Food Microbiology 49 (2015) 211e219

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Comparison of homo- and heterofermentative lactic acid bacteria for implementation of fermented wheat bran in bread Michael Prückler a, *, Cindy Lorenz b, Akihito Endo c, Manuel Kraler a, Klaus Dürrschmid d, Karel Hendriks a, Francisco Soares da Silva a, Eric Auterith e, Wolfgang Kneifel a, Herbert Michlmayr a, f a

Christian Doppler Laboratory for Innovative Bran Biorefinery, Department of Food Science and Technology, BOKU e University of Natural Resources and Life Sciences, Vienna, Austria Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU e University of Natural Resources and Life Sciences, Vienna, Austria c Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Japan d Food Sensory Science, Department of Food Science and Technology, BOKU e University of Natural Resources and Life Sciences, Vienna, Austria e GoodMills Austria, Schwechat, Austria f Department of Applied Genetics and Cell Biology, BOKU e University of Natural Resources and Life Sciences, Vienna, Austria b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 December 2014 Received in revised form 19 February 2015 Accepted 23 February 2015 Available online 6 March 2015

Despite its potential health benefits, the integration of wheat bran into the food sector is difficult due to several adverse technological and sensory properties such as bitterness and grittiness. Sourdough fermentation is a promising strategy to improve the sensory quality of bran without inducing severe changes to the bran matrix. Therefore, identification of species/strains with potential for industrial sourdough fermentations is important. We compared the effects of different representatives of species of lactic acid bacteria (LAB) on wheat bran. Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sanfranciscensis and Fructobacillus fructosus produced highly individual fermentation patterns as judged from carbohydrate consumption and organic acid production. Interestingly, fructose was released during all bran fermentations and possibly influenced the fermentation profiles of obligately heterofermentative species to varying degrees. Except for the reduction of ferulic acid by L. plantarum and L. pentosus, analyses of phenolic compounds and alkylresorcinols suggested that only minor changes thereof were induced by the LAB metabolism. Sensory analysis of breads baked with fermented bran did not reveal significant differences regarding perceived bitterness and aftertaste. We conclude that in addition to more traditionally used sourdough species such as L. sanfranciscensis and L. brevis, also facultatively heterofermentative species such as L. plantarum and L. pentosus possess potential for industrial wheat bran fermentations and should be considered in further investigations. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Wheat bran Bread Sourdough Lactobacillus Fructobacillus Fermentation

1. Introduction

Abbreviations: AACC, American Association of Cereal Chemists; DM, dry matter; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; HQ, high quality; LAB, lactic acid bacteria; OD, optical density; HPLC, high pressure liquid chromatography; PKP, phosphoketolase pathway; RP-HPLC, reversed phase high pressure liquid chromatography; TTA, total titratable acid; W700, Austrian wheat flour quality corresponding DIN10355 Type 550. * Corresponding author. Christian Doppler Laboratory for Innovative Bran Biorefinery, Department of Food Science and Technology, BOKU e University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria. Tel.: þ43 1 47654 6767. E-mail address: [email protected] (M. Prückler). http://dx.doi.org/10.1016/j.fm.2015.02.014 0740-0020/© 2015 Elsevier Ltd. All rights reserved.

In the course of milling, wheat is separated into flour, the germ and bran. The bran fraction contains the outer layers (aleurone and pericarp) of the wheat kernel and on average accounts for 15% of the grain mass (Hemery et al., 2007). Bran is regarded as an unavoidable by-product of the milling industry with little commercial value and so far found its main use as a supplement for animal feed. This is unfortunate because wheat bran has a high content of valuable secondary plant metabolites and is an excellent source of dietary fibre (Brouns et al., 2012; Prückler et al., 2014). Dietary fibre lowers the glycaemic index (Jensen et al., 2006), which reduces the risk of developing type 2 diabetes (Vogel et al., 2012) and also

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improves the insulin/glucose metabolism in type 2 diabetes patients (Brennan, 2005). Currently, the average intake of dietary fibre in the EU and the USA is below the daily intake recommended for adults (EFSA Panel on Dietetic Products Nutrition and Allergies (NDA), 2010; Trumbo et al., 2005). Reintegration of wheat bran into the food chain is currently much debated as a strategy to motivate consumers to increase their regular uptake of dietary fibre. Especially the baked goods sector offers ample opportunities to benefit from the health beneficial qualities of bran in food commodities. As such, addition of wheat bran is not only regarded as a means to improve public health but also meets the economic interests of the milling industry to increase the value of the bran fraction. However, poor baking performance and most severely, several unpleasant sensory properties are the major drawbacks in the production of bran containing food commodities (Challacombe et al., 2012; Savolainen et al., 2014). While the strawy or sandy mouth feeling caused by bran can readily be reduced by particle size reduction, the major challenge for the incorporation of wheat bran in food products is its unpleasant bitter taste accompanied by a long lasting bitter/astringent aftertaste. This circumstance finally leads the majority of consumers to reject whole meal products or products containing higher amounts of bran. These undesirable sensory properties are caused by a complex array of constituents and the exact chemical nature of this phenomenon has not been completely resolved to date. Plant secondary metabolites such as phenolic compounds and alkylresorcinols have been made responsible for bitterness and € et al., 2008). However, aftertaste of cereals (Bin et al., 2012; Heinio despite their adverse sensory effects, these metabolites are also of nutritional value due to several potential health benefits (Parikka et al., 2006). These include their antioxidant activities (Kozubek and Nienartowicz, 1995) and a possible influence on lowering the glycaemic index (Poutanen et al., 2009). Several processes on the market promise a debittering of wheat bran. The debittering is done by acidification and oxidation of bitter constituents with ozone (Monsalve-Gonzalez and Prakash, 2011) or a combination of hydrothermal treatment and fine grinding (Farigel process, Westhove, Limagrain). Extrusion, a process combining hydrothermal treatment, high shear force and drying has been reported to improve the taste of wheat pasta containing bran jtowicz and Moscicki, 2011). However, such processes are (Wo economically challenging due to the technological requirements and high energy demands. Furthermore, they possibly reduce the activity of health beneficial plant metabolites. A relatively simple, though effective, method to improve dough rheology and to modify bread taste is sourdough fermentation. Traditional Type I sourdoughs stem from spontaneous fermentations and contain complex cultures of symbiotic yeasts and lactic acid bacteria (LAB) that are continuously propagated by backslopping. Most of the LAB strains isolated from sourdough belong to the genus Lactobacillus. Lactobacillus sanfranciscensis, Lactobacillus brevis and Lactobacillus plantarum are among the key species €nzle et al., 2007). Isolated LAB of the sourdough microflora (Ga species are also crucial for industrial Type II and Type III sourdough technologies (Corsetti and Settanni, 2007). Increased CO2 retention of the dough is among the technological benefits of sourdough fermentations is thereby contributing to improved loaf volume, firmness and shelf life. Reduction of lipase activity retards fat hydrolysis and thus reduces rancid flavour (Gobbetti et al., 2014). Furthermore, lactic acid fermentation offers several health related benefits through the reduction of antinutritive factors such as phytic acid and improved bioavailability of antioxidants (Gobbetti et al., 2005). Reduced starch digestion through lactic acid and a prolonged gastric emptying rate through

acetic acid were reported to reduce the glycaemic index (Novotni et al., 2011; Poutanen et al., 2009). Fermentation of wheat bran with L. brevis was reported to improve the technological and sensory quality of the resulting breads (Katina, Salmenkallio-Marttila et al., 2006; SalmenkallioMarttila et al., 2001). It was further shown that acidification partially masks or reduces the unpleasant bran flavour. However, a detailed study comparing the effect of metabolically distinct LAB species on wheat bran has, to our awareness, not been performed. Therefore, the aim of this study was to investigate the effect of different LAB (i.e., Lactobacillus) species on the complex matrix of wheat bran and to determine which species may be of interest for industrial bran fermentations. This paper reports the effect of the LAB metabolism on carbohydrate, organic acid and phenol/alkyresorcinol profiles of wheat bran. These investigations were accompanied by a sensory evaluation as to whether a correlation of chemical changes with sensory characteristics can be made. 2. Materials and methods 2.1. Raw materials Fine wheat bran (food grade, from mechanically cleaned and peeled wheat) and wheat flour (W700/Type 550) (basic analytics see Table 1) were provided by GoodMills Group (Schwechat, Austria). The fine bran fraction was taken from the 500 mm sieve deck with 80% of the particles smaller than 250 mm. The ingredients for dough preparation, with exception for yeast (saf instant, S.I. Lesaffre, France), were obtained from a local supermarket (sunflower oil, Osana, Austria; fine sugar, Agrana, Austria; fine salt, iodized, Salinen Austria, Austria). 2.2. Fermentation of wheat bran All Lactobacillus strains used for fermentation (Table 2) were obtained from Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ, Braunschweig, Germany). Fructobacillus fructosus FF14-1 strain isolated from a flower was also included because of its specific metabolic characteristics (Endo and Salminen, 2013). Pre-cultures of each strain were prepared in De Man, Rogosa & Sharpe (MRS, DSMZ Medium 11) broth with 1% (w/v) glucose and incubated overnight at the optimal growth temperatures (30/ 37  C). For propagation of F. fructosus, fructose (1% w/v) was added in addition to glucose. The overnight cultures were suspended in sterile NaCl (0.9% w/v). Per kg of dry bran (91.6% dry matter), 37 mL of cell suspensions with OD600 ¼ 1 were added. This resembles a 2% (v/w) inoculum, taking into account that the water content was finally adjusted to 50%. The mixtures were homogenised for 10 min and compressed in plastic barrels. The barrels were filled to the top, closed and incubated for 18 h at 30  C. All fermentations were

Table 1 Basic analytics of food grade wheat bran and wheat flour. All values are means of triplicate determination and refer to dry matter. Material

Food grade bran fine

W700/high gluten flour

Dry matter [%] Ash [%] Crude fat [%] Crude protein [%] Starch [%] b-Glucan [%] Soluble dietary fibre [%] Insoluble dietary fibre [%]

91.6 5.8 5.7 15.5 19.3 2.7 3.4 40.5

87.3 0.8 1.3 13.9 65.1 0.3 1.1 3.4

M. Prückler et al. / Food Microbiology 49 (2015) 211e219 Table 2 Strains of lactic acid bacteria used in this study. Name

Strain designation

Lactobacillus acidophilusa Lactobacillus delbrueckii subsp. delbrueckiia Lactobacillus pentosusb Lactobacillus plantarum subsp. plantarumb Lactobacillus sanfranciscensisc Lactobacillus brevis subsp. brevisc Fructobacillus fructosusc, d

DSM 20079T DSM 20074T DSM 20314T DSM 20174T DSM 20451T DSM 20054T FF14-1

a b c d

Obligatorily homofermentative. Facultatively heterofermentative. Obligatorily heterofermentative. Endo and Salminen (2013).

performed in triplicate. After incubation, the content of each barrel was homogenised by mixing for a minute. Total titratable acid, dry matter and pH were measured immediately after fermentation. Samples for microbiological examination were taken at this time point too. Samples for further analysis and baking were stored at 30  C. 2.3. Determination of dry matter, total titratable acid & phytatephosphorus content Dry matter content was determined by drying the samples at 103  C (16 h) according to AACC 44-15A. For TTA determination, 10 g of sample were suspended in 100 mL of HQ water and titrated with 0.1 M NaOH until a pH of 8.5 was stable for 5 min, according to AACC 02-31. Phytate-phosphorus was determined according to AOAC method 986.11 with modifications according to Latta and Eskin (1980). 2.4. Determination of microbiological status The selective isolation and enumeration of Enterobacteriaceae was done on Violet Red Bile Dextrose (VRBD) agar for 24 h at 30  C. For the enumeration of yeasts and moulds Yeast Glucose Chloramphenicol (YGC) agar was used with an incubation time of 4 days at 25  C. Plate counts of LAB were done on de Man, Rogosa and Sharpe (MRS) agar and MRS agar including 50 mg/L vancomycin and enumerated after 3 days at 37  C under anaerobic conditions. All sample dilutions were prepared in duplicate. 2.5. Determination of sugars, organic acids and ethanol One gram samples of fermented or native wheat bran were placed in 15 mL tubes and extracted by continuous shaking for 10 min at room temperature with 5 mL of water. The samples were heated at 80  C in a water bath to inactivate microorganisms and to precipitate protein and then centrifuged at 4000 rpm for 10 min. A 1.5 mL aliquot of supernatant was centrifuged again at 20,800 g for 3 min to obtain a clear sample. Lactic, acetic and propionic acid as well as ethanol were determined by HPLC following a protocol by Lefebvre et al. (2002). The method employed a Dionex HPLC System (P600 pump, ASI1000 Autosampler, TCC100 column oven) and a Bio-Rad Aminex HPX87H column, 300  7.8 mm, under the following conditions: mobile phase 0.005 N H2SO4, isocratic, flow rate 0.5 mL/min, run time 80 min, column temperature 60  C. 20 mL of the sample were injected. Detection was performed by UVD170 at 210 nm and a RI101 refractive index detector. The identity of the substances was confirmed by retention times of pure standards and mixes. Maltose, glucose, fructose, mannitol, arabinose and xylose were quantified by a method modified after Cotte et al. (2003) with the

213

same HPLC system as described above employing a Dionex CarboPac PA-100 column, 250  4 mm, under the following conditions: mobile phase A: 100 mM NaOH, mobile phase B: 0.5 M sodium acetate in 100 mM NaOH, gradient: 0 min, 0% B, increasing over 20 min to 23% B, then 5 min 100% B and again 10 min 0% B. 20 mL of the sample were injected. Detection was performed by electrochemical detector ED40 with gold electrode. The working electrode was maintained at the following potentials and durations during the operation: E1 ¼ 0.1 V (t1 ¼ 0.40 s), E2 ¼ 2.0 V (t2 ¼ 0.03 s), E3 ¼ 0.6 V (t3 ¼ 0.01 s), E4 ¼ 0.1 V (t1 ¼ 0.06 s). The identity of the substances was confirmed by retention times of pure standards. Assay kits from Megazyme International (Wicklow, Ireland) were used to determine the contents of sucrose (K-SUFRG), fructan (K-FRUC), starch (K-TSTA), b-glucan (K-BGLU) and total dietary fibre (K-TDFR). 2.6. Determination of phenolic acid and flavanoid content Extraction of phenolic compounds was conducted as described by Mattila et al. (2005) with the following changes: a sample containing 0.5 g DM was extracted twice with 12 mL 80% methanol (Roth, HPLC grade) to obtain the free (extractable) phenolic acid fraction. The extract was clarified by centrifugation, the final volume measured, filtrated and used to determinate free phenolic acid and flavonoid contents. The residues of the above extraction were first mixed with HQwater (3 mL), then 5 M NaOH (5 mL) followed by 10 M NaOH (5 mL) and incubated for 16 h with agitation. After adjusting the pH to 2.0 with 8 M HCl, the samples were extracted 3 times with 12 mL of pure ethyl acetate. The organic layers were combined, evaporated to dryness and the residues dissolved in 4 mL 50% MeOH for measurement of the bound phenolic acids and flavonoid contents. Phenolic acids and flavonoids were quantified using a RP-HPLC procedure employing a Phenomenex Kinetex C18 column, 100 A, 150 mm  3 mm, 2.6 mm (Phenomenex, Aschaffenburg, Germany). Gradient elution was conducted with 0.05% trifluoroacetic acid in water (A) and 0.05% trifluoroacetic acid in acetonitrile (B). The solvent gradient was delivered using two LC-10ADVP pumps (Shimadzu, Korneuburg, Austria) and was programmed as follows: at 0 min, 5% B; increasing from 1 to 8 min to 10% B; from 8 to 16 min to 20% B; from 16 to 34 min to 44% B; 38e39 min to 80%; decreasing thereafter to 5% B within the next 3 min. The flow rate was 0.45 mL/ min. Phenolic acids and flavanoids were detected at 280 nm using a SPD-M10AVP photodiodearray detector (Shimadzu, Korneuburg, Austria). 20 mL injections were made in each run, and peak areas were used for all calculations. Phenolic acids and flavonoids were identified by UV-spectra and interpreted from pure phenolic acid & flavonoid standard curves. 2.7. Determination of alkylresorcinol content The alkylresorcinol content was quantified by a method after Kulawinek et al. (2008) One gram samples of fermented wheat bran were placed in 50 mL tubes and extracted by continuous shaking for 48 h at room temperature with 30 mL of acetone. The samples were centrifuged at 3200 g for 10 min, and the separated supernatant was evaporated to dryness in a rotavapor. The dry residues were then redissolved in 2 mL of ethyl acetate. Alkylresorcinols were identified and quantified by HPLC using the same instrument as for phenolic acids and flavonoids. The method employed a 150 mm  3.0 mm, 2.6 mm, Phenomenex Kinetex, RP-C18 column with water (A) and methanol (B) as the mobile phase, a flow rate of 0.3 mL/min, and UV detection at 280 nm. 20 mL of each ethyl acetate sample was injected into the

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chromatographic column tempered at room temperature. The mobile phase gradient was as follows: 90e92% B in 3 min, 92e95% B in 5 min, 95e97% in 40 min, hold at 97% B for 7 min, and finally 97 - 90% B in 5 min. The identities and areas were extrapolated from pure standards, 5-heptadecylresorcinol (C17:0), 5nonadecylresorcinol (C19:0), 5-heneicosylresorcinol (C21:0) and 5-tricosylresorcinol (C23:0), procured from Fluka Analytics. 2.8. Preparation of breads and measurement of loaf volume Breads were prepared such to give final bran content of 20% (dry matter based). Doughs were produced with 400 g of the flour (87% DM), 183 g of wet, fermented bran (50% DM), 9 g of dry yeast (saf instant, S.I. Lesaffre, France), 10 g of sodium chloride, 2 g of sugar, 7 g of sunflower oil and 270 g of 30  C tap water (corrected for moisture in flour and bran). The required volume of water was determined prior to baking by Farinograph measurement (Brabender, Duisburg, Germany) according to ICC 115/1. The dry ingredients were mixed for 1 min, then oil & water were added and mixed for 6 min more. The dough was left in a fermenting chamber (30  C, 85% relative humidity) for 30 min. Then it was divided into two pieces of 350 g each. These pieces were rolled out, moulded and placed in greased pans, which were again placed in the fermenting chamber (30  C, 85% relative humidity) for 60 min. Subsequently, the fermented dough pieces were baked at 220  C for 30 min with full humidification in an oven (Manz Backtechnik GmbH, Münster, Germany). After reaching room temperature, the loaf volume was determined by the rape seed displacement method according to AACC 10-05. The breads were stored at 20  C and 50% humidity until sensory evaluation was performed. 2.9. Sensory evaluation Intensities of the following three sensory attributes were rated by a trained panel using unstructured line scales: “bitterness”, “aftertaste” and “acidity”. The panel consisted of twenty-one trained assessors. The scales for aftertaste and bitter taste were anchored at each end with example breads. For the left end of the scale bread without bran was used (low intensity) and for the right end bread with 20% of untreated bran was used (high intensity of the attribute). The anchors of the acidity scale were described verbally as “low” and “high acidity”. The bread samples were presented to the assessors as 2 cm slices in a coded and randomised way. Each assessor was provided with the five bread samples and the two example breads and requested to cleanse the palate with the provided water after each sample. Scores were recorded computerised by Compusense five 5.2 (CSA, Computerized Sensory Analysis System, Compusense Inc., Canada) and statistical analysis was done by Statgraphics Centurion XVI (version 16.0.09, StatPoint Technologies, Inc.). 2.10. Statistical methods All statistical tests (including multiple regression, partial correlation and principal components analysis) were done using Statgraphics Centurion XVI (version 16.0.09, StatPoint Technologies, Inc.). A 0.05 alpha level was used as a significance level for all statistical tests. The following tests were done to all data: ANOVA, multiple comparisons of means and the Kruskal-Wallistest to check the validity of data. All means and standard deviations arise from 3 batches, with the exception of the unfermented native bran. Here the data arises from 3 measurements of one lot.

3. Results & discussion Members of homofermentative (Lactobacillus acidophilus, Lactobacillus delbrueckii) as well as facultatively (L. plantarum L. pentosus) and obligately (L. sanfranciscensis, L. brevis) heterofermentative Lactobacillus species were evaluated, as all of these species were reported to be associated with sourdough fermentation (Corsetti and Settanni, 2007). Instead of using industrial baking strains, we chose reference (Type) strains (Table 2) for this study. F. fructosus (Endo and Okada, 2008) was included as a rather “exotic” candidate. The species depends on fructose for optimal growth, and the main interest for its inclusion was to investigate its carbohydrate metabolism on wheat bran. 3.1. Acidity & microbiological status Judged from TTA and organic acid contents (Table 3), the highest acidification was obtained with L. sanfranciscensis, followed by L. plantarum and Lactobacillus pentosus. L. acidophilus and L. delbrueckii possess anaerobic to microaerophilic properties and have optimal growth temperatures of 37  C. Both strains failed to develop on wheat bran under the applied conditions, the resulting acidity profiles were comparable to those of controls 1 and 2. Due to the requirement of an external electron acceptor for glucose fermentation and the reported inability to ferment maltose (Endo and Okada, 2008; Endo et al., 2014), F. fructosus was not expected to show pronounced growth on wheat bran. However, acid production by F. fructosus was comparable to that of L. brevis. The viable LAB cell counts on MRS agar (þ/ vancomycin) were comparable in all samples. The high counts of controls 1 and 2 and for L. acidophilus/L. delbrueckii may result from the occurrence of autochthonous LAB in the bran. The viable cell counts of yeasts/moulds and Enterobacteriaceae (Table 3) indicate that fermentation with L. sanfranciscensis, L. plantarum and L. pentosus or acidification with lactic acid (control 3), completely inhibited growth/viability of the autochthonous microflora, L. brevis and F. fructosus could reduce the cell counts to levels below that of untreated bran. 3.2. Carbon sources and fermentation profiles 3.2.1. Carbohydrate analysis Carbohydrate analyses (Table 3) indicated a starch decrease in the range of 50 mg/g bran in all fermented samples compared to the co-incubated negative controls, the concentrations of maltose increased significantly during incubation. A rather unexpected finding was a clear increase of fructose in all samples during incubation. Part of fructose (and therefore, also of glucose) appears to originate from sucrose hydrolysis. A further possible source of fructose is hydrolysis of fructan. Karppinen et al. (2000) reported a fructan content of 44 mg/g in wheat bran. Our results show a similar level of 50 mg/g in untreated, dry bran (Table 3), which was reduced to 9e10 mg/g in all three controls. Higher fructan degradation appeared to occur in most of the LAB inoculated samples, with the lowest fructan concentrations found in bran fermented with L. sanfranciscensis and F. fructosus. Some lactobacilli have been zquez-Martínez et al., 2014). reported to metabolise fructan (Vela Since degradation of sucrose and fructan also occurred in all three controls, hydrolysis by wheat endogenous enzymes and/or acidic hydrolysis seems to be likely. Smaller amounts of the pentose sugars xylose and arabinose were also released during incubation, probably arising from partial arabinoxylan degradation. With few exceptions, the changes in maltose, glucose, fructose and pentose sugars (arabinose, xylose) during fermentation generally match the

50.6 ± 0.2c 2.9  103 1.7  106 1.4  106 2.0  103 5.5  104 5.3 53 ± 1e 12.3 ± 0.3a 6.2 ± 0.2a 2.3 ± 0.1d 154 ± 6c,d 11.4 ± 0.4e,f 2.4 ± 1.0d 32.3 ± 1.1d 1.1 ± 0.3a,b 0.9 ± 0.0a 2.8 ± 0.1f 2.0 ± 0.1d,e 6.2 ± 1.3c n.d. 51.0 ± 0.5 1.3  104 1.7  106 1.7  106 1.0  103 1.2  104 5.5 43 ± 4d 16.7 ± 1.6b 7.4 ± 0.8b 2.4 ± 0.1d 138 ± 4a 11.5 ± 3.1e,f, 1.8 ± 1.4c,d 41.3 ± 2.8f 3.0 ± 2.0a,b 13.8 ± 2.9c 0.7 ± 0.0b 2.4 ± 0.4f 3.9 ± 0.3a 1.2 ± 0.4a 51.4 ± 0.1 1.1  104 1.5  106 1.3  106 n.d. n.d. 4.3 83 ± 1i 37.6 ± 1.0c 8.2 ± 0.7c 1.5 ± 0.1b 149 ± 2b,c,d n.d. 0.4 ± 0.8a,b 16.7 ± 0.4b 0.4 ± 0.9a 2.7 ± 0.6b n.d. n.d. 4.7 ± 0.3b 10.2 ± 0.8b 50.8 ± 0.4 8.9  103 2.4  106 2.3  106 n.d. n.d. 4.3 75 ± 3h 42.2 ± 1.3d n.d. 2.8 ± 0.0e 144 ± 1a,b,c 2.2 ± 0.2b 0.1 ± 0.1a 22.9 ± 0.9c 1.2 ± 1.8a,b 31.4 ± 0.4g 3.7 ± 0.1h 1.9 ± 0.1d,e n.d. n.d.

Controls: moisture of bran adjusted to 50% with sterile 0.9% NaCl (Control 1), deionized water (Control 2) or sterile 0.9% NaCl and 48 mg/g lactic acid (Control 3). a

52.0 ± 0.1 5.2  103 2.1  106 2.1  106 n.d. n.d. 4.2 67 ± 2g 41.3 ± 1.1d n.d. 1.8 ± 0.0c 140 ± 0a,b 4.7 ± 0.1c 1.6 ± 1.0b,c,d 24.5 ± 0.8c 4.9 ± 0.4b,c 16.9 ± 0.4d 2.9 ± 0.1g 1.6 ± 0.1c n.d. n.d. 51.9 ± 0.2 1.2  103 2.0  103 1.8  103 2.0  103 2.4  107 6.3 25 ± 0b n.d. n.d. 2.3 ± 0.1d 168 ± 2e 12.2 ± 0.4f 0.9 ± 0.7a,b,c 40.8 ± 0.7f 11.4 ± 1.0d 28.3 ± 0.2f 3.7 ± 0.0h 2.1 ± 0.1e n.d. n.d. 50.6 ± 0.1 2.7  103 6.8  106 6.2  106 1.0  105 2.3  107 6.3 27 ± 0b n.d. n.d. 2.4 ± 0.2d 159 ± 9d,e 15.9 ± 0.7g 0.7 ± 0.3a,b,c 41.9 ± 0.5f 8.3 ± 2.3c,d 28.0 ± 0.3f 2.7 ± 0.1f 1.9 ± 0.1d n.d. n.d. 49.8 ± 0,0 n.d. n.d. n.d. n.d. n.d. 4.1 63 ± 1f 48 ± 0f n.d. 1.6 ± 0c,d 186 ± 6f 1.1 ± 0a,b n.d. 37.9 ± 0.2e 10.1 ± 2.1d 30.0 ± 0.1g 1.3 ± 0c n.d. n.d. n.d. 46.8 ± 0.0 n.d. 2.4  105 2.4  105 2.5  106 1.1  109 6.4 27 ± 0b n.d. n.d. 2.4 ± 0.1d 206 ± 5g 8.9 ± 0.1d 1.6 ± 0.6b,c,d 31.7 ± 0.3d 8.7 ± 0.8d 21.8 ± 0.1e 2.2 ± 0.0e 1.4 ± 0.1b,c n.d. n.d. 47.1 ± 0.0 n.d. 2.0  103 2.0  103 7.4  105 4.7  108 6.3 30 ± 1c n.d. n.d. 2.4 ± 0.3c 204 ± 4g 10.0 ± 0.1d,e 1.1 ± 0.7a,b,c,d 31.1 ± 0.4d 8.8 ± 2.2d 21.8 ± 0.3e 2.0 ± 0.0d 1.4 ± 0.1b n.d. n.d. 91.6 ± 0.1 n.d. n.d. n.d. 4.0  104 5.9  105 6.4 14 ± 0a n.d. n.d. n.d. 193 ± 5f 6.4 ± 0.7c 14.9 ± 0.3e 2.5 ± 0.2a 49.9 ± 5.5e 1.6 ± 0.1a,b n.d. n.d. n.d. n.d. DM [%] Initial density [cfu/g] Final density (MRS) [cfu/g] Final density (MRS þ vancomycin) [cfu/g] Yeasts & moulds [cfu/g] Total Enterobactericeae [cfu/g] pH TTA [ml 0.1 M NaOH/10 g] Lactic acid [mg/g] Acetic acid [mg/g] Propionic acid [mg/g] Raw starch [mg/g] Maltose [mg/g] Sucrose [mg/g] Glucose [mg/g] Fructan [mg/g] Fructose [mg/g] Arabinose [mg/g] Xylose [mg/g] Mannitol [mg/g] Ethanol [mg/g]

L. brevis

c

F. fructosus

3.2.2. L. plantarum and L. pentosus L. plantarum and L. pentosus are classified as facultatively homofermentative and are considered taxonomically very close (Stiles and Holzapfel, 1997). Both species generated purely homofermentative fermentation profiles as judged from high amounts of lactic acid, the absence of acetic acid and no evidence for pentose fermentation (Table 3). In further experiments with MRS broth (Supplemental Table S1), L. pentosus and especially L. plantarum showed low disposition to metabolise xylose, even when xylose was the only carbon source available.

e

L. sanfranciscensis L. pentosus

215

expected fermentation profiles of the individual LAB species (see detailed discussion below).

c d

L. plantarum L. delbrueckii

d,e c

L. acidophilus Control 3a

b a

Control 2a Control 1a

a f

Dry, milled bran

Table 3 Microbiological status, acidity and carbohydrate content of fermented wheat bran. All values are dry matter based and represent the means of triplicate determination ± SD, except for microbial counts which were determined in duplicate. Values labelled with the same superscript letter are not different at p < 0.05 in Fishers LSD multiple comparisons of means.

M. Prückler et al. / Food Microbiology 49 (2015) 211e219

3.2.3. L. sanfranciscensis and L. brevis L. sanfranciscensis prefers maltose over glucose as carbon source €nzle et al., 2007) and completely consumed the available (Ga maltose during bran fermentation. However, L. sanfranciscensis also displayed the highest glucose consumption of all strains. The data shown in Supplemental Table S1 indicate that the presence of fructose strongly increases the efficiency of glucose fermentation by L. sanfranciscensis. This suggests that an external electron acceptor is needed for glucose, but not for maltose metabolism of L. sanfranciscensis. Although it was reported that L. sanfranciscensis €nzle et al., 2007), our results is unable to ferment pentoses (Ga indicate complete consumption of available xylose and arabinose (Table 3 and S1). L. brevis seemingly only metabolised arabinose and fructose, and no reduction of maltose and glucose was evident. It is further possible that L. brevis is able to metabolise xylooligosaccharides and low molecular weight arabinoxylooligosaccharides as its genome contains several genes encoding functional b-D-xylosidases and a-L-arabinosidases (Michlmayr et al., 2013). The ratio of acetic acid/lactic acid also indicates a higher degree of pentose fermentation by L. brevis compared to L. sanfrancsiscensis: the molar ratio acetic acid/lactic acid (fermentation quotient) was 0.66 for L. brevis and 0.33 for L. sanfranciscensis. Obligately heterofermentative LAB species such as L. sanfranciscensis and L. brevis normally process sugars exclusively through the PKP (Endo and Dicks, 2014). A key characteristic of the PKP is that hexoses are fermented to lactic acid and ethanol, while pentose fermentation results in the equimolar production of lactic acid and acetic acid (Fig. 1). High ethanol production was detected in bran fermented with L. sanfranciscensis, lower amounts of ethanol were produced by L. brevis, which is a further indication of a higher rate of hexose fermentation by L. sanfranciscensis. 3.2.4. Fructose metabolism and F. fructosus Of particular interest is to clarify a possible influence of fructose on the observed fermentation profiles. Addition of fructose to sourdough fermented with L. sanfranciscensis was reported to increase the relative proportion of acetic acid (Gobbetti and Corsetti, 1997). While fructose is exclusively metabolised to lactic acid in the homofermentative pathway, the metabolism of fructose is more complex in obligately heterofermentative species. Fructose can be metabolised through the PKP, but it can also act as an external electron acceptor. The metabolism of hexoses (i.e., of glucose) through the PKP requires the additional reduction of two NADþ (Fig. 1). In order to regenerate these redox-equivalents, acetylphosphate is reduced to ethanol instead of being converted to acetate and ATP through the acetate kinase reaction. Alternatively, fructose can act as an external electron acceptor to regenerate NADþ and is thereby reduced to mannitol, resulting in the gain of an additional ATP and a shift towards acetate production. This is consistent with the observation that on bran, formation of mannitol and reduction of fructose co-occurred and were highest with the

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Fig. 1. Schematic of the key steps of hexose and pentose fermentation in the phosphoketolase pathway of lactic acid bacteria. Adapted from Gobbetti and Corsetti (1997) and Fuente-Hernandez et al. (2013). Phosphoketolase [1], acetaldehyde dehydrogenase [2], alcohol dehydrogenase [3], acetate kinase [4], mannitol dehydrogenase [5].

heterofermentative species L. sanfranciscensis, L. brevis and especially, F. fructosus (Table 3). A crucial question is therefore whether the release of fructose during fermentation of bran had a significant influence on fermentation profiles regarding a shift to acetic acid and to higher acid production in general. Stoichiometrically, the reduction of 2 mol of fructose to mannitol yields one additional mole of acetate. Considering the amounts of mannitol detected in fermented bran, a maximum of 10% of the total acetic acid formed by L. sanfranciscensis and L. brevis could be attributed to fructose reduction, implying a minor effect on the obtained fermentation profiles. In vitro assays with MRS media (Supplemental Table S1) were conducted to partially justify these observation/considerations. These results confirm that L. sanfranciscensis, L. brevis and F. fructosus metabolise fructose partially through the heterofermentative pathway and partially reduce it to mannitol. Since L. sanfranciscensis seems to require fructose for glucose fermentation, fructose may have contributed to the high acidification capacity of this strain on bran. F. fructosus is obligately heterofermentative and was previously reclassified from Leuconostoc fructosus (Endo and Okada, 2008). The organism is unable to ferment maltose and xylose, and due to a deficiency in alcohol/acetaldehyde dehydrogenase gene (Endo et al., 2014) it is unable to regenerate additional redoxequivalents occurring during glucose fermentation unless alternative electron acceptors such as fructose are present (Fig. 1). Therefore F. fructosus is unable to produce ethanol, and glucose can only efficiently be co-fermented with fructose or other external electron acceptor. The data shown in Supplemental Table S1 confirm the inability to produce ethanol, to metabolise xylose and maltose and further poor glucose fermentation in the absence of fructose.

48% to 85% compared to untreated bran (100%). The highest phytate reduction occurred in the controls and it appears that LAB fermentation had little or no effect on phytate degradation. This contradicts previous reports on phytase activity of several LAB species, including L. sanfranciscensis (De Angelis et al., 2003), L. brevis and L. plantarum (Fischer et al., 2014). It can be assumed that the observed phytate reduction was caused by different phytases of plant and microbial origin that are influenced by acidity. However, reviewing previous literature on the subject, it seems that the contribution of wheat endogenous phytases can be assumed to be minor (Lopez et al., 2001; Brejnholt et al., 2011). Similar to the results reported by Katina et al. (2007) and Liukkonen et al. (2003), fermentation only slightly changed the alkylresorcinol contents (Table 4). The contents of several extractable phenolic acids (gallic, ferulic, vanillic, caffeic acids, Table 5) increased during bran incubation. Analogous to the observations made with phytate and alkylresorcinols as discussed above, the effect seems not to be caused by LAB fermentation. An interesting exception was the low concentration of ferulic acid in bran fermented with L. plantarum and L. pentosus. Rodríguez et al. (2008) reported that L. plantarum is able to degrade hydroxycinnamic acids such as p-coumaric, m-coumaric, caffeic, and ferulic acid, the latter being decarboxylated to 4vinyl guaiacol. Since L. plantarum and L. pentosus are taxonomically close, a similar metabolism of hydroxycinnamic acids could be expected as well. No significant changes were detected in the concentrations of insoluble phenolic acids (Supplemental Table S2). Except for the reduction rutin, the changes recorded for soluble and insoluble flavonoids were minor as well (Supplemental Table S3). 3.4. Loaf volume & sensory evaluation

3.3. Phytate phosphorus, alkylresorcinols, phenolic acids & flavonoids The phytate content (Table 4), (determined as phytate-bound phosphorus), decreased in all samples to values ranging from

Breads prepared with untreated, milled bran had a specific loaf volume of 2.17 ± 0.03 g/cm3. The specific loaf volumes of breads produced with fermented bran including the controls were significantly different to that of untreated bran and ranged from 2.34 to

M. Prückler et al. / Food Microbiology 49 (2015) 211e219

217

Table 4 Phytate and alkylresorcinol contents in fermented wheat bran. All values are dry matter (DM) based and represent the means of triplicate determination ± SD. Values labelled with the same superscript letter are not different at p < 0.05 in Fishers LSD multiple comparisons of means. Phytate [mg/g]

Alkylresorcinols C17:0 [mg/g]

Untreated, dry bran Control 1a Control 2a L. acidophilus L. delbrueckii L. plantarum L. pentosus L. sanfranciscensis L. brevis F. fructosus a

10.4 5.4 5.0 6.7 5.0 6.2 8.9 6.2 6.9 5.7

± ± ± ± ± ± ± ± ± ±

e

0.18 0.14 0.16 0.22 0.21 0.23 0.21 0.22 0.30 0.22

0.1 0.4a,b 0.1a,b 0.4c 0.1a 0.3b,c 1.6d 0.4b,c 0.0c 0.9a,b,c

± ± ± ± ± ± ± ± ± ±

C19:0 [mg/g]

b

0.01 0.01a 0.01a,b 0.02c 0.00c 0.00c 0.00c 0.02c 0.03d 0.02c

0.84 0.44 0.50 0.77 0.79 0.85 0.70 0.99 0.76 1.06

± ± ± ± ± ± ± ± ± ±

b,c

0.05 0.04a 0.05a 0.02b 0.03b 0.01b,c 0.02b 0.24c,d 0.11b 0.14d

C21:0 [mg/g] 0.95 0.81 0.83 0.82 0.73 0.83 0.79 0.89 0.79 0.84

± ± ± ± ± ± ± ± ± ±

C23:0 [mg/g]

c

0.06 0.03a,b 0.07a,b 0.04a,b 0.01a 0.01a,b 0.02a,b 0.10b,c 0.09a,b 0.07b

0.26 0.16 0.19 0.22 0.19 0.22 0.22 0.19 0.44 0.16

± ± ± ± ± ± ± ± ± ±

Total [mg/g]

c

0.02 0.01a 0.00a,b 0.04b,c 0.03a,b 0.00b,c 0.00b,c 0.02a,b 0.05d 0.01a

2.2 1.6 1.7 2.0 1.9 2.1 1.9 2.3 2.3 2.3

± ± ± ± ± ± ± ± ± ±

0.1d 0.1a 0.0a,b 0.1c,d 0.1b,c 0.0c,d 0.0b,c 0.4d 0.1d 0.2d

Controls: moisture of bran adjusted to 50% with sterile 0.9% NaCl (Control 1) or deionized water (Control 2).

Table 5 Extractable phenolic acids in fermented wheat bran. All values are dry matter (DM) based and represent the means of triplicate determination ± SD. Values labelled with the same superscript letter are not different at p < 0.05 in Fishers LSD multiple comparisons of means. Gallic acid [mg/100 g] Untreated, dry bran Control 1a Control 2a L. acidophilus L. delbrueckii L. plantarum L. pentosus L. sanfranciscensis L. brevis F. fructosus a

10 ± 0a 45 39 42 54 43 40 24 27 39

± ± ± ± ± ± ± ± ±

1e 1c 6c,d,e 1f 1d,e 2c,d 3b 1b 1c,d

Vanillic acid [mg/100 g] 2 ± 0a 55 54 45 30 43 50 50 41 45

± ± ± ± ± ± ± ± ±

2g 2f,g 2c,d 1b 1c 4d,e 6e,f 3c 1c,d

Caffeic acid [mg/100 g]

Syringic acid [mg/100 g]

p-coumaric acid [mg/100 g]

n.d.

5 ± 0e

5 ± 0b,c

2 ± 1d,e 1 ± 0c,d,e 2 ± 0e 1 ± 0b,c n.d. 1 ± 0c,d 1 ± 0c,d 1 ± 0c,d 1 ± 0c,d

1 2 4 6 2 3 3 5 3

± ± ± ± ± ± ± ± ±

0a 0a,b 0e 0f 0a,b 0b,c 1d 0e 0c,d

5 5 4 7 5 7 4 4 4

± ± ± ± ± ± ± ± ±

0c 0b,c 0b 0d 1c 1d 1a 1b 0a

Ferulic acid [mg/100 g] 1 ± 0a 32 30 33 33 9 7 27 32 28

± ± ± ± ± ± ± ± ±

2d 2c,d 2d 2d 3b 0b 3c 3d 0c

o-coumaric acid [mg/100 g] 9 ± 0d 8 7 8 8 7 3 7 4 5

± ± ± ± ± ± ± ± ±

1c,d 0b,c 0c,d 1c,d 1c 1a 2b,c 1a 0b

Total soluble phenolic acids [mg/100 g] 24 ± 0a 142 132 132 131 103 109 110 111 121

± ± ± ± ± ± ± ± ±

5e 2d 5d 3d 2b 5b 13b 4b 1c

Controls: moisture of bran adjusted to 50% with sterile 0.9% NaCl (Control 1) or deionised water (Control 2).

2.41 g/cm3. The differences between fermented samples and the controls were not significant. Initial descriptive analysis of bread containing wheat bran was performed in order to identify the most detrimental sensory properties and to find a suited vocabulary for these attributes. At this stage, panels identified 20 attributes from flavour attributes (e.g. acidity, rancidity) to oral haptic characteristics (e.g. mouthfeeling, grittiness etc.) as important. Later, attributes were reduced to the most convincing three: bitterness, aftertaste and acidity,

since our interest was also to assess the influence of lactic acid fermentation on these parameters. Further, a finer milling fraction was used in subsequent procedures to avoid the influence of haptic factors like grittiness etc., which were described as being most unpleasant in early sessions. Breads prepared with bran inoculated with L. acidophilus and L. delbrueckii as well as the control breads were excluded from the final sensory panel due to their high counts in total Enterobacteriaceae. Since data analysis showed no significant differences for individual panellists, all data were included in statistical analysis. The differences in perceived acidity as described by the sensory panel were significant (p < 0.001). L. sanfranciscensis fermented bran bread received the highest score in acidity, followed by L. pentosus, F. fructosus, L. brevis and L. plantarum (Fig. 2 and Supplemental Fig. S1). With the exception of L. plantarum, these results correlate with the determination of pH, TTA and the sum of organic acids (Table 3). This is remarkable, because L. plantarum and L. pentosus produced highly similar acidity profiles (Table 3). Especially L. plantarum is well known for the degradation of bitter constituents in fermented plant food. In this context, it is worthwhile to consider whether the known metabolism of plant secondary metabolites by L. plantarum may have had an impact on perceived acidity. Since sensory analysis is a complex issue and naturally underlies subjective preferences of the panellists, a synergism between bitterness and perceived acidity cannot be excluded.

Fig. 2. Score means and least square differences (2-way ANOVA) of the sensory description of breads containing fermented wheat bran. 1: L. pentosus, 2: L. plantarum, 3: L. brevis, 4: L. sanfranciscensis, 5: F. fructosus. The differences in perceived acidity were significant (p < 0.001), while perceived bitterness and aftertaste were not significantly different (p > 0.05).

3.4.1. Statistical evaluation A multivariate approach including the data of chemical, microbiological and sensory analyses was applied to compare the results of the individual strains. Although bitterness and aftertaste were

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not significant in ANOVA and KolmogoroveSmirnov tests, multiple regressions after Box-Cox-transformation (Supplemental Table S4) revealed some parameters with a possible impact on bitterness. Partial correlation analysis (Supplemental Table S5) further suggests a significant negative correlation of perceived bitterness with the lactic acid content and a significant positive correlation with TTA. In other words, high acidity intensifies perceived bitterness while high lactic acid content seems to lower the bitter impression. Although this appears contradictive, it could explain the fact that samples that did not contain acetic acid (L. plantarum and L. pentosus) received the lowest scores in perceived bitterness/ aftertaste. Katina et al. (2006a) reported that acetic acid increased the intensity of the wheat bran aftertaste, a trend that might also be indicated here: perceived aftertaste significantly correlates with bitterness, suggesting similar perception of these two parameters. This further implies that the persistent aftertaste could negatively influence the sensory results by affecting perceived bitterness. Clearly, a different approach using a larger sensory panel will be required to decide on this question. The correlations between the individual parameters are also visualised in a Bi-plot of principle component analysis (Supplemental Fig. S2). This further illustrates the correlations between perceived acidity and lactic acid content/pH as well as the negative correlation of TTA with microbiological spoilage by Enterobacteriaceae, yeast and moulds. 4. Conclusions Fermentation of wheat bran with lactic acid bacteria is a promising strategy to usefully incorporate wheat bran in bread through a technologically simple pre-treatment procedure. However, particle size reduction appears to be a key requirement to reduce haptic problems such as grittiness that were perceived as highly problematic with unprocessed wheat bran. Although particle size reduction was also reported to be detrimental for baking performance, especially concerning loaf volume and dough stability (Noort et al., 2010), such effects can be partially compensated for by pre-moistening of bran (Lai et al., 1989). Our study implies that pre-moistening positively influences factors like bread loaf volume, phytate and (bitter) alkylresorcinol contents and increases the levels of free phenolic acids. However, moist incubation also increases the risk of microbial spoilage, and a tailored fermentation step is beneficial to repress the development of contaminating microorganisms. While our results provide an interesting view on the carbohydrate metabolism of the included strains, sensory description did not show clear differences in perceived bitterness and bran specific aftertaste. In agreement with previous findings and based on statistical analyses, we hypothesize that acetic acid as produced by obligately heterofermentative species (L. sanfranciscensis, L. brevis) could have an influence on perceived bitterness/aftertaste, and further investigation in this regard is warranted. We conclude that in addition to more traditional sourdough species such as L. sanfranciscensis and L. brevis, also the facultatively heterofementative species L. plantarum and L. pentosus possess potential for industrial wheat bran fermentations and should be considered in further investigations. Acknowledgements The Christian Doppler Forschungsgesellschaft, Austria, Bühler AG, Switzerland and GoodMills Group GmbH, Austria are acknowledged for their financial support. We also appreciate the technical support of A. Bogdan. F. Soares da Silva gratefully acknowledges the financial support of the Erasmus programme.

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