheat bran bread + FBM (WBB + FBM).

softer crumb and higher slice height (Arendt, Ryan, & Dal Bello, 2007).On the other hand, a moderate dough acidification also enhancedwheat flour proteinase activities that, at the optimal pH values (3.8–4.1), influence the extensibility of gluten and the final quality of bread(Schober, Dockery, & Arendt, 2003; Thiele, Gänzle, & Vogel, 2002). Thebeneficial effect of LAB observed in sourdough can be obtained by the ad-dition of LAB-derived acidifiers, even if their effects on physico-chemical,textural and microbiological quality of yeast-leavened bread formulatedwith wheat bran, needs to be further investigated. Thus, the aim of thecurrent work was to apply a L. brevis-based bioingredient in yeast-leavened wheat bread containing bran to improve the final quality ofenriched fibre bread.

2. Materials and methods

2.1. Bacterial cultures

L. brevis LMGP-25726was isolated from sourdough and deposited inthe Belgian Coordinated Collections of Microorganisms (BCCM/LGM,Gent, Belgium). For long-term storage, stock cultures were preparedby mixing 8 mL of a culture with 2 mL of Bacto glycerol (Difco, BectonDickinson Co., Sparks, MD, USA) and freezing 1 mL portions of thismixture 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.

2.2. LbBio bioingredient preparation

Thebioingredient (LbBio)was prepared inoculating 5 mLof anover-night (37 °C, 150 rpm) L. brevis LMG P-25726 culture in a flour-basedmedium obtained by a mixture of white wheat flour (100 g), water(500 mL), demineralised whey powder W714 (5 g) (Profile™ 90,Kerry Ingredients, Listowel, Ireland) and fructose (5 g) and incubatedat 37 °C, 150 rpm for 18 h. The final product was combined, insteadof water amount, with ingredients of wheat bread according the formu-lations reported in Table 1. As a control the flour-based medium (FBM)incubated in the same conditions (37 °C, 150 rpm, 18 h) but not inocu-lated with the L. brevis strain LMG P-25726, was used.

2.3. Bread production

The bread formulation was optimised and standardised within theEuropean Seventh Framework Programme project DREAM (Designand development of realistic food models with well characterisedmicro- and macro-structure and composition, http://dream.aaeuropae.org/AboutDREAM/tabid/56/Default.aspx). All ingredients were mixedand 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

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

WBB + LbBio WWB + FBM WBB + FBM

280 350 28070 – 706.3 6.3 6.310.5 10.5 10.55.25 5.25 5.2535 – 35210 – –

– 210 210

WB + LbBio), wheat bran bread + LbBio (WBB + LbBio), white wheat bread + FBM

Table 2Rope spoilage and total viable count (log CFU/g) of bacterial contaminants naturallyoccurring in white wheat bread (WWB) and wheat bran bread (WBB) containing ornot 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 T1 day T3 days Rope (T3 days)

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 thesame row indicate statistically significant differences between samples by the Tukey test(p b 0.05).

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

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in Table 1. Breads containing the FBM instead of the LbBio orwaterwerealso made as controls (WWB + FBM andWBB + FBM). Finally, doughpH and total titratable acidity (TTA) were determined after the leaven-ing step. TTA was measured according to AOAC Method No.: 981.12(AOAC, 1990). Loaves were stored for 3 days at 30 °C for microbiologi-cal 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 thesupernatant was freeze-dried, resuspended in HPLC mobile phase(0.005 mol/L H2SO4, Fluka, Deisenhofen, Germany) and passed througha micro-concentrator (Ultracel-3 k, Amicon, Danvers, MA, USA) with amolecular-mass cut-off of 3000 Da, by centrifugation (7000 ×g, 2 °C,1 h). Solutions were loaded onto the column. In the case of the dough,10-gramme portions were homogenised with 90 mL of sterile tapwater 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 passedthrough a 3000 Da cut-off micro-concentrator by centrifugation(7000 ×g, 2 °C, 1 h). The resulting solutions were loaded onto the col-umn. The analysis of organic acids (lactic, acetic, propionic, phenyllactic,hydroxyl-phenyllactic, valeric, isovaleric, butyric, and 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 phasewas pumped at a flow rate of 0.6 mL/min through the column heated to70 °C. Quantification of the organic acids was performed by integratingcalibration 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 checkedfor the presence of natural spore-forming contaminants as reportedin Valerio et al. (2012) with slight modifications. Briefly, 20 g of eachsample was diluted with 180 g of a sterile Maximum Recovery Diluent(MRD, Oxoid) and homogenised in a stomacher (Seward, London,United Kingdom) for 2 min. The suspensionwas filtered through sterileWhatman paper No. 4 (Whatman, Maidstone, United Kingdom) to re-move the coarse material and heat treated for 10 min at 80 °C to selectspores. Therefore, the suspensionwas pour plated (1 mL) and decimallydiluted and spread plated (100 μL) on Plate Count Agar (PCA, Oxoid). Inthe case of raw materials expected to be contaminated at low levels(b100 spores/g), the suspension was centrifuged (8422 ×g, 10 min)after heat treatment and the pellet resuspended in one-tenth of theoriginal volume.

To enumerate bacterial contaminants of bread samples producedin laboratory and stored at 30 °C, loaves were aseptically sliced afterbaking and cooling and slices were individually sealed in sterile poly-ethylene bags. At each sampling time (T0, T1 day and T3 days), 20 g ofeach sample was diluted with 180 g of sterile MRD and homogenisedin a stomacher for 2 min. The food homogenate was decimally dilutedand suspensions plated on PCA agar.

Plates were incubated for 24 h at 30 °C and the number colonyforming units (CFU) was counted. Detection limit of bacterial countwas 1 CFU/g.

Bread slices were also monitored during storage for rope appear-ance, by evaluating sweet rope odour, discoloration of the crumb andsticky threads.

2.6. Physico-chemical and firmness properties of bread

After cooking, the bread loaves not subjected to microbiologicalanalysis were cooled for 2 h and evaluated for their weight and for

volume and specific volume using the rapeseed displacement approvedmethod 10-05.01 (AACC International, 2010). Furthermore, the loaveswere sliced to obtain six transversal slices (25 mm-thickness) thatwere analysed for crumb firmness (defined as the force required tocompress the bread slice) by a Zwicki-line uniaxial testing machine(Zwick, Ulm, Germany) with a 500 N load cell and TestXpert version6.01 software was used. Briefly, all slices from each loaf were com-pressed by 6.25 and 10 mm (25 and 40% deformation, respectively)with a crosshead speed of 1.7 mm/s to measure the crumb hardnessaccording to the approved method 74-09, 2000 (AACC International,2000). These strains correspond to the common practice to squeezebread between the forefinger and opposed thumb to evaluate thecrumb freshness or between incisor teeth at the first bite (Bourne,2002). Water activity values were measured on the crumb of cookedloaves after cooling using a Decagon AquaLab Series 3 (Steroglass,Perugia, Italy) and the pH was recorded with a portable 110 pH meter(Oakton Instruments, Vernon Hills, IL, USA) supplied with Double PoreD electrode (Hamilton, Bonaduz, Switzerland).

2.7. Statistical analyses

Data are presented as mean values ± standard deviation. Compari-sons during time were done by using the one-way analysis of variance(ANOVA), while bread types were compared by using the two-wayANOVA (Hogg & Ledolter, 1987). The function “anovan” of the softwareMatlab R2012a (TheMathWorks, Inc.) was used for testing the effects ofthe two factors (presence of bran and/or LbBio) and their interactionson the mean values of the measured variables. To determine whichpairs of levels of the factors were significantly different, the Tukey'stest was adopted (Hochberg & Tamhane, 1987). Statistical significancewas assessed at a level of 5%.

3. Results

3.1. Microbiological quality of bread types during storage and organic acidquantification

Microbiological analysis of flour and bran batches used in this workindicated a low level of bacterial spores (b103 spores/g of flour or bran)even though, the total bacterial count in baked control bread types withand without bran (WBB andWWB) reached a concentration level closeto 5 log CFU/g after only 1 day of storage at 30 °C (Table 2). After baking(T0) some differences in bacterial counts among bread types wereobserved and could be related to the different batch used for each ex-periment. When bread formulation was modified replacing waterwith a relevant amount of the liquid bioingredient LbBio, the total bac-terial count as well as the ropy spoilage appearance were reduced after3 days only in bread preparedwithwhite wheat flour (WWB + LbBio),while the efficacy of LbBio was lowered when bread was prepared with

Table 3Organic 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 flourand 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 ± 5.23ba 4.37 ± 1.01a 0.05 ± 0.02a 0.02 ± 0.01aFBMc 3.90 ± 0.84b 9.11 ± 0.06b –db – b

Dough sample (mmol/kg)

WWB bDLea bDLa bDLa bDLa –

WWB + LbBio 9.31 ± 2.71b 3.43 ± 1.47a 0.02 ± 0.01b 0.01 ± 0.00b 2.71WWB + FBM bDLa 2.58 ± 2.40a bDLa bDLa 0.18WBB bDLa 11.13 ± 1.7b bDLa bDLa –

WBB + LbBio 9.36 ± 5.39b 4.60 ± 2.74ac 0.02 ± 0.01b 0.01 ± 0.01b 2.03WBB + FBM bDLa 8.32 ± 2.66bc bDLa bDLa 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 b 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), and 0.0008 mmol/kg (OH-PLA).

5F. Valerio et al. / Innovative Food Science and Emerging Technologies 25 (2014) 2–8

bran (WBB + LbBio) (Table 2).When aflour-basedmedium (FBM)wasused as a control instead of LbBio, no inhibition of bacterial count wasobserved.

The production of lactic, acetic, phenyllactic (PLA) and OH-phenyllactic (OH-PLA) acids was evaluated in LbBio, after growingL. brevis LMG P-25726 for 18 h, in FBM and in the resulting dough sam-ples (Table 3). No trace of propionic, valeric, isovaleric, butyric, andisobutyric acidswas foundboth in the LbBio, FBMand in dough samples.The concentration of lactic acid, acetic acid, PLA and OH-PLA producedby L. brevis LMG P-25726 in the bioingredient was found to be signifi-cantly higher (p N 0.05) than in the flour-based medium. Dough sam-ples with bioingredient contained all the acids and showed the highermolar 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).

In order to assess the influence of each factor (bran and/or LbBio)and their interaction on bread quality, a two-way ANOVA test wasperformed (Table 4). The analysis demonstrated the ability of LbBio tosignificantly affect the dough content of lactic acid, PLA and OH-PLAregardless of the presence of bran and this effect determined the re-duction of bacterial count after 3 days of storage. Whereas, significanteffect of bran and interaction between the two factors (LbBio andbran) were observed for the acetic acid in dough and for bread contam-ination (3 day-storage), although in these bread formulations (WBBand WBB + LbBio) bacterial counts were not modified (Table 2 andTable 4). These observations were explained by the multiple compari-son Tukey's test performed to establish which pairs of levels of the fac-tors were significantly different for each variable (Fig. 1). The analysisconfirmed that a significant inhibitory effect on bread contaminationwas exerted by the LbBio (Table 2; Fig. 1, comparison 2), but this abilitywas influenced by the presence of bran. In fact, the addition of LbBio tothe dough together with bran did not produce any significant changes

Table 4Two-way ANOVA test p values to measure the effect of the addition of bran and/or LbBioand their interaction on bacterial contamination of bread during storage at 30 °C for differ-ent times and on the organic acids content of dough.

Bacterial contaminationof bread

Organic acids content in dough

Factors T0 T1 day T3 days Lactic acid Acetic acid PLA OH-PLA

Bran 0.1616 0.7735 0.0008 0.9875 0.0001 0.8048 0.2396LbBio 0.3673 0.1081 0.0000 0.0009 0.1507 0.0012 0.0003Bran * LbBio 0.1842 0.9319 0.0195 0.9875 0.0005 0.8048 0.2396

on bread contamination (Table 2; Fig. 1, comparison 5). The additionof bran to the dough without LbBio did not modify its contaminationlevel (Table 2; Fig. 1, comparison 1), whereas bran added to the doughwith LbBio (WBB + LbBio) led to an increase in the bacterial count ofbaked bread with respect to the formulation lacking bran (WWB +LbBio) (Table 2; Fig. 1, comparison 6). At the same time LbBio signifi-cantly reduced the effect of bran on the acetic acid content in thedough (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 tex-tural properties of bread is reported in Tables 5 and 6, Figs. 1 and 2.Results indicated that white wheat bread (WWB) was significantlydifferent from bread containing wheat bran (WBB) in all propertiesexcept for water activity, bread pH and hardness at 25% deformation(Tables 5 and 6, Fig. 1, comparison 1). The use of LbBio significantlyaffected dough and bread pH values inWWBwhich resulted most acid-ifiedwhile this effectwas limited by the presence of bran (Table 5, Fig. 1,comparisons 2 and 5). At the same time, a significant increase in theTTA values was observed in both dough types after LbBio application(WWB + LbBio and WBB + LbBio). The two-way ANOVA confirmedthat the addition of LbBio significantly affected dough and bread pHand dough TTA values (Table 5) and that this effect was significantlyinfluenced by bran addition (see the interaction between factorsBran * LbBio in Table 5).

4. Discussion

Results of this work indicated an effective role of L. brevis LMGP-25726 in the LbBio technological and antimicrobial efficacy andconfirmed the applicability of LAB-based fermentation products asbioingredients in yeast-leavened bread.

The microbiological quality of breads during storage was improvedand the rope spoilage was delayed or hampered, in the presence ofLbBio, confirming the synergistic role of organic acids as previously ob-served (Mantzourani et al., 2014; Valerio et al., 2008). Although a highacetic acid content and FQ down to 1 can be associated to enhanced in-hibitory efficacy against rope producing bacteria (Röcken, 1996), thepresence of only acetic acid in breads not prepared with LbBio did notwarranty bacterial inhibition. Additionally, other authors demonstratedthat acetic acid can determine a shorter and harder gluten and that theproduction of lactic acid is advisable to increase elasticity of dough(Gobbetti, Corsetti, & Rossi, 1995). In fact, the high molar lactic/acetic

selbairavhguoDselbairavdaerB

Specific a

Bacterial contamination

Hardness 25%

Hardness 40%

Dough Dough volume

pH w

in bread deformation deformationTTA pH

Organic acids in dough

ComparisonsGrouping

variables (A)Grouping

variables (B)T0 T1 T3 T0 T1 T3 T0 T1 T3

Lactic acid

Acetic acid

PLA OH-PLA

0000000000BBWBWW1

2 WWB 0 0 0 0 0 0 0 0 0 0WWB + LbBio 0 0 0 0 0 0 0 0 0 0

000000oiBbL+BWWBBW4

3 WWB WBB + LbBio 0 0 0 0 0 0 0 0 0 0 0 0

5 WBB WBB + LbBio 0 0 0 0 0 0 0 0 0 0 0 0

00000000000oiBbL+BBWoiBbL+BWW6

Means no difference between the mean values of Group A and Group B

Means Group A mean value < Group B mean value

Means Group A mean value > Group B mean value

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

Table 5Physico-chemical properties of bread types and two-way ANOVA test p values to measure the effect of the addition of bran and/or LbBio on physico-chemical properties of bread anddough.

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 ± 0.50bWWB + LbBio 4.57 ± 0.14a 4.45 ± 0.07 3.10 ± 0.00 0.94 ± 0.01 4.82 ± 0.18bWBB 5.43 ± 0.15a 3.96 ± 0.06 2.65 ± 0.23 0.96 ± 0.01 5.49 ± 0.14aWBB + LbBio 5.17 ± 0.11a 5.70 ± 0.14 2.65 ± 0.35 0.95 ± 0.01 5.20 ± 0.16a

Two-way ANOVAp values

Factors

Bran 0.0000 0.0000 0.0341 0.0678 0.0705LbBio 0.0000 0.0000 0.8036 0.7384 0.000Bran * LbBio 0.0490 0.0452 0.8229 0.3396 0.0041

a Values represent volume (mL) of NaOH 0.1 N. TTA was determined using the AOAC method No. 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 b 0.05).

Table 6Crumb hardness (N) of bread containing or not bran and/or the bioingredient LbBio during storage at 25 °C for 3 days. Two-way ANOVA test p values to measure the effect of the additionof 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 T1 day T3 days T0 T1 day T3 days

WWB 6.49 ± 1.55aa 14.85 ± 1.39b 29.04 ± 1.28c 16.79 ± 2.35 a 22.37 ± 3.34a 35.08 ± 6.57bWWB + LbBio 8.28 ± 0.98a 11.32 ± 2.70a 23.28 ± 2.72b 14.99 ± 1.92a 21.70 ± 0.92b 29.27 ± 3.43cAWBB 16.81 ± 0.92a 20.78 ± 2.53a 32.85 ± 4.04b 19.49 ± 8.55a 35.65 ± 4.40b 46.38 ± 6.24cWBB + 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-way ANOVAp values

Factors

Bran 0.0000 0.0014 0.0002 0.1884 0.0000 0.0000LbBio 0.0003 0.0499 0.0057 0.5924 0.0942 0.0248Bran * 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 betweensampling times by the Tukey's test (p b 0.05).

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Fig. 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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acid ratio (FQ N 1) generally obtained in sourdough is associated topleasant odour, sensory properties and improved shelf-life. In ouryeast-leavened bread types, the presence of the LbBio allowed to obtainoptimal FQ, TTA and pH values comparable to those previously reportedfor sourdough bread (FQ N 1, TTA: 4.8–6.2; pH: 4.8–5.1) (Katina et al.,2002; Mantzourani et al., 2014).

Additionally to the influence onmicrobiological quality, the applica-tion of LAB bioingredients in bread-making could have positive effectson some physico-chemical and textural properties of bread as widelyreported (Komlenić. et al., 2010; Pepe, Ventorino, Cavella, Fagnano,& Brugno, 2013). Acids produced during fermentation can influencethe mixing behaviour of dough, since the gluten protein solubilitywas proved to cause an increase in softness and elasticity of gluten(Takeda, Matsumura, & Shimizu, 2001; Wehrle, Grau, & Arendt, 1997).In addition, the dough acidification could enhance enzymatic activityof wheat proteases, causing an increase in free amino acid content andimproved bread flavour (Clarke, Schober, Dockery, O'Sullivan, &Arendt, 2004; Rizzello, Coda, Mazzacane, Minervini, & Gobbetti, 2012;Thiele et al., 2002). In our study, a significant increase in TTA valueswas observed in both dough types containing LbBio and values werecomparable to those obtained in L. brevis sourdough (Komlenić. et al.,2010). Furthermore, the addition of bran significantly influenced theeffect of LbBio on pH and TTA values (see the interaction betweenfactors Bran * LbBio in Table 6). This result is consistent with the acidityincrease of dough when sourdough was prepared with bran fractions,than confirming an interaction occurring between LAB and dietaryfibres (Rizzello et al., 2012). Recently, Pepe et al. (2013) demonstratedthe contribute of LAB producing exopolysaccharides (EPS) in combina-tionwith immaturewheat grain flour containing fructo-oligosaccharides(FOS), in improving the nutritional and technological characteristicsof breads. The ability of LAB to produce EPS is frequently exploited inbread technology to reduce the amount of additives since these metabo-lites influence the viscoelastic characteristics and stabilise the rheologicalproperties of dough (Palomba et al., 2012).Moreover, the use of rich-FOSprebiotics can stimulate themicrobial metabolism leading to an increasein TTA (Pepe et al., 2013).

5. Conclusions

The bioingredient L. brevis-based LbBio, acting as a sourdough, coun-teract the negative effects of bran allowing to obtain an enriched-fibre

bread with overall quality comparable to that of the reference whitewheat bread. The study also indicated that bread formulation can mod-ulate bacterial behaviour during storage and consequently the breadspoilage. Further investigations are needed to better evaluate the posi-tive and negative interactions on bread quality occurringwhen differentlevels of dietary fibres are used in the presence of the bioingredientLbBio.

Acknowledgements

This research has received funding from the European Community'sSeventh Framework Programme (FP7/2007–2013) under the grantagreement no. FP7-222 654-DREAM.

N.A. and T.M.C were supported by Project FIRB CAROMICSRBAP11B2SX.

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