Exopolysaccharides Production during the Fermentation of Soybean

Article pubs.acs.org/JAFC

Exopolysaccharides Production during the Fermentation of Soybean and Fava Bean Flours by Leuconostoc mesenteroides DSM 20343 Yan Xu,* Rossana Coda, Qiao Shi, Paï vi Tuomainen, Kati Katina, and Maija Tenkanen Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, FI-00014 Helsinki, Finland S Supporting Information *

ABSTRACT: Consumption of legumes is highly recommended due to their beneficial properties. Thus, there is a great interest in developing new legume-based products with good texture. In situ produced microbial exopolysaccharides (EPS) are regarded as efficient texture modifiers in the food industry. In this study, soybean and fava bean flours with different levels of added sucrose were fermented by Leuconostoc mesenteroides DSM 20343. After fermentation, a significant increase in viscosity was observed. Sugars, glucans, fructans, mannitol, lactic acid, and acetic acid were quantified to follow the EPS and metabolite production. By treating the fermented doughs selectively with dextranase or levanase, the major role of glucans in viscosity improvement was confirmed. The roles of microbial fructansucrase and endogenous α-galactosidase in degradation of raffinose family oligosaccharides (RFO) were also investigated. This study shows the potential of Ln. mesenteroides DSM 20343 in tailoring viscosity and RFO profiles in soybean and fava bean flours. KEYWORDS: soybean, fava bean, Leuconostoc mesenteroides DSM 20343, exopolysaccharides, glucan, fructan, sucrose, raffinose

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INTRODUCTION Soybean (Glycine max), which originates from East Asia, is regarded as the most important legume for human consumption and animal feed in oriental countries.1 Fava bean (Vicia faba L.) is a legume growing in different climatic areas around the world, and it is a traditional crop for food and animal feed in Europe, Africa, and Asia.2,3 In recent years, interest in legume protein has grown not only because of its valuable nutritional composition but also because of its potential in replacing animal protein in the diet.4 Therefore, many recent studies have explored the use of legumes in different foods, including legume-based snacks, pasta, and gluten-free cakes.5−8 However, antinutritional factors (ANF) and the beany flavor of legumes have limited their application in the food industry.9,10 Raffinose family oligosaccharides (RFO; raffinose, stachyose, verbascose), one of the ANF, are a concern in increased legume consumption since they can cause flatulence and diarrhea.11 It has been reported that the endogenous α-galactosidase in soy and fava bean seeds was able to degrade RFO.12,13 Besides this, several processing methods have been addressed to reduce the content of ANF, such as heating, soaking, dehulling, extrusion, germination, enzyme treatment, and fermentation.14−18 Of these methods, fermentation has been most successful in decreasing ANF contents in different legumes.14,17,19 In particular, lactic acid bacteria (LAB) fermentation has been shown to effectively reduce the content of ANF and to enhance the content of free amino acids and in vitro protein digestibility of legumes.19,20 Furthermore, the effect of LAB fermentation on the overall bioactive potential of legumes has been reported.21 Exopolysaccharides (EPS) are long-chain polysaccharides produced by some microorganisms under certain conditions. They are associated with the cell surface in the form of a capsule or are secreted into the environment in the form of slime.22 According to their chemical composition, EPS are classified into homopolysaccharides, which are composed of only one type of © XXXX American Chemical Society

monosaccharide, or heteropolysaccharides, which contain different types of monosaccharides. Glucans (dextran, mutan, alternan, reuteran) and fructan (levan, inulin) are produced from sucrose by extracellular glucansucrases and fructansucrases, respectively. During the past decade, microbial EPS have been studied extensively for their potential health benefits and various industrial applications.23−25 In the food industry, microbial EPS are regarded as alternatives to plant polysaccharides because of their good performance in stabilization, emulsification, and texture modification.26 EPS can be synthesized by many food grade microbes, including LAB, propionibacteria and bifidobacteria.22 LAB have been of increasing interest, particularly in fermented cereal-based food production.26 Because of the long history of safe use of LAB, in situ produced EPS by LAB may have a great potential to substitute added food hydrocolloids in improving the texture and mouth feel of food products.9,27−29 Leuconostoc mesenteroides subsp. mesenteroides DSM 20343 (also known as ATCC 8293 and NRRL B-1118) has been shown to produce glucans as well as fructans in the presence of sucrose.30,31 These two polysaccharide types have been proven effective in texture modification.26 Both soluble and insoluble glucans can be produced by this strain, and three responsible dextransucrase encoding genes (Dsr) (NCBI accession GI_23024084, GI_53689207, and GI_53689431) have been identified from the genome of Ln. mesenteroides DSM 20343.31 DsrS (dextransucrase, soluble) is responsible for the formation of soluble glucans and DsrI (dextransucrase, insoluble) is responsible for the formation of insoluble glucans. However, the insoluble glucans of Ln. mesenteroides DSM 20343 are products of two or more enzymes. It has been reported that DsrI Received: Revised: Accepted: Published: A

December March 21, March 22, March 22,

7, 2016 2017 2017 2017 DOI: 10.1021/acs.jafc.6b05495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

first dissolved in water and then mixed thoroughly with the flour. The soybean and fava bean doughs were inoculated with Ln. mesenteroides DSM 20343 cell suspensions with an initial cell density of 6−7 log cfu/g and were fermented at 30 °C for 24 h. LAB counting was performed by homogenizing dough samples (10 g) with 90 mL of sterile saline in a Stomacher 400 lab blender (Seward Medical, London, England). Serial dilutions were made, and the diluted bacterial suspensions were plated on MRS agar. After 48 h of incubation at 30 °C, the bacterial colonies were counted. The pH values of samples were determined by a pH meter (model HI 99161, Hanna Instruments, Woonsocket, RI) with a food penetration probe. Viscosity Analysis. Viscosity values of soybean and fava bean doughs at different shear rates were measured directly at 20 °C before and after fermentation. Before measurement, doughs were mixed thoroughly, and 60 g of each was taken. Viscosity measurement was achieved by a RheolabQC rheometer (Anton Paar, Graz, Austria) under different shear rates, from 2 to 100 1/s (up and down sweeps). In order to evaluate the specific viscosity changes of the doughs after fermentation, the viscosity values at the shear rate of 100 1/s were compared. Analysis of Sugars, Mannitol, and Organic Acids. Sugars, mannitol, and organic acids in all samples were analyzed according to the methods reported in our previous paper.36 Degradation of RFO. The influence of endogenous α-galactosidase present in soybean and fava bean flours on the degradation of RFO was evaluated by comparing sugar profiles of doughs made with untreated or autoclaved flours after fermentation. In detail, soybean and fava bean flours were autoclaved and milled thoroughly. Then, they were mixed with only water or with raffinose and water according to Table 1. After this, doughs were fermented by Ln. mesenteroides DSM 20343 under the same conditions as other doughs made with untreated flour. After fermentation, doughs made with autoclaved flours were freeze-dried and treated for sugar analysis using the same HPAEC-PAD method as described earlier.36 As a comparison, two chemically acidified and aseptic doughs were prepared by using untreated soybean or fava bean flour. In total, 40 g of fava bean flour was added to 60 mL of distilled water, and 30 g of soybean flour was added to 70 mL of distilled water. The pH of the mixed doughs were adjusted to 4.5 by using a mixture of lactic and acetic acid in a molar ratio of 4:1. Cycloheximide (Sigma-Aldrich) and chloramphenicol (Sigma-Aldrich) were added to inhibit the growth of microbes at a concentration of 0.01% (w/w). After 24 h of incubation at 30 °C, these two doughs were freeze-dried and treated for sugar analysis using the same method as other doughs. Manninotriose (Carbosynth 176 Ltd., Berkshire, England) was used as one of the sugar standards for peak identification. EPS Analysis. The contents of soluble dextran produced during fermentation by Ln. mesenteroides DSM 20343 were analyzed by an enzyme-assisted method using a mixture of dextranase (Sigma-Aldrich) and α-glucosidase (Megazyme, Ireland) according to the method reported by Katina et al.37 Total glucan contents in fava bean doughs were analyzed according to the method reported earlier.36 Since there is no starch in soybean flour, the glucan contents in soybean doughs were analyzed similarly only without the starch degradation process. For fructan analysis, milder acid hydrolysis was used because 1.0 M sulfuric acid would destroy the released fructose. Freeze-dried samples (100 mg) were washed twice with aqueous ethanol (80%). Then, 1 mL of 0.5 M TFA was added, and samples were kept in 50 °C for 2 h. After this, 250 μL of 1.0 M Na2CO3 was added to stop the reaction. Samples were diluted before HPAEC-PAD analysis. Fructan was quantified by calculating the content of released fructose, and the produced fructan contents were calculated by subtracting the fructan contents in unfermented samples. Fructose was treated under the same conditions and was used as the standard for quantification. Role of EPS in Viscosity Improvement. In order to confirm the role of glucans and fructans in viscosity improvement, fermented soybean and fava bean doughs with 10% sucrose (S10 and F10) were hydrolyzed by dextranase (Sigma-Aldrich) or levanase (Novozymes, Denmark). In detail, fermented doughs were mixed thoroughly, and the

acts via acceptor reactions on soluble glucans formed by DsrS, producing insoluble glucans.32 The potential use of insoluble glucans for gels, films, and fibers30 has resulted in increased research interest in this strain. Additionally, it has been shown that Ln. mesenteroides DSM 20343 produces levan-type fructan,33 and three responsible levansucrase encoding genes (NCBI accession GI_23025176, GI_53689149, and GI_23025181) have been also identified.31 Because of the confirmed capability to produce both glucans and fructans, this strain was chosen for this study. The aim of this study was to evaluate the potential of Ln. mesenteroides DSM 20343 in EPS production and the viscosity improving ability of the in situ produced EPS in soybean and fava bean doughs with the addition of sucrose or raffinose. The growth of the starter, the acidification and the viscosity changes of the fermented doughs were analyzed. Sugars, EPS (glucans and fructans), organic acids, and mannitol were all quantified. The role of glucans and fructans in viscosity improvement was studied by hydrolyzing the fermented doughs with dextranase or levanase. Changes in RFO profiles of untreated and autoclaved soybean and fava bean flours after fermentation were investigated as well.

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MATERIALS AND METHODS

Bacterial Strain and Culture Conditions. Leuconostoc mesenteroides subsp. mesenteroides DSM 20343 (Leibniz Institute DSMZ, Braunschweig, Germany) was routinely propagated in MRS broth at 30 °C (Oxoid, Hampshire, England). When used for fermentation, this strain was cultivated in MRS broth with 2% (w/v) sucrose for 16 h. Microbial cells were obtained by centrifugation (10000g for 10 min) and successively washed twice with 0.05 M sodium phosphate buffer (pH 7.0). The cells were then resuspended in distilled water for inoculation. Dough Preparation and Fermentation. Soybean flour, milled from heat-treated soy seeds, was purchased from Cereform Ltd. (Northampton, England). Heat treatment was performed in order to ensure the shelf life and safety of the flour.34,35 Fava bean flour, milled from untreated fava bean seeds, was purchased from Cerealveneta, (Padova, Italy). Both flours were mixed with distilled water, sucrose (Merck, Germany) or raffinose (Sigma-Aldrich) in different ratios according to Table 1. During dough preparation, sucrose (raffinose) was

Table 1. Composition of Soybean and Fava Bean Doughs sample code

flour (g)

sucrose (raffinose) (g)

water (mL)

soybean dough without sucrose addition soybean dough with 5% sucrose soybean dough with 10% sucrose soybean dough with 15% sucrose soybean dough with 10% raffinose

S0

30

0

70

S5

25

5

70

S10

20

10

70

S15

15

15

70

SR10

20

10a

70

fava bean dough without sucrose addition fava bean dough with 5% sucrose Fava bean dough with 10% sucrose fava bean dough with 15% sucrose fava bean dough with 10% raffinose

F0

40

0

60

F5

35

5

60

F10

30

10

60

F15

25

15

60

30

a

60

a

FR10

10

Raffinose was added instead of sucrose. B

DOI: 10.1021/acs.jafc.6b05495 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 2. Lactic Acid Bacteria (LAB) Cell Density; Increase in LAB Cell Density; pH; Concentrations of Citric Acid, Lactic Acid and Acetic Acid; and the Ratio of Lactic and Acetic Acid (Fermentation Quotient, FQ) in Soybean and Fava Bean Doughs after Fermentationa sample codeb Soybean S0 S5 S10 S15 SR10 Fava bean F0 F5 F10 F15 FR10

Δlogc

pH

ΔpH

citric acid (mmol/100 g dough)

lactic acid

acetic acid

FQ

9.4 ± 0.0 a 9.5 ± 0.1 a 9.5 ± 0.1 a 9.4 ± 0.0 a 9.5 ± 0.1 a

3.1 3.2 2.9 2.8 2.8

4.8 ± 0.1 a 4.5 ± 0.0 b 4.5 ± 0.0 b 4.7 ± 0.0 c 4.8 ± 0.0 a

1.7 2.0 2.4 2.1 1.8

4.06 ± 0.04 a 3.31 ± 0.06 b 2.63 ± 0.04 c 1.96 ± 0.06 d 2.83 ± 0.04 e

9.42 ± 0.28 a 9.54 ± 0.15 a 8.10 ± 0.04 b 5.60 ± 0.10 c 7.41 ± 0.12 d

3.27 ± 0.37 a 8.32 ± 0.35 b 8.24 ± 0.53 b 5.96 ± 0.67 c 4.17 ± 0.12 a

2.88 1.15 0.98 0.93 1.78

8.5 ± 0.1 a 9.1 ± 0.3 b 9.5 ± 0.1 b,c 9.8 ± 0.1 c 9.7 ± 0.0 c

2.3 2.8 2.5 2.8 2.7

4.4 ± 0.0 a 4.3 ± 0.0 b 4.3 ± 0.0 b,c 4.2 ± 0.0 c 4.3 ± 0.0 b,c

1.9 2.0 2.2 2.4 1.9

1.58 ± 0.02 a 1.40 ± 0.03 b 1.13 ± 0.03 c 0.95 ± 0.01 d 1.17 ± 0.02 c

12.97 ± 0.21 a 9.05 ± 0.11 b 10.10 ± 0.21 c 8.30 ± 0.02 d 9.57 ± 0.14 e

3.17 ± 0.30 a 7.09 ± 0.89 b,d 10.02 ± 0.88 c 9.18 ± 0.65 b,c 6.06 ± 0.29 d

4.09 1.28 1.01 0.90 1.58

cell density (log cfu/g)

a c

Values in the same column with different letters (a−e) are significantly different (p < 0.05). bDetails about the sample code can be found in Table 1. Increase in LAB cell density after fermentation.

viscosity values were measurement at 20 °C under different shear rates from 2 to 100 1/s. Then, samples were separated into three beakers with the same weight (60 g). Next, 100 μL of dextranase or levanase (both 100 U, one unit was defined as the amount of enzyme releasing 1 μmol of glucose/fructose in 1 min) was added to the sample, mixed thoroughly and incubated at 30 °C for 2 h. As a blank control, distilled water (100 μL) was added, and the sample was incubated under the same conditions. After hydrolysis, viscosity values were measured again under the same conditions, and the values at 100 1/s were taken. Samples were hydrolyzed and analyzed in triplicate. Statistical Analysis. The numerical results of microbial and chemical analyses in this study are averages of three independent fermentations. Data were analyzed by one-way analysis of variance (ANOVA) using Origin 8.6 (OriginLab Inc.). Means comparison was determined by Tukey’s test (P < 0.05). Multivariate data analysis was performed with principal component analysis (PCA) to evaluate the correlations among different variables by Simca 13.5 (MKS Umetrics AB, Malmö, Sweden).

Dough Viscosity. Changes in dough texture after fermentation were evaluated by viscosity analysis. All tested fava bean doughs showed a typical shear thinning (pseudoplasticity) and thixotropic behavior (Figure 1). Generally, sucroseenriched doughs (F5, F10, and F15) showed a more clear viscosity increase than the dough without sucrose addition (F0) after fermentation. Actually, the sucrose-enriched doughs formed a thin and inhomogeneous suspension before fermentation due to their low flour contents. However, after fermentation, these doughs formed a thick, homogeneous, and viscous system, indicating the thickening and emulsifying ability of EPS. Compared with sucrose-enriched doughs, the texture modification of raffinose-enriched dough (FR10) was less pronounced, as shown by its lower viscosity values after fermentation (Figure 1E). Because of similar viscosity flow curves of soybean doughs before and after fermentation, these curves are presented in the Figure S1 (Supporting Information). In order to assess the specific viscosity changes after fermentation, viscosity values at the shear rate of 100 1/s were compared (Table 3). The initial viscosity of doughs varied because of their different flour contents. After fermentation, these values all increased to different extents. In soybean doughs, the viscosity increase ranged from 0.26 to 6.75 Pa s, with the highest increase in S10 and the lowest in SR10. Similarly, in fava bean doughs, the viscosity increase varied from 1.24 to 6.36 Pa s, with the highest increase in F10 and the lowest in FR10. The viscosity increases in 0, 5, 10, and 15% sucrose-enriched samples were similar in the soybean and fava bean doughs, while there was a clear difference between SR10 and FR10. Monosaccharides, Oligosaccharides, and Degradation of RFO. Sucrose, raffinose, and stachyose were detected in S0 before fermentation (S0_0h), and stachyose was the main RFO (Figure 2 and Table 4). After fermentation, these sugars were not detected while melibiose, which was formed from raffinose after fructose release by the activity of levansucrase, was present. Manninotriose, which was formed through a similar reaction from stachyose, was also present (Figure 2). Glucose and fructose were released from sucrose during the synthesis of fructans and glucans, respectively. With the increasing sucrose content, different contents of glucose and fructose were detected after fermentation. In S5, more glucose was present compared with S15 in which more fructose was present. In SR10, glucose

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RESULTS LAB Growth and Acidification of Doughs. After 24 h of fermentation, the LAB cell density increased from 2.8 to 3.1 log cycles in soybean doughs and from 2.3 to 2.8 log cycles in fava bean doughs (Table 2). With the addition of sucrose, the LAB cell density slightly increased only in fava bean doughs. As expected, after 24 h of fermentation, the pH values decreased by 1.7 to 2.4 units in all doughs, showing a slightly higher decrease in sucrose-enriched doughs. Generally, lactic and acetic acid concentrations were lower in soybean doughs than in fava bean doughs (Table 2). The addition of sugars induced the formation of acetic acid, reaching the highest concentration in S5 and F10 for soybean and fava bean doughs, respectively. As a consequence, the ratio of lactic and acetic acid (fermentation quotient, FQ) showed a decrease when sugars were added. In soybean doughs, the FQ decreased sharply from S0 to S5 and more slowly from S5 to S15. In SR10, the FQ was higher than those in sucrose-enriched doughs. A similar trend was also observed in fava bean doughs. Citric acid was detected in both soybean and fava bean doughs before fermentation (data not shown); the amounts detected after fermentation were almost the same. Therefore, it can be hypothesized that citric acid was not involved in the microbial metabolism, and the concentration decreased in the sugarenriched doughs primarily due to the reduced flour content (Table 1). C


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Figure 1. Viscosity flow curves of fava bean doughs before (0 h) and after fermentation (24 h) under different shear rates from 2 to 100 1/s (up and down sweeps). Fava bean dough (A) without sucrose addition (F0), (B) with 5% sucrose (F5), (C) with 10% sucrose (F10), (D) with 15% sucrose (F15), and (E) with 10% raffinose (FR10).

were detected in FR10, and only a small amount of raffinose (0.36%) was left after fermentation (Figure 2 and Table 4). Similar to soybean doughs, different contents of glucose and fructose were also detected in fava bean doughs with the increasing sucrose content after fermentation. Because of different sugar profiles of soybean and fava bean doughs on RFO degradation, the effect of endogenous αgalactosidase in both flours on RFO degradation was investigated by comparing the sugar profiles of aseptic doughs and doughs made with untreated or autoclaved flours before and after fermentation (Figure 2). The sugar profile in the chemically

and fructose were not detected but melibiose was detected and some raffinose (1.01%) was left after fermentation. In addition to sucrose, raffinose, stachyose, and verbascose were all present in F0 before fermentation (F0_0h). After fermentation, the contents of stachyose and verbascose decreased considerably while sucrose and raffinose were not detected any longer (Figure 2 and Table 4). Differing from S0, melibiose was not detected while some galactose was detected in F0. Particularly, in FR10, a higher content of galactose, originating most probably from raffinose by the activity of αgalactosidase, was detected. Meanwhile, glucose and melibiose D


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endogenous α-galactosidase in soybean flour. Meanwhile, the sugar profiles of untreated (S0_U) and autoclaved (S0_A) soybean doughs were the same, which was also observed in SR10_U and SR10_A (Figure 2A,B). However, in fava bean doughs, the sugar profiles changed after autoclaving (Figure 2C,D). In F0 made with autoclaved fava bean flour (F0_A), galactose, which previously appeared in F0_U, was not observed but two new peaks appeared. On the basis of a manninotriose standard and a previous study,38 these two peaks were identified as manninotriose and manninotetraose, indicating the activity of levansucrase and the absence of microbial α-galactosidase activity in F0_A. As expected, no galactose was detected in FR10 made with autoclaved flour (FR10_A). Compared with FR10_U, higher amounts of melibiose and raffinose were found in FR10_A, indicating that the added raffinose was mostly converted to melibiose by levansucrase. In addition, the appearance of galactose and the decrease of RFO load in F0_C revealed the role of endogenous α-galactosidase in RFO degradation in fava bean flour. Therefore, the major difference on RFO degradation in soy and fava bean doughs was mainly due to the endogenous α-galactosidase which was inactivated in soybean flour but not inactivated in fava bean flour. EPS and Mannitol. Generally, with the same sucrose content, EPS (glucan, dextran, and fructan) contents of soybean

Table 3. Initial and Final Viscosity and Viscosity Increase in Soybean and Fava Bean Doughsa sample codeb Soybean S0 S5 S10 S15 SR10 Fava bean F0 F5 F10 F15 FR10

initial viscosityc (Pa s)

final viscosityc (Pa s)

viscosity increase (Pa s)

3.93 ± 0.18 a 0.82 ± 0.05 b 0.14 ± 0.00 c 0.05 ± 0.00 c 0.14 ± 0.00 c

5.24 ± 0.12 a 5.34 ± 0.08 a 6.89 ± 0.28 b 5.07 ± 0.07 a 0.40 ± 0.00 c

1.31 4.52 6.75 5.02 0.26

0.82 ± 0.01 a 0.40 ± 0.01 b 0.29 ± 0.01 c 0.15 ± 0.01 d 0.16 ± 0.01 d

2.21 ± 0.11 a 4.55 ± 0.16 b 6.65 ± 0.08 c 5.33 ± 0.12 d 1.40 ± 0.03 e

1.39 4.15 6.36 5.18 1.24

a Values in the same column with different letters (a−e) are significantly different (p < 0.05). bDetails about the sample code can be found in Table 1. cThe viscosity values of all samples were obtained at a shear rate of 100 1/s.

acidified and aseptic soybean dough (S0_C) was the same as the unfermented dough (S0_0h), confirming the inactivation of

Figure 2. HPAEC-PAD chromatograms of S0 (A), SR10 (B), F0 (C), and FR10 (D) before and after fermentation (0 h, unfermented doughs; C, chemically acidified and aseptic dough; U, dough made with untreated flour; A, dough made with autoclaved flour; ISD, internal standard; Gal, galactose; Glc, glucose; Suc, sucrose; Mel, melibiose; Raf, raffinose; Sta, stachyose; Ver, verbascose). E


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Table 4. Contents of Monosaccharides and Oligosaccharides in Soybean and Fava Bean Doughs before and after Fermentation sugarsb (%, w/w, wet matter) sample code

a

S0_0hc S0 S5 S10 S15 SR10 F0_0he F0 F5 F10 F15 FR10

Glc

Fru

Suc

Mel

Raf

Sta

Ver

Gal

ndd ndd 0.68 ± 0.05 1.31 ± 0.02 1.94 ± 0.03 ndd 0.06 ± 0.00 ndd 0.67 ± 0.02 0.94 ± 0.02 1.19 ± 0.03 0.26 ± 0.01

ndd ndd 0.09 ± 0.00 1.51 ± 0.03 3.94 ± 0.09 ndd ndd ndd ndd 0.51 ± 0.01 2.42 ± 0.05 ndd

1.93 ± 0.04 ndd ndd ndd ndd ndd 1.46 ± 0.01 ndd ndd ndd ndd ndd

nd 0.05 ± 0.01 0.09 ± 0.00 0.06 ± 0.00 0.07 ± 0.00 4.91 ± 0.08 ndd ndd ndd ndd ndd 1.88 ± 0.02

0.17 ± 0.00 ndd ndd ndd ndd 1.01 ± 0.03 0.08 ± 0.00 ndd ndd ndd ndd 0.36 ± 0.02

1.60 ± 0.04 ndd ndd ndd ndd ndd 0.91 ± 0.01 0.11 ± 0.00 0.08 ± 0.00 ndd ndd 0.16 ± 0.04

ndd ndd ndd ndd ndd ndd 1.73 ± 0.00 0.14 ± 0.00 0.18 ± 0.00 0.21 ± 0.00 0.19 ± 0.01 0.32 ± 0.03

ndd ndd ndd ndd ndd ndd 0.07 ± 0.00 0.36 ± 0.01 0.55 ± 0.01 0.41 ± 0.01 0.30 ± 0.01 1.49 ± 0.01

a Details about the sample code can be found in Table 1. bPercentages were calculated on a wet weight basis. Sugars: Glc, glucose; Fru, fructose; Suc, sucrose; Mel, melibiose; Raf, raffinose; Sta, stachyose; Ver, verbascose; Gal, galactose. cS0_0h, soybean dough without sucrose addition before fermentation. dnd = not detected. eF0_0h, fava bean dough without sucrose addition before fermentation.

Table 5. Contents of Glucan, Dextran, Fructan, and Mannitol in Soybean and Fava Bean Doughs after Fermentationa sample codeb Soybean S0 S5 S10 S15 SR10 Fava bean F0 F5 F10 F15 FR10

glucanc (%)

dextranc (%)

fructanc (%)

mannitolc (%)

EPSc (%)

yieldd (%)

dextran/glucan

fructan/glucan

0.50 ± 0.07 a 1.79 ± 0.24 b 3.33 ± 0.40 c 4.20 ± 0.30 d 0.42 ± 0.03 a

0.29 ± 0.01 a 1.20 ± 0.03 b 1.79 ± 0.03 c 2.32 ± 0.03 d 0.25 ± 0.00 a

0.38 ± 0.02 a 1.38 ± 0.08 b 2.09 ± 0.12 c 2.31 ± 0.09 c 2.79 ± 0.08 d

0.78 ± 0.05 a 2.37 ± 0.42 b 2.40 ± 0.36 b 1.83 ± 0.31 b,c 0.97 ± 0.10 a,c

0.88 3.17 5.42 6.51 3.21

90.72 95.92 96.01 81.53 88.19

0.58 0.67 0.54 0.55 0.59

0.76 0.77 0.63 0.55 6.64

0.49 ± 0.10 a 1.13 ± 0.12 b 2.29 ± 0.18 c 3.05 ± 0.25 d 1.04 ± 0.16 b

0.21 ± 0.01 a 0.83 ± 0.01 b 1.22 ± 0.01 c 1.38 ± 0.02 d 0.68 ± 0.03 e

0.20 ± 0.01 a 1.11 ± 0.06 b 1.58 ± 0.12 c 1.76 ± 0.08 c 1.71 ± 0.07 c

0.24 ± 0.02 a 2.33 ± 0.21 b,d 3.04 ± 0.32 b,c 3.09 ± 0.30 c 1.77 ± 0.04 d

0.69 2.24 3.87 4.81 2.75

94.52 71.37 69.76 59.77 73.09

0.43 0.73 0.53 0.45 0.65

0.41 0.98 0.69 0.58 1.64

a EPS content, yield of EPS, ratio of dextran to glucan, and ratio of fructan to glucan are also presented. Values in the same column with different letters are significantly different (p < 0.05). bDetails about the sample code can be found in Table 1. cPercentages were calculated on a wet weight basis. dYield is the percentage ratio between real EPS content and theoretical EPS content.

PCA. PCA modeling visualized how the variables correlated with each other and has been used in different food systems to evaluate the relationships among various parameters.39−41 For soybean doughs (Figure 3A), the principal component 1 (PC1) mainly explained the model (54.7%) and PC2 had less role in the model (25.6%). Also, for fava bean doughs (Figure 3B), the PC1 mainly explained the model (63.2%) and PC2 had less of a role in the model (25.7%). As shown in Figure 3A, the consumed sucrose (ΔSuc) in soybean doughs was more correlated with the produced glucans but less correlated with the produced fructans. A similar result was also found in fava bean doughs (Figure 3B). The viscosity increase (ΔVis) was more correlated with dextran and glucan contents but less correlated with fructan contents in both soybean and fava bean doughs. Additionally, dextran and glucan contents were strongly correlated with the acetic acid and mannitol contents in both doughs. The consumed raffinose (ΔRaf) content was strongly correlated with the content of melibiose and the ratio of fructans to glucans in both cases. However, in soybean doughs, the LAB cell density increase (Δlog) was positively correlated with the EPS yield and the lactic acid content; but in fava bean doughs, the LAB cell density increase was negatively correlated with the EPS yield and the lactic acid content while positively correlated with the ΔSuc, ΔpH, and the acetic acid content.

doughs were higher than those of fava bean doughs, and the contents increased with the rising sucrose content (Table 5). The glucan content was lowest in SR10, and it was comparable to S0. However, in FR10, the glucan content was higher than F0 and was comparable to F5. The ratio of water-soluble dextran to total glucans varied from 0.54 to 0.67 in soybean doughs and from 0.43 to 0.73 in fava bean doughs. As expected, the highest fructan content in soybean doughs was found in SR10. However, in fava bean doughs, the highest fructan content was found in F15, and no significant difference was observed between F15 and FR10. The types of EPS produced and thus the ratio of fructan to glucan varied from 0.55 to 0.77 in sucrose-enriched soybean doughs and from 0.58 to 0.98 in the corresponding fava bean doughs (Table 5). More glucans rather than fructans were produced with increasing sucrose levels in both doughs. The addition of sucrose facilitated mannitol formation in both doughs, which can be observed from the mannitol contents in S10 and F10. (Table 5). In SR10, the mannitol content was comparable to S0, while in FR10 the mannitol content was comparable to F5. Compared with sucrose-enriched doughs (S10 and F10), the mannitol contents of raffinose-enriched doughs (SR10 and FR10) were much lower in both cases. F


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Figure 3. PCA biplot of soybean (A) and fava bean doughs (B) (ΔSuc, consumed sucrose; ΔRaf, consumed raffinose; ΔpH, pH drop; ΔVis, viscosity increase; Δlog, LAB cell density increase; Dex, dextran; Gl, glucan; Fr, fructan; Glc, glucose; Fru, fructose; Mel, melibiose; Lac, lactic acid; Ace, acetic acid; Man, mannitol).

Role of Glucan and Fructan in Viscosity Improvement. In order to reveal the role of glucans and fructans in viscosity improvement, soybean and fava bean doughs with 10% sucrose (S10 and F10) were both hydrolyzed by dextranase or levanase. After dextranase hydrolysis, the viscosity of S10 decreased considerably, from 7.69 to 0.37 Pa s (Figure 4). However, after levanase hydrolysis, the viscosity of S10 decreased much less, from 7.69 to 6.14 Pa s. A similar phenomenon was also observed in F10. The viscosity dropped from 5.51 to 0.79 Pa s after dextranase hydrolysis and from 5.51 to 4.78 Pa s after levanase hydrolysis. As blank controls, samples with added water showed the least viscosity drop, from 7.69 to 6.87 Pa s in S10 and from 5.51 to 5.19 Pa s in F10.

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DISCUSSION EPS produced by LAB have the potential to replace plant polysaccharides, which are commonly used as conditioners, softeners, and antistaling agents in food products.26 In our study, EPS were produced by Ln. mesenteroides DSM 20343 in soybean and fava bean flours. The pronounced viscosity increase, together with the glucan and fructan contents in all doughs, indicated that the microbial activity during fermentation is attributable only to Ln. mesenteroides DSM 20343. The two flours used in this study have a great potential in the food industry due to their various

Figure 4. Viscosity changes of soybean and fava bean doughs with 10% sucrose (S10 and F10) before and after the hydrolysis of dextranase or levanase. (The same volume of water was added to the sample as a blank control.)

beneficial properties. The main difference between these two flours consisted of the inactivation of the endogenous enzymes of G


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Figure 5. Two degradation ways of RFO: levansucrase-involved way (A) and α-galactosidase- and glucansucrase-involved way (B). Levansucrase and glucansucrase are both from Ln. mesenteroides DSM 20343 while α-galactosidase is from fava bean flour.

soybean flour, providing α-galactosidase activity and, presumably, other glycosyltransferases and glycosyl hydrolases,46 contributing to the modification of the sugar content during fermentation and impacting the metabolic activity of the starter. Generally, the specific response of LAB to different environmental/nutrient conditions involve the hierarchical and/or simultaneous use of various energy sources and electron acceptors. Furthermore, interactions between LAB and the endogenous enzymes also play an important role in the adaptive responses.47 Sucrose content also influenced the formation of insoluble glucans, which was shown by the ratio of dextran and glucan contents (Table 5). More soluble dextrans were formed under low sucrose content, while more insoluble dextrans were formed under high sucrose content (above 5%). This could be partially explained by the formation mechanism of insoluble glucans, as they were formed based on soluble dextrans.32 Meanwhile, the added sucrose changed the ratio of fructan and glucan as well. On the basis of the same sucrose content, glucan was preferably formed in comparison to fructan in all doughs. This trend was also revealed by PCA, which indicated a stronger correlation between the consumed sucrose and glucans rather than fructans. Because of the formation of EPS, a clear viscosity increase was observed in both soybean and fava bean doughs after fermentation. As hydrocolloids, EPS are able to bind water and interact with proteins in doughs, leading to an increased viscosity. The interactions between polysaccharides and proteins are wellknown.48 However, the interactions between legume proteins and EPS in a complex system are still poorly studied. Under the conditions of our study, glucans and fructans showed different effects on viscosity improvement. By hydrolyzing S10 (soybean dough) and F10 (fava bean dough) with dextranase and levanase

soybean flour as a consequence of heat treatment of soy seeds prior to milling, necessary to inactivate antinutritional factors, enhance quality of soybean products, and to ensure the safe storage.34,35 In soybean doughs, the increase of LAB cell density (Δlog) was independent of sucrose or raffinose addition. However, in fava bean doughs, the addition of sugars seemed to affect the LAB cell density increase. Generally, the total amount of lactic acid and acetic acid in fava bean doughs was higher than that in soybean doughs. Lactic acid was the dominant acid in doughs without sugar addition. In the presence of sugars, more acetic acid was formed since Leuconostoc spp. can use fructose liberated from sucrose as the electron acceptor to produce mannitol, contributing to acetic acid formation.42 The addition of sucrose enabled EPS formation. Theoretically, half of the sucrose could be used for EPS production. By calculating the percentage ratio between the real and theoretical EPS content, we found that EPS were produced efficiently, especially in soybean doughs (Table 5). The efficiency of EPS production was affected by the sucrose content. A higher EPS yield was observed under a lower sucrose content in both doughs, indicating a possible substrate inhibition of dextransucrase and levansucrase by the high sucrose content.43,44 Furthermore, compared with soybean doughs, a sharper decrease in EPS yield was found in fava bean doughs, pointing to the effects of endogenous enzymes in fava bean doughs on EPS yield. Actually, the EPS yields of fermented soy and fava bean doughs were relatively high if compared to the EPS yields of wheat bran.45 However, some of the fermentation-related variables of fava bean doughs, like Δlog and ΔpH, were negatively correlated with the EPS yield. A possible explanation lies in the endogenous enzymatic activities of fava bean flour, which is destroyed in H


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In conclusion, Ln. mesenteroides DSM 20343 showed a potential in EPS production in soybean and fava bean doughs, resulting in a pronounced viscosity increase after fermentation. Glucans rather than fructans played the main role in viscosity improvement in this study. Different degradation ways of RFO were found due to the effect of endogenous α-galactosidase. The viscous legume-based doughs containing in situ EPS may meet consumers’ needs for reduced use of food additives, and they have several potential applications in the baking industry, in gluten-free food manufacturing, and for meat substitution. Further work can be done on the mechanism of the differences between glucans and fructans in viscosity improvement, the effects of flour pretreatment on the structure and content of EPS, and the applications of fermented legumes with in situ EPS in the food industry.

separately, the major role of glucans in viscosity improvement was confirmed. PCA results also revealed a stronger correlation between glucan contents and viscosity increases. The lower viscosity-improving ability of fructans might be due to its low molecular weight,31 but further research is needed to clarify this effect. It has been reported that levansucrase, sucrose phosphorylase, and α-galactosidase are the main enzymes contributing to the degradation of RFO in Lactobacillus.38 In our work, the contents of RFO decreased after fermentation, as expected. The αgalactosidase activity of Ln. mesenteroides DSM 20343 was not detected in our study although it has been reported that many LAB, including Leuconostoc species, produce α-galactosidase and have been used to eliminate RFO in food products prepared from soybean, pinto bean, field pea, and pearl millet.11,49,50 By analyzing the sugar profiles of chemically acidified and aseptic doughs and fermented doughs made with untreated or autoclaved flours, two different degradation ways of RFO could be hypothesized, based on the enzymatic activities of microbial levansucrase or plant-derived α-galactosidase (Figure 5). The hydrolyzing or polymerizing activity of levansucrase on raffinose, stachyose, and verbascose resulted in the formation of melibiose, manninotriose, and manninotetraose (Figure 5A). Meanwhile, the activity of plant-derived α-galactosidase from fava bean flour on raffinose, stachyose, and verbascose could lead to the formation of galactose and sucrose. The resulted sucrose could be further converted into glucan and free fructose through the activity of glucansucrase from Ln. mesenteroides DSM 20343 (Figure 5B). The first way was supported by the appearance of melibiose, manninotriose, and manninotetraose in fermented doughs and by the detection of fructan in SR10 after fermentation. The second way was supported by the appearance of galactose in the aseptic fava bean dough and fermented fava bean doughs and by the high glucan content in FR10. Since the previous inactivation of endogenous α-galactosidase in soybean flour, no galactose was detected in the aseptic soybean dough or soybean doughs made with untreated or autoclaved flour. In contrast, galactose appeared in F0_C (chemically acidified and aseptic) and F0_U (untreated) since the endogenous α-galactosidase in fava bean flour was not inactivated previously. The presence of galactose together with melibiose and mannotriose in FR10_U indicated the involvement of both microbial levansucrase and plant-derived αgalactosidase in RFO degradation, suggesting the induction effect of the bacterial levansucrase by raffinose.38 Because of the sole appearance of galactose in untreated F0 and the joint appearance of galactose and melibiose in untreated FR10, it could be hypothesized that the content of raffinose could affect the action of microbial levansucrase. Furthermore, as manninotriose and manninotetraose appeared in autoclaved F0, melibiose and manninotriose appeared in autoclaved FR10, it could be hypothesized that the plant-derived α-galactosidase could also affect the action of microbial levansucrase. On one hand, if the plant-derived α-galactosidase was activated and there was little raffinose, microbial levansucrase may not act on the RFO. However, if there was enough raffinose and the plant-derived αgalactosidase was activated as well, microbial levansucrase might act on the RFO together with the plant-derived α-galactosidase. On the other hand, if the plant-derived α-galactosidase was inactivated, microbial levansucrase may act on the RFO even with little raffinose. However, more work is still needed on the expression of levansucrase and the activity of α-galactosidase in fava bean flour in order to confirm this speculation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05495. Viscosity flow curves of soybean doughs (S0, S5, S10, S15, and SR10) under different shear rates from 2 to 100 1/s, up and down sweeps (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +358 46 5446527. Fax: +358 919158475. E-mail: xu.z. yan@helsinki.fi. ORCID

Yan Xu: 0000-0001-8960-5505 Funding

The China Scholarship Council is greatly acknowledged for its financial support of this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Jose Martin Ramos Diaz is acknowledged for his kind help in statistical analysis (PCA).

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ABBREVIATIONS USED EPS, exopolysaccharides; LAB, lactic acid bacteria; ANF, antinutritional factors; RFO, raffinose family oligosaccharides; Dsr, dextransucrases; HPAEC-PAD, high-performance anion exchange chromatography with pulse amperometric detection; PCA, principal component analysis; FQ, fermentation quotient

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REFERENCES

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Exopolysaccharides Production during the Fermentation of Soybean

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