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Food Control

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A novel lactic acid bacterium for improving the quality and shelf life ofwhole wheat bread

Lei Sun, Xiangfei Li, Yingyue Zhang, Wenjian Yang, Gaoxing Ma, Ning Ma, Qiuhui Hu, Fei Pei∗

College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Centre for Modern Grain Circulation and Safety, Nanjing,210023, People's Republic of China

A R T I C L E I N F O

Keywords:Whole wheatSourdoughLactic acid bacteriaQualityShelf life

A B S T R A C T

The aim of this study was to screen for a lactic acid bacterium (LAB) that could improve the quality of wholewheat bread (WWB) and extend its shelf life. The LABs with strong antifungal activity were screened amongtwenty LABs. Both sourdough properties (rheology, tensility, water mobility and gluten structure) and WWBqualities (texture, volatile flavour and shelf life) were determined and compared. The results showed that theLactobacillus plantarum LB-1, F-3 and F-50 exhibited stronger antifungal activity among the twenty tested LABs.Moreover, compared to the other LABs, the sourdough fermented by LB-1 demonstrated significantly betterviscoelasticity, extensibility, and water holding capacity, as well as a more ordered gluten secondary structure(6.49% more than the control). Meanwhile, Lactobacillus plantarum LB-1 can remarkably improve the texturecharacteristics, enrich the aroma volatile compounds, and prolong the shelf life of WWB from 3 days to 6 dayscompared to the control group. Above all, Lactobacillus plantarum LB-1 was recommended for WWB fermentationto improve its quality and to extend the shelf life.

1. Introduction

Whole wheat bread (WWB) is a kind of bread made from wholewheat flour without removing the bran and wheat germ. Compared torefined wheat bread, it is rich in higher levels of dietary fibres, mi-nerals, phytochemicals, antioxidants and vitamins (Tebben, Shen, & Li,2018). The American Institute for Cancer Research (AICR) and theWorld Cancer Research Fund (WCRF) reported that eating whole grainfoods such as WWB daily can reduce the risk of colorectal cancer (Lafay& Ancellin, 2015) and serum cholesterol (Slavin & Joanne, 2004).Nowadays, consumers worldwide have shown increasing interests inreducing disease risks and managing chronic diseases by eating wholegrain food (Niu, Hou, Kindelspire, Krishnan, & Zhao, 2017).

However, compared to refined wheat bread, WWB may be moresusceptible to fungal infections because it contains the intact grain andepidermal part (Zhang, Pei, Fang, Li, Zhao, Shen, et al., 2019). Mostfungi were located in the outer layer of wheat kernels. These fungi canact as a potential reservoir of contamination and produce mycotoxins(including aflatoxin, zearalenone and deoxynivalenol) in the manu-facturing environment, which cause more safety hazards (Saladino, Luz,Manyes, Fernández-Franzón, & Meca, 2016). Moreover, the productacceptability of WWB is lower due to the crude fibre content (Bin &Peterson, 2016). The compounds and origin of bitterness in WWB

including amadori rearrangement product (ARP) and 5-(hydro-xymethyl) furfural (HMF) can seriously affect its taste (Jiang &Peterson, 2013).

Recently, the improvement of WWB is mainly based on using che-mical leavening and preservative agents. Hydrocolloids, such as hy-droxypropyl methyl cellulose, carboxymethyl cellulose, locust beangum and xanthan gum, have been widely used to improve the water-absorbing and gas-holding capacities of bread (Anna-Sophie & Arendt,2013; Matuda, Chevallier, de Alcântara Pessôa Filho, LeBail, & Tadini,2008). Moreover, Katsinis, Lohano, Sheikh, and Shahnawaz (2009)assessed the chemical preservatives calcium propionate for its effect onmaintaining the quality of bread, proving that calcium propionate couldextend its shelf life significantly. However, these chemical additivesmay cause changes in the natural components of whole wheat, some ofwhich may be toxic to the human body (Ginocchio et al., 1979).Therefore, it is of great significance to develop natural functional doughimprovers.

Since chemical additives have anti-nutritional and toxic effects, LABas a natural starter has been paid widespread attention. In a study byDal Bello, Clarke, Ryan, Ulmer, and Schober (2007), lactic acid, phe-nyllactic acid and cyclic dipeptides cyclo produced by Lactobacillusplantarum FST 1.7 showed consistent inhibition against Fusarium speciesand had the potential to extend the shelf life of bread. In addition,

https://doi.org/10.1016/j.foodcont.2019.106914Received 28 June 2019; Received in revised form 25 August 2019; Accepted 21 September 2019

∗ Corresponding author.E-mail address: feipei87@163.com (F. Pei).

Food Control 109 (2020) 106914

Available online 24 September 20190956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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Wang, Hwang, Tzeng, Hwang, and Mau (2012) suggested that the totalvolatile content including alcohols, esters and carbonyls of bread madewith Lactobacillus delbrueckii subsp. Delbrueckii was significantly higherthan those in the blank group. However, most of these studies werelimited to refined wheat, while fungal contamination and poor qualityare far more serious problems in whole wheat. Therefore, the overallpurpose of this study was finding a novel LAB to simultaneously solvethe problem of fungal contamination and distastefulness in WWB.

In this study, specific LABs with high antifungal activity werescreened from a total of twenty LABs. Subsequently, the effects of theselected LABs on dough improvement were evaluated via rheologicalmeasurements, tensile tests, water mobility determination, gluten sec-ondary structures quantification and microstructure observation. Thesourdoughs were then processed into breads, and their texture, volatilecompounds and shelf life were also analysed to verify the applicabilityof the LABs as starters for WWB.

2. Materials and methods

2.1. Strains and culture conditions

Twenty-six strains, comprised of twenty LABs (F-1, F-3, F-6, F-13, F-17, F-24, F-48, F-50 and F-59 were obtained from the Key Laboratory ofGrains and Oils Quality Control and Processing. LB-1, LB-2, LB-3, LB-4and LB-5 were isolated from local pickles in Nanjing, China.Lactobacillus rhamnosus (BNCC 136673), Leuconostoc mesenteroides(BNCC 195309), Lactobacillus plantarum (BNCC 194165) andLactobacillus casei (BNCC 134415) were purchased from the BeNaCulture Collection. Lactobacillus rhamnosus strain GG (ATCC 53103)was purchased from the American Type Culture Collection), and sixfungi (Penicillium citrinum, Aspergillus niger, Aspergillus flavus, Aspergillusochraceus, Aspergillus fumigatus and Fusarium graminearum were isolatedfrom whole wheat) were studied in this experiment. LAB strains werepreserved in sterile 25% glycerol at −80 °C before use.

2.2. LAB suspension and fungus spore suspension preparation

Single LAB strains (2% v/v) were inoculated into MRS broth at 37 °Cfor 20 h. The cells were harvested by centrifugation (5500 g for10min at 4 °C) and the supernatant was removed. The obtained LABcells were suspended in sterile water and counted, varying from 107 to109 CFU/mL. The fungi were inoculated in PDA medium at 28 °C for7–10 days. The spores were harvested from Petri plates in sterile waterto prepare a suspension containing 1×104 spores/mL. The LAB sus-pension and fungus spore suspension were preserved at 4 °C before use.

2.3. Antifungal activity in vitro

The dual culture method described by Quattrini et al. (2018) wasmodified and used to test the antifungal activity of LABs. Briefly, 10 μLof the LAB suspension (109 CFU/mL) was dropped on the centre of MRSagar plates and incubated at 37 °C for 48 h. Subsequently, 10mL of PDAcontaining 104 spores/mL was cooled to 50 °C and poured on the plates.Plates were incubated at 28 °C for 4 days, and the sizes of the inhibitionzone were measured using an electronic digital calliper (Guanglu,Guilin, China). The antifungal activity of the LABs was calculated onthe basis of the inhibition zone as no inhibition (−) for an inhibitionzone smaller than 0.05 mm, weak inhibition (+) for an inhibition zonesmaller than 10 mm, moderate inhibition (++) for an inhibition zonein the range of 10–20 mm, and strong inhibition (+++) for an in-hibition zone larger than 20 mm.

2.4. LAB identification

DNA was isolated from selected strains using an EasyPure GenomicDNA Kit (Transgen Biotech, Beijing, China) according to the

instructions. The primer sets 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for amplifi-cation of the gene. PCR conditions reported by Joo et al. (2015) wereset as follows: 95 °C for 5min followed by 30 cycles of 95 °C for 1min,52 °C for 30 s, and 72 °C for 1min 30 s, with a final extension at 72 °Cfor 7min. The amplified DNAs were purified and sequenced using anABI 3730XL DNA analyser (Life Technologies, New York, USA). The 16SrRNA sequences were blasted against the NCBI GenBank database(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.5. Sourdough fermentation and bread manufacture

Whole wheat was obtained from the China Grain Reserves Group(Sinograin, Nanjing, China). The sourdough recipe according to GB/T14611–2008 included 200 g of whole wheat, 3.6 g of yeast powder(Angel Yeast, Inner Mongolia, China), 3 g of salt, 3 g of butter, 12 g ofsugar (Herunhua FOOD, Jiangsu, China) and 120 g of LAB suspension(108 CFU/g). Dough with the same quantity of water (containing noLAB) was set as the control group. The sourdoughs were kneaded by adough mixer for 6min and then placed in a fermentation box (tem-perature was 30 ± 1 °C and humidity was 85%–90%) for 5 h.Subsequently, the sourdough was divided into 100 g portion and bakedin a JKLZ4 oven (Fude Technology, Beijing, China) at 200 °C for 20min.

2.6. Rheological measurements

Dynamic rheological properties of the sourdough were measured bya rheometer (Anton-Paar-Strasse, Austria) using oscillation sweep tests.Sourdough was placed on the centre of the rheometer plate and left for5min for relaxing the residual stress. Mineral oil was daubed aroundthe edge of the sourdough to prevent moisture loss. A strain of 0.5% anda measure position of 2mm were applied in this test. The test tem-perature was 25 ± 1 °C and the test frequency was in the range of0.1–40 Hz (Hao et al., 2008; Inglett, Chen, Liu, & Lee, 2014).

2.7. Tensile properties tests

The tensile properties of the sourdough were measured using a TA-XT Plus texture analyser (Stable Micro Systems, London, England) withan A/KIE probe. A trigger force of 5 g and a test distance of 50mm wereapplied to perform this test. The pre-test speed, test speed and postspeed were 2mm/s, 3.3 mm/s and 10mm/s, respectively. The sour-dough was rested in the fermentation box (temperature was 30 ± 1 °Cand humidity was 85%–90%) for 10min before the test. Each groupwas tested six times (Suchy, Lukow, & Ingelin, 2000).

2.8. Water mobility determination

Water mobility of the sourdough was detected using low field nu-clear magnetic resonance (LF-NMR) (MesoMR, Niumag Corporation,China). The sourdough was evenly stuffed into the sample vials andthen inserted in the NMR probe. Carr-Purcell-Meiboom-Gill (CPMG)sequences were employed to measure spin-spin relaxation time (T2)(Ding et al., 2015). Test pulse parameters were as follows:SW=200 KHz, SF=19MHz, RFD=0.5ms, O1=948164.27 Hz,RG1=20 db, P1=13 us, DRG1=3, TD=300150, TW=1500ms,P2= 25 us, TE= 0.2ms, NECH=7500 and NS=32. T21 representsthe bound water, T23 represents free water, and T22 represents the ad-sorbed water between the bound water and free water (Doona & Moo-Yeol, 2007).

2.9. Fourier transform infrared spectroscopy (FTIR) and gluten secondarystructure

Gluten was isolated and collected from the dough according to theAACCI Approved Method 38–10.01. The gluten was dried using an oven

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at 40 °C. Then, the dried gluten was ground and sieved (75 μm).Subsequently, gluten mixed with KBr (1% (w/w) per gram of KBr) wasground into a uniform powder again and pressed into sheets using apowder pressing machine (769 YP-15A, Tianjin, China). A total of 64scans were run for each analysis at an interval of 4 cm−1 in the range of400–4000 cm−1. The curve of the Amide I region was selected andfitted by a module (AutoFit Peaks II Second Derivative) of Peakfit V4.12software. Then, the percentage of peak areas corresponding to thegluten secondary structure was given (Chen et al., 2019). The spectralregions were assigned as 1612–1640 cm−1 and 1670–1694 cm−1 for β-sheets, 1640–1650 cm−1 for random coils, 1648–1660 cm−1 for α-he-lices, and 1662–1684 cm−1 for β-turn structures. The second derivativearea for each secondary structural region was divided by the total areaof the amide I region (Carbonaro & Nucara, 2010; Goormaghtigh,Cabiaux, & Ruysschaert, 1994; Pelton & McLean, 2000).

2.10. Scanning electron microscopy (SEM)

Sourdough flakes were fixed in glutaraldehyde (2.5%) for 12 h anddehydrated in increasing grades of ethanol (25%, 50%, 75%, 95% and100%). Then, the sourdough flakes were coated with gold particlesusing a MSP-1S Sputter Coater (Hitachi, Tokyo, Japan) after lyophili-sation. Microstructures of the different sourdoughs were photographedusing a TM3000 SEM (Hitachi, Tokyo, Japan) at 250× and1000×magnification (Liu et al., 2015).

2.11. Gas chromatography-mass spectrometry

Aroma volatile compounds were analysed by headspace gas chro-matography mass spectrometry (GC-MS) using the solid-phase micro-extraction (SPME) method as previously described (Plessas et al.,2008). Briefly, 2 g of a bread sample was placed in a 20mL glass vialand incubated in a water bath at 60 °C. The SPME fibre (50/30 μmDVB/CAR/PDMS, Stable Flex Supelco, Bellefonte, PA, USA) was ex-posed to the headspace for 60min. Desorption of volatiles was in theinjector port (280 °C) of the gas chromatograph (Agilent7890A, PaloAlto, CA, USA) for 5min in splitless mode. An Agilent DB-5MS capillarycolumn (0.25 μm film thickness, 30m×250 μm) was used. The GCtemperature program (total run time 51.73min) was as follows: 35 °Cfor 5min, then increased by 5 °C/min to 50 °C (held for 5min),

increased by 5.5 °C/min to 230 °C (held for 5min). The carrier gas wasHe at a flow rate of 2mL/min. The mass spectrum was recorded byelectronic impact (EI) at 70 eV. The scan mode was in the range of m/z33–200. Compounds were identified by the MS database (NIST 98)combined with Kovats indexes (KI) comparison. Match quality higherthan 80% was considered to be reliable. Experimental KI was based onn-alkanes (C7 to C30, o2si smart solutions, USA). Compounds werequantified by a peak area normalization method.

2.12. Textural measurement and fungi spoilage analysis

The textural properties of the WWB were measured using a TA-XT2itexture analyser (Stable Micro Systems, London, England) with a P/36Rprobe. Each sample was cut into 1 cm thick slices and placed for 1 h forcooling. Test parameters according to AACCI74-09 were as follows: pre-test speed, 3 mm/s; test speed, 1 mm/s; post speed, 5 mm/s; triggertype, auto-10 g; target mode, distance-5 mm; interval between twocompressions, 10 s. For the fungi spoilage analysis, bread samples werepacked in polyethylene bags and stored at room temperature(23 ± 1 °C). Fungal colonies on the bread surface were monitoreddaily for one week. Contamination was calculated as follows: no visiblecolonies (−), one colony (+), two (++), and three or more (+++)(Bartkiene, Bartkevics, Lele, Pugajeva, Zavistanaviciute, Mickiene,et al., 2018).

2.13. Statistical analysis

All of the experiments were repeated at least three times. Means andstandard deviations were calculated. The results were analysed by SPSSversion 23 software for Windows (SPSS Inc., Chicago, IL, USA). Duncantests at a significance level of P < 0.05 were performed for significanceanalysis.

3. Results and discussion

3.1. Selection of LABs with strong antifungal activity and LABsidentification

A screen of the twenty LAB strains was carried out by measuringantifungal activity against six susceptible fungi of wheat: Penicillium

Table 1Antifungal activity of LAB strains against different fungi.

Strains Antifungal activitya

Penicillium citrinum Aspergillus niger Aspergillus flavus Aspergillus ochraceus Aspergillus fumigatus Fusarium graminearum

F-50 +++ ++ +++ ++ +++ +++F-3 +++ ++ +++ ++ +++ +++LB-1 +++ ++ ++ ++ +++ +++LB-2 +++ + ++ ++ ++ +LB-3 +++ ++ ++ + +++ +LB-4 +++ – +++ + ++ +LB-5 + + + – – –LGG +++ – ++ – +++ ++F-1 ++ + ++ + ++ +F-6 + – – – – +F-13 + – – – – –F-14 – – ++ – + ++F-17 – – – – ++ ++F-24 – – ++ – +++ –F-48 – + +++ – ++ –F-59 +++ – ++ + +++ ++

BNCC 136673 +++ + +++ + +++ +++BNCC 195309 +++ + + + +++ ++BNCC 194165 +++ + +++ ++ ++ +BNCC 134415 +++ – ++ – + –

a Calculation of antifungal activity: smaller than 0.05 mm diameter clearing zone (−), 10 mm diameter clearing zone (+), 20 mm diameter clearing zone (++),and more than 20 mm diameter clearing zone (+++).

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citrinum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus,Aspergillus fumigatus and Fusarium graminearum. These fungi are con-sidered to be the most common spoilage agents in bakery products dueto their high prevalence in wheat flour and their xerotolerance to xer-ophilic behaviour, which can act as a potential reservoir of con-tamination in the manufacturing environment (Saladino et al., 2016).The antifungal activity of LAB was calculated on the basis of the in-hibition zone, and the results are shown in Table 1. Most of the LABstrains demonstrated strong inhibition against Penicillium citrinum, As-pergillus flavus, Aspergillus fumigatus and Fusarium graminearum, whileAspergillus niger and Aspergillus ochraceus were inhibited only by certainkinds of LAB strains. Remarkably, compared to the other LAB strains,the LB-1, F-3 and F-50 strains possessed at least moderate inhibitoryactivity (++) against all six susceptible fungi. Therefore, LB-1, F-3 andF-50 were selected to further compare their effects on the qualities ofWWB.

The 16S rRNA sequencing results of the selected LAB strains LB-1, F-3 and F-50 are shown in Table S1, S2 and S3. The 16S rRNA genes of theselected LAB strains LB-1, F-3 and F-50 were identical (100%) to theLactobacillus plantarum NCU116 (Accession: CP016071.1), Lactobacillusplantarum CB5 (Accession: MK687387.1) and Lactobacillus plantarum

EM (Accession: CP037429.1) in 1400, 1407 and 1449 nucleotides, re-spectively (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Therefore, thethree selected LAB strains were classified as Lactobacillus plantarum LB-1, Lactobacillus plantarum F-3 and Lactobacillus plantarum F-50, respec-tively.

3.2. Effect of LABs on rheology, tensility and water mobility of sourdoughs

The sourdough properties fermented by the selected LAB strainswere evaluated via rheology, tensility and water mobility determina-tion (Fig. 1). The G′ and G″ of different sourdoughs at a frequency of0.1–40 Hz are shown in Fig. 1A and B. Storage modulus (G′) representsthe ability of materials to store elastic deformation energy, and lossmodulus (G″) represents the viscous portion (Meyers & Chawla, 1990).It could be seen that both moduli (G′ and G″) of all sourdoughs in-creased with the increase of frequency. Moduli (G′ and G″) of sour-dough LB-1 and F-3 were significantly higher than that of F-50 and thecontrol group, which indicated that sourdoughs fermented by LB-1 andF-3 were more viscous and elastic. Ghodke and Laxmi (2007) found thatexopolysaccharides (EPS) produced by Lactobacillus buchneri FUA3154can replace hydrocolloids to influence the rheological properties of

Fig. 1. Effect of different LABs on rheology (A and B), tensile property (C), water mobility (D) during sourdough fermentation.

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sourdoughs. The greater viscosity and elasticity in sourdough LB-1 andF-3 may be attributed to the cementability and stability of EPS.

Tensile properties are important indices in dough baking and areclosely related to gluten network formation and dough gas-holdingcapacity (McCann, Le Gall, & Day, 2016). Fig. 1C shows the tensileresistance and extensibility of the different sourdoughs. It can be seenthat the tensile resistances of the three fermented sourdoughs weresignificantly higher (P < 0.05) than that of the control group. Mean-while, sourdough LB-1 and F-50 demonstrated better tensile resistancecompared to sourdough F-3. Additionally, the extensibilities of sour-dough F-3 and LB-1 were significantly higher (P < 0.05) than those ofF-50 and the control group. The results indicated that the extensibilityand strength of the gluten network structure in sourdough LB-1 and F-3were enhanced. A previous study reported by Schober, Bean, and Boyle(2007) showed that proteolysis during sourdough fermentation couldreduce the interference of protein with starch and result in strongerstarch gel strength, which was consistent with the findings of this study.

Water mobility is one of the main characteristics of dough and playsan important role in the dough quality. Continuous distributions ofspin-spin relaxation time (T2) are shown in Fig. 1D. As shown inFig. 1D, water in the dough mainly existed in the form of adsorbedwater (T22). However, two distinct peaks appearing at T21 representedthe bound water in sourdough LB-1 and F-3, which can be regarded as

the water closely bound to protein and starch, influencing the forma-tion of the gluten network structure in dough (Assifaoui, Champion,Chiotelli, & Verel, 2006). Therefore, it can be concluded that thesourdough LB-1 and F-3 possessed stronger water-holding capacitycompared to the control group. Zhang, Doehlert, and Moore (1997)studied the effect of β-glucan on oat flour, and they found that theviscosity and the water binding capacity of oat flour was positivelycorrelated with the content of β-glucan. LABs can produce β-glucan inthe process of growth and metabolism. Therefore, sourdough absorbedmore water and became more viscous due to the presence of β-glucan,which showed strong agreements with rheological measurements(Fig. 1A) in this study.

3.3. Secondary structure of gluten and SEM observation

The gluten network structure is the backbone structure of glutenincomplexes formed by glutenin subunits crosslinking through disulfidebond ends (Ooms & Delcour, 2019). Therefore, from a microcosmicperspective, the secondary structures of gluten and the exterior shape ofthe sourdoughs were studied to explain the orderliness of sourdoughsby FTIR and SEM respectively. The FTIR spectra of the different samplesare shown in Fig. 2A. Amide I region (1600–1700 cm−1) has been usedto study the secondary structure of gluten because it is almost entirelyattributable to C]O stretch vibrations and is sensitive to gluten con-formation (Robertson, Gregorski, & Cao, 2006). The curve of the AmideI region was selected and fitted to calculate the secondary structures ofgluten using Peakfit V4.12 software. As seen in Fig. 2B, the LAB strainshad a great influence on the secondary structures of sourdoughs. β-sheets and α-helices were the main secondary structures of the gluten,which is consistent with a previous study (Wang et al., 2016). With theaddition of the LAB strains, there was a dramatic decrease in randomcoils together with significant increases in β-turns, indicating con-formational changes in the gluten. Compared to the control group, thesum of β-sheets and α-helices for sourdoughs LB-1, F-3 and F-50 in-creased by 6.49%, 4.49% and 0.12%, respectively. Previous studies ongluten secondary structures proved that β-sheets and α-helices enableddough to form a more ordered network structure, while random coilswere considered as disordered structures (Marti, Bock, Pagani, Ismail, &Seetharaman, 2016; Mecozzi & Sturchio, 2015). The increase in β-sheets and α-helices indicated that sourdough LB-1 formed a moreregular and orderly gluten structure.

The microstructures of the sourdoughs were detected by SEM atmagnifications of 250× and 1000× to further analyse the glutenstructure. As shown in Fig. 3A, discontinuity of the gluten networkstructure could be obviously observed in the control group. Starchgranules aggregated in clusters and did not disperse evenly in thenetwork structure (Fig. 3a). This was due to the addition of bran, whichcan cause water redistribution and a partial dehydration of gluten (Bock& Damodaran, 2013). For the sourdoughs F-50 and F-3, a break injunctions of the membrane-like gluten matrix could be clearly observed(Fig. 3C) and most starch granules were aggregated into clusters andexposed to stomata (Fig. 3d). Compared to the other groups, the sour-dough LB-1 (Fig. 3B) formed a more ordered and compact gluten net-work structure, and the starch granules were wrapped tightly andevenly in the membrane-like gluten matrix (Fig. 3b). This phenomenonindicated that the gluten network of sourdough, which was wellformed, possessed good ductility when fermented by LB-1.

3.4. Effect of different LABs on textural properties

After evaluating the rheology, tensility and water mobility of thesourdoughs, we made the sourdoughs into WWB according to GB/T14611–2008 and tested the texture characteristics of WWB, includinghardness, springiness, cohesiveness, gumminess and chewiness(Table 2). It was observed that compared to the control, F-3 and F-50groups, the hardness, gumminess and chewiness of WWB LB-1 were

Fig. 2. ATR-FTIR spectrum of gluten in different sourdoughs (A), and fittingresults of Amide I region for secondary structure content of gluten protein (B).

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significantly (P < 0.05) lower, and the cohesiveness was significantly(P < 0.05) higher. A large number of studies have proven that hard-ness, gumminess and chewiness are negatively correlated with breadquality, while springiness and cohesiveness are positive attributes (Al-Farga, Zhang, Siddeeg, Chamba, Kimani, Hassanin, et al., 2016;

Wronkowska, Jadacka, Soral-Śmietana, Zander, Dajnowiec,Banaszczyk, et al., 2015). The results obtained from TPA indicated thatthe WWB fermented by Lactobacillus plantarum LB-1 tasted springier,softer and more refreshing, which more closely caters to consumertastes compared to other groups.

Fig. 3. Scanning electron micrographs of sourdoughs (Control: A= 250× , a=1000× ; LB-1: B= 250× , b=1000× ; F-50: C= 250× , c=1000× ; F-3:D= 250× , d= 1000× ).

Table 2The texture properties of WWB made with Lactobacillus plantarum LB-1, F-3 and F-50 (n=8).

Sample Hardness Springiness Cohesiveness Gumminess Chewiness

Control 383.57 ± 8.45a 0.99 ± 0.01a 0.91 ± 0.02b 365.59 ± 5.67a 363.29 ± 7.30aLB-1 253.77 ± 10.83b 0.97 ± 0.03a 0.95 ± 0.01a 236.12 ± 5.48b 230.17 ± 12.90bF-3 376.27 ± 15.15a 0.96 ± 0.03a 0.92 ± 0.02 ab 345.12 ± 13.87a 330.52 ± 14.89aF-50 364.80 ± 17.87a 0.95 ± 0.02a 0.91 ± 0.02b 330.83 ± 19.80a 316.27 ± 15.24a

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3.5. Aroma volatile compounds analysis

Sixty-two aroma volatile compounds of the WWBs were detected bySPME-GC-MS and the results are shown in Table 3. It is noteworthy thatafter fermentation by LB-1, F-3 and F-50, the aroma volatile compounds(60, 54 and 55 kinds of volatile compounds, respectively) increased andwere enriched compared to the control group (37 kinds of volatilecompounds). Meanwhile, carboxylic acids of the control group (1.87%)were significantly lower than those of the WWB LB-1, F-3 and F-50groups (24.70%, 21.61% and 22.13%, respectively), and the content ofacetic acid was the highest among them. This is consistent with aprevious study on the flavour of sourdough bread by Cavallo et al.(2017), who reported that acetic acid could be generated during theprocessing of bread fermentation. Moreover, Su et al. (2019) studiedorganic acids on bread quality improvement, finding that acetic acidcould give bread a higher specific volume, a lower pH value and adecreased hardness. In addition, the acetic acid content determined theefficiency of sourdough as a possible preservative agent against themicrobial spoilage of bread (Martínez-Anaya, Llin, Macías, & Collar,1994).

3.6. Effect of different LABs on fungus infection during WWB storage

The fungus colonies on the bread surface were monitored dailyduring a test of a one week shelf life (Table 4). Two visible funguscolonies were first observed in WWB F-50 on the third day and thenobserved in the WWB F-3 on the fifth day. In the control group, visiblefungus colonies were detectable on the fourth day, whereas in the WWBfermented with LB-1, visible fungus colonies were not seen until theseventh day. According to the fungal infection state on the seventh day,the fungal spores grew vigorously and showed their original colour inthe WWB F-3, F-50 and the control group, which indicated that thefungi were not significantly inhibited. However, in the WWB LB-1, onlytwo small colonies were observed, and the growth of fungal spores wasalmost completely inhibited. Compared to the control group, the ex-tended three-day shelf life of WWB LB-1 may be attributed to the lacticacid and acetic acid produced by the LAB, since as previously reported,lactic acid and acetic acid exhibit strong antifungal activity(Mantzourani et al., 2014).

4. Conclusions

This study provides a new strategy to improve the quality andprolong the shelf life of WWB with great application prospect. Our re-sults showed that the Lactobacillus plantarum LB-1, which isolated fromlocal pickles (Nanjing, China), demonstrated the extraordinary poten-tial for improving the qualities of sourdough and WWB. Specifically,sourdough fermented by Lactobacillus plantarum LB-1 possessed betterqualities of in terms of higher viscoelasticity, stronger extensibility andwater holding capacity. Meanwhile, the ordered secondary structures(β-sheets and α-helices) quantified by FTIR were 6.49% more thancontrol group. In addition, during WWB production, the texture char-acteristics analysis and aroma volatiles determination indicated thatLactobacillus plantarum LB-1 enabled WWB a better taste and a richer

Table 3Aroma volatile compounds of different WWB made with Lactobacillus plantarumLB-1, F-3 and F-50.

Compound KIa IDb Concentration (%)

Control LB-1 F-3 F-50

AlcoholsEthanol 427 MS 37.8 20.14 21.28 22.321-Propanol 536 MS ndc 1.16 0.65 1.032-Methyl-1-propanol 625 MS,RI nd 0.58 0.37 0.541-Butanol 658 MS 0.45 1.29 1.08 1.12Isopentyl alcohol 727 MS,RI 5.22 1.45 1.91 1.231-Pentanol 769 MS,RI nd 1.62 0.73 0.412,3-Butanediol 782 MS 2.32 0.11 0.15 0.132-Butanone 803 MS,RI 6.23 nd 1.13 nd1-Hexanol 869 MS,RI nd 2.78 0.56 2.012-Heptanol 903 MS nd 3.51 2.73 2.942-Ethyl-1-hexanol 1029 MS,RI 0.76 3.37 1.97 2.35Benzyl alcohol 1057 MS,RI nd 0.08 0.09 0.04Phenylethyl Alcohol 1107 MS 3.69 5.12 5.14 6.34Total 56.47 41.21 37.79 40.46EstersEthyl Acetate 608 MS 7.08 4.78 5.41 5.71Ethyl pentanoate 915 MS 0.34 nd 0.14 0.97Ethyl hexanoate 997 MS,RI 0.37 0.27 0.97 0.13Ethyl caprylate 1173 MS,RI 1.46 1.41 1.67 1.24β-Phenylethyl acetate 1245 MS,RI nd 0.56 0.42 0.49Ethyl nonylate 1296 MS,RI nd 0.10 0.12 0.02Ethyl dec-9-enoate 1390 MS,RI 0.16 0.16 0.09 0.03Ethyl decanoate 1396 MS 0.72 0.85 0.78 0.71Ethyl dodecanoate 1579 MS,RI 0.20 0.06 0.15 0.08Ethyl octadecanoate 2188 MS,RI nd 0.15 0.11 0.07Total 10.33 8.34 9.86 9.45Ketones2,3-Butanedione 600 MS 0.72 5.18 4.35 3.932-Butanone 622 MS,RI 1.72 0.18 1.29 0.252,3-Pentanedione 696 MS nd 0.05 0.20 0.176-Methyl-5-heptene-2-one 989 MS,RI nd 0.13 0.36 0.042-Octanone 994 MS nd 0.16 0.72 0.13Total 2.44 5.70 6.92 4.52AldehydesButanal, 3-methyl- 655 MS 3.35 1.98 2.50 1.352-Butenal 657 MS nd 0.32 0.28 0.15Butanal, 2-methyl- 661 MS,RI 0.31 0.08 0.04 ndHexanal 807 MS,RI 0.36 0.12 0.43 0.31Furfural 830 MS,RI 3.80 3.17 2.36 2.353- Furfural 837 MS,RI 3.12 4.10 2.74 3.34Octanal 980 MS 0.23 0.20 0.30 0.25Benzeneacetaldehyde 1043 MS,RI nd 0.28 0.90 0.732-Nonenal 1160 MS,RI nd 0.46 0.59 0.54Decanal 1207 MS 0.24 0.18 0.12 0.072,4-Decadienal 1284 MS,RI 0.26 nd 0.22 0.11Tridecanal 1518 MS,RI nd 0.10 0.71 0.36Tetradecanal 1625 MS,RI 0.84 0.73 0.88 0.41Total 12.51 11.72 12.07 9.97Carboxylic acidsAcetic acid 646 MS 0.90 19.1 15.41 11.14Propanoic acid 700 MS nd 1.18 1.07 1.23Methacrylic acid 711 MS nd 1.19 1.15 1.212-Methylpropanoic acid 770 MS,RI nd 0.14 nd ndHexanoic acid 1024 MS,RI nd 1.62 1.38 1.51Octanoic Acid 1180 MS 0.40 0.37 0.33 2.30Benzoic acid 1199 MS 0.15 0.21 1.27 2.15Nonanoic acid 1273 MS,RI nd 0.27 0.43 ndn-Decanoic acid 1371 MS,RI 0.42 0.62 0.57 2.59Total 1.87 24.70 21.61 22.13Heterocyclic compoundsFuran, 2-methyl- 603 MS 2.02 1.12 nd 1.15Furan, 2-ethyl- 702 MS,RI 1.98 0.77 nd 1.46Pyrazine 747 MS,RI nd 0.29 nd nd2,5-Dimethylpyrazine 910 MS,RI 1.17 1.95 nd 1.83Pyrazine, ethyl- 928 MS 2.13 1.96 1.74 1.945-Methyl-2-furfural 970 MS,RI 1.64 0.59 2.53 1.57Furan, 2-pentyl 991 MS 1.31 0.44 2.47 1.41Pyrrole-2-aldehyde 1036 MS 2.17 0.35 1.8 1.76Maltol 1133 MS nd 0.27 nd ndAnethole 1265 MS 1.27 0.42 1.82 2.29

Table 3 (continued)

Compound KIa IDb Concentration (%)

Control LB-1 F-3 F-50

Nonan-1,4-olide 1363 MS,RI 1.62 0.17 1.04 ndButylated Hydroxytoluene 1483 MS 1.07 nd 0.35 0.06Total 16.38 8.33 11.75 13.47

a KI: Kovats index.b MS: identification by MS data; RI: identification by reference value of KI.c nd: not detected.

L. Sun, et al. Food Control 109 (2020) 106914

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flavour. Moreover, the shelf life of WWB was extended from 3 days to 6days compared to the control group. Therefore, Lactobacillus plantarumLB-1, replacing traditional chemical additions, can be successfully usedas a starter for WWB quality improvement and fungi contaminationprevention.

Declaration of interests

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge the financial support providedby the Jiangsu Agriculture Science and Technology Innovation Fund(JASTIF, Grant No. CX(17)1003), Qing Lan Project, and the PriorityAcademic Program Development of Jiangsu Higher EducationInstitutions (PAPD).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodcont.2019.106914.

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