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A novel lactic acid bacterium for improving the quality and shelf life of whole wheat bread
Food Control 109 (2020) 106914
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A novel lactic acid bacterium for improving the quality and shelf life of whole wheat bread
T
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 LE I N FO
A B S T R A C T
Keywords: Whole wheat Sourdough Lactic acid bacteria Quality Shelf life
The aim of this study was to screen for a lactic acid bacterium (LAB) that could improve the quality of whole wheat bread (WWB) and extend its shelf life. The LABs with strong antifungal activity were screened among twenty LABs. Both sourdough properties (rheology, tensility, water mobility and gluten structure) and WWB qualities (texture, volatile flavour and shelf life) were determined and compared. The results showed that the Lactobacillus 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 better viscoelasticity, 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 texture characteristics, enrich the aroma volatile compounds, and prolong the shelf life of WWB from 3 days to 6 days compared to the control group. Above all, Lactobacillus plantarum LB-1 was recommended for WWB fermentation to improve its quality and to extend the shelf life.
1. Introduction Whole wheat bread (WWB) is a kind of bread made from whole wheat flour without removing the bran and wheat germ. Compared to refined wheat bread, it is rich in higher levels of dietary fibres, minerals, phytochemicals, antioxidants and vitamins (Tebben, Shen, & Li, 2018). The American Institute for Cancer Research (AICR) and the World Cancer Research Fund (WCRF) reported that eating whole grain foods 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 in reducing disease risks and managing chronic diseases by eating whole grain food (Niu, Hou, Kindelspire, Krishnan, & Zhao, 2017). However, compared to refined wheat bread, WWB may be more susceptible to fungal infections because it contains the intact grain and epidermal part (Zhang, Pei, Fang, Li, Zhao, Shen, et al., 2019). Most fungi were located in the outer layer of wheat kernels. These fungi can act as a potential reservoir of contamination and produce mycotoxins (including aflatoxin, zearalenone and deoxynivalenol) in the manufacturing environment, which cause more safety hazards (Saladino, Luz, Manyes, Fernández-Franzón, & Meca, 2016). Moreover, the product acceptability 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-(hydroxymethyl) furfural (HMF) can seriously affect its taste (Jiang & Peterson, 2013). Recently, the improvement of WWB is mainly based on using chemical leavening and preservative agents. Hydrocolloids, such as hydroxypropyl methyl cellulose, carboxymethyl cellulose, locust bean gum and xanthan gum, have been widely used to improve the waterabsorbing 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 on maintaining the quality of bread, proving that calcium propionate could extend its shelf life significantly. However, these chemical additives may cause changes in the natural components of whole wheat, some of which may be toxic to the human body (Ginocchio et al., 1979). Therefore, it is of great significance to develop natural functional dough improvers. Since chemical additives have anti-nutritional and toxic effects, LAB as a natural starter has been paid widespread attention. In a study by Dal Bello, Clarke, Ryan, Ulmer, and Schober (2007), lactic acid, phenyllactic acid and cyclic dipeptides cyclo produced by Lactobacillus plantarum FST 1.7 showed consistent inhibition against Fusarium species and had the potential to extend the shelf life of bread. In addition,
Corresponding author. E-mail address: [email protected] (F. Pei).
https://doi.org/10.1016/j.foodcont.2019.106914 Received 28 June 2019; Received in revised form 25 August 2019; Accepted 21 September 2019 Available online 24 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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instructions. The primer sets 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for amplification of the gene. PCR conditions reported by Joo et al. (2015) were set as follows: 95 °C for 5 min followed by 30 cycles of 95 °C for 1 min, 52 °C for 30 s, and 72 °C for 1 min 30 s, with a final extension at 72 °C for 7 min. The amplified DNAs were purified and sequenced using an ABI 3730XL DNA analyser (Life Technologies, New York, USA). The 16S rRNA sequences were blasted against the NCBI GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Wang, Hwang, Tzeng, Hwang, and Mau (2012) suggested that the total volatile content including alcohols, esters and carbonyls of bread made with Lactobacillus delbrueckii subsp. Delbrueckii was significantly higher than those in the blank group. However, most of these studies were limited to refined wheat, while fungal contamination and poor quality are far more serious problems in whole wheat. Therefore, the overall purpose of this study was finding a novel LAB to simultaneously solve the problem of fungal contamination and distastefulness in WWB. In this study, specific LABs with high antifungal activity were screened from a total of twenty LABs. Subsequently, the effects of the selected LABs on dough improvement were evaluated via rheological measurements, tensile tests, water mobility determination, gluten secondary structures quantification and microstructure observation. The sourdoughs were then processed into breads, and their texture, volatile compounds and shelf life were also analysed to verify the applicability of the LABs as starters for WWB.
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/T 14611–2008 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 of sugar (Herunhua FOOD, Jiangsu, China) and 120 g of LAB suspension (108 CFU/g). Dough with the same quantity of water (containing no LAB) was set as the control group. The sourdoughs were kneaded by a dough mixer for 6 min and then placed in a fermentation box (temperature was 30 ± 1 °C and humidity was 85%–90%) for 5 h. Subsequently, the sourdough was divided into 100 g portion and baked in a JKLZ4 oven (Fude Technology, Beijing, China) at 200 °C for 20 min.
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, F17, F-24, F-48, F-50 and F-59 were obtained from the Key Laboratory of Grains and Oils Quality Control and Processing. LB-1, LB-2, LB-3, LB-4 and LB-5 were isolated from local pickles in Nanjing, China. Lactobacillus rhamnosus (BNCC 136673), Leuconostoc mesenteroides (BNCC 195309), Lactobacillus plantarum (BNCC 194165) and Lactobacillus casei (BNCC 134415) were purchased from the BeNa Culture Collection. Lactobacillus rhamnosus strain GG (ATCC 53103) was purchased from the American Type Culture Collection), and six fungi (Penicillium citrinum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus fumigatus and Fusarium graminearum were isolated from whole wheat) were studied in this experiment. LAB strains were preserved in sterile 25% glycerol at −80 °C before use.
2.6. Rheological measurements Dynamic rheological properties of the sourdough were measured by a rheometer (Anton-Paar-Strasse, Austria) using oscillation sweep tests. Sourdough was placed on the centre of the rheometer plate and left for 5 min for relaxing the residual stress. Mineral oil was daubed around the edge of the sourdough to prevent moisture loss. A strain of 0.5% and a measure position of 2 mm were applied in this test. The test temperature was 25 ± 1 °C and the test frequency was in the range of 0.1–40 Hz (Hao et al., 2008; Inglett, Chen, Liu, & Lee, 2014).
2.2. LAB suspension and fungus spore suspension preparation
2.7. Tensile properties tests
Single LAB strains (2% v/v) were inoculated into MRS broth at 37 °C for 20 h. The cells were harvested by centrifugation (5500 g for 10 min at 4 °C) and the supernatant was removed. The obtained LAB cells were suspended in sterile water and counted, varying from 107 to 109 CFU/mL. The fungi were inoculated in PDA medium at 28 °C for 7–10 days. The spores were harvested from Petri plates in sterile water to prepare a suspension containing 1 × 104 spores/mL. The LAB suspension and fungus spore suspension were preserved at 4 °C before use.
The tensile properties of the sourdough were measured using a TAXT Plus texture analyser (Stable Micro Systems, London, England) with an A/KIE probe. A trigger force of 5 g and a test distance of 50 mm were applied to perform this test. The pre-test speed, test speed and post speed were 2 mm/s, 3.3 mm/s and 10 mm/s, respectively. The sourdough was rested in the fermentation box (temperature was 30 ± 1 °C and humidity was 85%–90%) for 10 min before the test. Each group was tested six times (Suchy, Lukow, & Ingelin, 2000).
2.3. Antifungal activity in vitro
2.8. Water mobility determination
The dual culture method described by Quattrini et al. (2018) was modified and used to test the antifungal activity of LABs. Briefly, 10 μL of the LAB suspension (109 CFU/mL) was dropped on the centre of MRS agar plates and incubated at 37 °C for 48 h. Subsequently, 10 mL of PDA containing 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 inhibition zone were measured using an electronic digital calliper (Guanglu, Guilin, China). The antifungal activity of the LABs was calculated on the basis of the inhibition zone as no inhibition (−) for an inhibition zone smaller than 0.05 mm, weak inhibition (+) for an inhibition zone smaller than 10 mm, moderate inhibition (++) for an inhibition zone in the range of 10–20 mm, and strong inhibition (+++) for an inhibition zone larger than 20 mm.
Water mobility of the sourdough was detected using low field nuclear magnetic resonance (LF-NMR) (MesoMR, Niumag Corporation, China). The sourdough was evenly stuffed into the sample vials and then 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 = 19 MHz, RFD = 0.5 ms, O1 = 948164.27 Hz, RG1 = 20 db, P1 = 13 us, DRG1 = 3, TD = 300150, TW = 1500 ms, P2 = 25 us, TE = 0.2 ms, NECH = 7500 and NS = 32. T21 represents the bound water, T23 represents free water, and T22 represents the adsorbed water between the bound water and free water (Doona & MooYeol, 2007).
2.4. LAB identification
2.9. Fourier transform infrared spectroscopy (FTIR) and gluten secondary structure
DNA was isolated from selected strains using an EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China) according to the
Gluten was isolated and collected from the dough according to the AACCI Approved Method 38–10.01. The gluten was dried using an oven 2
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Table 1 Antifungal activity of LAB strains against different fungi. Strains
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
Antifungal activitya Penicillium citrinum
Aspergillus niger
Aspergillus flavus
Aspergillus ochraceus
Aspergillus fumigatus
Fusarium graminearum
+++ +++ +++ +++ +++ +++ + +++ ++ + + – – – – +++ +++ +++ +++ +++
++ ++ ++ + ++ – + – + – – – – – + – + + + –
+++ +++ ++ ++ ++ +++ + ++ ++ – – ++ – ++ +++ ++ +++ + +++ ++
++ ++ ++ ++ + + – – + – – – – – – + + + ++ –
+++ +++ +++ ++ +++ ++ – +++ ++ – – + ++ +++ ++ +++ +++ +++ ++ +
+++ +++ +++ + + + – ++ + + – ++ ++ – – ++ +++ ++ + –
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 (+++).
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) was ground into a uniform powder again and pressed into sheets using a powder pressing machine (769 YP-15A, Tianjin, China). A total of 64 scans were run for each analysis at an interval of 4 cm−1 in the range of 400–4000 cm−1. The curve of the Amide I region was selected and fitted by a module (AutoFit Peaks II Second Derivative) of Peakfit V4.12 software. Then, the percentage of peak areas corresponding to the gluten secondary structure was given (Chen et al., 2019). The spectral regions were assigned as 1612–1640 cm−1 and 1670–1694 cm−1 for βsheets, 1640–1650 cm−1 for random coils, 1648–1660 cm−1 for α-helices, and 1662–1684 cm−1 for β-turn structures. The second derivative area for each secondary structural region was divided by the total area of the amide I region (Carbonaro & Nucara, 2010; Goormaghtigh, Cabiaux, & Ruysschaert, 1994; Pelton & McLean, 2000).
increased by 5.5 °C/min to 230 °C (held for 5 min). The carrier gas was He at a flow rate of 2 mL/min. The mass spectrum was recorded by electronic impact (EI) at 70 eV. The scan mode was in the range of m/z 33–200. Compounds were identified by the MS database (NIST 98) combined with Kovats indexes (KI) comparison. Match quality higher than 80% was considered to be reliable. Experimental KI was based on n-alkanes (C7 to C30, o2si smart solutions, USA). Compounds were quantified 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-XT2i texture analyser (Stable Micro Systems, London, England) with a P/36R probe. Each sample was cut into 1 cm thick slices and placed for 1 h for cooling. Test parameters according to AACCI74-09 were as follows: pretest speed, 3 mm/s; test speed, 1 mm/s; post speed, 5 mm/s; trigger type, auto-10 g; target mode, distance-5 mm; interval between two compressions, 10 s. For the fungi spoilage analysis, bread samples were packed in polyethylene bags and stored at room temperature (23 ± 1 °C). Fungal colonies on the bread surface were monitored daily for one week. Contamination was calculated as follows: no visible colonies (−), one colony (+), two (++), and three or more (+++) (Bartkiene, Bartkevics, Lele, Pugajeva, Zavistanaviciute, Mickiene, et al., 2018).
2.10. Scanning electron microscopy (SEM) Sourdough flakes were fixed in glutaraldehyde (2.5%) for 12 h and dehydrated in increasing grades of ethanol (25%, 50%, 75%, 95% and 100%). Then, the sourdough flakes were coated with gold particles using a MSP-1S Sputter Coater (Hitachi, Tokyo, Japan) after lyophilisation. Microstructures of the different sourdoughs were photographed using a TM3000 SEM (Hitachi, Tokyo, Japan) at 250 × and 1000 × magnification (Liu et al., 2015).
2.13. Statistical analysis 2.11. Gas chromatography-mass spectrometry All of the experiments were repeated at least three times. Means and standard deviations were calculated. The results were analysed by SPSS version 23 software for Windows (SPSS Inc., Chicago, IL, USA). Duncan tests at a significance level of P < 0.05 were performed for significance analysis.
Aroma volatile compounds were analysed by headspace gas chromatography mass spectrometry (GC-MS) using the solid-phase microextraction (SPME) method as previously described (Plessas et al., 2008). Briefly, 2 g of a bread sample was placed in a 20 mL glass vial and incubated in a water bath at 60 °C. The SPME fibre (50/30 μm DVB/CAR/PDMS, Stable Flex Supelco, Bellefonte, PA, USA) was exposed to the headspace for 60 min. Desorption of volatiles was in the injector port (280 °C) of the gas chromatograph (Agilent7890A, Palo Alto, CA, USA) for 5 min in splitless mode. An Agilent DB-5MS capillary column (0.25 μm film thickness, 30 m × 250 μm) was used. The GC temperature program (total run time 51.73 min) was as follows: 35 °C for 5 min, then increased by 5 °C/min to 50 °C (held for 5 min),
3. Results and discussion 3.1. Selection of LABs with strong antifungal activity and LABs identification A screen of the twenty LAB strains was carried out by measuring antifungal activity against six susceptible fungi of wheat: Penicillium 3
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Fig. 1. Effect of different LABs on rheology (A and B), tensile property (C), water mobility (D) during sourdough fermentation.
citrinum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus fumigatus and Fusarium graminearum. These fungi are considered to be the most common spoilage agents in bakery products due to their high prevalence in wheat flour and their xerotolerance to xerophilic behaviour, which can act as a potential reservoir of contamination in the manufacturing environment (Saladino et al., 2016). The antifungal activity of LAB was calculated on the basis of the inhibition zone, and the results are shown in Table 1. Most of the LAB strains demonstrated strong inhibition against Penicillium citrinum, Aspergillus flavus, Aspergillus fumigatus and Fusarium graminearum, while Aspergillus niger and Aspergillus ochraceus were inhibited only by certain kinds of LAB strains. Remarkably, compared to the other LAB strains, the LB-1, F-3 and F-50 strains possessed at least moderate inhibitory activity (++) against all six susceptible fungi. Therefore, LB-1, F-3 and F-50 were selected to further compare their effects on the qualities of WWB. The 16S rRNA sequencing results of the selected LAB strains LB-1, F3 and F-50 are shown in Table S1, S2 and S3. The 16S rRNA genes of the selected LAB strains LB-1, F-3 and F-50 were identical (100%) to the Lactobacillus plantarum NCU116 (Accession: CP016071.1), Lactobacillus plantarum CB5 (Accession: MK687387.1) and Lactobacillus plantarum
EM (Accession: CP037429.1) in 1400, 1407 and 1449 nucleotides, respectively (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Therefore, the three selected LAB strains were classified as Lactobacillus plantarum LB1, Lactobacillus plantarum F-3 and Lactobacillus plantarum F-50, respectively.
3.2. Effect of LABs on rheology, tensility and water mobility of sourdoughs The sourdough properties fermented by the selected LAB strains were evaluated via rheology, tensility and water mobility determination (Fig. 1). The G′ and G″ of different sourdoughs at a frequency of 0.1–40 Hz are shown in Fig. 1A and B. Storage modulus (G′) represents the ability of materials to store elastic deformation energy, and loss modulus (G″) represents the viscous portion (Meyers & Chawla, 1990). It could be seen that both moduli (G′ and G″) of all sourdoughs increased with the increase of frequency. Moduli (G′ and G″) of sourdough LB-1 and F-3 were significantly higher than that of F-50 and the control group, which indicated that sourdoughs fermented by LB-1 and F-3 were more viscous and elastic. Ghodke and Laxmi (2007) found that exopolysaccharides (EPS) produced by Lactobacillus buchneri FUA3154 can replace hydrocolloids to influence the rheological properties of 4
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the water closely bound to protein and starch, influencing the formation of the gluten network structure in dough (Assifaoui, Champion, Chiotelli, & Verel, 2006). Therefore, it can be concluded that the sourdough LB-1 and F-3 possessed stronger water-holding capacity compared to the control group. Zhang, Doehlert, and Moore (1997) studied the effect of β-glucan on oat flour, and they found that the viscosity and the water binding capacity of oat flour was positively correlated with the content of β-glucan. LABs can produce β-glucan in the process of growth and metabolism. Therefore, sourdough absorbed more 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 glutenin complexes formed by glutenin subunits crosslinking through disulfide bond ends (Ooms & Delcour, 2019). Therefore, from a microcosmic perspective, the secondary structures of gluten and the exterior shape of the sourdoughs were studied to explain the orderliness of sourdoughs by FTIR and SEM respectively. The FTIR spectra of the different samples are shown in Fig. 2A. Amide I region (1600–1700 cm−1) has been used to study the secondary structure of gluten because it is almost entirely attributable to C]O stretch vibrations and is sensitive to gluten conformation (Robertson, Gregorski, & Cao, 2006). The curve of the Amide I region was selected and fitted to calculate the secondary structures of gluten using Peakfit V4.12 software. As seen in Fig. 2B, the LAB strains had a great influence on the secondary structures of sourdoughs. βsheets and α-helices were the main secondary structures of the gluten, which is consistent with a previous study (Wang et al., 2016). With the addition of the LAB strains, there was a dramatic decrease in random coils together with significant increases in β-turns, indicating conformational changes in the gluten. Compared to the control group, the sum of β-sheets and α-helices for sourdoughs LB-1, F-3 and F-50 increased by 6.49%, 4.49% and 0.12%, respectively. Previous studies on gluten secondary structures proved that β-sheets and α-helices enabled dough to form a more ordered network structure, while random coils were considered as disordered structures (Marti, Bock, Pagani, Ismail, & Seetharaman, 2016; Mecozzi & Sturchio, 2015). The increase in βsheets and α-helices indicated that sourdough LB-1 formed a more regular and orderly gluten structure. The microstructures of the sourdoughs were detected by SEM at magnifications of 250 × and 1000 × to further analyse the gluten structure. As shown in Fig. 3A, discontinuity of the gluten network structure could be obviously observed in the control group. Starch granules aggregated in clusters and did not disperse evenly in the network structure (Fig. 3a). This was due to the addition of bran, which can cause water redistribution and a partial dehydration of gluten (Bock & Damodaran, 2013). For the sourdoughs F-50 and F-3, a break in junctions of the membrane-like gluten matrix could be clearly observed (Fig. 3C) and most starch granules were aggregated into clusters and exposed to stomata (Fig. 3d). Compared to the other groups, the sourdough LB-1 (Fig. 3B) formed a more ordered and compact gluten network structure, and the starch granules were wrapped tightly and evenly in the membrane-like gluten matrix (Fig. 3b). This phenomenon indicated that the gluten network of sourdough, which was well formed, possessed good ductility when fermented by LB-1.
Fig. 2. ATR-FTIR spectrum of gluten in different sourdoughs (A), and fitting results of Amide I region for secondary structure content of gluten protein (B).
sourdoughs. The greater viscosity and elasticity in sourdough LB-1 and F-3 may be attributed to the cementability and stability of EPS. Tensile properties are important indices in dough baking and are closely related to gluten network formation and dough gas-holding capacity (McCann, Le Gall, & Day, 2016). Fig. 1C shows the tensile resistance and extensibility of the different sourdoughs. It can be seen that the tensile resistances of the three fermented sourdoughs were significantly higher (P < 0.05) than that of the control group. Meanwhile, sourdough LB-1 and F-50 demonstrated better tensile resistance compared to sourdough F-3. Additionally, the extensibilities of sourdough F-3 and LB-1 were significantly higher (P < 0.05) than those of F-50 and the control group. The results indicated that the extensibility and strength of the gluten network structure in sourdough LB-1 and F-3 were enhanced. A previous study reported by Schober, Bean, and Boyle (2007) showed that proteolysis during sourdough fermentation could reduce the interference of protein with starch and result in stronger starch gel strength, which was consistent with the findings of this study. Water mobility is one of the main characteristics of dough and plays an important role in the dough quality. Continuous distributions of spin-spin relaxation time (T2) are shown in Fig. 1D. As shown in Fig. 1D, water in the dough mainly existed in the form of adsorbed water (T22). However, two distinct peaks appearing at T21 represented the bound water in sourdough LB-1 and F-3, which can be regarded as
3.4. Effect of different LABs on textural properties After evaluating the rheology, tensility and water mobility of the sourdoughs, we made the sourdoughs into WWB according to GB/T 14611–2008 and tested the texture characteristics of WWB, including hardness, springiness, cohesiveness, gumminess and chewiness (Table 2). It was observed that compared to the control, F-3 and F-50 groups, the hardness, gumminess and chewiness of WWB LB-1 were 5
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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 2 The texture properties of WWB made with Lactobacillus plantarum LB-1, F-3 and F-50 (n = 8). Sample
Hardness
Control LB-1 F-3 F-50
383.57 253.77 376.27 364.80
± ± ± ±
8.45a 10.83b 15.15a 17.87a
Springiness
Cohesiveness
Gumminess
Chewiness
0.99 0.97 0.96 0.95
0.91 0.95 0.92 0.91
365.59 236.12 345.12 330.83
363.29 230.17 330.52 316.27
± ± ± ±
0.01a 0.03a 0.03a 0.02a
± ± ± ±
0.02b 0.01a 0.02 ab 0.02b
± ± ± ±
5.67a 5.48b 13.87a 19.80a
± ± ± ±
7.30a 12.90b 14.89a 15.24a
Wronkowska, Jadacka, Soral-Śmietana, Zander, Dajnowiec, Banaszczyk, et al., 2015). The results obtained from TPA indicated that the WWB fermented by Lactobacillus plantarum LB-1 tasted springier, softer and more refreshing, which more closely caters to consumer tastes compared to other groups.
significantly (P < 0.05) lower, and the cohesiveness was significantly (P < 0.05) higher. A large number of studies have proven that hardness, gumminess and chewiness are negatively correlated with bread quality, while springiness and cohesiveness are positive attributes (AlFarga, Zhang, Siddeeg, Chamba, Kimani, Hassanin, et al., 2016; 6
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Table 3 (continued)
Table 3 Aroma volatile compounds of different WWB made with Lactobacillus plantarum LB-1, F-3 and F-50. Compound
Alcohols Ethanol 1-Propanol 2-Methyl-1-propanol 1-Butanol Isopentyl alcohol 1-Pentanol 2,3-Butanediol 2-Butanone 1-Hexanol 2-Heptanol 2-Ethyl-1-hexanol Benzyl alcohol Phenylethyl Alcohol Total Esters Ethyl Acetate Ethyl pentanoate Ethyl hexanoate Ethyl caprylate β-Phenylethyl acetate Ethyl nonylate Ethyl dec-9-enoate Ethyl decanoate Ethyl dodecanoate Ethyl octadecanoate Total Ketones 2,3-Butanedione 2-Butanone 2,3-Pentanedione 6-Methyl-5-heptene-2-one 2-Octanone Total Aldehydes Butanal, 3-methyl2-Butenal Butanal, 2-methylHexanal Furfural 3- Furfural Octanal Benzeneacetaldehyde 2-Nonenal Decanal 2,4-Decadienal Tridecanal Tetradecanal Total Carboxylic acids Acetic acid Propanoic acid Methacrylic acid 2-Methylpropanoic acid Hexanoic acid Octanoic Acid Benzoic acid Nonanoic acid n-Decanoic acid Total Heterocyclic compounds Furan, 2-methylFuran, 2-ethylPyrazine 2,5-Dimethylpyrazine Pyrazine, ethyl5-Methyl-2-furfural Furan, 2-pentyl Pyrrole-2-aldehyde Maltol Anethole
KI
a
ID
b
Compound
KIa
IDb
Concentration (%) Control
LB-1
F-3
F-50
427 536 625 658 727 769 782 803 869 903 1029 1057 1107
MS MS MS,RI MS MS,RI MS,RI MS MS,RI MS,RI MS MS,RI MS,RI MS
37.8 ndc nd 0.45 5.22 nd 2.32 6.23 nd nd 0.76 nd 3.69 56.47
20.14 1.16 0.58 1.29 1.45 1.62 0.11 nd 2.78 3.51 3.37 0.08 5.12 41.21
21.28 0.65 0.37 1.08 1.91 0.73 0.15 1.13 0.56 2.73 1.97 0.09 5.14 37.79
22.32 1.03 0.54 1.12 1.23 0.41 0.13 nd 2.01 2.94 2.35 0.04 6.34 40.46
608 915 997 1173 1245 1296 1390 1396 1579 2188
MS MS MS,RI MS,RI MS,RI MS,RI MS,RI MS MS,RI MS,RI
7.08 0.34 0.37 1.46 nd nd 0.16 0.72 0.20 nd 10.33
4.78 nd 0.27 1.41 0.56 0.10 0.16 0.85 0.06 0.15 8.34
5.41 0.14 0.97 1.67 0.42 0.12 0.09 0.78 0.15 0.11 9.86
5.71 0.97 0.13 1.24 0.49 0.02 0.03 0.71 0.08 0.07 9.45
600 622 696 989 994
MS MS,RI MS MS,RI MS
0.72 1.72 nd nd nd 2.44
5.18 0.18 0.05 0.13 0.16 5.70
4.35 1.29 0.20 0.36 0.72 6.92
3.93 0.25 0.17 0.04 0.13 4.52
655 657 661 807 830 837 980 1043 1160 1207 1284 1518 1625
MS MS MS,RI MS,RI MS,RI MS,RI MS MS,RI MS,RI MS MS,RI MS,RI MS,RI
3.35 nd 0.31 0.36 3.80 3.12 0.23 nd nd 0.24 0.26 nd 0.84 12.51
1.98 0.32 0.08 0.12 3.17 4.10 0.20 0.28 0.46 0.18 nd 0.10 0.73 11.72
2.50 0.28 0.04 0.43 2.36 2.74 0.30 0.90 0.59 0.12 0.22 0.71 0.88 12.07
1.35 0.15 nd 0.31 2.35 3.34 0.25 0.73 0.54 0.07 0.11 0.36 0.41 9.97
646 700 711 770 1024 1180 1199 1273 1371
MS MS MS MS,RI MS,RI MS MS MS,RI MS,RI
0.90 nd nd nd nd 0.40 0.15 nd 0.42 1.87
19.1 1.18 1.19 0.14 1.62 0.37 0.21 0.27 0.62 24.70
15.41 1.07 1.15 nd 1.38 0.33 1.27 0.43 0.57 21.61
11.14 1.23 1.21 nd 1.51 2.30 2.15 nd 2.59 22.13
603 702 747 910 928 970 991 1036 1133 1265
MS MS,RI MS,RI MS,RI MS MS,RI MS MS MS MS
2.02 1.98 nd 1.17 2.13 1.64 1.31 2.17 nd 1.27
1.12 0.77 0.29 1.95 1.96 0.59 0.44 0.35 0.27 0.42
nd nd nd nd 1.74 2.53 2.47 1.8 nd 1.82
1.15 1.46 nd 1.83 1.94 1.57 1.41 1.76 nd 2.29
Nonan-1,4-olide Butylated Hydroxytoluene Total a b c
1363 1483
MS,RI MS
Concentration (%) Control
LB-1
F-3
F-50
1.62 1.07 16.38
0.17 nd 8.33
1.04 0.35 11.75
nd 0.06 13.47
KI: Kovats index. MS: identification by MS data; RI: identification by reference value of KI. nd: not detected.
3.5. Aroma volatile compounds analysis Sixty-two aroma volatile compounds of the WWBs were detected by SPME-GC-MS and the results are shown in Table 3. It is noteworthy that after fermentation by LB-1, F-3 and F-50, the aroma volatile compounds (60, 54 and 55 kinds of volatile compounds, respectively) increased and were enriched compared to the control group (37 kinds of volatile compounds). Meanwhile, carboxylic acids of the control group (1.87%) were significantly lower than those of the WWB LB-1, F-3 and F-50 groups (24.70%, 21.61% and 22.13%, respectively), and the content of acetic acid was the highest among them. This is consistent with a previous study on the flavour of sourdough bread by Cavallo et al. (2017), who reported that acetic acid could be generated during the processing of bread fermentation. Moreover, Su et al. (2019) studied organic acids on bread quality improvement, finding that acetic acid could give bread a higher specific volume, a lower pH value and a decreased hardness. In addition, the acetic acid content determined the efficiency of sourdough as a possible preservative agent against the microbial 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 daily during a test of a one week shelf life (Table 4). Two visible fungus colonies were first observed in WWB F-50 on the third day and then observed in the WWB F-3 on the fifth day. In the control group, visible fungus colonies were detectable on the fourth day, whereas in the WWB fermented with LB-1, visible fungus colonies were not seen until the seventh day. According to the fungal infection state on the seventh day, the fungal spores grew vigorously and showed their original colour in the WWB F-3, F-50 and the control group, which indicated that the fungi were not significantly inhibited. However, in the WWB LB-1, only two small colonies were observed, and the growth of fungal spores was almost completely inhibited. Compared to the control group, the extended three-day shelf life of WWB LB-1 may be attributed to the lactic acid 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 and prolong the shelf life of WWB with great application prospect. Our results showed that the Lactobacillus plantarum LB-1, which isolated from local pickles (Nanjing, China), demonstrated the extraordinary potential for improving the qualities of sourdough and WWB. Specifically, sourdough fermented by Lactobacillus plantarum LB-1 possessed better qualities of in terms of higher viscoelasticity, stronger extensibility and water holding capacity. Meanwhile, the ordered secondary structures (β-sheets and α-helices) quantified by FTIR were 6.49% more than control group. In addition, during WWB production, the texture characteristics analysis and aroma volatiles determination indicated that Lactobacillus plantarum LB-1 enabled WWB a better taste and a richer 7
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Table 4 Effect of different LABs on fungal infection during WWB storage. Sample
Days of storage
Control LB-1 F-3 F-50 Fungal infection on the seventh day
Control a
1
2
3
4
5
6
7
a
– – – –
– – – ++
++ – – +++
+++ – + +++
+++ – +++ +++
+++ ++ +++ +++
– – –
LB-1
F-3
F-50
Contamination was calculated as follows: no visible colonies (−), one colony (+), two (++), and three or more (+++).
flavour. Moreover, the shelf life of WWB was extended from 3 days to 6 days compared to the control group. Therefore, Lactobacillus plantarum LB-1, replacing traditional chemical additions, can be successfully used as a starter for WWB quality improvement and fungi contamination prevention.
(2017). Microbial cell-free extracts affect the biochemical characteristics and sensorial quality of sourdough bread. Food Chemistry, 237, 159–168. Chen, G., Ehmke, L., Sharma, C., Miller, R., Faa, P., Smith, G., et al. (2019). Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt. Food Chemistry, 275, 569–576. Dal Bello, F., Clarke, C. I., Ryan, L. A. M., Ulmer, H., & Schober, T. J. (2007). Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7. Journal of Cereal Science, 45(3), 309–318. Ding, X., Zhang, H., Wang, L., Qian, H., Qi, X., & Xiao, J. (2015). Effect of barley antifreeze protein on thermal properties and water state of dough during freezing and freeze-thaw cycles. Food Hydrocolloids, 47, 32–40. Doona, C. J., & Moo-Yeol, B. (2007). Molecular mobility in model dough systems studied by time-domain nuclear magnetic resonance spectroscopy. Journal of Cereal Science, 45(3), 257–262. Ghodke, S. K., & Laxmi, A. (2007). Influence of additives on rheological characteristics of whole-wheat dough and quality of Chapatti (Indian unleavened Flat bread) Part Ihydrocolloids. Food Hydrocolloids, 21(1), 110–117. Ginocchio, A. V., Waite, V., Hardy, J., Fisher, N., Hutchinson, J. B., & Berry, R. (1979). Long-term toxicity and carcinogenicity studies of the bread improver potassium bromate 2. Studies in mice. Food and Cosmetics Toxicology, 17(1), 41–47. Goormaghtigh, E., Cabiaux, V., & Ruysschaert, J.-M. (1994). Determination of soluble and membrane protein structure by fourier transform infrared spectroscopy. Sub-Cellular Biochemistry, 23, 363–403. Hao, C. C., Wang, L. J., Li, D., Özkan, N., Wang, D. C., Chen, X. D., et al. (2008). Influence of alfalfa powder concentration and granularity on rheological properties of alfalfawheat dough. Journal of Food Engineering, 89(2), 137–141. Inglett, G. E., Chen, D., Liu, S. X., & Lee, S. (2014). Pasting and rheological properties of oat products dry-blended with ground chia seeds. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 55(1), 148–156. Jiang, D., & Peterson, D. G. (2013). Identification of bitter compounds in whole wheat bread. Food Chemistry, 141(2), 1345–1353. Joo, H. J., Kim, H. Y., Kim, L. H., Lee, S., Ryu, J. G., & Lee, T. (2015). A Brevibacillus sp. antagonistic to mycotoxigenic. Fusarium spp. Biological Control, 87, 64–70. Katsinis, G., Lohano, D. K., Sheikh, S. A., & Shahnawaz, M. (2009). Effect of chemical preservatives on the shelf life of bread at various temperatures. Pakistan Journal of Nutrition, 9(3), 279–283. Lafay, L., & Ancellin, R. (2015). Alimentation et cancer colorectal. Cahiers de Nutrition et de Diététique, 50(5), 262–270. Liu, R., Xing, Y., Zhang, Y., Zhang, B., Jiang, X., & Wei, Y. (2015). Effect of mixing time on the structural characteristics of noodle dough under vacuum. Food Chemistry, 188, 328–336. Mantzourani, I., Plessas, S., Saxami, G., Alexopoulos, A., Galanis, A., & Bezirtzoglou, E. (2014). Study of kefir grains application in sourdough bread regarding rope spoilage caused by Bacillus spp. Food Chemistry, 143, 17–21. Marti, A., Bock, J. E., Pagani, M. A., Ismail, B., & Seetharaman, K. (2016). Structural characterization of proteins in wheat flour doughs enriched with intermediate wheatgrass (Thinopyrum intermedium) flour. Food Chemistry, 194, 994–1002. Martínez-Anaya, M. A., Llin, M. L., Macías, M. P., & Collar, C. (1994). Regulation of acetic acid production by homo- and heterofermentative lactobacilli in whole-wheat sourdoughs. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 199(3), 186–190. Matuda, T. G., Chevallier, S., de Alcântara Pessôa Filho, P., LeBail, A., & Tadini, C. C. (2008). Impact of guar and xanthan gums on proofing and calorimetric parameters of frozen bread dough. Journal of Cereal Science, 48(3), 741–746. McCann, T. H., Le Gall, M., & Day, L. (2016). Extensional dough rheology - impact of flour composition and extension speed. Journal of Cereal Science, 69, 228–237. Mecozzi, M., & Sturchio, E. (2015). Effects of essential oil treatments on the secondary protein structure of Vicia faba: A mid-infrared spectroscopic study supported by twodimensional correlation analysis. Spectrochimica Acta Part A: Molecular Spectroscopy,
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors gratefully acknowledge the financial support provided by the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF, Grant No. CX(17)1003), Qing Lan Project, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.106914. References Al-Farga, A., Zhang, H., Siddeeg, A., Chamba, M. V. M., Kimani, B. G., Hassanin, H., et al. (2016). Effect of the addition of alhydwan seed flour on the dough rheology, bread quality, texture profile and microstructure of wheat bread. Journal of Texture Studies, 47(6), 484–495. Anna-Sophie, H., & Arendt, E. K. (2013). Influence of hydroxypropylmethylcellulose (HPMC), xanthan gum and their combination on loaf specific volume, crumb hardness and crumb grain characteristics of gluten-free breads based on rice, maize, teff and buckwheat. Food Hydrocolloids, 32(1), 195–203. Assifaoui, A., Champion, D., Chiotelli, E., & Verel, A. (2006). Characterization of water mobility in biscuit dough using a low-field 1H NMR technique. Carbohydrate Polymers, 64(2), 197–204. Bartkiene, E., Bartkevics, V., Lele, V., Pugajeva, I., Zavistanaviciute, P., Mickiene, R., et al. (2018). A concept of mould spoilage prevention and acrylamide reduction in wheat bread: Application of lactobacilli in combination with a cranberry coating. Food Control, 91, 284–293. Bin, Q., & Peterson, D. G. (2016). Identification of bitter compounds in whole wheat bread crumb. Food Chemistry, 203, 8–15. Bock, J. E., & Damodaran, S. (2013). Bran-induced changes in water structure and gluten conformation in model gluten dough studied by Fourier transform infrared spectroscopy. Food Hydrocolloids, 31(2), 146–155. Carbonaro, M., & Nucara, A. (2010). Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region. Amino Acids, 38(3), 679–690. Cavallo, N., De Angelis, M., Calasso, M., Quinto, M., Mentana, A., Minervini, F., et al.
8
Food Control 109 (2020) 106914
L. Sun, et al.
Journal of Agricultural and Food Chemistry, 55(13), 5137–5146. Slavin, & Joanne (2004). Whole grains and human health. Nutrition Research Reviews, 17(1), 99–110. Suchy, J., Lukow, O. M., & Ingelin, M. E. (2000). Dough microextensibility method using a 2-g mixograph and a texture analyzer. Cereal Chemistry, 77(1), 39–43. Su, X., Wu, F., Zhang, Y., Yang, N., Chen, F., Jin, Z., et al. (2019). Effect of organic acids on bread quality improvement. Food Chemistry, 278, 267–275. Tebben, L., Shen, Y., & Li, Y. (2018). Improvers and functional ingredients in whole wheat bread: A review of their effects on dough properties and bread quality. Trends in Food Science & Technology, 81, 10–24. Wang, H. E., Hwang, C. F., Tzeng, Y. M., Hwang, W. Z., & Mau, J. L. (2012). Quality of white bread made from lactic acid bacteria-enriched dough. Journal of Food Processing and Preservation, 36(6), 553–559. Wang, K., Luo, S., Cai, J., Sun, Q., Zhao, Y., Zhong, X., et al. (2016). Effects of partial hydrolysis and subsequent cross-linking on wheat gluten physicochemical properties and structure. Food Chemistry, 197, 168–174. Wronkowska, M., Jadacka, M., Soral-Śmietana, M., Zander, L., Dajnowiec, F., Banaszczyk, P., et al. (2015). ACID whey concentrated by ultrafiltration a tool for modeling bread properties. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 61(1), 172–176. Zhang, D., Doehlert, D. C., & Moore, W. R. (1997). Factors affecting viscosity of slurries of oat groat flours. Cereal Chemistry, 74(6), 722–726. Zhang, Y., Pei, F., Fang, Y., Li, P., Zhao, Y., Shen, F., et al. (2019). Comparison of concentration and health risks of 9 Fusarium mycotoxins in commercial whole wheat flour and refined wheat flour by multi-IAC-HPLC. Food Chemistry, 275, 763–769.
137, 90–98. Meyers, M. A., & Chawla, K. K. (1990). Mechanical behavior of materials. Mechanical behavior of materials. Niu, M., Hou, G. G., Kindelspire, J., Krishnan, P., & Zhao, S. (2017). Microstructural, textural, and sensory properties of whole-wheat noodle modified by enzymes and emulsifiers. Food Chemistry, 223, 16–24. Ooms, N., & Delcour, J. A. (2019). How to impact gluten protein network formation during wheat flour dough making. Current Opinion in Food Science, 25, 88–97. Pelton, J. T., & McLean, L. R. (2000). Spectroscopic methods for analysis of protein secondary structure. Analytical Biochemistry, 277(2), 167–176. Plessas, S., Fisher, A., Koureta, K., Psarianos, C., Nigam, P., & Koutinas, A. A. (2008). Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making. Food Chemistry, 106(3), 985–990. Quattrini, M., Bernardi, C., Stuknyte, M., Masotti, F., Passera, A., Ricci, G., et al. (2018). Functional characterization of Lactobacillus plantarum item 17215: A potential biocontrol agent of fungi with plant growth promoting traits, able to enhance the nutritional value of cereal products. Food Reserch International, 106, 936–944. Robertson, G. H., Gregorski, K. S., & Cao, T. K. (2006). Changes in secondary protein structures during mixing development of high absorption (90%) flour and water mixtures. Cereal Chemistry Journal, 83(2), 136–142. Saladino, F., Luz, C., Manyes, L., Fernández-Franzón, M., & Meca, G. (2016). In vitro antifungal activity of lactic acid bacteria against mycotoxigenic fungi and their application in loaf bread shelf life improvement. Food Control, 67, 273–277. Schober, T. J., Bean, S. R., & Boyle, D. L. (2007). Gluten-free sorghum bread improved by sourdough fermentation: Biochemical, rheological, and microstructural background.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
A novel lactic acid bacterium for improving the quality and shelf life of whole wheat bread
T
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 LE I N FO
A B S T R A C T
Keywords: Whole wheat Sourdough Lactic acid bacteria Quality Shelf life
The aim of this study was to screen for a lactic acid bacterium (LAB) that could improve the quality of whole wheat bread (WWB) and extend its shelf life. The LABs with strong antifungal activity were screened among twenty LABs. Both sourdough properties (rheology, tensility, water mobility and gluten structure) and WWB qualities (texture, volatile flavour and shelf life) were determined and compared. The results showed that the Lactobacillus 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 better viscoelasticity, 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 texture characteristics, enrich the aroma volatile compounds, and prolong the shelf life of WWB from 3 days to 6 days compared to the control group. Above all, Lactobacillus plantarum LB-1 was recommended for WWB fermentation to improve its quality and to extend the shelf life.
1. Introduction Whole wheat bread (WWB) is a kind of bread made from whole wheat flour without removing the bran and wheat germ. Compared to refined wheat bread, it is rich in higher levels of dietary fibres, minerals, phytochemicals, antioxidants and vitamins (Tebben, Shen, & Li, 2018). The American Institute for Cancer Research (AICR) and the World Cancer Research Fund (WCRF) reported that eating whole grain foods 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 in reducing disease risks and managing chronic diseases by eating whole grain food (Niu, Hou, Kindelspire, Krishnan, & Zhao, 2017). However, compared to refined wheat bread, WWB may be more susceptible to fungal infections because it contains the intact grain and epidermal part (Zhang, Pei, Fang, Li, Zhao, Shen, et al., 2019). Most fungi were located in the outer layer of wheat kernels. These fungi can act as a potential reservoir of contamination and produce mycotoxins (including aflatoxin, zearalenone and deoxynivalenol) in the manufacturing environment, which cause more safety hazards (Saladino, Luz, Manyes, Fernández-Franzón, & Meca, 2016). Moreover, the product acceptability 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-(hydroxymethyl) furfural (HMF) can seriously affect its taste (Jiang & Peterson, 2013). Recently, the improvement of WWB is mainly based on using chemical leavening and preservative agents. Hydrocolloids, such as hydroxypropyl methyl cellulose, carboxymethyl cellulose, locust bean gum and xanthan gum, have been widely used to improve the waterabsorbing 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 on maintaining the quality of bread, proving that calcium propionate could extend its shelf life significantly. However, these chemical additives may cause changes in the natural components of whole wheat, some of which may be toxic to the human body (Ginocchio et al., 1979). Therefore, it is of great significance to develop natural functional dough improvers. Since chemical additives have anti-nutritional and toxic effects, LAB as a natural starter has been paid widespread attention. In a study by Dal Bello, Clarke, Ryan, Ulmer, and Schober (2007), lactic acid, phenyllactic acid and cyclic dipeptides cyclo produced by Lactobacillus plantarum FST 1.7 showed consistent inhibition against Fusarium species and had the potential to extend the shelf life of bread. In addition,
Corresponding author. E-mail address: [email protected] (F. Pei).
https://doi.org/10.1016/j.foodcont.2019.106914 Received 28 June 2019; Received in revised form 25 August 2019; Accepted 21 September 2019 Available online 24 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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instructions. The primer sets 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for amplification of the gene. PCR conditions reported by Joo et al. (2015) were set as follows: 95 °C for 5 min followed by 30 cycles of 95 °C for 1 min, 52 °C for 30 s, and 72 °C for 1 min 30 s, with a final extension at 72 °C for 7 min. The amplified DNAs were purified and sequenced using an ABI 3730XL DNA analyser (Life Technologies, New York, USA). The 16S rRNA sequences were blasted against the NCBI GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Wang, Hwang, Tzeng, Hwang, and Mau (2012) suggested that the total volatile content including alcohols, esters and carbonyls of bread made with Lactobacillus delbrueckii subsp. Delbrueckii was significantly higher than those in the blank group. However, most of these studies were limited to refined wheat, while fungal contamination and poor quality are far more serious problems in whole wheat. Therefore, the overall purpose of this study was finding a novel LAB to simultaneously solve the problem of fungal contamination and distastefulness in WWB. In this study, specific LABs with high antifungal activity were screened from a total of twenty LABs. Subsequently, the effects of the selected LABs on dough improvement were evaluated via rheological measurements, tensile tests, water mobility determination, gluten secondary structures quantification and microstructure observation. The sourdoughs were then processed into breads, and their texture, volatile compounds and shelf life were also analysed to verify the applicability of the LABs as starters for WWB.
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/T 14611–2008 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 of sugar (Herunhua FOOD, Jiangsu, China) and 120 g of LAB suspension (108 CFU/g). Dough with the same quantity of water (containing no LAB) was set as the control group. The sourdoughs were kneaded by a dough mixer for 6 min and then placed in a fermentation box (temperature was 30 ± 1 °C and humidity was 85%–90%) for 5 h. Subsequently, the sourdough was divided into 100 g portion and baked in a JKLZ4 oven (Fude Technology, Beijing, China) at 200 °C for 20 min.
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, F17, F-24, F-48, F-50 and F-59 were obtained from the Key Laboratory of Grains and Oils Quality Control and Processing. LB-1, LB-2, LB-3, LB-4 and LB-5 were isolated from local pickles in Nanjing, China. Lactobacillus rhamnosus (BNCC 136673), Leuconostoc mesenteroides (BNCC 195309), Lactobacillus plantarum (BNCC 194165) and Lactobacillus casei (BNCC 134415) were purchased from the BeNa Culture Collection. Lactobacillus rhamnosus strain GG (ATCC 53103) was purchased from the American Type Culture Collection), and six fungi (Penicillium citrinum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus fumigatus and Fusarium graminearum were isolated from whole wheat) were studied in this experiment. LAB strains were preserved in sterile 25% glycerol at −80 °C before use.
2.6. Rheological measurements Dynamic rheological properties of the sourdough were measured by a rheometer (Anton-Paar-Strasse, Austria) using oscillation sweep tests. Sourdough was placed on the centre of the rheometer plate and left for 5 min for relaxing the residual stress. Mineral oil was daubed around the edge of the sourdough to prevent moisture loss. A strain of 0.5% and a measure position of 2 mm were applied in this test. The test temperature was 25 ± 1 °C and the test frequency was in the range of 0.1–40 Hz (Hao et al., 2008; Inglett, Chen, Liu, & Lee, 2014).
2.2. LAB suspension and fungus spore suspension preparation
2.7. Tensile properties tests
Single LAB strains (2% v/v) were inoculated into MRS broth at 37 °C for 20 h. The cells were harvested by centrifugation (5500 g for 10 min at 4 °C) and the supernatant was removed. The obtained LAB cells were suspended in sterile water and counted, varying from 107 to 109 CFU/mL. The fungi were inoculated in PDA medium at 28 °C for 7–10 days. The spores were harvested from Petri plates in sterile water to prepare a suspension containing 1 × 104 spores/mL. The LAB suspension and fungus spore suspension were preserved at 4 °C before use.
The tensile properties of the sourdough were measured using a TAXT Plus texture analyser (Stable Micro Systems, London, England) with an A/KIE probe. A trigger force of 5 g and a test distance of 50 mm were applied to perform this test. The pre-test speed, test speed and post speed were 2 mm/s, 3.3 mm/s and 10 mm/s, respectively. The sourdough was rested in the fermentation box (temperature was 30 ± 1 °C and humidity was 85%–90%) for 10 min before the test. Each group was tested six times (Suchy, Lukow, & Ingelin, 2000).
2.3. Antifungal activity in vitro
2.8. Water mobility determination
The dual culture method described by Quattrini et al. (2018) was modified and used to test the antifungal activity of LABs. Briefly, 10 μL of the LAB suspension (109 CFU/mL) was dropped on the centre of MRS agar plates and incubated at 37 °C for 48 h. Subsequently, 10 mL of PDA containing 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 inhibition zone were measured using an electronic digital calliper (Guanglu, Guilin, China). The antifungal activity of the LABs was calculated on the basis of the inhibition zone as no inhibition (−) for an inhibition zone smaller than 0.05 mm, weak inhibition (+) for an inhibition zone smaller than 10 mm, moderate inhibition (++) for an inhibition zone in the range of 10–20 mm, and strong inhibition (+++) for an inhibition zone larger than 20 mm.
Water mobility of the sourdough was detected using low field nuclear magnetic resonance (LF-NMR) (MesoMR, Niumag Corporation, China). The sourdough was evenly stuffed into the sample vials and then 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 = 19 MHz, RFD = 0.5 ms, O1 = 948164.27 Hz, RG1 = 20 db, P1 = 13 us, DRG1 = 3, TD = 300150, TW = 1500 ms, P2 = 25 us, TE = 0.2 ms, NECH = 7500 and NS = 32. T21 represents the bound water, T23 represents free water, and T22 represents the adsorbed water between the bound water and free water (Doona & MooYeol, 2007).
2.4. LAB identification
2.9. Fourier transform infrared spectroscopy (FTIR) and gluten secondary structure
DNA was isolated from selected strains using an EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China) according to the
Gluten was isolated and collected from the dough according to the AACCI Approved Method 38–10.01. The gluten was dried using an oven 2
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Table 1 Antifungal activity of LAB strains against different fungi. Strains
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
Antifungal activitya Penicillium citrinum
Aspergillus niger
Aspergillus flavus
Aspergillus ochraceus
Aspergillus fumigatus
Fusarium graminearum
+++ +++ +++ +++ +++ +++ + +++ ++ + + – – – – +++ +++ +++ +++ +++
++ ++ ++ + ++ – + – + – – – – – + – + + + –
+++ +++ ++ ++ ++ +++ + ++ ++ – – ++ – ++ +++ ++ +++ + +++ ++
++ ++ ++ ++ + + – – + – – – – – – + + + ++ –
+++ +++ +++ ++ +++ ++ – +++ ++ – – + ++ +++ ++ +++ +++ +++ ++ +
+++ +++ +++ + + + – ++ + + – ++ ++ – – ++ +++ ++ + –
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 (+++).
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) was ground into a uniform powder again and pressed into sheets using a powder pressing machine (769 YP-15A, Tianjin, China). A total of 64 scans were run for each analysis at an interval of 4 cm−1 in the range of 400–4000 cm−1. The curve of the Amide I region was selected and fitted by a module (AutoFit Peaks II Second Derivative) of Peakfit V4.12 software. Then, the percentage of peak areas corresponding to the gluten secondary structure was given (Chen et al., 2019). The spectral regions were assigned as 1612–1640 cm−1 and 1670–1694 cm−1 for βsheets, 1640–1650 cm−1 for random coils, 1648–1660 cm−1 for α-helices, and 1662–1684 cm−1 for β-turn structures. The second derivative area for each secondary structural region was divided by the total area of the amide I region (Carbonaro & Nucara, 2010; Goormaghtigh, Cabiaux, & Ruysschaert, 1994; Pelton & McLean, 2000).
increased by 5.5 °C/min to 230 °C (held for 5 min). The carrier gas was He at a flow rate of 2 mL/min. The mass spectrum was recorded by electronic impact (EI) at 70 eV. The scan mode was in the range of m/z 33–200. Compounds were identified by the MS database (NIST 98) combined with Kovats indexes (KI) comparison. Match quality higher than 80% was considered to be reliable. Experimental KI was based on n-alkanes (C7 to C30, o2si smart solutions, USA). Compounds were quantified 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-XT2i texture analyser (Stable Micro Systems, London, England) with a P/36R probe. Each sample was cut into 1 cm thick slices and placed for 1 h for cooling. Test parameters according to AACCI74-09 were as follows: pretest speed, 3 mm/s; test speed, 1 mm/s; post speed, 5 mm/s; trigger type, auto-10 g; target mode, distance-5 mm; interval between two compressions, 10 s. For the fungi spoilage analysis, bread samples were packed in polyethylene bags and stored at room temperature (23 ± 1 °C). Fungal colonies on the bread surface were monitored daily for one week. Contamination was calculated as follows: no visible colonies (−), one colony (+), two (++), and three or more (+++) (Bartkiene, Bartkevics, Lele, Pugajeva, Zavistanaviciute, Mickiene, et al., 2018).
2.10. Scanning electron microscopy (SEM) Sourdough flakes were fixed in glutaraldehyde (2.5%) for 12 h and dehydrated in increasing grades of ethanol (25%, 50%, 75%, 95% and 100%). Then, the sourdough flakes were coated with gold particles using a MSP-1S Sputter Coater (Hitachi, Tokyo, Japan) after lyophilisation. Microstructures of the different sourdoughs were photographed using a TM3000 SEM (Hitachi, Tokyo, Japan) at 250 × and 1000 × magnification (Liu et al., 2015).
2.13. Statistical analysis 2.11. Gas chromatography-mass spectrometry All of the experiments were repeated at least three times. Means and standard deviations were calculated. The results were analysed by SPSS version 23 software for Windows (SPSS Inc., Chicago, IL, USA). Duncan tests at a significance level of P < 0.05 were performed for significance analysis.
Aroma volatile compounds were analysed by headspace gas chromatography mass spectrometry (GC-MS) using the solid-phase microextraction (SPME) method as previously described (Plessas et al., 2008). Briefly, 2 g of a bread sample was placed in a 20 mL glass vial and incubated in a water bath at 60 °C. The SPME fibre (50/30 μm DVB/CAR/PDMS, Stable Flex Supelco, Bellefonte, PA, USA) was exposed to the headspace for 60 min. Desorption of volatiles was in the injector port (280 °C) of the gas chromatograph (Agilent7890A, Palo Alto, CA, USA) for 5 min in splitless mode. An Agilent DB-5MS capillary column (0.25 μm film thickness, 30 m × 250 μm) was used. The GC temperature program (total run time 51.73 min) was as follows: 35 °C for 5 min, then increased by 5 °C/min to 50 °C (held for 5 min),
3. Results and discussion 3.1. Selection of LABs with strong antifungal activity and LABs identification A screen of the twenty LAB strains was carried out by measuring antifungal activity against six susceptible fungi of wheat: Penicillium 3
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Fig. 1. Effect of different LABs on rheology (A and B), tensile property (C), water mobility (D) during sourdough fermentation.
citrinum, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus fumigatus and Fusarium graminearum. These fungi are considered to be the most common spoilage agents in bakery products due to their high prevalence in wheat flour and their xerotolerance to xerophilic behaviour, which can act as a potential reservoir of contamination in the manufacturing environment (Saladino et al., 2016). The antifungal activity of LAB was calculated on the basis of the inhibition zone, and the results are shown in Table 1. Most of the LAB strains demonstrated strong inhibition against Penicillium citrinum, Aspergillus flavus, Aspergillus fumigatus and Fusarium graminearum, while Aspergillus niger and Aspergillus ochraceus were inhibited only by certain kinds of LAB strains. Remarkably, compared to the other LAB strains, the LB-1, F-3 and F-50 strains possessed at least moderate inhibitory activity (++) against all six susceptible fungi. Therefore, LB-1, F-3 and F-50 were selected to further compare their effects on the qualities of WWB. The 16S rRNA sequencing results of the selected LAB strains LB-1, F3 and F-50 are shown in Table S1, S2 and S3. The 16S rRNA genes of the selected LAB strains LB-1, F-3 and F-50 were identical (100%) to the Lactobacillus plantarum NCU116 (Accession: CP016071.1), Lactobacillus plantarum CB5 (Accession: MK687387.1) and Lactobacillus plantarum
EM (Accession: CP037429.1) in 1400, 1407 and 1449 nucleotides, respectively (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Therefore, the three selected LAB strains were classified as Lactobacillus plantarum LB1, Lactobacillus plantarum F-3 and Lactobacillus plantarum F-50, respectively.
3.2. Effect of LABs on rheology, tensility and water mobility of sourdoughs The sourdough properties fermented by the selected LAB strains were evaluated via rheology, tensility and water mobility determination (Fig. 1). The G′ and G″ of different sourdoughs at a frequency of 0.1–40 Hz are shown in Fig. 1A and B. Storage modulus (G′) represents the ability of materials to store elastic deformation energy, and loss modulus (G″) represents the viscous portion (Meyers & Chawla, 1990). It could be seen that both moduli (G′ and G″) of all sourdoughs increased with the increase of frequency. Moduli (G′ and G″) of sourdough LB-1 and F-3 were significantly higher than that of F-50 and the control group, which indicated that sourdoughs fermented by LB-1 and F-3 were more viscous and elastic. Ghodke and Laxmi (2007) found that exopolysaccharides (EPS) produced by Lactobacillus buchneri FUA3154 can replace hydrocolloids to influence the rheological properties of 4
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the water closely bound to protein and starch, influencing the formation of the gluten network structure in dough (Assifaoui, Champion, Chiotelli, & Verel, 2006). Therefore, it can be concluded that the sourdough LB-1 and F-3 possessed stronger water-holding capacity compared to the control group. Zhang, Doehlert, and Moore (1997) studied the effect of β-glucan on oat flour, and they found that the viscosity and the water binding capacity of oat flour was positively correlated with the content of β-glucan. LABs can produce β-glucan in the process of growth and metabolism. Therefore, sourdough absorbed more 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 glutenin complexes formed by glutenin subunits crosslinking through disulfide bond ends (Ooms & Delcour, 2019). Therefore, from a microcosmic perspective, the secondary structures of gluten and the exterior shape of the sourdoughs were studied to explain the orderliness of sourdoughs by FTIR and SEM respectively. The FTIR spectra of the different samples are shown in Fig. 2A. Amide I region (1600–1700 cm−1) has been used to study the secondary structure of gluten because it is almost entirely attributable to C]O stretch vibrations and is sensitive to gluten conformation (Robertson, Gregorski, & Cao, 2006). The curve of the Amide I region was selected and fitted to calculate the secondary structures of gluten using Peakfit V4.12 software. As seen in Fig. 2B, the LAB strains had a great influence on the secondary structures of sourdoughs. βsheets and α-helices were the main secondary structures of the gluten, which is consistent with a previous study (Wang et al., 2016). With the addition of the LAB strains, there was a dramatic decrease in random coils together with significant increases in β-turns, indicating conformational changes in the gluten. Compared to the control group, the sum of β-sheets and α-helices for sourdoughs LB-1, F-3 and F-50 increased by 6.49%, 4.49% and 0.12%, respectively. Previous studies on gluten secondary structures proved that β-sheets and α-helices enabled dough to form a more ordered network structure, while random coils were considered as disordered structures (Marti, Bock, Pagani, Ismail, & Seetharaman, 2016; Mecozzi & Sturchio, 2015). The increase in βsheets and α-helices indicated that sourdough LB-1 formed a more regular and orderly gluten structure. The microstructures of the sourdoughs were detected by SEM at magnifications of 250 × and 1000 × to further analyse the gluten structure. As shown in Fig. 3A, discontinuity of the gluten network structure could be obviously observed in the control group. Starch granules aggregated in clusters and did not disperse evenly in the network structure (Fig. 3a). This was due to the addition of bran, which can cause water redistribution and a partial dehydration of gluten (Bock & Damodaran, 2013). For the sourdoughs F-50 and F-3, a break in junctions of the membrane-like gluten matrix could be clearly observed (Fig. 3C) and most starch granules were aggregated into clusters and exposed to stomata (Fig. 3d). Compared to the other groups, the sourdough LB-1 (Fig. 3B) formed a more ordered and compact gluten network structure, and the starch granules were wrapped tightly and evenly in the membrane-like gluten matrix (Fig. 3b). This phenomenon indicated that the gluten network of sourdough, which was well formed, possessed good ductility when fermented by LB-1.
Fig. 2. ATR-FTIR spectrum of gluten in different sourdoughs (A), and fitting results of Amide I region for secondary structure content of gluten protein (B).
sourdoughs. The greater viscosity and elasticity in sourdough LB-1 and F-3 may be attributed to the cementability and stability of EPS. Tensile properties are important indices in dough baking and are closely related to gluten network formation and dough gas-holding capacity (McCann, Le Gall, & Day, 2016). Fig. 1C shows the tensile resistance and extensibility of the different sourdoughs. It can be seen that the tensile resistances of the three fermented sourdoughs were significantly higher (P < 0.05) than that of the control group. Meanwhile, sourdough LB-1 and F-50 demonstrated better tensile resistance compared to sourdough F-3. Additionally, the extensibilities of sourdough F-3 and LB-1 were significantly higher (P < 0.05) than those of F-50 and the control group. The results indicated that the extensibility and strength of the gluten network structure in sourdough LB-1 and F-3 were enhanced. A previous study reported by Schober, Bean, and Boyle (2007) showed that proteolysis during sourdough fermentation could reduce the interference of protein with starch and result in stronger starch gel strength, which was consistent with the findings of this study. Water mobility is one of the main characteristics of dough and plays an important role in the dough quality. Continuous distributions of spin-spin relaxation time (T2) are shown in Fig. 1D. As shown in Fig. 1D, water in the dough mainly existed in the form of adsorbed water (T22). However, two distinct peaks appearing at T21 represented the bound water in sourdough LB-1 and F-3, which can be regarded as
3.4. Effect of different LABs on textural properties After evaluating the rheology, tensility and water mobility of the sourdoughs, we made the sourdoughs into WWB according to GB/T 14611–2008 and tested the texture characteristics of WWB, including hardness, springiness, cohesiveness, gumminess and chewiness (Table 2). It was observed that compared to the control, F-3 and F-50 groups, the hardness, gumminess and chewiness of WWB LB-1 were 5
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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 2 The texture properties of WWB made with Lactobacillus plantarum LB-1, F-3 and F-50 (n = 8). Sample
Hardness
Control LB-1 F-3 F-50
383.57 253.77 376.27 364.80
± ± ± ±
8.45a 10.83b 15.15a 17.87a
Springiness
Cohesiveness
Gumminess
Chewiness
0.99 0.97 0.96 0.95
0.91 0.95 0.92 0.91
365.59 236.12 345.12 330.83
363.29 230.17 330.52 316.27
± ± ± ±
0.01a 0.03a 0.03a 0.02a
± ± ± ±
0.02b 0.01a 0.02 ab 0.02b
± ± ± ±
5.67a 5.48b 13.87a 19.80a
± ± ± ±
7.30a 12.90b 14.89a 15.24a
Wronkowska, Jadacka, Soral-Śmietana, Zander, Dajnowiec, Banaszczyk, et al., 2015). The results obtained from TPA indicated that the WWB fermented by Lactobacillus plantarum LB-1 tasted springier, softer and more refreshing, which more closely caters to consumer tastes compared to other groups.
significantly (P < 0.05) lower, and the cohesiveness was significantly (P < 0.05) higher. A large number of studies have proven that hardness, gumminess and chewiness are negatively correlated with bread quality, while springiness and cohesiveness are positive attributes (AlFarga, Zhang, Siddeeg, Chamba, Kimani, Hassanin, et al., 2016; 6
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Table 3 (continued)
Table 3 Aroma volatile compounds of different WWB made with Lactobacillus plantarum LB-1, F-3 and F-50. Compound
Alcohols Ethanol 1-Propanol 2-Methyl-1-propanol 1-Butanol Isopentyl alcohol 1-Pentanol 2,3-Butanediol 2-Butanone 1-Hexanol 2-Heptanol 2-Ethyl-1-hexanol Benzyl alcohol Phenylethyl Alcohol Total Esters Ethyl Acetate Ethyl pentanoate Ethyl hexanoate Ethyl caprylate β-Phenylethyl acetate Ethyl nonylate Ethyl dec-9-enoate Ethyl decanoate Ethyl dodecanoate Ethyl octadecanoate Total Ketones 2,3-Butanedione 2-Butanone 2,3-Pentanedione 6-Methyl-5-heptene-2-one 2-Octanone Total Aldehydes Butanal, 3-methyl2-Butenal Butanal, 2-methylHexanal Furfural 3- Furfural Octanal Benzeneacetaldehyde 2-Nonenal Decanal 2,4-Decadienal Tridecanal Tetradecanal Total Carboxylic acids Acetic acid Propanoic acid Methacrylic acid 2-Methylpropanoic acid Hexanoic acid Octanoic Acid Benzoic acid Nonanoic acid n-Decanoic acid Total Heterocyclic compounds Furan, 2-methylFuran, 2-ethylPyrazine 2,5-Dimethylpyrazine Pyrazine, ethyl5-Methyl-2-furfural Furan, 2-pentyl Pyrrole-2-aldehyde Maltol Anethole
KI
a
ID
b
Compound
KIa
IDb
Concentration (%) Control
LB-1
F-3
F-50
427 536 625 658 727 769 782 803 869 903 1029 1057 1107
MS MS MS,RI MS MS,RI MS,RI MS MS,RI MS,RI MS MS,RI MS,RI MS
37.8 ndc nd 0.45 5.22 nd 2.32 6.23 nd nd 0.76 nd 3.69 56.47
20.14 1.16 0.58 1.29 1.45 1.62 0.11 nd 2.78 3.51 3.37 0.08 5.12 41.21
21.28 0.65 0.37 1.08 1.91 0.73 0.15 1.13 0.56 2.73 1.97 0.09 5.14 37.79
22.32 1.03 0.54 1.12 1.23 0.41 0.13 nd 2.01 2.94 2.35 0.04 6.34 40.46
608 915 997 1173 1245 1296 1390 1396 1579 2188
MS MS MS,RI MS,RI MS,RI MS,RI MS,RI MS MS,RI MS,RI
7.08 0.34 0.37 1.46 nd nd 0.16 0.72 0.20 nd 10.33
4.78 nd 0.27 1.41 0.56 0.10 0.16 0.85 0.06 0.15 8.34
5.41 0.14 0.97 1.67 0.42 0.12 0.09 0.78 0.15 0.11 9.86
5.71 0.97 0.13 1.24 0.49 0.02 0.03 0.71 0.08 0.07 9.45
600 622 696 989 994
MS MS,RI MS MS,RI MS
0.72 1.72 nd nd nd 2.44
5.18 0.18 0.05 0.13 0.16 5.70
4.35 1.29 0.20 0.36 0.72 6.92
3.93 0.25 0.17 0.04 0.13 4.52
655 657 661 807 830 837 980 1043 1160 1207 1284 1518 1625
MS MS MS,RI MS,RI MS,RI MS,RI MS MS,RI MS,RI MS MS,RI MS,RI MS,RI
3.35 nd 0.31 0.36 3.80 3.12 0.23 nd nd 0.24 0.26 nd 0.84 12.51
1.98 0.32 0.08 0.12 3.17 4.10 0.20 0.28 0.46 0.18 nd 0.10 0.73 11.72
2.50 0.28 0.04 0.43 2.36 2.74 0.30 0.90 0.59 0.12 0.22 0.71 0.88 12.07
1.35 0.15 nd 0.31 2.35 3.34 0.25 0.73 0.54 0.07 0.11 0.36 0.41 9.97
646 700 711 770 1024 1180 1199 1273 1371
MS MS MS MS,RI MS,RI MS MS MS,RI MS,RI
0.90 nd nd nd nd 0.40 0.15 nd 0.42 1.87
19.1 1.18 1.19 0.14 1.62 0.37 0.21 0.27 0.62 24.70
15.41 1.07 1.15 nd 1.38 0.33 1.27 0.43 0.57 21.61
11.14 1.23 1.21 nd 1.51 2.30 2.15 nd 2.59 22.13
603 702 747 910 928 970 991 1036 1133 1265
MS MS,RI MS,RI MS,RI MS MS,RI MS MS MS MS
2.02 1.98 nd 1.17 2.13 1.64 1.31 2.17 nd 1.27
1.12 0.77 0.29 1.95 1.96 0.59 0.44 0.35 0.27 0.42
nd nd nd nd 1.74 2.53 2.47 1.8 nd 1.82
1.15 1.46 nd 1.83 1.94 1.57 1.41 1.76 nd 2.29
Nonan-1,4-olide Butylated Hydroxytoluene Total a b c
1363 1483
MS,RI MS
Concentration (%) Control
LB-1
F-3
F-50
1.62 1.07 16.38
0.17 nd 8.33
1.04 0.35 11.75
nd 0.06 13.47
KI: Kovats index. MS: identification by MS data; RI: identification by reference value of KI. nd: not detected.
3.5. Aroma volatile compounds analysis Sixty-two aroma volatile compounds of the WWBs were detected by SPME-GC-MS and the results are shown in Table 3. It is noteworthy that after fermentation by LB-1, F-3 and F-50, the aroma volatile compounds (60, 54 and 55 kinds of volatile compounds, respectively) increased and were enriched compared to the control group (37 kinds of volatile compounds). Meanwhile, carboxylic acids of the control group (1.87%) were significantly lower than those of the WWB LB-1, F-3 and F-50 groups (24.70%, 21.61% and 22.13%, respectively), and the content of acetic acid was the highest among them. This is consistent with a previous study on the flavour of sourdough bread by Cavallo et al. (2017), who reported that acetic acid could be generated during the processing of bread fermentation. Moreover, Su et al. (2019) studied organic acids on bread quality improvement, finding that acetic acid could give bread a higher specific volume, a lower pH value and a decreased hardness. In addition, the acetic acid content determined the efficiency of sourdough as a possible preservative agent against the microbial 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 daily during a test of a one week shelf life (Table 4). Two visible fungus colonies were first observed in WWB F-50 on the third day and then observed in the WWB F-3 on the fifth day. In the control group, visible fungus colonies were detectable on the fourth day, whereas in the WWB fermented with LB-1, visible fungus colonies were not seen until the seventh day. According to the fungal infection state on the seventh day, the fungal spores grew vigorously and showed their original colour in the WWB F-3, F-50 and the control group, which indicated that the fungi were not significantly inhibited. However, in the WWB LB-1, only two small colonies were observed, and the growth of fungal spores was almost completely inhibited. Compared to the control group, the extended three-day shelf life of WWB LB-1 may be attributed to the lactic acid 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 and prolong the shelf life of WWB with great application prospect. Our results showed that the Lactobacillus plantarum LB-1, which isolated from local pickles (Nanjing, China), demonstrated the extraordinary potential for improving the qualities of sourdough and WWB. Specifically, sourdough fermented by Lactobacillus plantarum LB-1 possessed better qualities of in terms of higher viscoelasticity, stronger extensibility and water holding capacity. Meanwhile, the ordered secondary structures (β-sheets and α-helices) quantified by FTIR were 6.49% more than control group. In addition, during WWB production, the texture characteristics analysis and aroma volatiles determination indicated that Lactobacillus plantarum LB-1 enabled WWB a better taste and a richer 7
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Table 4 Effect of different LABs on fungal infection during WWB storage. Sample
Days of storage
Control LB-1 F-3 F-50 Fungal infection on the seventh day
Control a
1
2
3
4
5
6
7
a
– – – –
– – – ++
++ – – +++
+++ – + +++
+++ – +++ +++
+++ ++ +++ +++
– – –
LB-1
F-3
F-50
Contamination was calculated as follows: no visible colonies (−), one colony (+), two (++), and three or more (+++).
flavour. Moreover, the shelf life of WWB was extended from 3 days to 6 days compared to the control group. Therefore, Lactobacillus plantarum LB-1, replacing traditional chemical additions, can be successfully used as a starter for WWB quality improvement and fungi contamination prevention.
(2017). Microbial cell-free extracts affect the biochemical characteristics and sensorial quality of sourdough bread. Food Chemistry, 237, 159–168. Chen, G., Ehmke, L., Sharma, C., Miller, R., Faa, P., Smith, G., et al. (2019). Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt. Food Chemistry, 275, 569–576. Dal Bello, F., Clarke, C. I., Ryan, L. A. M., Ulmer, H., & Schober, T. J. (2007). Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7. Journal of Cereal Science, 45(3), 309–318. Ding, X., Zhang, H., Wang, L., Qian, H., Qi, X., & Xiao, J. (2015). Effect of barley antifreeze protein on thermal properties and water state of dough during freezing and freeze-thaw cycles. Food Hydrocolloids, 47, 32–40. Doona, C. J., & Moo-Yeol, B. (2007). Molecular mobility in model dough systems studied by time-domain nuclear magnetic resonance spectroscopy. Journal of Cereal Science, 45(3), 257–262. Ghodke, S. K., & Laxmi, A. (2007). Influence of additives on rheological characteristics of whole-wheat dough and quality of Chapatti (Indian unleavened Flat bread) Part Ihydrocolloids. Food Hydrocolloids, 21(1), 110–117. Ginocchio, A. V., Waite, V., Hardy, J., Fisher, N., Hutchinson, J. B., & Berry, R. (1979). Long-term toxicity and carcinogenicity studies of the bread improver potassium bromate 2. Studies in mice. Food and Cosmetics Toxicology, 17(1), 41–47. Goormaghtigh, E., Cabiaux, V., & Ruysschaert, J.-M. (1994). Determination of soluble and membrane protein structure by fourier transform infrared spectroscopy. Sub-Cellular Biochemistry, 23, 363–403. Hao, C. C., Wang, L. J., Li, D., Özkan, N., Wang, D. C., Chen, X. D., et al. (2008). Influence of alfalfa powder concentration and granularity on rheological properties of alfalfawheat dough. Journal of Food Engineering, 89(2), 137–141. Inglett, G. E., Chen, D., Liu, S. X., & Lee, S. (2014). Pasting and rheological properties of oat products dry-blended with ground chia seeds. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 55(1), 148–156. Jiang, D., & Peterson, D. G. (2013). Identification of bitter compounds in whole wheat bread. Food Chemistry, 141(2), 1345–1353. Joo, H. J., Kim, H. Y., Kim, L. H., Lee, S., Ryu, J. G., & Lee, T. (2015). A Brevibacillus sp. antagonistic to mycotoxigenic. Fusarium spp. Biological Control, 87, 64–70. Katsinis, G., Lohano, D. K., Sheikh, S. A., & Shahnawaz, M. (2009). Effect of chemical preservatives on the shelf life of bread at various temperatures. Pakistan Journal of Nutrition, 9(3), 279–283. Lafay, L., & Ancellin, R. (2015). Alimentation et cancer colorectal. Cahiers de Nutrition et de Diététique, 50(5), 262–270. Liu, R., Xing, Y., Zhang, Y., Zhang, B., Jiang, X., & Wei, Y. (2015). Effect of mixing time on the structural characteristics of noodle dough under vacuum. Food Chemistry, 188, 328–336. Mantzourani, I., Plessas, S., Saxami, G., Alexopoulos, A., Galanis, A., & Bezirtzoglou, E. (2014). Study of kefir grains application in sourdough bread regarding rope spoilage caused by Bacillus spp. Food Chemistry, 143, 17–21. Marti, A., Bock, J. E., Pagani, M. A., Ismail, B., & Seetharaman, K. (2016). Structural characterization of proteins in wheat flour doughs enriched with intermediate wheatgrass (Thinopyrum intermedium) flour. Food Chemistry, 194, 994–1002. Martínez-Anaya, M. A., Llin, M. L., Macías, M. P., & Collar, C. (1994). Regulation of acetic acid production by homo- and heterofermentative lactobacilli in whole-wheat sourdoughs. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 199(3), 186–190. Matuda, T. G., Chevallier, S., de Alcântara Pessôa Filho, P., LeBail, A., & Tadini, C. C. (2008). Impact of guar and xanthan gums on proofing and calorimetric parameters of frozen bread dough. Journal of Cereal Science, 48(3), 741–746. McCann, T. H., Le Gall, M., & Day, L. (2016). Extensional dough rheology - impact of flour composition and extension speed. Journal of Cereal Science, 69, 228–237. Mecozzi, M., & Sturchio, E. (2015). Effects of essential oil treatments on the secondary protein structure of Vicia faba: A mid-infrared spectroscopic study supported by twodimensional correlation analysis. Spectrochimica Acta Part A: Molecular Spectroscopy,
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors gratefully acknowledge the financial support provided by the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF, Grant No. CX(17)1003), Qing Lan Project, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.106914. References Al-Farga, A., Zhang, H., Siddeeg, A., Chamba, M. V. M., Kimani, B. G., Hassanin, H., et al. (2016). Effect of the addition of alhydwan seed flour on the dough rheology, bread quality, texture profile and microstructure of wheat bread. Journal of Texture Studies, 47(6), 484–495. Anna-Sophie, H., & Arendt, E. K. (2013). Influence of hydroxypropylmethylcellulose (HPMC), xanthan gum and their combination on loaf specific volume, crumb hardness and crumb grain characteristics of gluten-free breads based on rice, maize, teff and buckwheat. Food Hydrocolloids, 32(1), 195–203. Assifaoui, A., Champion, D., Chiotelli, E., & Verel, A. (2006). Characterization of water mobility in biscuit dough using a low-field 1H NMR technique. Carbohydrate Polymers, 64(2), 197–204. Bartkiene, E., Bartkevics, V., Lele, V., Pugajeva, I., Zavistanaviciute, P., Mickiene, R., et al. (2018). A concept of mould spoilage prevention and acrylamide reduction in wheat bread: Application of lactobacilli in combination with a cranberry coating. Food Control, 91, 284–293. Bin, Q., & Peterson, D. G. (2016). Identification of bitter compounds in whole wheat bread crumb. Food Chemistry, 203, 8–15. Bock, J. E., & Damodaran, S. (2013). Bran-induced changes in water structure and gluten conformation in model gluten dough studied by Fourier transform infrared spectroscopy. Food Hydrocolloids, 31(2), 146–155. Carbonaro, M., & Nucara, A. (2010). Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region. Amino Acids, 38(3), 679–690. Cavallo, N., De Angelis, M., Calasso, M., Quinto, M., Mentana, A., Minervini, F., et al.
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Food Control 109 (2020) 106914
L. Sun, et al.
Journal of Agricultural and Food Chemistry, 55(13), 5137–5146. Slavin, & Joanne (2004). Whole grains and human health. Nutrition Research Reviews, 17(1), 99–110. Suchy, J., Lukow, O. M., & Ingelin, M. E. (2000). Dough microextensibility method using a 2-g mixograph and a texture analyzer. Cereal Chemistry, 77(1), 39–43. Su, X., Wu, F., Zhang, Y., Yang, N., Chen, F., Jin, Z., et al. (2019). Effect of organic acids on bread quality improvement. Food Chemistry, 278, 267–275. Tebben, L., Shen, Y., & Li, Y. (2018). Improvers and functional ingredients in whole wheat bread: A review of their effects on dough properties and bread quality. Trends in Food Science & Technology, 81, 10–24. Wang, H. E., Hwang, C. F., Tzeng, Y. M., Hwang, W. Z., & Mau, J. L. (2012). Quality of white bread made from lactic acid bacteria-enriched dough. Journal of Food Processing and Preservation, 36(6), 553–559. Wang, K., Luo, S., Cai, J., Sun, Q., Zhao, Y., Zhong, X., et al. (2016). Effects of partial hydrolysis and subsequent cross-linking on wheat gluten physicochemical properties and structure. Food Chemistry, 197, 168–174. Wronkowska, M., Jadacka, M., Soral-Śmietana, M., Zander, L., Dajnowiec, F., Banaszczyk, P., et al. (2015). ACID whey concentrated by ultrafiltration a tool for modeling bread properties. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 61(1), 172–176. Zhang, D., Doehlert, D. C., & Moore, W. R. (1997). Factors affecting viscosity of slurries of oat groat flours. Cereal Chemistry, 74(6), 722–726. Zhang, Y., Pei, F., Fang, Y., Li, P., Zhao, Y., Shen, F., et al. (2019). Comparison of concentration and health risks of 9 Fusarium mycotoxins in commercial whole wheat flour and refined wheat flour by multi-IAC-HPLC. Food Chemistry, 275, 763–769.
137, 90–98. Meyers, M. A., & Chawla, K. K. (1990). Mechanical behavior of materials. Mechanical behavior of materials. Niu, M., Hou, G. G., Kindelspire, J., Krishnan, P., & Zhao, S. (2017). Microstructural, textural, and sensory properties of whole-wheat noodle modified by enzymes and emulsifiers. Food Chemistry, 223, 16–24. Ooms, N., & Delcour, J. A. (2019). How to impact gluten protein network formation during wheat flour dough making. Current Opinion in Food Science, 25, 88–97. Pelton, J. T., & McLean, L. R. (2000). Spectroscopic methods for analysis of protein secondary structure. Analytical Biochemistry, 277(2), 167–176. Plessas, S., Fisher, A., Koureta, K., Psarianos, C., Nigam, P., & Koutinas, A. A. (2008). Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making. Food Chemistry, 106(3), 985–990. Quattrini, M., Bernardi, C., Stuknyte, M., Masotti, F., Passera, A., Ricci, G., et al. (2018). Functional characterization of Lactobacillus plantarum item 17215: A potential biocontrol agent of fungi with plant growth promoting traits, able to enhance the nutritional value of cereal products. Food Reserch International, 106, 936–944. Robertson, G. H., Gregorski, K. S., & Cao, T. K. (2006). Changes in secondary protein structures during mixing development of high absorption (90%) flour and water mixtures. Cereal Chemistry Journal, 83(2), 136–142. Saladino, F., Luz, C., Manyes, L., Fernández-Franzón, M., & Meca, G. (2016). In vitro antifungal activity of lactic acid bacteria against mycotoxigenic fungi and their application in loaf bread shelf life improvement. Food Control, 67, 273–277. Schober, T. J., Bean, S. R., & Boyle, D. L. (2007). Gluten-free sorghum bread improved by sourdough fermentation: Biochemical, rheological, and microstructural background.
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