Note
Making sugar-free bread with wheat flour and halotolerant Hyphopichia burtonii-enriched culture
2023 Volume 29 Issue 2 Pages 123-128
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2023 Volume 29 Issue 2 Pages 123-128
We re-evaluated the feasibility of fermentation seeds made from Inaniwa udon, a traditional wheat noodle. Inaniwa udon dough was cultivated in a 5 % saline medium at 29 °C for three days (hereinafter ‘Hyphopichia-enriched starter’). Without adding sugar or yeast, this Hyphopichia-enriched starter could be used to make a primitive type of bread whose ingredients were limited to flour, salt and a starter. A typical strain of this culture, Hyphopichia burtonii M2, had much higher α-amylase activity than the type culture of H. burtonii, NBRC 10837T. When used for bread making, both strong and weak flour yielded bread with a specific volume of 2.0–2.2 cm3/g. The fresh bread crumb tended to be dark in color, with an L* of 63–70.
A sourdough fermentation seed, or starter, is a paste-like fermented product obtained by naturally fermenting wheat flour, rye flour and the like (Fujimoto, 2012). World-famous fermentation seeds include hops (UK), levain (France), panettone (Italy), San Francisco sour (USA) and lye sour (Germany). In Japan, there is a fermentation seed called sakadane, which is made by adding water and rice to koji (cooked rice inoculated with Aspergillus oryzae) (Kawai, 1996). Onishi et al. (2001) developed a novel fermentation seed using a cold-growing lactic acid bacterium (LAB), Lactobacillus sakei, derived from lactic acid-producing microorganisms used in the traditional manufacture of sake. The raw materials and manufacturing methods employed vary across the country, but all are based on LAB and yeast, which play an important role in bread making. Usually, the ratio of LAB to yeast in the fermented seed is 100:1 (Fujimoto, 2017). The addition of fermentation seed to bread dough increases not only the specific volume and softness of the bread, but also produces new nutritionally active compounds, such as organic acids and amino acid derivatives (Gobbetti et al., 2014; Inomata et al., 2019). The authors have found a new fermentation seed (which we term ‘Hyphopichia-enriched starterx2019;) that differs from traditional sourdough (Ohisa et al., 2022). It is mainly composed of Hyphopichia burtonii, a halotolerant yeast, and is characterized by a low proportion of LAB. The combination of Hyphopichia-enriched starter with conventional bread yeast increased the specific volume and softness of the resulting bread (Ohisa et al., 2020). Ordinary baker's yeast has lost its a-amylase; thus, sugar must be added to the dough to ferment it. It was thought that if H. burtonii or its enrichment culture had amylolytic activity, it would be possible to ferment flour dough without adding sugar. We therefore attempted to make bread with a simple composition consisting only of wheat flour and Hyphopichia-enriched starter.
Materials Two types of strong flour (Nippon Flour Mills, protein content 12 and 13 %), one type of medium-strength flour (Nisshin Flour Mills, protein content 10 %) and one type of weak flour (Nisshin Flour Mills, protein content 8 %) were used.
The halotolerant H. burtonii yeast strains M2 (NITE Biological Resource Center, Deposit No. NBRC 114656) (Ohisa et al., 2019) and NBRC 10837T (CBS 2352) were used in this study.
Inaniwa udon dough culture (Hyphopichia-enriched starter) One gram of Inaniwa udon dough (Kumagai Noodle Industry Co., Ltd.) was cultivated in 100 ml of a 5 % salt medium at 29 ° C for three days. Twenty-one of the 29 isolates were identified as H. burtonii, a halotolerant yeast. No other yeasts were found in higher dilutions of this saline culture (Ohisa et al., 2022). We termed the Inaniwa udon dough in a salt-containing medium as Hyphopichia-enriched starter. The medium contained (w/w) 5 % salt, 1 % glucose, 0.2 % yeast extract (Kyokuto Pharmaceutical Industry Co., Ltd.) and 0.2 % peptone (Nihon Pharmaceutical Co., Ltd.).
Dough rising test Twenty grams of flour (weak and strong flours), 4 ml of Hyphopichia-enriched starter and 8 ml of water were used. These were mixed and packed in a 200-ml graduated cylinder. The surface of the dough was flattened, and the upper part of the graduated cylinder was covered with plastic wrap. This graduated cylinder was placed in an incubator (MI-100G, Sansyo Co., Ltd.) set at 30 °C. The upper surface after fermentation was read on the scale and used as the dough volume.
Prototype bread The dough contained 300 g of flour, 60 ml of Hyphopichia-enriched starter and 110 – 120 g of distilled water (Table 1). The dough was mixed, kneaded and fermented using the bread program (room temperature) of a rice cake making machine (PFC-20FK, Toshiba). About 70 g of the dough was taken and made into a roll. The dough roll was placed on a circular truncated cone tray (paper, 13.4 cm in diameter) and 2 g of shortening was applied to the inside of the tray to prevent water leakage. The top of the tray was covered with plastic wrap. The dough was fermented for 18–28 hours in an incubator (MI-100G, Sansyo Co., Ltd.) set at 30 °C. Finally, the fermented dough was baked at 200 °C in an electric oven (ER-C7 microwave oven, Toshiba) for 13 minutes. The prototype bread was allowed to cool naturally for one and a half hours, and after the internal temperature had dropped to almost room temperature, it was placed in a polyethylene bag (Asahi Kasei Home Products Co., Ltd., Ziploc) (Wan et al., 1997). After storage at room temperature for 24 hours, the weight, volume and color of the bread were measured. The volume was calculated using the rapeseed replacement method, and the specific volume of the bread (cm3/g) was calculated (n = 3 – 4). The lightness (L*), redness (a*) and yellowness (b*) of the bread crumb were measured using a color difference meter (CR410, Konica Minolta). The measurement was performed five times, and the measured value was expressed as the average value and standard deviation. A cross-section of the bread was photographed using a digital camera (Cyber-shot SX20, Sony).
Culture of H. burtonii The growth medium consisted of 0.2 g of yeast extract, 0.2 g peptone, 5.0 g salt and 1.0 g glucose, which were mixed in distilled water and autoclaved at 121 °C for 15 minutes. From a slope culture (H. burtonii M2, NBRC 10837T) stored in PDA medium (Nissui Pharmaceutical Co., Ltd.) containing 5% salt, loops were aseptically inoculated into the growth medium. This was cultured statically at 30 °C for two days. A dough rising test of these pure cultures was also performed using the method described in the dough rising test, replacing the Hyphopichia-enriched starter with H. burtonii M2 or NBRC 10837T culture.
Measurement of amylase activity A culture of halotolerant yeast (100 ml) was collected and suspended in 4.0 ml of 0.1 M phosphate-buffered saline (pH 7.4). Glass beads (20 mg) were added to the yeast suspension (2 ml), which was then processed in a grinder (BHA-6 Bead Grinding Machine, AS ONE). The crushed suspension was centrifuged at 12,600 rpm at 4 °C for 30 minutes to obtain a crude enzyme solution. The protein content of this crude enzyme solution was quantified using a Bradford Protein Assay Kit (Takara Bio Inc.). The α-amylase activity was tested using an α-amylase activity kit (Kikkoman Biochemifa Co., Ltd.). The effect of releasing 1 µmol of CNP from N3-G5-β-CNP in 1 min is defined as 1 U. The glucoamylase activity was examined by quantifying the glucose produced by reacting 0.1% soluble starch with the crude enzyme solution at 40 °C for 60 minutes using a Glucose CII Test Wako (Fujifilm). The effect of producing 1 mg of glucose from soluble starch at 40 °C for 60 min is defined as 1 U.
The soluble starch-digesting activity of Hyphopichia-enriched starter was examined using the paper disc method. The disc (8 mm, Advantec) was placed on an agar plate containing 1% soluble starch. A 20-µl aliquot of Hyphopichia-enriched starter was then applied to the disc and the agar plate was incubated at room temperature for 24 hours. Halos were confirmed by spraying iodine solution onto the agar plate.
Statistical processing The differences between the average values of bread specific volume were tested using R (4.2.1 for Windows) by one-way ANOVA and Tukey's multiple comparison test.
Dough rising The fermentation ability of the dough with Hyphopichia-enriched starter was investigated. It was found that the dough rose slowly without the addition of yeast or sugar (Fig. 1). The dough rose faster with the weak flour than the strong flour for up to 30 hours. However, the dough without Hyphopichia-enriched starter had not risen after 40 hours. The Hyphopichia-enriched starter was thus able to ferment the flour and cause the dough to rise.
Volume change of dough with weak and strong flours using Hyphopichia-enriched starter. ◆, control (strong flour without Hyphopichia-enriched starter); ▲, strong flour (protein 12 %); ○, weak flour (protein 8 %).
The Hyphopichia-enriched starter produced a 3-mm halo on soluble starch agar plates, as indicated by iodine, suggesting soluble starch-digesting activity.
H. burtonii M2, a common strain in Hyphopichia- enriched starter, showed stronger a-amylase activity than NBRC 10837T (CBS 2352) (type culture) (Table 2). On the other hand, glucoamylase activity was stronger in NBRC 10837T.
| Amylase activity | H. burtonii NBRC 10837T (CBS 2352) | H. burtonii M2 |
|---|---|---|
| α-Amylase* | 1.23 U/mg | 5.21 U/mg |
| Glucoamylase** | 1.78 U/mg | 0.37 U/mg |
Since a volume change was observed in udon dough (Ohisa et al., 2012), the volume change of the dough with two types of yeast was investigated using medium-strength flour. As shown in Fig. 2, dough containing H. burtonii M2 rose without the addition of sugar. However, with H. burtonii NBRC 10837T, only slight rising of the dough was observed after 45 hours of incubation.
Volume change of medium-strength flour dough using Hyphopichia burtonii M2 and Hyphopichia burtonii NBRC 10837T. ○, Hyphopichia burtonii M2; ◆, Hyphopichia burtonii NBRC 10837T.
In experiments investigating enzyme activity, no starch was added to the culture medium (Culture of H. burtonii), suggesting that H. burtonii M2 and NBRC 10837T naturally retain the enzyme. Strain NBRC 10837T could not utilize wheat starch because of its weak a-amylase activity (Table 2) or because it does not release a-amylase to the exterior.
More than 20 kinds of yeast species have been isolated from sourdough, including Saccharomyces cerevisiae, Candida humilis, Kazachstania exigua, Pichia kudriavzevii, Wickerhamomyces anomalus and Torulaspora delbrueckii (Vuyst and Neysens, 2005; Vuyst et al., 2016). Of these, a P. kudriavzevii strain was halotolerant and could degrade Acid Red B (Feng et al., 2014). However, halotolerant H. burtonii has not hitherto been reported in sourdough. It appears that the high-salinity processing of Inaniwa udon dough has led to the accumulation of H. burtonii with amylase activity.
Prototype bread The strong flour dough with Hyphopichia-enriched starter was baked after lengthening the fermentation time from 18 to 28 hours. With increasing fermentation time, the specific volume of the bread increased from 1.7 to 2.3 cm3/g (Table 3). The fermentation time was then set to 24 hours for making a prototype. Figure 2 shows the cut surface of the prototype bread. Bread could be made not only with strong flour (protein content 12, 13 %) but also with weak flour (protein content 8 %). As shown in Table 3, there was no significant difference in the specific volume of the bread among the four flour varieties. Since the generation of carbon dioxide gas was slow, even weak flour with low gas retention could be used. However, the use of weak flour tended to result in a flatter loaf (Fig. 3A).
| Fermentation time (h) | Flour type | Specific volume (cm3/g) |
|---|---|---|
| 18 | Strong (12 %) | 1.70 ± 0.06 c |
| 20 | Strong (12 %) | 1.87 ± 0.05 b |
| 22 | Strong (12 %) | 1.97 ± 0.08 b |
| 25 | Strong (12 %) | 2.20 ± 0.03 a |
| 28 | Strong (12 %) | 2.30 ± 0.04 a |
| 24 | Strong (13 %) | 2.07 ± 0.09 |
| 24 | Strong (12 %) | 2.03 ± 0.08 |
| 24 | Medium-strength (10 %) | 2.17 ± 0.06 |
| 24 | Weak (8 %) | 2.12 ± 0.08 |
Average ± standard deviation (n = 3 – 4). Different letters within rows are significant at p = 0.05 by Tukey'; multiple comparison test.
Cross-section of bread made from wheat flour with different protein contents. Bread dough with Hyphopichia-enriched starters were fermented 24 hours at 29 °C. A, weak flour (protein 8 %); B, medium-strength flour (protein 10 %); C, strong flour (protein 12 %); D, strong flour (protein 13 %). Numerical values in the figure indicate centimeter.
Bread made with both dry yeast and H. burtonii M2 also tended to result in a flatter loaf, with a bread height-to-width ratio of 0.4–0.5 (Ohisa et al., 2020). We conclude that the dough was softened by the α-amylase produced by H. burtonii. α-Amylase plays an important role in improving dough properties and bread quality (Pritchard, 1992; Kim et al., 2006; Struyf et al., 2017).
The color characteristics of the bread crumb are shown in Table 4. The brightness (L*) of the prototype bread was 63–70, lower than previously reported for white bread (L* 75–82) (Tsutsui and Kanai, 1999; Tachi, 2010; Yamada et al., 2017).
| Flour type | L* | a* | b* |
|---|---|---|---|
| Strong (13 %) | 65.36 ± 0.40 b | 0.43 ± 0.16 a | 10.35 ± 0.42 b |
| Strong (12 %) | 70.26 ± 0.44 a | −0.94 ± 0.09 b | 11.25 ± 0.54 a |
| Medium-strength (10 %) | 70.29 ± 0.56 a | −0.88 ± 0.12 b | 11.50 ± 0.32 a |
| Weak (8 %) | 63.54 ± 1.14 c | 0.38 ± 0.07 a | 10.48 ± 0.54 ab |
Parentheses indicate the protein contents. Average ± standard deviation (n = 5). Different letters within rows are significant at p = 0.05 by Tukey's multiple comparison test.
L* indicates the degree of reflection of the surface of the object, suggesting insufficient bubble formation and low specific volume during baking.
Compared to commercially-available bread, the fermentation time was longer at 24 hours, and the specific volume was smaller. The bread after baking was hard, but it was characterized by a delicious baked bread flour scent. After 2 days, it was so hard that it could not be eaten unless toasted.
In a previous report, H. burtonii was used to make bread containing sucrose. The bread's specific volume ranged from 2.42–2.62 (cm3/g) (Ohisa et al., 2019). In these experiments, Hyphopichia-enriched starter was used as a fermentation seed to make bread without sugar (Table 1). The specific volume of the bread was 1.7–2.3 cm3/g (Table 3). Hyphopichia-enriched starter appears to be usable in the production of bread with or without sugar.
Acknowledgements We would like to thank President Jun Kumagai of Kumagai Noodle Industry Co., Ltd. for providing the Inaniwa udon dough.
Conflict of interest There are no conflicts of interest to declare.
