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Partial purification of components in rye water extractables which improve the quality of oat bread
Journal of Cereal Science 79 (2018) 141e147
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Partial purification of components in rye water extractables which improve the quality of oat bread Anneleen Pauly, Jan A. Delcour* Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 October 2016 Accepted 15 October 2017 Available online 17 October 2017
Unlike wheat bread, the dough of which has a visco-elastic network and high gas-holding capacity, oat bread generally has a low volume and a dense structure. We showed earlier that including rye waterextractable components in an oat bread batter recipe increases loaf volume by ca. 30% (Pauly and Delcour, submitted as back-to-back publication). We here report on efforts to identify the active factor(s). Anion exchange chromatography allowed enriching the active factor(s). This and the fact that only a limited volume increase was observed when oat batter was supplemented with boiled rye extract indicate that proteins are likely the most important components responsible for the volume increase. While the most active factor(s) had a pI below 4.5, components with pI values between 4.5 and 8.5 also contributed to oat loaf volume. Alkaline rye components (pI > 8.5) or rye arabinoxylan had no impact. Rye water-extractable components smaller than 6e8 kDa also had a positive impact on loaf volume. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Gluten-free/oat bread Ion exchange chromatography Water-extractable components Loaf volume
1. Introduction Celiac disease is an autoimmune-mediated disease triggered by ingesting wheat gluten or related proteins. Its reported prevalence has increased over the last 30 years. It probably affects more than 1% of the world population (Lohi et al., 2007; Hüttner and Arendt, 2010). The present only effective treatment is a life-long and strict adherence to a gluten-free diet. Although initially developed for patients suffering from celiac disease, gluten-free diets are also followed e.g. as part of a life-style choice. In the United States, only 7% of the gluten-free products on the market are purchased by celiac patients (Watson, 2012). Furthermore, consumers nowadays increasingly choose bread prepared from cereals other than wheat because of nutrition related reasons (e.g. levels of dietary fiber, essential amino acids, minerals) (Dewettinck et al., 2008). However, the absence of wheat gluten presents technological challenges. Indeed, wheat gluten has unique visco-elastic properties which
Abbreviations: AEC, anion exchange chromatography; CEC, cation exchange chromatography; dm, dry matter; E440, extinction at 440 nm; E590, extinction at 590 nm; MM, molecular mass; MWCO, molecular weight cut-off; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)-aminomethane. * Corresponding author. E-mail address: [email protected] (J.A. Delcour). https://doi.org/10.1016/j.jcs.2017.10.007 0733-5210/© 2017 Elsevier Ltd. All rights reserved.
confer upon bread its desired structure (Delcour et al., 2012). The quality of non-wheat bread products is, hence, much inferior to that of their counterparts containing gluten. Non-wheat bread has low loaf volume, an uneven distribution of large gas cells in the crumb and firm and crumbly texture (Hager et al., 2012; Houben et al., 2012). To overcome such quality deficiencies, additives are frequently used in non-wheat bread recipes. Hydrocolloids (e.g. xanthan gum, cellulose derivatives such as hydroxypropyl methyl cellulose), surfactants (e.g. diacetyl tartaric esters of mono- and diacylglycerols, sodium stearoyl lactylate) and enzymes (e.g. amylases, transglutaminases) are well-studied additives in non-wheat bread making (Renzetti et al., 2008, 2010; Hüttner and Arendt, 2010; Sciarini et al., 2012). Cereal water extractables contain several components which may well increase batter stability. Non-starch polysaccharides may act by virtue of their water binding and viscosity increasing capacity (Courtin and Delcour, 2002; Izydorczyk and Dexter, 2008). Also, much as some surfactants, some water-extractable surface active proteins can stabilize gas cells (MacRitchie, 1976; Pauly et al., 2014). Cereal aqueous extracts also contain various enzymes. In a previous paper, we included water extracts from barley, rye, oat and wheat flour in simple oat bread recipes (Pauly and Delcour, submitted as back-to-back publication). Oat contains high levels of the dietary fiber (1 / 3, 1 / 4)-b-D-glucan (further referred to as b-glucan) (Shewry et al., 2008). Sufficient high intake of b-glucan
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can reduce postprandial blood glucose, insulin and serum cholesterol levels (Braaten et al., 1994a, 1994b; Kerckhoffs et al., 2003). Other oat nutrients include essential amino acids, vitamins, minerals and antioxidants (Dewettinck et al., 2008). Oat can also be used as raw material for gluten-free products, although some controversy exists on the matter (Hüttner and Arendt, 2010). Replacing an equal weight of oat flour by rye extract [5.0% on dry matter basis (dm)] resulted in a larger loaf volume increase (þ27%) than did replacing oat flour by barley (þ21%), wheat (þ14%) or oat (þ5%) extracts in a similar way. Furthermore, we showed that replacing only 2.0% dm oat flour by rye extract made crumb significantly softer, while it had no impact on crumb mean cell area and number of cell per surface unit. Amongst the four extracts, that from rye contained the highest arabinoxylan levels and enzyme activities. High b-glucan levels and extract viscosity are probably not the main factors contributing to the volume increasing effect of rye extract (Pauly and Delcour, submitted as back-to-back publication). In the present paper, we contribute to the identification of the active component(s) in the rye extract responsible for oat loaf volume increases. To that end, the rye extract is treated in order to enrich the active component(s) and the impact on oat loaf volume is evaluated. We here report on the outcome of our work. 2. Materials and methods 2.1. Materials Commercial oat flour (moisture, protein, and ash contents on dm respectively 10.1%, 12.4%, and 1.67%) and rye flour (corresponding data 12.9%, 7.2%, and 0.82%) were from Raisio Nutrition (Raisio, Finland) and from Koopmans (Leeuwarden, The Netherlands), respectively. Dry yeast was from Puratos (Groot-Bijgaarden, Belgium). Sugar and salt were commercial food grade products. SP Sepharose Big Beads and Q Sepharose Fast Flow ion exchange matrices for batch and column fractionations were from GE Healthcare (Uppsala, Sweden). All other chemicals, solvents and reagents were from Sigma-Aldrich (Bornem, Belgium) and were at least analytical grade, unless specified otherwise. 2.2. Experimental 2.2.1. Boiling of the rye extract Aqueous extracts of rye flour were prepared as in Pauly and Delcour (submitted as back-to-back publication). For some experiments, the freeze-dried extract (3.0 g) was suspended in 100 mL deionized water, boiled for 15 min, cooled to room temperature and centrifuged (5000 g, 15 min). The supernatant was then freezedried. 2.2.2. Batch ion exchange fractionation Fractionation into bound and unbound material was performed with batch anion exchange chromatography (AEC) or cation exchange chromatography (CEC) on SP Sepharose Big Beads or Q Sepharose Fast Flow, respectively. The ion exchange matrices (ca. 500 mL) were equilibrated with 25 mM tris-(hydroxymethyl)aminomethane (Tris) - HCl (pH 8.5) or 25 mM sodium acetate (pH 4.5) for AEC or CEC, respectively. The suspensions of matrix and extract (3.0 g in 100 mL of these media) were shaken overnight at 6 C and then brought onto a P1 glass filter. The matrix was rinsed with 25 mM Tris-HCl (AEC) or 25 mM sodium acetate (CEC) to remove unbound material. Bound material was eluted with 1.0 L 1.0 M NaCl. The four obtained fractions (unbound and eluate of AEC or CEC) were dialyzed [molecular weight cut-off (MWCO) 6e8 kDa] against deionized water and freeze-dried. The untreated extract
was also dialyzed (MWCO 6e8 kDa) against deionized water to investigate the impact of removing small rye components. 2.2.3. Column anion exchange chromatography Extracts [3.0 g in 150 mL 25 mM Tris-HCl (pH 8.5)] were separated by AEC on a Q Sepharose Fast Flow column (diameter 16 mm, length 230 mm). They were suspended in loading buffer [25 mM Tris-HCl (pH 8.5)] and centrifuged (5000 g, 15 min, 4 C). The supernatants were loaded onto the column, equilibrated with loading buffer, at a flow rate of 0.3 mL/min. After rinsing the column with loading buffer (flow rate 1.0 mL/min) to remove unbound material (¼ run-through fraction), bound molecules were eluted using a gradient of 1.0 M NaCl in 25 mM Tris-HCl (pH 8.5) at a flow rate of 1.0 mL/min. The NaCl concentration was first increased linearly from 0 to 300 mM NaCl in 150 m), and then further increased to 800 mM in 500 mL. This elution gradient was chosen based on preliminary tests. The fractions detected at 280 nm were dialyzed (MWCO 6e8 kDa) against deionized water and freeze-dried. 2.2.4. Protein electrophoresis Protein composition was analyzed with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Freeze-dried extracts or extract fractions were dissolved in sample buffer [12.5 mM Tris-HCl; 4.0% (w/v) SDS; 30% (v/v) glycerol; 0.004% (w/v) bromophenol blue; pH 6.8] to obtain a protein concentration of approximately 2.0 mg/mL. After boiling (5 min) and centrifugation (10,900 g; 3 min), the proteins in the supernatant were separated under non-reducing conditions with a PhastSystem (GE Healthcare) according to GE Healthcare separation technique file 110. Molecular mass (MM) markers (LMW-SDS Marker Kit, GE Healthcare) were also analyzed. Gels were stained with sensitive silver staining as described in the GE Healthcare development technique file 210. 2.2.5. Enzyme activities Enzyme activities were determined at pH 5.4 [i.e. average batter pH during fermentation (Pauly and Delcour, submitted as back-toback publication)] and 30 C (i.e. fermentation temperature). aAmylase, endoxylanase and endo-b-glucanase activity levels were determined in triplicate with the Amylazyme, Xylazyme AX and bGlucazyme methods (Megazyme, Bray, Ireland), respectively. Freeze-dried extracts (50 mg) were suspended in 25 mM 10.0 mL sodium maleate buffer (pH 5.4) containing 5.0 mM CaCl2 for the Amylazyme method or 25 mM sodium acetate buffer (pH 5.4) for the Xylazyme AX and b-Glucazyme methods. After centrifugation (5000 g, 15 min), the supernatants (1.0 mL) were equilibrated for 10 min at 30 C before adding an Amylazyme, Xylazyme AX or a bGlucazyme tablet, respectively. After 90 min incubation for aamylase, 18 h for endoxylanase and 5 h for endo-b-glucanase activity levels, the reactions were stopped by adding 10.0 mL of a Tris solution (2.0% or 1.0% for the Amylazyme and b-Glucazyme or Xylazyme AX methods, respectively). The solutions were vigorously vortexed, filtered through a MN 615 filter (Macherey-Nagel, Düren, Germany) and their extinction at 590 nm (E590) measured. Control samples were extract suspensions incubated without the respective tablets. Corrections were made for non-enzymatic color release by the respective tablets. a-Amylase, endoxylanase and endo-bglucanase activities were expressed as units per gram dm extract. One unit is the enzyme activity which increases E590 by 1.00 per hour of incubation under the conditions of the test. Endopeptidase activity levels were determined in triplicate using azocasein substrate (Brijs et al., 1999). Freeze-dried extracts (50 mg) were suspended in 10.0 mL 25 mM sodium acetate buffer (pH 5.4). After centrifugation (5000 g, 15 min), 250 mL supernatant was combined with 350 mL 1.4% (w/v) azocasein. After 24 h
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incubation at 30 C, 500 mL cool (7 C) 10.0% (w/v) trichloroacetic acid was added. The precipitated proteins were removed by centrifugation (9600 g, 10 min). Finally, 500 mL 0.5 M sodium hydroxide was added to 500 mL supernatant and the extinction at 440 nm (E440) was measured. Activity was expressed as endopeptidase units per gram dm. One unit is the enzyme activity which increases E440 by 1.00 per hour of incubation under the experimental conditions. 2.2.6. Foaming properties The foaming properties of the freeze-dried boiled extract were determined in duplicate for 50.0 mL solutions with the whipping method of Caessens et al. (1997), but with small modifications. DL Solutions were poured in a graduated glass cylinder (internal diameter: 60 mm) and whipped for 70 s using a rotating propeller (outer diameter: 45.0 mm, thickness: 0.4 mm, diameter cylindrical bar: 6.0 mm) at 2000 rpm. The initial foam volume was measured 2 min after the start of whipping, whereas foam volume was monitored during 60 min. 2.2.7. Small scale oat bread making A simple oat bread recipe based on Hager et al. (2014) was used. The baking procedure was adapted to 25 g batter to minimize the amounts of treated and fractionated samples needed. Batter was prepared with oat flour, 1.5% salt, 6.0% sugar, 3.0% yeast and 90% deionized water (percentages based on flour weight at 14.0% moisture). When treated extracts or extract fractions were tested (levels up to 2.0%), they replaced an equal weight of oat flour. The yield of the treated or fractionated samples was taken into account when dosing in order to allow drawing conclusions on enrichment of the active factor(s). For example, when a fraction was obtained in a yield of 50%, it was dosed at 1.0% (¼ 50 % 2.0 %) to be comparable with a 2.0% dosage of the untreated rye extract. The optimal water addition level was determined by preliminary baking trials. Total batter weight was 150 g. Yeast was suspended in the water (preincubated at 30 C) and rested for 10 min in a fermentation cabinet (National Manufacturing, Lincoln, NE, USA) at 30 C and a relative humidity of 90%. The yeast suspension was then added to the premixed dry ingredients (flour, sugar, salt and, when tested, freeze-dried extract). Mixing was at speed 1 for 60 s with a KitchenAid (St. Joseph, MI, USA) KPM5 mixer and, after the bowl content was scraped down, further mixed for 90 s at speed 4. Slightly greased baking tins were filled with 25 g batter and placed in the fermentation cabinet for 30 min at 30 C and 90% relative humidity. Fermented batters were then baked in a Condilux deck oven (Hein, Strasse, Luxemburg) for 30 min at 195 C. After cooling for 120 min, bread samples were weighed and loaf volumes determined with a Volscan Profiler (Stable Microsystems, Godalming, Surrey, UK). Baking trials were performed at least in fourfold. 2.2.8. Rheofermentometer analysis Rheofermentometer analyses of control oat batter or oat batter of which 2.0% flour weight was replaced by rye extract were performed. Batter (300 g, prepared as described in Section 2.2.7) was placed in the basket of the Rheofermentometer (Chopin, Villeneuve-La-Garenne, France) and covered with the 254 g piston (without additional weight). The analyses were performed at 30 C during 90 min. Total carbon dioxide production was derived from the gas release curve. 2.2.9. Statistical analysis Significant differences (a < 0.05) for several variables, based on at least triplicate replications, were determined using the one-way ANOVA procedure with JMP® Pro 11.2.0 (SAS institute, Cary, NC, USA).
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3. Results and discussion 3.1. Boiling of rye extract We earlier showed that replacing only 2.0% dm oat flour by water-extractable rye flour components sufficed for a maximum volume increase of 34% (Pauly and Delcour, submitted as back-toback publication). In the present study, dosages were based on this replacement level. In a first step, the rye extract was boiled to denature proteins and inactivate enzymes. The coagulated material was removed by centrifugation and accounted for ca. 20% dm (Table 1). Boiling the rye extract resulted in almost complete loss of activity (6.2% volume increase, data not shown). This indicates that arabinoxylan is not the major component responsible for the volume increase as it does not lose functionality upon boiling (Delcour et al., 1998). However, it may account for the significant volume increase of ca. 6% when oat bread was supplemented with the boiled extract. Silver-stained SDS-PAGE gels did not reveal a strong impact of boiling on the protein composition. Small differences in the bands of high and low MM proteins (disappearance of bands larger than ca. 45 kDa and smaller than ca. 30 kDa) were observed. a-Amylase, endoxylanase and endo-b-glucanase activities were strongly reduced, while endopeptidase activity was not affected by boiling (data not shown). Pauly and Delcour (submitted as back-toback publication) noted that heat-treated oat flour contains active endopeptidases but no active a-amylase, endoxylanase and endob-glucanase. Foam formation capacity of the rye extract was not affected by boiling, whereas the foam stability after 30 min of the boiled rye extract was only ca. 7% lower than that of the nontreated extract (data not shown). Based on the above, it seems that enzyme activities are more important than the foaming properties of the extract for the oat bread volume increase. 3.2. Batch ion exchange fractionation of the rye aqueous extract Crude batch CEC and AEC of the extract yielded their unbound and eluate fractions. Since dialysis not only removes buffer salts but also low molecular weight (<6e8 kDa) extract components, the unfractionated extract was also dialyzed to produce an additional control sample. This removed about half of the extract (Table 1). The dialyzed extract resulted in a smaller oat bread volume increase than the untreated one (Fig. 1.A), indicating that components with MM below 6e8 kDa play a role in the oat bread improving effect of the rye extract. Such low MM components include mono-, di- and oligosaccharides, amino acids, oligopeptides and small proteins, minerals, … (Delcour and Hoseney, 2010). It is very unlikely that the sugars in the extract would have caused the volume increase as the oat bread recipe already contained 6.0% (on flour weight basis) sucrose and thus sufficient fermentable sugars for the complete bread making process. While the other low MM components such as amino acids, peptides, minerals and vitamins could serve as nutrient for yeast and thereby enhance its activity (Belitz et al., 2009), Rheofermentometer analyses showed that total carbon dioxide production in batter to which rye extract was added was not significantly different (p < 0.05) from that by the control batter (data not shown). This indicates that rye extract components of low MM did not affect yeast functionality in oat batter. As a result of using AEC or CEC, most rye extract components ended up in the unbound fractions (Table 1). Since the ion exchange matrices mainly bind proteins, proteins were enriched in the eluate fractions, whereas both unbound fractions had lower protein concentrations (Table 1). Among the fractions obtained with ion exchange chromatography, inclusion of the eluate from AEC and the unbound fraction in CEC resulted in the largest volume increases (Fig. 1.A). Both fractions partially contain the same components
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Table 1 Yields, protein levels and dosages used in oat bread making of the rye extract fractions obtain after boiling, dialysis and anion or cation exchange chromatography (AEC or CEC, respectively). See also Fig. 3 for identity of fractions IeIV. Fraction
Yield (%)a
Protein (%)a
Dosage used in oat bread making (%)b
Untreated Boiled Dialyzed Batch ion exchange AEC unbound fraction AEC eluate CEC unbound fraction CEC eluate Column AEC I II III IV
100.0 81.4 53.0
19.7 (±0.4) 16.2 (±0.3) 27.4 (±0.9)
2.00 1.63 1.06
40.3 7.9 34.2 6.3
17.8 (±0.2) 65.8 (±1.7) 6.4 (±0.3) 74.8 (±0.3)
0.81 0.16 0.68 0.13
5.1 1.2 0.5 0.6
64.8 66.1 73.8 63.6
0.10 0.04 0.02 0.02
a b
(±2.0) (±0.2) (±0.5) (±2.1)
and and and and
0.30 0.12 0.06 0.06
Expressed on dry matter basis. Expressed on dry matter basis, replacing an equal weight of oat flour.
Fig. 1. Specific loaf volumes of oat breads supplemented with untreated and (A) boiled extract; (B) batch anion (AEC) and cation (CEC) exchange chromatography run-through and elution fractions and (C) Q-SFF AEC elution fractions (see Fig. 3 for identity of fractions IeIV). Specific loaf volume of control oat bread (without added extract) was 2.15 cm3/g.
since those with a pI below 4.5 are present in the AEC eluate and CEC unbound fraction (Supplementary Fig. 1). The major protein band in both fractions had a MM between 50 and 60 kDa as shown with silver-stained SDS-PAGE (Fig. 2.B). This was the most clear protein band in the run-through fraction obtained with CEC, but
many other minor proteins of varying MM were also present. The volume increase obtained by adding the AEC eluate was comparable to that obtained with 1.0% oat flour replacement by rye extract (Pauly and Delcour, submitted as back-to-back publication). The AEC eluate dosage was only 0.16%, indicating enrichment of active
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between 4.5 and 8.5 can also contribute to oat bread quality (Supplementary Fig. 1). The two major protein bands in the unbound AEC fraction had MMs of ca. 35e40 kDa and 70e80 kDa (Fig. 2.B). In addition, arabinoxylan was strongly enriched in this unbound fraction (data not shown) while it had no impact on loaf volume. Hence, ion exchange chromatography gave further evidence that arabinoxylan plays only a limited role in improving oat bread quality. Delcour et al. (1998) earlier reported on the impact of rye waterextractable components on wheat bread quality. They showed, also with ion exchange chromatography, that the most active fractions in bread making contained (glyco)proteins with a pI higher than 7.5. Based on this observation and on what was shown here, the components responsible for the volume increase in oat bread differ from those causing beneficial effects in wheat bread making as studied earlier by Delcour et al. (1998). 3.3. Separation of the rye aqueous extract with column AEC
Fig. 2. Silver-stained SDS-PAGE profiles of untreated and batch anion (AEC) and cation (CEC) exchange chromatography unbound and elution fractions (A) and Q-SFF AEC elution fractions (B). Lanes M: molecular mass marker (kDa); lane 1: untreated rye extract; lane 2: boiled rye extract; lane 3: batch AEC run-through; lane 4: batch AEC eluate; lane 5: batch CEC run-through; lane 6: batch CEC eluate; lane 7: Q-SFF fraction I: lane 8: Q-SFF fraction II; lane 9: Q-SFF fraction III; lane 10: Q-SFF fraction IV.
component(s) in this fraction. The volume improving effects of the AEC eluate and CEC runthrough suggest that the most active factor(s) in the rye extract has/have a pI lower than 4.5. However, adding the eluate from CEC still resulted in a loaf volume increase (Fig. 1.A). This, in combination with the fact that supplementation with the unbound fraction from AEC (i.e. components with a pI above 8.5) had no impact on oat loaf volume (Fig. 1.A), indicates that component(s) having a pI
Since (i) adding only a low dosage of the AEC eluate already resulted in a large volume increase, thus indicating enrichment of the active factor(s) in the eluate, and since (ii) AEC yielded a good separation of active and non-active components (Section 3.2), it was used to further separate proteins in the rye extract. This way, the eluate was divided into four fractions (I to IV) (Fig. 3). The NaCl gradient and division into fractions was based on preliminary experiments which showed that the most active components elute at concentrations above 0.3 M NaCl (data not shown). When fractions I to IV were used in bread making in dosages similar to those of the untreated extract and taking the yields into account, no volume increases were obtained (data not shown). When the dosages used in bread making were tripled (Table 1), volume increases of 16.8%, 12.5% and 10.5% were noted for fractions III, I and II, respectively (Fig. 1.B). Adding fraction IV had no impact on oat bread loaf volume. That higher dosages were needed to obtain volume increases indicates that (i) more than one active factor was responsible for the volume increase and that they were now divided over fractions I to III, or that (ii) the active factor was not completely enriched in one fraction. SDS-PAGE showed that most proteins were present in more than one fraction (Fig. 2.C). This was also expected because the peaks in the elution profile were not well resolved (Fig. 3). The most intense protein band in fraction III, which gave the most pronounced volume increase while it was dosed at only 0.06%, had a MM of around 70 kDa. This band was also affected when the rye extract was boiled (Section 3.1 and Fig. 2.A). The protein with a MM between 50 and 60 kDa, which showed up as the only clear band in the SDS-PAGE profile of the CEC run-through (Section 3.2 and Fig. 2.B) was more abundant in fraction I than in fractions II and III, while the dosage of fraction I was 2.5 and 5 times higher than that of fractions II and III, respectively. This indicates that this protein is likely not the (only) active factor. The same reasoning holds for proteins with MMs of ca. 45 kDa [which might be an a-amylase (Muralikrishna and Nirmala, 2005)] and those with MMs below 30 kDa. 4. Conclusion In order to gain more information on the identity of the active factor(s) responsible for increasing oat loaf volume, rye aqueous extract was fractionated with ion exchange chromatography. Since several fractions had a positive effect on loaf volume, the volume increase is likely due to more than one component. Because boiling destroyed almost all activity and fractions enriched in the active factor(s) were obtained with AEC, we conclude that mainly protein is responsible for the volume increase. The most active factor(s) in
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Fig. 3. Anion exchange chromatography profile of the water extract isolated from rye flour with indication of the pooled fractions (IeIV). The extinction at 280 nm ( concentration ( ) are shown.
the extract had a pI lower than 4.5, but also component(s) with a pI between 4.5 and 8.5 increased oat loaf volume, while those having a pI higher above 8.5 did not contribute to loaf volume. Enzymes seem to be more important than surface active proteins, but the most active factor is probably neither an a-amylase nor a peptidase. Components smaller than 6e8 kDa also contribute to oat loaf volume, since addition of the dialyzed extract resulted in a lower volume increase than when the untreated extract was added. Arabinoxylan did not appear to be important. Acknowledgements The authors thank Ir. Astrid Maes for technical assistance and Dr. Ellen Fierens for fruitful discussions. Anneleen Pauly gratefully acknowledges the Research Foundation - Flanders (FWO - Vlaanderen, Brussels, Belgium) for a position as postdoctoral researcher. This work is part of the Methusalem programme ‘Food for the future’ (2007e2021). Jan A. Delcour is W.K. Kellogg Chair in Cereal Science and Nutrition at the KU Leuven. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jcs.2017.10.007. References Belitz, H.-D., Grosch, W., Schieberle, P., 2009. Food Chemistry. Springer-Verlag, Berlin Heidelberg. , D., Braaten, J.T., Scott, F.W., Wood, P.J., Riedel, K.D., Wolynetz, M.S., Brule Collins, M.W., 1994a. High b-glucan oat bran and oat gum reduce postprandial blood glucose and insulin in subjects with and without type 2 diabetes. Diabet. Med. 11, 312e318. Braaten, J.T., Wood, P.J., Scott, F.W., Wolynetz, M.S., Lowe, M.K., Bradley-White, P., Collins, M.W., 1994b. Oat b-glucan reduces cholesterol concentration in hypercholesterolemic subjects. Eur. J. Clin. Nutr. 48, 465e474. Brijs, K., Bleukx, W., Delcour, J.A., 1999. Proteolytic activities in dormant rye (Secale cereale L.) grain. J. Agric. Food Chem. 47, 3572e3578. Caessens, P.W.J.R., Gruppen, H., Visser, S., van Aken, G.A., Voragen, A.G.J., 1997. Plasmin hydrolysis of beta-casein: foaming and emulsifying properties of the fractionated hydrolysate. J. Agric. Food Chem. 45, 2935e2941.
) and NaCl
Courtin, C.M., Delcour, J.A., 2002. Arabinoxylans and endoxylanases in wheat flour bread-making. J. Cereal Sci. 35, 225e243. Delcour, J.A., Hoseney, R.C., 2010. Principles of Cereal Science and Technology. AACCI, St. Paul, MN, USA. Delcour, J.A., Joye, I.J., Pareyt, B., Wilderjans, E., Brijs, K., Lagrain, B., 2012. Wheat gluten functionality as a quality determinant in cereal-based food products. Annu. Rev. Food Sci. Technol. 3, 1e24. Delcour, J.A., Van Haesendonck, I.P., Cleemput, G., Rogers, D.E., Hoseney, R.C., 1998. Partial purification of a water-extractable rye (Secale cereale) protein capable of improving the quality of wheat bread. Cereal Chem. 75, 403e407. Dewettinck, K., Van Bockstaele, F., Kühne, B., Van de Walle, D., Courtens, T.M., Gellynck, X., 2008. Nutritional value of bread: influence of processing, food interaction and consumer perception. J. Cereal Sci. 48, 243e257. Hager, A.-S., Bosmans, G.M., Delcour, J.A., 2014. Physical and molecular changes during the storage of gluten-free rice and oat bread. J. Agric. Food Chem. 62, 5682e5689. Hager, A.-S., Wolter, A., Czerny, M., Bez, J., Zannini, E., Arendt, E.K., Czerny, M., 2012. Investigation of product quality, sensory profile and ultrastructure of breads made from a range of commercial gluten-free flours compared to their wheat counterparts. Eur. Food Res. Technol. 235, 333e344. €chsto €tter, A., Becker, T., 2012. Possibilities to increase the quality in Houben, A., Ho gluten-free bread production: an overview. Eur. Food Res. Technol. 235, 195e208. Hüttner, E.K., Arendt, E.K., 2010. Recent advances in gluten-free baking and the current status of oats. Trends Food Sci. Technol. 21, 303e312. Izydorczyk, M.S., Dexter, J.E., 2008. Barley b-glucans and arabinoxylans: molecular structure, physicochemical properties, and uses in food products - a review. Food Res. Int. 41, 850e868. Kerckhoffs, D., Hornstra, G., Mensink, R., 2003. Cholesterol lowering effect of bglucan from oat bran in mildly hypercholesterolemic subjects may decrease when b-glucan is incorporated into bread and cookies. Am. J. Clin. Nutr. 78, 221e227. Lohi, S., Mustalahti, K., Kaukinen, K., Laurila, K., Collin, P., Rissanen, H., Lohi, O., €ki, M., 2007. Increasing prevalence of Bravi, E., Gasparin, M., Reunanen, A., Ma coeliac disease over time. Alimentary Pharmacol. Ther. 26. MacRitchie, F., 1976. Liquid phase of dough and its role in baking. Cereal Chem. 53, 318e326. Muralikrishna, G., Nirmala, M., 2005. Cereal a-amylases - an overview. Carbohydr. Polym. 60, 163e173. Pauly, A., Delcour, J. A., submitted as back-to-back publication. Impact of cereal water-extractable components on oat bread quality. Pauly, A., Pareyt, B., Fierens, E., Delcour, J.A., 2014. Fermentation affects the composition and foaming properties of the aqueous phase of dough from soft wheat flour. Food Hydrocoll. 37, 221e228. Renzetti, S., Courtin, C.M., Delcour, J.A., Arendt, E.K., 2010. Oxidative and proteolytic enzyme preparations as promising improvers for oat bread formulations: rheological, biochemical and microstructural background. Food Chem. 119, 1465e1473. Renzetti, S., Dal Bello, F., Arendt, E.K., 2008. Microstructure, fundamental rheology and baking characteristics of batters and breads from different gluten-free
A. Pauly, J.A. Delcour / Journal of Cereal Science 79 (2018) 141e147 flours treated with a microbial transglutaminase. J. Cereal Sci. 48, 33e45. Sciarini, L.S., Ribotta, P.D., Leon, A.E., Perez, G.T., 2012. Incorporation of several additives into gluten free breads: effect on dough properties and bread quality. J. Food Eng. 111, 590e597. € m, L., Li, L., Rakszegi, M., Fras, A., Shewry, P.R., Piironen, V., Lampi, A.-M., Nystro Boros, D., Gebruers, K., Courtin, C.M., Delcour, J.A., Andersson, A.A.M.,
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Dimberg, L., Bedo, Z., Ward, J.L., 2008. Phytochemical and fiber components in oat varieties in the HEALTHGRAIN diversity screen. J. Agric. Food Chem. 56, 9777e9784. Watson, E., 2012. The rise and rise of gluten-free: but can the meteoric growth continue? http://www.foodnavigator-usa.com/Markets/The-rise-and-rise-ofgluten-free-But-can-the-meteoric-growth-continue online, 8 February 2016.
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Partial purification of components in rye water extractables which improve the quality of oat bread Anneleen Pauly, Jan A. Delcour* Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 October 2016 Accepted 15 October 2017 Available online 17 October 2017
Unlike wheat bread, the dough of which has a visco-elastic network and high gas-holding capacity, oat bread generally has a low volume and a dense structure. We showed earlier that including rye waterextractable components in an oat bread batter recipe increases loaf volume by ca. 30% (Pauly and Delcour, submitted as back-to-back publication). We here report on efforts to identify the active factor(s). Anion exchange chromatography allowed enriching the active factor(s). This and the fact that only a limited volume increase was observed when oat batter was supplemented with boiled rye extract indicate that proteins are likely the most important components responsible for the volume increase. While the most active factor(s) had a pI below 4.5, components with pI values between 4.5 and 8.5 also contributed to oat loaf volume. Alkaline rye components (pI > 8.5) or rye arabinoxylan had no impact. Rye water-extractable components smaller than 6e8 kDa also had a positive impact on loaf volume. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Gluten-free/oat bread Ion exchange chromatography Water-extractable components Loaf volume
1. Introduction Celiac disease is an autoimmune-mediated disease triggered by ingesting wheat gluten or related proteins. Its reported prevalence has increased over the last 30 years. It probably affects more than 1% of the world population (Lohi et al., 2007; Hüttner and Arendt, 2010). The present only effective treatment is a life-long and strict adherence to a gluten-free diet. Although initially developed for patients suffering from celiac disease, gluten-free diets are also followed e.g. as part of a life-style choice. In the United States, only 7% of the gluten-free products on the market are purchased by celiac patients (Watson, 2012). Furthermore, consumers nowadays increasingly choose bread prepared from cereals other than wheat because of nutrition related reasons (e.g. levels of dietary fiber, essential amino acids, minerals) (Dewettinck et al., 2008). However, the absence of wheat gluten presents technological challenges. Indeed, wheat gluten has unique visco-elastic properties which
Abbreviations: AEC, anion exchange chromatography; CEC, cation exchange chromatography; dm, dry matter; E440, extinction at 440 nm; E590, extinction at 590 nm; MM, molecular mass; MWCO, molecular weight cut-off; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)-aminomethane. * Corresponding author. E-mail address: [email protected] (J.A. Delcour). https://doi.org/10.1016/j.jcs.2017.10.007 0733-5210/© 2017 Elsevier Ltd. All rights reserved.
confer upon bread its desired structure (Delcour et al., 2012). The quality of non-wheat bread products is, hence, much inferior to that of their counterparts containing gluten. Non-wheat bread has low loaf volume, an uneven distribution of large gas cells in the crumb and firm and crumbly texture (Hager et al., 2012; Houben et al., 2012). To overcome such quality deficiencies, additives are frequently used in non-wheat bread recipes. Hydrocolloids (e.g. xanthan gum, cellulose derivatives such as hydroxypropyl methyl cellulose), surfactants (e.g. diacetyl tartaric esters of mono- and diacylglycerols, sodium stearoyl lactylate) and enzymes (e.g. amylases, transglutaminases) are well-studied additives in non-wheat bread making (Renzetti et al., 2008, 2010; Hüttner and Arendt, 2010; Sciarini et al., 2012). Cereal water extractables contain several components which may well increase batter stability. Non-starch polysaccharides may act by virtue of their water binding and viscosity increasing capacity (Courtin and Delcour, 2002; Izydorczyk and Dexter, 2008). Also, much as some surfactants, some water-extractable surface active proteins can stabilize gas cells (MacRitchie, 1976; Pauly et al., 2014). Cereal aqueous extracts also contain various enzymes. In a previous paper, we included water extracts from barley, rye, oat and wheat flour in simple oat bread recipes (Pauly and Delcour, submitted as back-to-back publication). Oat contains high levels of the dietary fiber (1 / 3, 1 / 4)-b-D-glucan (further referred to as b-glucan) (Shewry et al., 2008). Sufficient high intake of b-glucan
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can reduce postprandial blood glucose, insulin and serum cholesterol levels (Braaten et al., 1994a, 1994b; Kerckhoffs et al., 2003). Other oat nutrients include essential amino acids, vitamins, minerals and antioxidants (Dewettinck et al., 2008). Oat can also be used as raw material for gluten-free products, although some controversy exists on the matter (Hüttner and Arendt, 2010). Replacing an equal weight of oat flour by rye extract [5.0% on dry matter basis (dm)] resulted in a larger loaf volume increase (þ27%) than did replacing oat flour by barley (þ21%), wheat (þ14%) or oat (þ5%) extracts in a similar way. Furthermore, we showed that replacing only 2.0% dm oat flour by rye extract made crumb significantly softer, while it had no impact on crumb mean cell area and number of cell per surface unit. Amongst the four extracts, that from rye contained the highest arabinoxylan levels and enzyme activities. High b-glucan levels and extract viscosity are probably not the main factors contributing to the volume increasing effect of rye extract (Pauly and Delcour, submitted as back-to-back publication). In the present paper, we contribute to the identification of the active component(s) in the rye extract responsible for oat loaf volume increases. To that end, the rye extract is treated in order to enrich the active component(s) and the impact on oat loaf volume is evaluated. We here report on the outcome of our work. 2. Materials and methods 2.1. Materials Commercial oat flour (moisture, protein, and ash contents on dm respectively 10.1%, 12.4%, and 1.67%) and rye flour (corresponding data 12.9%, 7.2%, and 0.82%) were from Raisio Nutrition (Raisio, Finland) and from Koopmans (Leeuwarden, The Netherlands), respectively. Dry yeast was from Puratos (Groot-Bijgaarden, Belgium). Sugar and salt were commercial food grade products. SP Sepharose Big Beads and Q Sepharose Fast Flow ion exchange matrices for batch and column fractionations were from GE Healthcare (Uppsala, Sweden). All other chemicals, solvents and reagents were from Sigma-Aldrich (Bornem, Belgium) and were at least analytical grade, unless specified otherwise. 2.2. Experimental 2.2.1. Boiling of the rye extract Aqueous extracts of rye flour were prepared as in Pauly and Delcour (submitted as back-to-back publication). For some experiments, the freeze-dried extract (3.0 g) was suspended in 100 mL deionized water, boiled for 15 min, cooled to room temperature and centrifuged (5000 g, 15 min). The supernatant was then freezedried. 2.2.2. Batch ion exchange fractionation Fractionation into bound and unbound material was performed with batch anion exchange chromatography (AEC) or cation exchange chromatography (CEC) on SP Sepharose Big Beads or Q Sepharose Fast Flow, respectively. The ion exchange matrices (ca. 500 mL) were equilibrated with 25 mM tris-(hydroxymethyl)aminomethane (Tris) - HCl (pH 8.5) or 25 mM sodium acetate (pH 4.5) for AEC or CEC, respectively. The suspensions of matrix and extract (3.0 g in 100 mL of these media) were shaken overnight at 6 C and then brought onto a P1 glass filter. The matrix was rinsed with 25 mM Tris-HCl (AEC) or 25 mM sodium acetate (CEC) to remove unbound material. Bound material was eluted with 1.0 L 1.0 M NaCl. The four obtained fractions (unbound and eluate of AEC or CEC) were dialyzed [molecular weight cut-off (MWCO) 6e8 kDa] against deionized water and freeze-dried. The untreated extract
was also dialyzed (MWCO 6e8 kDa) against deionized water to investigate the impact of removing small rye components. 2.2.3. Column anion exchange chromatography Extracts [3.0 g in 150 mL 25 mM Tris-HCl (pH 8.5)] were separated by AEC on a Q Sepharose Fast Flow column (diameter 16 mm, length 230 mm). They were suspended in loading buffer [25 mM Tris-HCl (pH 8.5)] and centrifuged (5000 g, 15 min, 4 C). The supernatants were loaded onto the column, equilibrated with loading buffer, at a flow rate of 0.3 mL/min. After rinsing the column with loading buffer (flow rate 1.0 mL/min) to remove unbound material (¼ run-through fraction), bound molecules were eluted using a gradient of 1.0 M NaCl in 25 mM Tris-HCl (pH 8.5) at a flow rate of 1.0 mL/min. The NaCl concentration was first increased linearly from 0 to 300 mM NaCl in 150 m), and then further increased to 800 mM in 500 mL. This elution gradient was chosen based on preliminary tests. The fractions detected at 280 nm were dialyzed (MWCO 6e8 kDa) against deionized water and freeze-dried. 2.2.4. Protein electrophoresis Protein composition was analyzed with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Freeze-dried extracts or extract fractions were dissolved in sample buffer [12.5 mM Tris-HCl; 4.0% (w/v) SDS; 30% (v/v) glycerol; 0.004% (w/v) bromophenol blue; pH 6.8] to obtain a protein concentration of approximately 2.0 mg/mL. After boiling (5 min) and centrifugation (10,900 g; 3 min), the proteins in the supernatant were separated under non-reducing conditions with a PhastSystem (GE Healthcare) according to GE Healthcare separation technique file 110. Molecular mass (MM) markers (LMW-SDS Marker Kit, GE Healthcare) were also analyzed. Gels were stained with sensitive silver staining as described in the GE Healthcare development technique file 210. 2.2.5. Enzyme activities Enzyme activities were determined at pH 5.4 [i.e. average batter pH during fermentation (Pauly and Delcour, submitted as back-toback publication)] and 30 C (i.e. fermentation temperature). aAmylase, endoxylanase and endo-b-glucanase activity levels were determined in triplicate with the Amylazyme, Xylazyme AX and bGlucazyme methods (Megazyme, Bray, Ireland), respectively. Freeze-dried extracts (50 mg) were suspended in 25 mM 10.0 mL sodium maleate buffer (pH 5.4) containing 5.0 mM CaCl2 for the Amylazyme method or 25 mM sodium acetate buffer (pH 5.4) for the Xylazyme AX and b-Glucazyme methods. After centrifugation (5000 g, 15 min), the supernatants (1.0 mL) were equilibrated for 10 min at 30 C before adding an Amylazyme, Xylazyme AX or a bGlucazyme tablet, respectively. After 90 min incubation for aamylase, 18 h for endoxylanase and 5 h for endo-b-glucanase activity levels, the reactions were stopped by adding 10.0 mL of a Tris solution (2.0% or 1.0% for the Amylazyme and b-Glucazyme or Xylazyme AX methods, respectively). The solutions were vigorously vortexed, filtered through a MN 615 filter (Macherey-Nagel, Düren, Germany) and their extinction at 590 nm (E590) measured. Control samples were extract suspensions incubated without the respective tablets. Corrections were made for non-enzymatic color release by the respective tablets. a-Amylase, endoxylanase and endo-bglucanase activities were expressed as units per gram dm extract. One unit is the enzyme activity which increases E590 by 1.00 per hour of incubation under the conditions of the test. Endopeptidase activity levels were determined in triplicate using azocasein substrate (Brijs et al., 1999). Freeze-dried extracts (50 mg) were suspended in 10.0 mL 25 mM sodium acetate buffer (pH 5.4). After centrifugation (5000 g, 15 min), 250 mL supernatant was combined with 350 mL 1.4% (w/v) azocasein. After 24 h
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incubation at 30 C, 500 mL cool (7 C) 10.0% (w/v) trichloroacetic acid was added. The precipitated proteins were removed by centrifugation (9600 g, 10 min). Finally, 500 mL 0.5 M sodium hydroxide was added to 500 mL supernatant and the extinction at 440 nm (E440) was measured. Activity was expressed as endopeptidase units per gram dm. One unit is the enzyme activity which increases E440 by 1.00 per hour of incubation under the experimental conditions. 2.2.6. Foaming properties The foaming properties of the freeze-dried boiled extract were determined in duplicate for 50.0 mL solutions with the whipping method of Caessens et al. (1997), but with small modifications. DL Solutions were poured in a graduated glass cylinder (internal diameter: 60 mm) and whipped for 70 s using a rotating propeller (outer diameter: 45.0 mm, thickness: 0.4 mm, diameter cylindrical bar: 6.0 mm) at 2000 rpm. The initial foam volume was measured 2 min after the start of whipping, whereas foam volume was monitored during 60 min. 2.2.7. Small scale oat bread making A simple oat bread recipe based on Hager et al. (2014) was used. The baking procedure was adapted to 25 g batter to minimize the amounts of treated and fractionated samples needed. Batter was prepared with oat flour, 1.5% salt, 6.0% sugar, 3.0% yeast and 90% deionized water (percentages based on flour weight at 14.0% moisture). When treated extracts or extract fractions were tested (levels up to 2.0%), they replaced an equal weight of oat flour. The yield of the treated or fractionated samples was taken into account when dosing in order to allow drawing conclusions on enrichment of the active factor(s). For example, when a fraction was obtained in a yield of 50%, it was dosed at 1.0% (¼ 50 % 2.0 %) to be comparable with a 2.0% dosage of the untreated rye extract. The optimal water addition level was determined by preliminary baking trials. Total batter weight was 150 g. Yeast was suspended in the water (preincubated at 30 C) and rested for 10 min in a fermentation cabinet (National Manufacturing, Lincoln, NE, USA) at 30 C and a relative humidity of 90%. The yeast suspension was then added to the premixed dry ingredients (flour, sugar, salt and, when tested, freeze-dried extract). Mixing was at speed 1 for 60 s with a KitchenAid (St. Joseph, MI, USA) KPM5 mixer and, after the bowl content was scraped down, further mixed for 90 s at speed 4. Slightly greased baking tins were filled with 25 g batter and placed in the fermentation cabinet for 30 min at 30 C and 90% relative humidity. Fermented batters were then baked in a Condilux deck oven (Hein, Strasse, Luxemburg) for 30 min at 195 C. After cooling for 120 min, bread samples were weighed and loaf volumes determined with a Volscan Profiler (Stable Microsystems, Godalming, Surrey, UK). Baking trials were performed at least in fourfold. 2.2.8. Rheofermentometer analysis Rheofermentometer analyses of control oat batter or oat batter of which 2.0% flour weight was replaced by rye extract were performed. Batter (300 g, prepared as described in Section 2.2.7) was placed in the basket of the Rheofermentometer (Chopin, Villeneuve-La-Garenne, France) and covered with the 254 g piston (without additional weight). The analyses were performed at 30 C during 90 min. Total carbon dioxide production was derived from the gas release curve. 2.2.9. Statistical analysis Significant differences (a < 0.05) for several variables, based on at least triplicate replications, were determined using the one-way ANOVA procedure with JMP® Pro 11.2.0 (SAS institute, Cary, NC, USA).
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3. Results and discussion 3.1. Boiling of rye extract We earlier showed that replacing only 2.0% dm oat flour by water-extractable rye flour components sufficed for a maximum volume increase of 34% (Pauly and Delcour, submitted as back-toback publication). In the present study, dosages were based on this replacement level. In a first step, the rye extract was boiled to denature proteins and inactivate enzymes. The coagulated material was removed by centrifugation and accounted for ca. 20% dm (Table 1). Boiling the rye extract resulted in almost complete loss of activity (6.2% volume increase, data not shown). This indicates that arabinoxylan is not the major component responsible for the volume increase as it does not lose functionality upon boiling (Delcour et al., 1998). However, it may account for the significant volume increase of ca. 6% when oat bread was supplemented with the boiled extract. Silver-stained SDS-PAGE gels did not reveal a strong impact of boiling on the protein composition. Small differences in the bands of high and low MM proteins (disappearance of bands larger than ca. 45 kDa and smaller than ca. 30 kDa) were observed. a-Amylase, endoxylanase and endo-b-glucanase activities were strongly reduced, while endopeptidase activity was not affected by boiling (data not shown). Pauly and Delcour (submitted as back-toback publication) noted that heat-treated oat flour contains active endopeptidases but no active a-amylase, endoxylanase and endob-glucanase. Foam formation capacity of the rye extract was not affected by boiling, whereas the foam stability after 30 min of the boiled rye extract was only ca. 7% lower than that of the nontreated extract (data not shown). Based on the above, it seems that enzyme activities are more important than the foaming properties of the extract for the oat bread volume increase. 3.2. Batch ion exchange fractionation of the rye aqueous extract Crude batch CEC and AEC of the extract yielded their unbound and eluate fractions. Since dialysis not only removes buffer salts but also low molecular weight (<6e8 kDa) extract components, the unfractionated extract was also dialyzed to produce an additional control sample. This removed about half of the extract (Table 1). The dialyzed extract resulted in a smaller oat bread volume increase than the untreated one (Fig. 1.A), indicating that components with MM below 6e8 kDa play a role in the oat bread improving effect of the rye extract. Such low MM components include mono-, di- and oligosaccharides, amino acids, oligopeptides and small proteins, minerals, … (Delcour and Hoseney, 2010). It is very unlikely that the sugars in the extract would have caused the volume increase as the oat bread recipe already contained 6.0% (on flour weight basis) sucrose and thus sufficient fermentable sugars for the complete bread making process. While the other low MM components such as amino acids, peptides, minerals and vitamins could serve as nutrient for yeast and thereby enhance its activity (Belitz et al., 2009), Rheofermentometer analyses showed that total carbon dioxide production in batter to which rye extract was added was not significantly different (p < 0.05) from that by the control batter (data not shown). This indicates that rye extract components of low MM did not affect yeast functionality in oat batter. As a result of using AEC or CEC, most rye extract components ended up in the unbound fractions (Table 1). Since the ion exchange matrices mainly bind proteins, proteins were enriched in the eluate fractions, whereas both unbound fractions had lower protein concentrations (Table 1). Among the fractions obtained with ion exchange chromatography, inclusion of the eluate from AEC and the unbound fraction in CEC resulted in the largest volume increases (Fig. 1.A). Both fractions partially contain the same components
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Table 1 Yields, protein levels and dosages used in oat bread making of the rye extract fractions obtain after boiling, dialysis and anion or cation exchange chromatography (AEC or CEC, respectively). See also Fig. 3 for identity of fractions IeIV. Fraction
Yield (%)a
Protein (%)a
Dosage used in oat bread making (%)b
Untreated Boiled Dialyzed Batch ion exchange AEC unbound fraction AEC eluate CEC unbound fraction CEC eluate Column AEC I II III IV
100.0 81.4 53.0
19.7 (±0.4) 16.2 (±0.3) 27.4 (±0.9)
2.00 1.63 1.06
40.3 7.9 34.2 6.3
17.8 (±0.2) 65.8 (±1.7) 6.4 (±0.3) 74.8 (±0.3)
0.81 0.16 0.68 0.13
5.1 1.2 0.5 0.6
64.8 66.1 73.8 63.6
0.10 0.04 0.02 0.02
a b
(±2.0) (±0.2) (±0.5) (±2.1)
and and and and
0.30 0.12 0.06 0.06
Expressed on dry matter basis. Expressed on dry matter basis, replacing an equal weight of oat flour.
Fig. 1. Specific loaf volumes of oat breads supplemented with untreated and (A) boiled extract; (B) batch anion (AEC) and cation (CEC) exchange chromatography run-through and elution fractions and (C) Q-SFF AEC elution fractions (see Fig. 3 for identity of fractions IeIV). Specific loaf volume of control oat bread (without added extract) was 2.15 cm3/g.
since those with a pI below 4.5 are present in the AEC eluate and CEC unbound fraction (Supplementary Fig. 1). The major protein band in both fractions had a MM between 50 and 60 kDa as shown with silver-stained SDS-PAGE (Fig. 2.B). This was the most clear protein band in the run-through fraction obtained with CEC, but
many other minor proteins of varying MM were also present. The volume increase obtained by adding the AEC eluate was comparable to that obtained with 1.0% oat flour replacement by rye extract (Pauly and Delcour, submitted as back-to-back publication). The AEC eluate dosage was only 0.16%, indicating enrichment of active
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between 4.5 and 8.5 can also contribute to oat bread quality (Supplementary Fig. 1). The two major protein bands in the unbound AEC fraction had MMs of ca. 35e40 kDa and 70e80 kDa (Fig. 2.B). In addition, arabinoxylan was strongly enriched in this unbound fraction (data not shown) while it had no impact on loaf volume. Hence, ion exchange chromatography gave further evidence that arabinoxylan plays only a limited role in improving oat bread quality. Delcour et al. (1998) earlier reported on the impact of rye waterextractable components on wheat bread quality. They showed, also with ion exchange chromatography, that the most active fractions in bread making contained (glyco)proteins with a pI higher than 7.5. Based on this observation and on what was shown here, the components responsible for the volume increase in oat bread differ from those causing beneficial effects in wheat bread making as studied earlier by Delcour et al. (1998). 3.3. Separation of the rye aqueous extract with column AEC
Fig. 2. Silver-stained SDS-PAGE profiles of untreated and batch anion (AEC) and cation (CEC) exchange chromatography unbound and elution fractions (A) and Q-SFF AEC elution fractions (B). Lanes M: molecular mass marker (kDa); lane 1: untreated rye extract; lane 2: boiled rye extract; lane 3: batch AEC run-through; lane 4: batch AEC eluate; lane 5: batch CEC run-through; lane 6: batch CEC eluate; lane 7: Q-SFF fraction I: lane 8: Q-SFF fraction II; lane 9: Q-SFF fraction III; lane 10: Q-SFF fraction IV.
component(s) in this fraction. The volume improving effects of the AEC eluate and CEC runthrough suggest that the most active factor(s) in the rye extract has/have a pI lower than 4.5. However, adding the eluate from CEC still resulted in a loaf volume increase (Fig. 1.A). This, in combination with the fact that supplementation with the unbound fraction from AEC (i.e. components with a pI above 8.5) had no impact on oat loaf volume (Fig. 1.A), indicates that component(s) having a pI
Since (i) adding only a low dosage of the AEC eluate already resulted in a large volume increase, thus indicating enrichment of the active factor(s) in the eluate, and since (ii) AEC yielded a good separation of active and non-active components (Section 3.2), it was used to further separate proteins in the rye extract. This way, the eluate was divided into four fractions (I to IV) (Fig. 3). The NaCl gradient and division into fractions was based on preliminary experiments which showed that the most active components elute at concentrations above 0.3 M NaCl (data not shown). When fractions I to IV were used in bread making in dosages similar to those of the untreated extract and taking the yields into account, no volume increases were obtained (data not shown). When the dosages used in bread making were tripled (Table 1), volume increases of 16.8%, 12.5% and 10.5% were noted for fractions III, I and II, respectively (Fig. 1.B). Adding fraction IV had no impact on oat bread loaf volume. That higher dosages were needed to obtain volume increases indicates that (i) more than one active factor was responsible for the volume increase and that they were now divided over fractions I to III, or that (ii) the active factor was not completely enriched in one fraction. SDS-PAGE showed that most proteins were present in more than one fraction (Fig. 2.C). This was also expected because the peaks in the elution profile were not well resolved (Fig. 3). The most intense protein band in fraction III, which gave the most pronounced volume increase while it was dosed at only 0.06%, had a MM of around 70 kDa. This band was also affected when the rye extract was boiled (Section 3.1 and Fig. 2.A). The protein with a MM between 50 and 60 kDa, which showed up as the only clear band in the SDS-PAGE profile of the CEC run-through (Section 3.2 and Fig. 2.B) was more abundant in fraction I than in fractions II and III, while the dosage of fraction I was 2.5 and 5 times higher than that of fractions II and III, respectively. This indicates that this protein is likely not the (only) active factor. The same reasoning holds for proteins with MMs of ca. 45 kDa [which might be an a-amylase (Muralikrishna and Nirmala, 2005)] and those with MMs below 30 kDa. 4. Conclusion In order to gain more information on the identity of the active factor(s) responsible for increasing oat loaf volume, rye aqueous extract was fractionated with ion exchange chromatography. Since several fractions had a positive effect on loaf volume, the volume increase is likely due to more than one component. Because boiling destroyed almost all activity and fractions enriched in the active factor(s) were obtained with AEC, we conclude that mainly protein is responsible for the volume increase. The most active factor(s) in
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Fig. 3. Anion exchange chromatography profile of the water extract isolated from rye flour with indication of the pooled fractions (IeIV). The extinction at 280 nm ( concentration ( ) are shown.
the extract had a pI lower than 4.5, but also component(s) with a pI between 4.5 and 8.5 increased oat loaf volume, while those having a pI higher above 8.5 did not contribute to loaf volume. Enzymes seem to be more important than surface active proteins, but the most active factor is probably neither an a-amylase nor a peptidase. Components smaller than 6e8 kDa also contribute to oat loaf volume, since addition of the dialyzed extract resulted in a lower volume increase than when the untreated extract was added. Arabinoxylan did not appear to be important. Acknowledgements The authors thank Ir. Astrid Maes for technical assistance and Dr. Ellen Fierens for fruitful discussions. Anneleen Pauly gratefully acknowledges the Research Foundation - Flanders (FWO - Vlaanderen, Brussels, Belgium) for a position as postdoctoral researcher. This work is part of the Methusalem programme ‘Food for the future’ (2007e2021). Jan A. Delcour is W.K. Kellogg Chair in Cereal Science and Nutrition at the KU Leuven. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jcs.2017.10.007. References Belitz, H.-D., Grosch, W., Schieberle, P., 2009. Food Chemistry. Springer-Verlag, Berlin Heidelberg. , D., Braaten, J.T., Scott, F.W., Wood, P.J., Riedel, K.D., Wolynetz, M.S., Brule Collins, M.W., 1994a. High b-glucan oat bran and oat gum reduce postprandial blood glucose and insulin in subjects with and without type 2 diabetes. Diabet. Med. 11, 312e318. Braaten, J.T., Wood, P.J., Scott, F.W., Wolynetz, M.S., Lowe, M.K., Bradley-White, P., Collins, M.W., 1994b. Oat b-glucan reduces cholesterol concentration in hypercholesterolemic subjects. Eur. J. Clin. Nutr. 48, 465e474. Brijs, K., Bleukx, W., Delcour, J.A., 1999. Proteolytic activities in dormant rye (Secale cereale L.) grain. J. Agric. Food Chem. 47, 3572e3578. Caessens, P.W.J.R., Gruppen, H., Visser, S., van Aken, G.A., Voragen, A.G.J., 1997. Plasmin hydrolysis of beta-casein: foaming and emulsifying properties of the fractionated hydrolysate. J. Agric. Food Chem. 45, 2935e2941.
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