Note
Availability of two water-soluble fractions in ripe banana on gluten-free breadmaking
2024 Volume 30 Issue 1 Pages 117-123
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2024 Volume 30 Issue 1 Pages 117-123
Gluten is a component of wheat flour and has a vital function in the manufacturing of processed food via the formation of a three-dimensional network. However, gluten is associated with several diseases, such as wheat allergy and celiac disease, and some humans with gluten hypersensitivity are not able to consume wheat products. To address this problem, not only gluten-free cereal flours but also hydrocolloids and gums as gluten substitutes are used as ingredients for gluten-free products. In this study, gluten-free breadmaking was attempted using only ripe banana flour (RBF) and wheat starch as the materials. To understand the role of RBF for gluten-free breadmaking, unripe banana was ripened and RBF was fractionated into three fractions. It was clarified that the water-soluble low molecular fraction (mainly sugars) in RBF acted as a carbon source for fermentation in the dough and the water-soluble high molecular fraction acted as a gluten substitute. Pectin in the water-soluble high molecular fraction was further analyzed for four fractions separated based on differential solvent solubility. The main pectin was chelate-soluble pectin. The RBF was shown to be a good material for gluten-free breadmaking.
Gluten, which is contained in wheat, can cause several diseases, such as wheat allergy, celiac disease, and gluten intolerance (Casper and Atwell, 2014; Kurppa et al., 2014). The development of gluten-free foods is needed for the growing number of people unable to consume wheat products. To eliminate gluten from diets, strategies include using ingredients without gluten and gluten substitutes. However, as gluten has a vital function in the manufacturing of processed food, the production of processed food without gluten presents a challenge. To overcome this problem, various gluten substitutes, such as hydrocolloids and gums, have been developed (Padalino et al., 2016). Further, various gluten-free bread formulations have been proposed (Monteiro, 2021). The use of hydrocolloids, such as hydroxypropyl methylcellulose (HPMC) and xanthan gum, as gluten substitutes has been reported (Lazaridou et al., 2007; Hager and Arendt, 2013). On the other hand, there are reports of several natural sources of ingredients serving as gluten substitutes being used for the manufacture of gluten-free bread, such as banana (Seguchi et al., 2014; Hosokawa et al., 2020), yam (Seguchi et al., 2012), and gagome kelp (Seguchi et al., 2018). In the case of overripe banana, wheat starch, ripe banana, sucrose, and yeast were used as raw materials for gluten-free breadmaking (Seguchi et al., 2014). In the case of ripe banana, only wheat starch, ripe banana, and yeast were used as raw materials for gluten-free breadmaking (Hosokawa et al., 2020). Notably, in the latter case, a high specific volume (SV) was reported, greater than 5 cm3/g. These results show that ripe banana has high potential as a gluten substitute. Accordingly, it is important to clarify the properties of ripe banana in its application to gluten-free breadmaking. With regard to its components, banana is known to undergo several chemical changes during ripening (Loesecke, 1950a). For example, the most conspicuous change is the conversion of starch into sugars during ripening (Loesecke, 1950b; Phillips et al., 2021). In addition, protopectin is converted to pectin and finally into pectic acid. Alterations in the former play a role in the sweetness of banana fruit, while those of the latter participate in the softness and viscosity of banana. However, how these chemical changes affect breadmaking remains to be clarified. Seguchi et al. (2014) reported on the favorable properties for gluten-free breadmaking of overripe banana and that the combination of low and high molecular weight components was important.
There are two important events in breadmaking: first is the generation of carbon dioxide during fermentation by yeast and second is the retention of gas generated in the gluten network of dough. However, it is unknown how the changes in ripe banana participate in breadmaking. The objective of the present study is to clarify how the ripening of banana is related to its utility in gluten-free breadmaking.
Materials Unripe banana fruit (20 kg) treated with ethylene gas was purchased from commercial sources [Musa acuminate (AAA subgroup, Cavendish subgroup), “Giant Cavendish” cultivated in the Philippines]. Ripe banana flour (RBF) was prepared from bananas stored at 20 °C for 0-4 weeks in an incubator (ICB-151LN; AGC Techno Glass Co., Ltd., Tokyo, Japan) according to the methods of a previous study (Hosokawa et al., 2020). Wheat starch WS-525 (Chiba Flour Milling Co., Ltd., Chiba, Japan) was used for breadmaking. Bread flour (Camellia; Nisshin Flour Milling Co., Ltd., Tokyo, Japan) and instant dry yeast (Super Camellia; Nisshin Foods Inc., Tokyo, Japan) were purchased from a local market in Japan. All chemicals used were of reagent grade and were purchased from Fujifilm Wako Chemicals Co. (Osaka, Japan).
Analysis of sugars in RBFs A 1-g portion of each 0–4-week RBF was suspended in 30 mL of 50 % ethanol using an ultrasonic generator (sonicator) for 30 min in an ice bath; the solution was then filled up to 50 mL with 50 % ethanol and filtered using filter paper. The filtrate was dried using a rotary evaporator at 40 °C. The solid sample was then re-dissolved in distilled water and filled up to 50 mL with distilled water. This sample was used for measurement of maltose. On the other hand, the sample was diluted 10 times for measurement of glucose, fructose, and sucrose. The four sugars were analyzed using high-performance liquid chromatography (HPLC). For glucose, fructose, and sucrose, the extracted sugars were separated on an Inertsil NH2 column (3.0 mm × 150 mm, 5 μm; GL Sciences, Tokyo, Japan) incorporated into the HPLC system (LC-20AD; Shimadzu Co., Kyoto, Japan) with a refractive index detector (RID-20A; Shimadzu Co.). The eluents were passed through the column at a flow rate of 0.7 mL·min−1 with isocratic elution of 5 % acetonitrile (v/v) at 25 °C. Maltose was separated on a HPLC system equipped with a TSKgel Sugar AXI column (4.6 mm × 150 mm; Tosoh Co., Tokyo, Japan) at a 0.7 mL · min−1 flow rate and 60 °C. The eluents were reacted with 1 % 1-asparagine at 150 °C, and maltose was detected with a high-sensitivity detection unit (RID-20A; Shimadzu Co.) based on a post-column system. HPLC grade solvents were used (Fujifilm Wako Chemicals Co.).
Size exclusion fractionation of 2-week RBF A 600-g portion of 2-week RBF was suspended in 3 L of distilled water and then dialyzed using a dialysis membrane (molecular weight cut off (MWCO) 6–8 kDa, Spectra/Por 1; Repligen Co., Waltham, MA, USA) against distilled water at 4 °C, 6 times. The dialyzable fraction (F1) was freeze-dried. The non-dialyzable fraction was centrifuged at 14 000 g for 5 min at 4 °C to separate the water-soluble (F2) and water-insoluble fractions (F3); both fractions were freeze-dried.
Dough preparation and breadmaking The straight dough method was employed according to the method of a previous report (Hosokawa et al., 2020). For breadmaking using RBFs, the following ingredients were used in the formulas: 108 g of starch, 12 g of RBF, 2.4 g of dry yeast, and 84 g of water. For breadmaking using fractions prepared from 2-week RBF, the following ingredients were used in the formulas: 108 g of starch, each fraction/combination of fractions (F1, 9.4 g; F2, 0.9 g; and F3, 1.0 g, *in which the weight of each fraction was equivalent to the weight prepared from 12 g of RBF), 2.4 g of dry yeast, and 84 g of water. For breadmaking using F2 and sucrose, the following ingredients were used in the formulas: 108 g of starch, 0.9 g of F2, 5.4 g of sucrose, 2.4 g of dry yeast, and 84 g of water. In the case of breadmaking without RBF, the formula was as follows: 108 g of starch, 2.4 g of dry yeast, and 84 g of water. For breadmaking using bread flour as the control, 108 g of starch and 12 g of RBF were replaced with 120 g of bread flour.
Starch, RBF, fractions, and bread flour were passed through a sifter (20 mesh). The dry yeast was rehydrated by adding water to the dough. The breadmaking was performed according to the method of Hosokawa et al. (2020). The baked loaves were removed from the pans and cooled for 60 min at room temperature. Volume (cm3) of one loaf was measured by the rapeseed displacement method and loaf weight (g) was measured according to the method of Yamauchi et al. (1992). SV [cm3/g] of bread was defined as the quotient between loaf volume (cm3) and loaf weight (g). Each experiment was conducted in triplicate.
Fermentation ability The total gas volume generated at 37 °C for 1 h under the various conditions of this experiment was measured with a Fermograph II AF-1101-10W (ATTO Co., Tokyo, Japan). The half weight of dough used for the above breadmaking was used for measuring fermentation ability. The dough was placed in glass bottles and the volume of gas generated at 37 °C for 1 h was recorded. Each experiment was conducted in triplicate.
Pectin analysis Alcohol insoluble solid (AIS) was prepared from 2-week RBF. A 5-g portion of 2-week RBF was suspended in 20 mL of distilled water and 60 mL of ethanol was added. The suspension was filtrated using a glass filter, followed by washing with 70 % ethanol 10 times, 99 % ethanol 3 times, acetone 3 times, and dimethyl ether 3 times. Then the sample (AIS) obtained as a precipitate was dried under reduced pressure. A 10-mg portion of AIS was immersed using a small amount of ethanol in a 50-mL test tube with a screw cap and 10 mL of 0.05 N HCl was added. The solution was heated for 40 min in boiling water, followed by adding 1 mL of 0.5 N NaOH, standing for 40 min at room temperature, and then the solution was diluted up to 100 mL with distilled water. The solution filtrated with paper filter was used for measurement of pectin. Pectin content in RBF was analyzed on a half scale using the 3,5-dimethylphenol method (Ralpf and Scott, 1979) and expressed as galacturonic acid. Each experiment was conducted in triplicate.
The prepared AIS was further analyzed using four fractions [water-soluble pectin (WSP), chelate-soluble pectin (CSP), acid-soluble pectin (ASP), and insoluble pectin (ISP)] separated based on differential solvent solubility. The fractionation procedure is shown in Fig. 1. The pectin content was analyzed according to the method described above.
Procedure of fractionation for classification of pectic substance based on solubility difference from AIS.
Statistical analysis The results were statistically examined. Statistical differences were analyzed by Tukey's test. p < 0.05 was considered to be statistically significant.
Sugars in RBF Total sugars in RBFs reached a maximum amount (81.0 %) at 1 week of ripening and thereafter gradually decreased (Table 1). Changes in the amount of each sugar were as follows. Sucrose reached maximum (46.0 %) at 1 week of ripening, as observed for total sugars. On the other hand, the amounts of glucose and fructose reached maximum at 3 weeks of ripening and thereafter decreased. It is known that starch in banana is broken down to soluble sugars during ripening and the sugars (mainly sucrose) accumulate in the fruit (Loesecke, 1950a; Phillips et al., 2021). Although the starch-to-sugar metabolism during banana ripening has been studied in detail (Cordenunsi-Lysenko et al., 2019; Hubbard et al., 1990; Beaudry et al., 1989), the total sugars and the composition of each soluble sugar during banana ripening were diverse in previous reports (Phillips et al., 2021; Komiyama et al., 1985; Shitasue et al., 2015). Although a very similar tendency was observed in the present study, the amount of total sugars decreased markedly in a ripening period-dependent manner.
| Ripening Period (week) |
Glucose | Fructose | Sugars (%, DW) Sucrose |
Maltose | Total |
|---|---|---|---|---|---|
| 0 | 4.0 | 3.9 | 35.5 | 0.1 | 43.5 |
| 1 | 17.3 | 17.7 | 46.0 | 0.0 | 81.0 |
| 2 | 25.7 | 27.9 | 24.8 | 0.0 | 78.4 |
| 3 | 32.1 | 36.4 | 7.0 | 0.0 | 75.5 |
| 4 | 26.1 | 35.7 | 4.5 | 0.0 | 66.3 |
Effects of banana ripening period on breadmaking The ripening period of unripe banana was important for breadmaking. The maximum SV was achieved when the unripe banana was treated for more than 2 weeks at 20 °C (Fig. 2). Breadcrumbs of breads with 1–4-week RBFs contained small gas cells and a tight crumb structure, while those made with bread flour and 0-week RBF contained large gas cells and an open structure (Fig. 3). On the other hand, gas cells in the breadcrumb of bread without RBF were hardly observed. It is known that the components in banana fruit change during banana ripening. Two factors are necessary for breadmaking; one is a carbon source for fermentation by yeast and the other is the formation of a three-dimensional network by gluten for gas retention in the dough. The SVs of breadmaking using 0-and 1-week RBFs were inferior to those of 2–4-week RBFs (Fig. 2). Although 0-week RBF contained sugars to some extent (43.5 %, Table 1), the dough hardly rose. The SV of 1-week RBF was slightly inferior to those of 2–4-week RBFs, although 1-week RBF contained the most sugar (81.0%, Table 1) of 0–4-week RBFs. The total sugar contents in each dough were 4.1, 7.5, 7.1, 6.7, and 5.7 % (w/w, wheat starch – RBF basis) for 0–4-week RBF, respectively. The results indicated that sufficient fermentation was achieved in the dough prepared from 2–4-week RBFs and the gas generated was retained within the dough.
Effects of banana fruit ripening period on gluten-free bread making.
Non-RBF: RBF was not used in the bread making. Values with different letters are significantly different (p < 0.05). The error bar indicates standard deviation (n = 3).
Cross-sectional views of breads baked using wheat flour and wheat starch with additional RBFs.
Scale bar = 1 cm. A: bread flour. B: RBF was not used in the bread making. C: 0-week RBF. D: 1-week RBF. E: 2-week RBF. F: 3-week RBF. G: 4-week RBF.
It was estimated that, in the dough prepared from 0-week RBF, the volume of gas generated was lower than for the other conditions and most of the gas generated leaked out from the dough. In the dough prepared from 1-week RBF, the gas generated leaked out slightly from the dough, although sufficient gas was generated. As a result, its SV was inferior to those of 2–4-week RBFs. Accordingly, it was speculated that the components that participate in gas retention might change during ripening.
Fermentation ability The gas generated by yeast during breadmaking was retained in the dough and the dough rose. The Fermograph was used to measure the volume of gas generated in the dough.
Gas generated in the dough was highest in the dough prepared from 1–4-week RBF (Fig. 2). There were no significant differences among these values. It was speculated that the generated gas was not sufficiently retained in the dough from 1-week RBF because the gluten substitute components were not yet sufficiently biosynthesized in the banana fruit. On the other hand, low gas generation was observed in 0-week RBF (84.6 mL/h, Fig. 2). The concentration of sugars necessary for sufficient fermentation in dough was estimated to be more than 5.7 % (w/w, wheat starch – RBF basis) in 4-week RBF. The reason for the low fermentation ability in 0-week RBF was attributed to the lack of available sugars for fermentation. Next, it was suggested that the reason for the low SV in 0-week RBF was that the gluten substitute components were not biosynthesized in banana fruit at all. From these results, it was speculated that components involved in gas retention in the dough are formed during banana ripening. However, it was thought that these components might have not yet been formed in sufficient quantity in the 1-week RBF.
Fractionation of RBF To clarify other factors necessary for breadmaking using RBF, RBF was fractionated according to solubility in water and membrane fractionation. The breads were made using each fraction and their combinations, and evaluated by SV (Fig. 4). The experiments using 2-week RBF in Figs. 2 and 4 were conducted under the same conditions. Although the SVs were slightly different, there was no significant difference between the two values at a 5 % level. Of the individual fractions, F1 exhibited the highest SV (4.93 cm3/g). Of the four combinations of fractions, the F1 and F2 combination showed a superior SV (5.36 cm3/g). Individual variation in gas cell size was observed for the breadcrumbs of breads from each fraction/combination of fractions (Fig. 5). The SVs of combinations containing F1 were higher, while those of combinations without F1 were lower. On the other hand, in bread made with F2 and added sucrose instead of F1, the SV was 5.12 ± 0.33 cm3/g. This result showed that F1 and F2 might act as a carbon source for yeast fermentation and gluten substitute, respectively.
Effects of each fraction and their combinations on gluten-free bread making.
2w-RBF: Ripe banana flour prepared from bananas stored at 20 °C for 2 weeks, Fraction: F1 (water soluble fraction below molecular weight 6–8 kD), F2 (water soluble fraction over molecular weight 6–8 kD), F3 (water insoluble fraction). Values with different letters are significantly different (p < 0.05). The error bar indicates standard deviation (n = 3).
Cross-sectional views of breads baked using wheat starch with additional RBF, each fraction and their combinations.
Scale bar = 1 cm. Abbreviations are the same as in Fig. 4.
A full understanding of the properties of RBF is important for its application to gluten-free breadmaking. While the contributions of certain identified factors in RBF were demonstrated, it is necessary to pursue further studies aimed at understanding the mechanisms underlying gluten-free breadmaking using RBF.
Pectic substances in RBF In order to estimate the components in RBF effective for gas retention in the dough, pectin was analyzed, as pectin is known to increase viscosity. Pectin contents in 0- to 4-week RBF were almost the same (Table 2). It was reported that pectin content decreased during ripening (Kawabata and Sawayama 1974; Kojima 1994). Then, gluten-free breadbaking using AIS extracted from 2-week RBF was performed and an SV of 4.98 ± 0.15 cm3/g was observed. This result showed the possibility that pectin may act as a gluten substitute, although more detailed research is needed.
| Ripening Period (week) |
Pectin* (%, dry base) |
|---|---|
| 0 | 3.02 ± 0.21 |
| 1 | 3.00 ± 0.09 |
| 2 | 3.28 ± 0.07 |
| 3 | 3.14 ± 0.11 |
| 4 | 3.37 ± 0.16 |
Hydrocolloids, such as HPMC and xanthan gum etc., have been widely researched as gluten substitutes for gluten-free breadmaking (Monteiro, 2021; Hager and Arendt, 2013; Mir et al., 2016; Encina-Zelada et al., 2019; Belorio and Gómez, 2020) and are used in commercial products (Masure et al., 2016; Roman et al., 2018). Pectin, a hydrocolloid, was less effective than HPMC on influencing loaf SV (Jang et al., 2018). In this study, however, ripe banana pectin was used with wheat starch without gluten for gluten-free breadmaking and produced a higher SV than bread flour.
In order to demonstrate the type of pectin contained in RBF, four types of pectin were fractionated based on differential solvent solubility. The contents of the four types of pectin were: WSP (0.61 %), CSP (2.37 %), ASP (0.85 %), and ISP (0.00 %) (Table 3). Kawabata et al. (1974) reported that the contents of three types of pectin (WSP, CSP, and ASP) varied depending on the origin, even with the same banana (M. cavendishi). In this study, CSP as a main pectin might act as a gluten substitute.
| Type of pectin* | Pectin (%, dry base of 2-week RBF) |
|---|---|
| WSP | 0.61 ± 0.02 |
| CSP | 2.37 ± 0.06 |
| ASP | 0.85 ± 0.01 |
| ISP | 0.00 ± 0.00 |
A full understanding of the properties of RBF is important for its application to gluten-free breadmaking. While the contribution of certain identified factors in RBF was demonstrated, it is necessary to pursue further studies aimed at understanding the mechanisms underlying gluten-free breadmaking using RBF. In the forthcoming research, a comparison among 0–2-week RBF will be important for clarification of the properties of RBF.
It has been demonstrated that ripe bananas are applicable to the production of gluten-free bread. The sugars contained in the F1 fraction (water-soluble low molecular fraction) from RBF serve as a carbon source for dough fermentation. Further, the components in the F2 fraction (water-soluble high molecular fraction) from RBF retain the gas generated in the dough, allowing the dough to expand. It is conceivable that the components in the latter fraction could serve as alternative substitutes for gluten.
Conflict of interest There are no conflicts of interest to declare.
