Fitness of Jiaozi starter for steamed bread production using a two‐stage procedure

Abstract Steamed bread is a popular staple food in China. Jiaozi shows many advantages as a starter for dough fermentation and is frequently used for steamed bread production. The knowledge about the dough fermentation process using Jiaozi is helpful for production management and quality improvement of the final product. In this study, the applicability of Jiaozi for steamed bread production was investigated. Some important factors involved in dough fermentation were carefully examined and analyzed, including the evolution and diversity of major bacteria and fungi, acidity change, reducing sugar utilization, CO 2 production and retention, and different full dough fermentation periods. Combined with the quality evaluation of the final product, the results displayed that traditional Jiaozi was suitable as starter for steamed bread production using a two‐stage procedure with a wide range of full fermentation time and also provided more insights into steamed bread production by Jiaozi.

Recently, the bacterial diversity of Jiaozi and dough during fermentation started by Jiaozi was evaluated by high-throughput sequencing method. The results revealed that the profiles of microorganisms of Jiaozi and dough were similar when the dough was fermented for 8 hr and 24 hr using Jiaozi starter. The predominant bacteria were some species from Lactobacillus, Weissella, and Leuconostoc in Jiaozi and dough, and the species from Lactobacillus were dominated in the fermented dough (Li et al., 2017). The similar pattern of bacterial community indicated a better adaptation of microbes to the dough conditions. However, little work has been conducted to investigate the influence of the full dough fermentation on the quality of steamed bread. When a starter is selected to use for dough fermentation in industry, its stability is as important as functional features (Minervini et al., 2010). Understanding the stability of Jiaozi for dough fermentation will be of great help for the production management and improvement of the quality and stability of the final product. In addition, the yeast communities responsible for carbon dioxide (CO 2 ) production and flavor formation have not been documented.
In this study, the possible factors involved in steamed bead production by Jiaozi starter during wheat dough fermentation were carefully examined, including the evolution and diversity of bacteria and fungi, acidity change, reducing sugar utilization and CO 2 production. In addition, the quality of steamed bread prepared by Jiaozi with different full dough fermentation times was also evaluated.

| Materials
Traditional Jiaozi starter was made from maize flour, muskmelon and Chinese Daqu, and dry powder of Jiaozi was obtained from Shangqiu, Henan, China (Li et al., 2017). White wheat flour with 10.72% protein, 0.36% ash, and 13.89% moisture was supplied by Jinyuan Flour Co., Ltd. (Zhengzhou, China). Other chemical reagents were of analytical grade.

| Dough fermentation
Fifty grams of Jiaozi starter was ground into powder and mixed with 500 g of wheat flour and 225 ml of water. After stirring for 13 min in SZM5 mixing machine (Xunzhong Co. Ltd., Guangzhou), the dough was fermented at 35°C and 85% relative humidity for 24 hr in a controlled fermentation cabinet (HWS180; Bilon Instrument Co. Ltd., Zhengzhou, China).

| Reducing sugar utilization and acidification
Ten grams of dough samples was collected from the fermented dough and homogenized with 90 ml of distilled water on ice for 15 min. Then, the samples were centrifuged with 5000 r/min at 4°C for 10 min. The supernatant fraction was used for reducing sugar analysis by the 3,5-dinitrosalicylic acid method, and the standard curve was plotted using various concentrations of maltose (Miller, 1959). Before sugar analysis, a Carrez precipitation was performed to eliminate proteins from the dough samples (Sterr, Weiss, & Schmidt, 2009). In brief, 0.5 ml of Carrez I solution [3.6% (wt/vol) K 4 Fe(CN) 6 •3H 2 O] was added into 8 ml of samples and incubated at room temperature for 1 min, followed by addition of 0.5 ml Carrez II solution [7.2% (wt/vol) ZnSO 4 •7H 2 O] and 1 ml of 100 mM NaOH and incubation at room temperature for another 5 min. Then, the samples were centrifuged at 8000 r/min at 4°C for 30 min and the supernatant fractions were used for sugar analysis.

| pH and the titratable acid assay (TTA)
The pH and total TTA values of the dough samples were determined as previously described (Edema & Sanni, 2008). Briefly, 10 g of the sample was homogenized with 90 ml of sterile distilled water. The values of pH were recorded and the acidity was titrated with 0.1 N NaOH to a final pH 8.5. The TTA was expressed in milliliter of 0.1 N NaOH.

| Microbial enumeration
Numbers of colony-forming units (CFUs) were determined by classical spread-plating method on YPD agar plates supplemented with 0.1 g/L chloramphenicol for yeasts and on MRS agar plates containing 0.1 g/L of cycloheximide for LAB, respectively (Li et al., 2016).

| CO 2 production and retention
The Rheofermentometer F3 (Chopin, Villeneuve-La-Garenne Cedex, France) was used to examine the total CO 2 release and retention during fermentation process. Immediately after mixing, 315 g of the dough samples prepared according to Section 2.3 was placed in the fermentation cabinet with an additional 2.0 kg of disk on the top of the dough samples. The proofing cabinet was closed tightly, and dough fermentation was performed at 35°C for 6 hr. The changes in the fermenting dough were measured in terms of CO 2 production and retention from the dough.

| Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis
PCR-DGGE analysis was conducted as previously reported (Li et al., 2016). Briefly, after DNA extraction from Jiaozi and dough samples, the V3 region of the 16S rRNA gene of bacteria was amplified using the universal primers (GC-338F and 518R) (Nakatsu, Torsvik, & Øvreås, 2000). The specific 18S rRNA gene of fungi was amplified by the universal primers (NS1 and GC-fung) (Hoshino & Morimoto, 2008). The PCR products were separated in an 8% polyacrylamide gel with a 30%-55% urea-formamide linear denaturing gradient for DGGE analysis of bacteria and 20%-45% denaturing gradient for specific DGGE analysis of fungi. Electrophoresis was conducted in 1× TAE buffer at a constant voltage of 150 V at 60°C for 4 hr for the V3 region of the 16S rRNA gene of bacteria and at a constant voltage of 50 V at 60°C for 20 hr for the 18S rRNA gene of fungi.
Following silver staining, the bands were excised, recycled using Poly-Gel DNA Extraction Kit of OMEGA, and re-amplified. The identity of the microorganisms was revealed by sequencing selected bands from the profiles of DGGE. The homology comparison was performed using BLAST via the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST).

| Steamed bread production
Two-stage fermentation method was used for steamed bread production according to the previously described with small modification (Li et al., 2015). Firstly, the dough was prepared according to Section 2.3 and fermented at 35°C and 85% relative humidity for 6 hr-8 hr in a cabinet (HWS-180, BiLon Instrument Co. Ltd.). Secondly, 200 g of wheat flour and 70 ml of water was added into the dough prepared in the firststage and mixed for 12 min. Then, the dough was sheeted 10 times on the surface pressure machine (YT-350; Yinying Instrument Technology Co. Ltd., Shandong, China) and split into 100 g portions. The chunks were formed into round shape by hand and fermented at 35°C and 85% relative humidity for 50 min. Then, the proofed dough was steamed for 30 min in a pot using a steam tray and boiling water.
The steamed bread was also made by one-stage fermentation method (a straight dough process) (Yeh et al., 2009). The dough after mixing according to Section 2.3 was sheeted 10 times on the surface pressure machine (YT-350, Yinying Instrument Technology Co. Ltd.) and split into 100 g portions. The chunks were formed into round shape by hand and fermented at 35°C and 85% relative humidity for 1 hr in a cabinet (HWS-180, Bilon Instrument Co. Ltd.) and then steamed for 30 min.

| Quality evaluation of steamed bread
After cooling at room temperature for 1 hr, the quality of steamed bread was evaluated. Hardness of steamed bread was determined as previously reported using a Texture Analyzer (TA.XT2i; Stable Micro Systems, Ltd., Godalming, UK) (Li et al., 2015). Specific volume of steamed bread was measured using the rape seed displacement method, and the whiteness was determined by whiteness meter (WGB-IV, Tasan Co. Ltd., Hangzhou, China) (Li et al., 2015;Sim, Noor Aziah, & Cheng, 2011).

| Statistical analysis
The data reported in this article were subjected to analysis of variance (ANOVA) by Duncan's multiple-range test (p < 0.05) using SPSS software (SPSS 19.0, SPSS Inc., Chicago, IL, USA) wherever applicable.

| DGGE profiles of bacteria and fungi
The diversity of the major bacteria and fungi associated with dough fermentation using Jiaozi as the starter was analyzed using  (Figure 1a and Table 1). The result was consistent with the analysis of high-throughput sequencing (Li et al., 2017). Some microbes, such as the species belonged to Lactobacillus, Weissella, and Pediococcus, were identified by both methods. However, some discrepancies were also observed. Using the high-throughput sequencing method, genus Acetobacter was found to be predominant in the Jiaozi starter, but it was not detected using the PCR-DGGE method. This discordance might be because the two analyses based on PCR arise different biases due to PCR amplification selectivity of different primers, and the hypothesis was in accordance with the previous results (Li et al., 2016;Madoroba et al., 2011;Mukisa et al., 2012). The results highlighted the necessity of a combined usage of varied approaches for detection of microbial communities within complex matrices.
In addition, band 5 in Figure 1a identified as W. paramesenteroides in the dough was not detected in the starter (Figure 1a and Table 1), indicating W. paramesenteroides detected at the dough fermentation stage could originate from the wheat flour. Thus, the initial wheat flour powder could be the potential sources of bacteria for dough fermentation. The hypothesis was consistent with the previous result, which has found wheat flour was an important inoculum for some LAB strains (Alfonzo et al., 2013). PCR-DGGE analysis showed two common bands in the starter identified as chloroplast DNA from Triticum turgidum and Vigna radiata var. Sublobata, respectively, which might be from the raw materials.
Band identification showed the dominant yeast species to be S. cerevisiae and W. anomalus, and they remained stable during dough fermentation ( Figure 1b and Table 1  The observation was in accordance with the previous results (Chao, Huang, Kang, Watanabe, & Tsai, 2013;Madoroba et al., 2011).

| Reducing sugar utilization during fermentation
The changes in the terms of reducing sugar during dough fermentation are shown in Figure 2. in accordance with the previous study (Gobbetti, Corsetti, & Rossi, 1994).

| Acidity changes and TTA analysis
The acidity analysis of the dough fermented by Jiaozi showed that in the beginning of the fermentation, the pH value of dough was 5.7 and slowly decreased in the first 4 hr (Figure 3). After 4 hr, the pH value rapidly dropped and stopped dropping at around 12 hr.
Subsequently, the pH value slowly decreased. Correspondingly, the TTA accumulated as fermentation proceeded and it rapidly developed to 9.6 after 16 hr of fermentation ( Figure 3). The values of pH and TTA were similar with the previous report of dough fermented by co-culture of yeasts and LAB (Edema & Sanni, 2008). The dough became acidic and indicated that the acid-producing bacteria gradually dominated the dough microflora and a large number of carbon sources were used to produce acid. Enumeration of numbers of microorganisms showed that the LAB number increased from 6.80 to 8.38 log CFU/g dough, whereas the yeast cell number only increased from 6.97 to 7.60 log CFU/g dough.

| CO 2 production and retention
The characteristics of CO 2 production and retention of the dough started with Jiaozi are illustrated in Figure 4. The total volume of CO 2 production increased rapidly after the addition of Jiaozi. The CO 2 formation rate increased exponentially before running asymptotically toward zero in the first 2 h, and then, a relatively constant rate of CO 2 production was observed (Figure 4). The CO 2 production kinetics indicated that the yeast metabolism was activated gradually in the first 2 hr and high yeast activity was kept during the whole fermentation time or for a long time, indicating a stable fermentative capability.
The previous study has shown that gas formation rate is directly related to carbohydrates concentration and activity of yeast cells (Verheyen, Albrecht, Elgeti, Jekle, & Becker, 2015). It was impossible for CO 2 formation rate to increased unrestrictedly due to the limitation of yeast metabolites potential and substrate availability.
As above mentioned in Section 3.2, the substrate present in the F I G U R E 2 Evolution of reducing sugar during dough fermentation incubated with or without Jiaozi F I G U R E 3 pH and TTA profiles of the dough fermented by the Jiaozi dough was replete, and this might be one of the reasons of the deceased rate of CO 2 production with the time. It has been reported that little oxygen is present in dough and is quickly exhausted shortly after kneading (Joye, Draganski, Delcour, & Ludescher, 2012), and yeast metabolism in dough is influenced by anaerobic conditions and only one third of CO 2 is synthesized during the anaerobic fermentation compared with aerobic condition (Jayaram et al., 2013;Verheyen, Jekle, & Becker, 2014). Therefore, the results also indicated that the metabolism or fermentative activity of yeast in the dough could reach the maximum after 2 hr. Besides, the metabolism of other microorganisms could also affect the CO 2 production of yeasts. This was in accordance with the previous report (Gobbetti et al., 1994).
The total CO 2 produced was almost totally retained by dough in the first 3 hr (Figure 4), which indicated that the porosity did not formed (upper and lower lines were superimposed) and it was beneficial to gas retention. Even after fermenting for 6 hr, it also showed a significantly higher retention coefficient (84.9%). Lactic acid, mainly produced by LAB in Jiaozi, can partially account for the more elastic gluten structure (Gobbetti et al., 1994) and gave the high gas retention ability. Although a high level of gas production during fermentation is crucial for sufficient leavening of steamed bread, the volume might still be impaired if the produced CO 2 cannot be stabilized during the production process.

| Steamed bread quality evaluation
The effects of the fermentation method and time on the quality of steamed bread were evaluated. The results in Table 2 demonstrated that there was no significant difference in the specific volume, hardness, and sensory score between the steamed breads prepared by twostage method with 6 hr and 8 hr fermentation, respectively. Higher whiteness of the steamed bread prepared by two-stage method with 6 hr fermentation was observed. A higher specific volume, whiteness, and sensory score of the steamed bread made by two-stage method were obtained compared with that of one-stage method.
The specific volume, hardness, and sensory score of steamed bread are closely related to the CO 2 production and metabolite by microorganisms and the gluten network structure of dough. The similar yeast activity and dough structure could be the reason for the comparable specific volume and hardness of steamed bread prepared by two-stage method with 6-hr and 8-hr fermentation, respectively. A higher whiteness of steamed bread with 6-hr dough fermentation might be related to the changed interior microstructure and metabolite influenced by the microbial activity (Li et al., 2015). When one-stage method was used to ferment the dough, no porosity was formed and the gas cells could not well expanded and developed due to the lower gas production as described above, which could explain the unsatisfactory breadcrumb texture, lowerspecific volume, higher hardness, smaller compact gas cells, and denser crumb of the final product.
Based on the data shown in Table 2, the quality of the steamed bread prepared by two-stage fermentation method was of better quality than the steamed bread prepared by one-stage fermentation method. It should be noted that although the specific volume of steamed bread by one-stage method using Jiaozi as starter was lower than that by two-stage method, it still reached the national standard of China that the specific volume of the steamed bread is higher than 1.7 (SAC, 2007).

| CON CLUS ION
Some key factors involving in the dough fermentation process using Jiaozi starter were investigated. The results found that the predominant yeast and LAB species remained at a relatively constant ratio during the whole process of dough fermentation. The high rate of CO 2 production with the fermentation time could be due to the high yeast activity for gas production as the carbohydrates were adequate Means with different superscript letters within the same column are significantly different (p < 0.05).
during the first 8 hr of dough fermentation. Together with the quality evaluation of the final product, the results displayed the suitability and convenience of Jiaozi as starter in steamed bread production using a two-stage procedure with flexible full fermentation periods.
The work is helpful for the production procedure management and improvement of the quality and stability of final product in future.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interest and the study does not involve any human or animal testing.