- Email: [email protected]
Microbiological characterization of traditional dough fermentation starter (Jiaozi) for steamed bread making by culture-dependent and culture-independent methods
International Journal of Food Microbiology 234 (2016) 9–14
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Microbiological characterization of traditional dough fermentation starter (Jiaozi) for steamed bread making by culture-dependent and culture-independent methods Zhijian Li a, Haifeng Li b,⁎, Ke Bian a,⁎ a b
College of Food Science and Technology, Henan University of Technology, Zhengzhou 450052, China College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China
a r t i c l e
i n f o
Article history: Received 6 April 2016 Received in revised form 17 June 2016 Accepted 21 June 2016 Available online 23 June 2016 Keywords: Jiaozi PCR-DGGE Yeast Bacteria Dough fermentation starter
a b s t r a c t In this study, the microbial composition of two types of Jiaozi (a dough fermentation starter in making steamed bread) was investigated using both culture-dependent and culture-independent (PCR-DGGE) methods. The numbers of the cultivable bacteria on MRS at 30 °C and yeast on YPD at 28 °C in the maize flour Jiaozi (MFJ) were 9.21 ± 0.16 Log CFU/g and 9.18 ± 0.05 Log CFU/g, respectively, which were higher than that in the rice flour Jiaozi (RFJ) (P b 0.05). A total of 140 bacteria and 124 yeasts were isolated and identified on the basis of the sequences of their 16S rRNA gene and ITS region. The culture-dependent method showed that Acetobacter tropicalis and Enterococcus durans were the predominant bacteria strains in MFJ, and accounted for 45.7% and 25.7% of the bacteria, and Lactobacillus plantarum and Pediococcus pentosaceus represented 12.8% and 8.6%. In the RFJ sample, the most prominent isolate was P. pentosaceus (38.6%), followed by L. plantarum (24.3%), A. tropicalis (22.8%), and E. durans (5.7%). P. pentosaceus and L. plantarum were also detected in both starters by PCR-DGGE, while some bacteria species such as A. tropicalis and E. durans, recovered as pure cultures, were not detected by direct PCR-DGGE. On the other hand, Lactobacillus brevis, Weissella sp. and Lactobacillus alimentarius detected by PCR-DGGE were not recovered in any of the media and conditions used. In the MFJ sample, the isolated main yeast species were identified as Wickerhamomyces anomalus (67.2%), Saccharomyces cerevisiae (27.9%) and Torulaspora delbrueckii (4.9%). In addition to S. cerevisiae (42.9%), W. anomalus (27.0%) and T. delbrueckii (7.9%), Saccharomycopsis fibuligera was also identified as the predominant isolate in RFJ samples and accounted for 22.2%. PCR-DGGE also indicated the presence of W. anomalus and S. cerevisiae in both MFJ and RFJ starters and S. fibuligera was also detected in RFJ, but T. delbrueckii was not detected in both samples. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Steamed bread is a traditional fermented staple food largely consumed in China (Li et al., 2015a). Although instant active dry yeast is widely used for dough fermentation in making steamed bread, traditional Jiaozi is still applied in many areas due to the unique flavour and taste of the product made with it (Li et al., 2014). Jiaozi is different from traditional sourdough because non-wheat flours were used as the main material for its preparation. In particular, Chinese Qu, which is a traditional starter for Chinese liquor production, is sometimes added in the preparation of Jiaozi (Li et al., 2014; Zheng et al., 2012). Based on the raw materials used, Jiaozi can be mainly categorized into maize flour Jiaozi (MFJ) and rice flour Jiaozi (RFJ). MFJ is naturally produced using maize flour, muskmelon and wheat flour as raw materials, while RFJ is manufactured by rice flour, rice wine and Qu. Although partly
⁎ Corresponding authors. E-mail address: hfl[email protected] (H. Li).
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.06.024 0168-1605/© 2016 Elsevier B.V. All rights reserved.
mechanized, the production process of Jiaozi is still based on empirical knowledge and relies on back-slopping and spontaneous fermentation. Therefore, the product quality and stability are usually variable due to the uncontrolled non-sterile fermentation conditions as well as occasional change of environmental conditions, such as temperature and water content of fermentation. Application of defined starter cultures is one of the approaches that could be used to improve indigenous technologies and ensure starter quality consistence (Mukisa et al., 2012). A prerequisite for the commercial production of Jiaozi from appropriate starter cultures is a thorough insight of the microbial composition behind it. Furthermore, having more information about the microbial structure existing in the starter is also important to produce product with high quality (Li et al., 2015b; Plessas et al., 2011). Although Jiaozi has been manufactured for hundreds of years, only few studies have been performed to characterize it from the microbiological point of view. Earlier microbiological work only studied RFJ using conventional culture-dependent techniques (Li et al., 2014). Several LAB and yeasts have been isolated from Jiaozi (Li et al., 2014); however, the study does not include
10
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
quantitative data of the strains isolated, even though this step is important to understand the ecological significance of these microorganisms. Furthermore, to date, no study investigated the microbial populations in Jiaozi by molecular method. In recent years, studies have shown that culture-dependent methods may lead to the underestimation of microbial diversity and failure to isolate the dominant flora in some cases. Therefore, complementing culture-dependent methods with culture-independent methods has attracted a lot of attention, and is recommended as a strategy to reduce bias and generate a better view of microbial diversity (Iacumin et al., 2009; Ongol and Asano, 2009). Various studies have shown that polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE) is one reliable and reproducible technique to study the ecology of traditional dough fermentation starters, such as sourdoughs (Cocolin et al., 2013). In this study, the diversity of the representative microbial populations associated with two types of Jiaozi was investigated and compared using a combination of culture-dependent and cultureindependent (PCR-DGGE) methods. This study is the first to extensively investigate the bacterial and yeast diversity in the Jiaozi, and is relevant for future development of effective defined starter cultures. 2. Materials and methods
applicable. Data were analyzed using SPSS software (SPSS 19.0, SPSS Inc., Chicago, USA).
2.3. DNA extraction from pure culture and PCR amplification Genomic DNA of bacteria and fungi was extracted by Bacteria and Yeast Genomic DNA Purification Kit (Tiangen, Beijing, China), respectively, according to the manufacturer's instructions. The 16S rRNA gene was amplified using forward primer P0 (5′-GAG AGT TTG ATC CTG GCT CAG-3′) and reverse primer P6 (5′-CTA CGG CTA CCT TGT TAC-3′) (Quero et al., 2014). The ITS regions were amplified by forward primer ITS4 (5′-TCC TCC GCT GAC TAA TAT GC-3′) and reverse primer ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) (Li et al., 2013). Amplifications were performed in a MasterCycler Gradient thermal cycler (Eppendorf, Germany). The PCR mixture (25 μL) contained 2.5 μL of 10× PCR buffer, 10 ng of template DNA, 0.5 μL of each primer (10 μM), 0.5 μL of dNTP mixture (10 mM each) and 2.5 U of Taq polymerase (Sangon Biotech Co. Ltd., Shanghai, China). Template DNA was denatured for 5 min at 94 °C, 30 cycles of 94 °C for 30 s, 54 °C for 45 s, 72 °C for 2 min and a final extension of 72 °C for 5 min. The same PCR amplification conditions were used for both bacterial and fungal pairs of primers. After purification, the PCR products were sent to a commercial facility for sequencing (Sangon Biotech Co. Ltd., Shanghai, China). Sequences were aligned with those in GeneBank with the BLAST program to determine the closest known relatives.
2.1. Sample collection Two kinds of Jiaozi final products (2.0 kg of each sample) were collected from a local market. The RFJ made from rice wine, Chinese Qu and rice flour was obtained from Nanyang, Henan, China. The MFJ made from maize flour, muskmelon and wheat flour was obtained from Shangqiu, Henan, China. The dimensions of RFJ and MFJ blocks were about 8 × 5 × 3 cm and 3 × 3 × 3 cm, respectively. Jiaozi blocks (500 g) were randomly selected and ground to powder in an alcoholdisinfected grinder; the powder was kept in plastic jars at 4 °C. 2.2. Microbiological analysis Samples of Jiaozi (10 g each) were homogenized with 90 mL sterile PPS (peptone physiological salt) solution containing 0.1% peptone and 0.85% NaCl. Appropriate serial dilutions were prepared using the same diluent. Bacteria were enumerated on plate count agar (PCA), de Man– Rogosa–Sharpe agar (MRS), medium 17 agar (M17) and modified Chalmers agar (MC) with 0.1% (w/v) natamycin to prevent yeast growth (Li et al., 2010; Pepe et al., 2001; Terzaghi and Sandine, 1975). Plates were incubated at 30 °C and 42 °C under aerobic or anaerobic conditions for 3 days. Anaerobic conditions were obtained via incubation in anaerobic jars (AnaeroPack Rectangular Jar™ Mitsubishi Gas Chemical Co., Tokyo, Japan) containing an O2 absorbing and CO2 releasing pack (AnaeroPack ™, Mitsubishi Gas Chemical Co., Tokyo, Japan) (Ongol and Asano, 2009). Fungi were enumerated on three different media, namely, Czapek– Dox agar (CDA), yeast peptone dextrose agar (YPD) and Rose Bengal agar (RBA), to which 100 mg/L chloramphenicol was added (Li et al., 2010, 2013, 2014). The diluted suspension (0.2 mL) was added to make spread plates which were incubated at 28 °C for 2 days. From plates with 20–200 colonies, the representative colonies of yeasts and bacteria were randomly selected according to their macromorphological aspects and isolated in proportion to their frequencies. The results reported in this article are the average values ± S.D. The significant differences between two treatments with different media were detected using one-way analysis of variance (ANOVA) by the Tukey's range test (P b 0.05). Student's t-test was used to compare the means of microorganism number between two Jiaozi samples wherever
2.4. PCR-DGGE analyses DNA from Jiaozi samples was extracted as previously described (Lv et al., 2013). The V3 region of the 16S rRNA gene of the total bacteria was amplified using the universal primers: GC-338F (CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGG CCT ACG GGA GGC AGC AG) and 518R (ATT ACC GCG GCT GCT GG) (Nakatsu et al., 2000). The Eucarya-specific 18S rRNA genes were amplified by one set of universal primers: NS1 (GTA GTC ATA TGC TTG TCT C) and GCfung (CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCC CAT TCC CCG TTA CCC GTT G) (Hoshino and Morimoto, 2008). PCR reactions were carried out using a PTC220 thermal cycler (Bio-Rad, USA). The reaction mixture (50 μL) contained 5 μL of 10× PCR buffer, 50 ng of template DNA, 1 μL of each primer (10 μM), 3.2 μL of dNTP (2.5 mM) and 2.5 U of Taq polymerase. The amplification program was 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 55 °C for 45 s and 72 °C for 1 min and a final extension at 72 °C for 10 min. The sizes and quantities of the PCR products were determined using 1.5% agarose gel electrophoresis. The gradient gel preparation and electrophoresis were performed using the Bio-Rad Dcode Universal Mutation Detection System according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). The PCR products were separated on an 8% polyacrylamide gel with a 30%–55% urea–formamide linear denaturing gradient for bacteria DGGE analysis and 20%–45% denaturing gradient for fungi DGGE analysis. The 100% denaturing solution consisted of 40% (v/v) formamide deionized and 7.0 M urea. Electrophoresis was conducted in 1× TAE buffer at a constant voltage of 150 V at 60 °C for 4 h for bacteria and at a constant voltage of 50 V at 60 °C for 20 h for fungi. Following electrophoresis, the gels were stained by silver staining (Zhang et al., 2011). The gel was scanned with “Quantity One” software using a calibrated imaging Gel-Doc2000 (Bio-Rad, CA, USA). Stained bands were excised and recycled using Poly-Gel DNA Extraction Kit of OMEGA and re-amplified with the primer without the GC clamp under the same conditions as described above. The product was cloned into pGEM-T easy vector and the inserts of the positive clones were sequenced. Sequencing of DNA was carried out by Sangon Biotech Co. Ltd., Shanghai, China and analysis of sequence data was performed using BLAST program as describe above.
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
3. Results and discussion
Table 2 Identities of bacteria and yeast isolates from two Jiaozi samples on MRS, M17, MC, YPD, RBA and CDA.
3.1. Enumeration of bacteria and yeast on selective media Traditionally, culture media are only partially selective and exclude parts of the microbial community. In order to find out more kinds of bacteria from Jiaozi, enumeration of total bacteria from the two Jiaozi samples was carried out on four different types of media (PCA, MRS, M17 and MC) at 30 °C or 42 °C under aerobic or anaerobic cultivation. The results of the microbiological analysis are shown in Table 1. The total aerobic bacteria count in MFJ was about 9.20 Log CFU/g on PCA, MRS and M17 media at 30 °C, which was higher than that on MC medium (P b 0.05). The bacteria number decreased to 5.94–6.70 Log CFU/g at 42 °C on these three media. The total anaerobic bacteria in MFJ were counted about 8.48–8.86 Log CFU/g on these three media. In RFJ sample, aerobic, anaerobic and thermophilic bacteria counts were comparable on MRS, which were higher than that on M17 and MC. In general, bacterial counts in the RFJ were lower than that in the MFJ on each of the four media used at 30 °C (P b 0.05). In addition, the high level of thermophilic bacteria in RFJ on MRS and M17 media at 42 °C makes this starter different from MFJ (P b 0.05). In this study, three mycological media (YPD, RBA and CDA) were used to understand the diverse yeast biota of Jiaozi. The yeast counts were present at levels of 8.83–9.18 Log CFU/g on three media in MFJ, which were about ten times higher than that in RFJ. The data obtained by traditional microbiological counting showed that the number of bacteria was comparable to the number of yeasts in each Jiaozi sample, especially in MFJ. This high level of yeast number in Jiaozi makes this kind of starter different from other types of traditional starters, such as sourdough and starter prepared by fed batch fermentation of which the yeast/LAB ratio is generally 1:100 or 1:10 (Iacumin et al., 2009; Li et al., 2014).
3.2. Identification of isolates A total of 140 bacterial isolates were randomly selected and identified from the two Jiaozi samples. The identities of the isolated strains are presented in Table 2. The result indicated that LAB and acetic acid bacteria (AAB) were the dominant bacterial groups in Jiaozi. Acetobacter tropicalis, Enterococcus durans, Lactobacillus plantarum and Pediococcus pentosaceus were found in both samples, whereas Acetobacter orleanensis, Acetobacter cerevisiae and Escherichia coli were only observed in MFJ and Enterococcus faecalis, Bacillus cereus and Staphylococcus warneri were only found in RFJ samples. Table 1 Microbiological analysis of the two Jiaozi samples. Microbial groups
Media and incubation conditions
MFJ (Log CFU/g)
RFJ (Log CFU/g)
Bacteria PCA (30 °C) MRS (30 °C) MRS (anaerobic conditions, 30
9.20 ± 0.02a 9.21 ± 0.16a 8.86 ± 0.03b
8.95 ± 0.04a 8.37 ± 0.06b 8.31 ± 0.11b
°C) MRS (42 °C) M17 (30 °C) M17 (anaerobic conditions, 30
6.69 ± 0.04c 9.20 ± 0.01a 8.58 ± 0.02d
8.46 ± 0.06b 7.85 ± 0.02cd 8.08 ± 0.12ef
5.94 ± 0.01e 8.62 ± 0.04d 8.48 ± 0.03d
7.68 ± 0.06c 6.69 ± 0.04g 7.82 ± 0.03cd
6.70 ± 0.06c 9.18 ± 0.05a 8.83 ± 0.02b 8.84 ± 0.03b
6.09 ± 0.05h 7.91 ± 0.05de 8.12 ± 0.04f 7.43 ± 0.04i
°C) M17 (42 °C) MC (30 °C) MC (anaerobic conditions, 30 ° C) MC (42 °C) Yeasts YPD (28 °C) RBA (28 °C) CDA (28 °C)
11
Means with different superscript letters within the same column are significantly different (P b 0.05).
Microbial groups
Species identification
Bacteria Acetobacter tropicalis Enterococcus durans Lactobacillus plantarum Pediococcus pentosaceus Acetobacter orleanensis Acetobacter cerevisiae Enterococcus faecium Bacillus cereus Escherichia coli Staphylococcus warneri Yeasts Wickerhamomyces anomalus Saccharomyces cerevisiae Torulaspora delbrueckii Saccharomycopsis fibuligera
MFJ
RFJ
(% of the 70 isolates) 45.7 25.7 12.8 8.6 2.9 2.9
(% of the 70 isolates) 22.8 5.7 24.3 38.6
1.4 2.9 1.4 (% of the 61 isolates) 67.2 27.9 4.9
4.3 (% of the 63 isolates) 27.0 42.9 7.9 22.2
In MFJ, the most commonly isolated species were A. tropicalis and E. durans constituting 45.7% and 25.7% of the total bacterial colonies, respectively (Table 2), and L. plantarum and P. pentosaceus represented 12.8% and 8.6%, respectively. P. pentosaceus, L. plantarum and A. tropicalis represented 38.6%, 24.3% and 22.8%, respectively, of the total number of isolates from RFJ. The isolates identified as E. coli in MFJ and B. cereus, E. faecium and S. warneri in RFJ constituted a minor proportion of the total number of isolates. B. cereus could originate from Qu, which was a material used for making RFJ, because it was revealed that Bacillus sp. was an abundant bacterial species in that ingredient (Zheng et al., 2012). The presence of E. coli in MFJ, E. faecium and S. warneri in RFJ was probably caused by contamination from the raw material or environment during Jiaozi manufacturing process. It was interesting to find a similarity of the predominant bacteria species between the two different kinds of Jiaozi samples from different regions, but their percentages in each Jiaozi were completely different. Although it has been reported that LAB play an important role in traditional dough fermentation starters (Alfonzo et al., 2013; Zhang et al., 2011), there is limited report indicating that AAB could be of importance in dough starter (Minervini et al., 2012). This report highlights the involvement of AAB in traditional dough fermentation starters. Yeast species composition in Jiaozi was also analyzed by the culturedependent analysis and the isolation percentages of the yeast species are shown in Table 2. Wickerhamomyces anomalus was the most commonly isolated species in sample MFJ (67.2%), followed by Saccharomyces cerevisiae (27.9%). S. cerevisiae, W. anomalus and Saccharomycopsis fibuligera were the three most abundant yeast species in RFJ, constituting 42.9%, 27.0% and 22.2% of the total isolates, respectively. Torulaspora delbrueckii was identified in both Jiaozi samples, but it only accounted for a small proportion. The analysis indicated that the dominant yeasts were in a different pattern and their ratio also varied between two types of Jiaozi. The key difference between the two samples can be attributed to S. fibuligera, which was only isolated in the RFJ sample. The variations could probably be influenced by differences in yeast flora of the raw materials and the production technologies used by the different producers. Various authors have shown that S. cerevisiae and W. anomalus are prevalent yeasts typically associated with sourdoughs (Garofalo et al., 2011; Daniel et al., 2011; Iacumin et al., 2009). The presence of S. fibuligera observed in RFJ could be related to the use of Qu for RFJ making (Lv et al., 2013; Zheng et al., 2012). These results indicate that non-
12
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
Saccharomyces yeasts are part of the microbial community in Jiaozi and could be adjunct cultures for dough fermentation. 3.3. PCR-DGGE analysis The microbial diversity of two types of Jiaozi was further studied using culture-independent technique (PCR-DGGE). The results of the diversity of the bacteria and fungi associated with two Jiaozi are reported in Fig. 1 and Table 3. DGGE profiles also displayed some similarities of dominant bacteria flora in two Jiaozi. Sequencing and identification of
Fig. 1. Bacterial (A) and fungal (B) PCR-DGGE profiles of MFJ and RFJ.
the 20 DNA bands revealed that more microbial species existed in RFJ than that in MFJ. Besides the P. pentosaceus, L. plantarum, Lactobacillus brevis and Lactobacillus alimentarius in both samples (Table 3), Lactobacillus farciminis, Weissella sp. and Bacillus sp. were also identified in RFJ. The results showed that direct PCR-DGGE of total community DNA yielded a different description of bacteria composition in the starter compared with culturing results. Although L. plantarum and P. pentosaceus were identified by both methods, L. brevis, L. farciminis, Weissella sp. and L. alimentarius were not recovered by cultivation techniques. L. brevis and W. cibaria are commonly associated with sourdoughs and L. plantarum particularly codominated with these LAB in sourdough production (Settanni et al., 2013; Ventimiglia et al., 2015). The possible explanation for the failure to isolate them might be due to their scarcity to be selected from the plates or due to the selectiveness of the culture media and culturing condition. In contrast, it was especially surprising that the other two cultivable species, A. tropicalis and E. durans that had been identified as the dominant species in MFJ and RFJ by culture-dependent analysis, were not detected by the PCRDGGE. The discrepancy could be attributed to the efficiency of DNA extraction or the selective PCR amplification (Madoroba et al., 2011; Mukisa et al., 2012; Reale et al., 2011). It has been reported that DNA extraction yields can vary significantly, even for closely related strains (Miambi et al., 2003). In addition, some community members may affect the detection of certain species due to competition during PCR (Miambi et al., 2003). Discrepancies between culturing and PCR-DGGE results have also been observed for sourdough (Iacumin et al., 2009; Zhang et al., 2011). This highlights different limitations of culturing and PCR-DGGE in studying microbial diversity. Therefore, a combination of both methods is indeed useful for studying microbial diversity of traditional starters. Bands 2, 17 and 20 were identified as a chloroplast DNA of plant and band 18 could not be identified using the GenBank database. The problematic multiple banding pattern observed for L. plantarum, L. brevis and P. pentosaceus could be related to the presence of multicopies of the 16S rRNA gene for these microorganisms as reported in previous studies (Chao et al., 2013; Miguel et al., 2010). Combining the results of culture-dependent and culture-independent analysis, it can be found that the Jiaozi has a broader microbial diversity and higher numbers of microorganisms. LAB strains are mainly responsible for the dough acidification (Li et al., 2015b). They contributed to the flavour development of steamed bread due to the formation of organic acids and other flavour compounds (Li et al., 2014). The results of sequencing of the fungal DGGE bands are reported in Table 3. The fungal results revealed by culture-independent method were almost the same as that of culture-dependent method. S. cerevisiae and W. anomalus were identified by both methods and were the dominant species in two Jiaozi samples. S. fibuligera observed in RFJ by PCRDGGE further confirmed the results observed by culture method. Conversely, T. delbrueckii was not detected by PCR-DGGE in both samples. Possible reasons might be that a small fraction of its DNA was extracted which was lower than the detection limit of PCR-DGGE (Chao et al., 2013; Mukisa et al., 2012). On the other hand, large amounts of DNA from other yeast species had the potential to interfere with the specific binding of the primers to the template of this species (Chen et al., 2008; Tu et al., 2010). The presence of Mucor indicus was probably caused by contamination from the raw material or environment (Zheng et al., 2012). Several bands derived from a single species may be due to the amplification of the interspecific or intraspecific heterogeneous nature of the ITS region targeted (Madoroba et al., 2011), as the case for the bacteria detected in this study. The simple and similar diversity of dominant yeasts in two types of Jiaozi indicates a long-term natural selection process of the yeasts originating from ancient times. It is highly likely that a constant recycling of inoculum, as with back-slopping, results in the dominance of these yeast species. These results are in agreement with the generally accepted concept that traditional fermentation products are dominated by a
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
13
Table 3 Identification of the bands obtained by PCR-DGGE. Microbial groups
Band(s)a
Closest relative
Accession number
Identity (%)
Sample
AB889691 KM352501.1 HG798506 KF668014 KC753454 KC753459 KF746906 NR_044701 HG670306.1 GU301189 AP014692.1
99 100 100 100 100 99 99 99 100 99 100
RFJ MFJ MFJ and RFJ RFJ MFJ and RFJ MFJ and RFJ RFJ MFJ and RFJ MFJ RFJ MFJ and RFJ
NR132222 KJ659884 KP119822 AY054699
99 99 99 100
MFJ and RFJ MFJ and RFJ RFJ MFJ and RFJ
Bacteria 1 2 3, 4, 5, 7, 8, 9 6 10, 19 11, 12, 14, 16 13 15 17 18 20
Lactobacillus farciminis Triticum turgidum subsp. durum cultivar Langdon chloroplast Lactobacillus plantarum Weissella sp. FB12 Lactobacillus brevis Pediococcus pentosaceus Bacillus sp. M-237-24 Lactobacillus alimentarius Triticum aestivum uncultured bacterium Vigna radiata var. sublobata chloroplast
Yeasts 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 12, 16 11, 13 14, 15 a
Saccharomyces cerevisiae Wickerhamomyces anomalus Saccharomycopsis fibuligera Mucor indicus SDM-13
Bands are numbered as indicated on the DGGE gels shown in Fig. 1.
few microbial species that are selected during the course of fermentation as a result of physiological and metabolic adaptation to the food matrix (Madoroba et al., 2011). S. cerevisiae is the most effective ethanol producer in the fermentation of cereals and alcoholic beverages (Lv et al., 2013). It also plays a leading role in the fermentation of wheat and maize dough and is normally reported as being predominant such that it forms the basis for a variety of different foods (Greppi et al., 2013; Iacumin et al., 2009). This could explain the predominance of S. cerevisiae in both Jiaozi, especially in RFJ, which is normally produced from rice wine. S. cerevisiae is positively affected by the presence of L. plantarum, which produced much more CO2 than when in association with Lactobacillus sanfranciscensis (Iacumin et al., 2009). W. anomalus was observed in Jiaozi as well by both detection approaches. This yeast species is common in cereal fermentations (wheat dough and Qu) and in combination with LAB, has been associated with the production of flavour and ethanol (Iacumin et al., 2009; Zheng et al., 2012). S. fibuligera, another abundant yeast species isolated in RFJ in this study, can secrete a large amount of α-amylase, which may facilitate the conversion of starch into fermentable carbohydrates, thus generating a suitable substrate for dough fermentation (Lv et al., 2013; Zheng et al., 2012). T. delbrueckii isolated in this study showed high activity of maltase (data not shown). Although it was detected in low percentage by culture dependent method in both samples, this yeast could play an important role to provide sufficient carbon source in the dough fermentation. In recent years, the significance of non-Saccharomyces yeasts in winemaking has attracted the interest of winemaking researchers (Lv et al., 2013). Nonetheless, the function of non-Saccharomyces yeasts in Jiaozi is still poorly understood. The functionality of the yeast biota needs further investigation. The results in this study reveal that the distribution profiles of microorganisms in Jiaozi products have a common pattern comprising of above predominant species only if special raw materials was used. The results enrich our knowledge of dough starter-related microorganisms, and could help in narrowing down the isolates that can be evaluated for future studies to generate effective defined starter cultures. Furthermore, the present study highlights a combined use of culture-dependent and -independent approaches for detection of microbial communities, especially bacteria within complex matrices. Acknowledgements This research was supported by the Fundamental Research Funds for the Henan Provincial Colleges and Universities in Henan University of Technology (2015QNJH01 and 2014YWQQ18), Special Fund for Agro-
scientific Research in the Public Interest (grant no. 201303070), National Natural Science Foundation of China (31400103) and Science and Technology Project of Henan Province (132300410423). References Alfonzo, A., Ventimiglia, G., Corona, O., Di Gerlando, R., Gaglio, R., Francesca, N., Moschetti, G., Settanni, L., 2013. Diversity and technological potential of lactic acid bacteria of wheat flours. Food Microbiol. 36, 343–354. Chao, S.H., Huang, H.Y., Kang, Y.H., Watanabe, K., Tsai, Y.C., 2013. The diversity of lactic acid bacteria in a traditional Taiwanese millet alcoholic beverage during fermentation. LWT Food Sci. Technol. 51, 135–142. Chen, H., Wang, S., Chen, M., 2008. Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods. Food Microbiol. 25, 492–501. Cocolin, L., Alessandria, V., Dolci, P., Gorra, R., Rantsiou, K., 2013. Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation. Int. J. Food Microbiol. 167, 29–43. Daniel, H.M., Moons, M.C., Huret, S., Vrancken, G., De Vuyst, L., 2011. Wickerhamomyces anomalus in the sourdough microbial ecosystem. Antonie Van Leeuwenhoek 99, 63–73. Garofalo, C., Aquilanti, L., Clementi, F., 2011. The biodiversity of the microbiota of traditional Italian sourdoughs. In: Almeida, M.T. (Ed.), Wheat: Genetics, Crops and Food Production. Nova Science Publishers, Inc., New York, pp. 366–392. Greppi, A., Rantisou, K., Padonou, W., Hounhouigan, J., Jespersen, L., Jakobsen, M., Cocolin, L., 2013. Yeast dynamics during spontaneous fermentation of mawè and tchoukoutou, two traditional products from Benin. Int. J. Food Microbiol. 165, 200–207. Hoshino, Y.T., Morimoto, S., 2008. Comparison of 18S rDNA primers for estimating fungal diversity in agricultural soils using polymerase chain reaction-denaturing gradient gel electrophoresis. Soil Sci. Plant Nutr. 54, 701–710. Iacumin, L., Cecchini, F., Manzano, M., Osualdini, M., Boscolo, D., Orlic, S., Comi, G., 2009. Description of the microflora of sourdoughs by culture-dependent and culture-independent methods. Food Microbiol. 26, 128–135. Li, Z., Li, H., Deng, C., Liu, C., 2014. Effect of mixed strain starter culture on rheological properties of wheat dough and quality of steamed bread. J. Texture Stud. 45, 180–186. Li, Z., Deng, C., Li, H., Liu, C., Bian, K., 2015a. Characteristics of remixed fermentation dough and its influence on the quality of steamed bread. Food Chem. 179, 257–262. Li, Z., Li, H., Deng, C., Bian, K., Liu, C., 2015b. Effect of Lactobacillus plantarum DM616 on dough fermentation and Chinese steamed bread quality. J. Food Process. Preserv. 39, 30–37. Li, L., Feng, L., Yi, J., Hua, C., Chen, F., Liao, X., Wang, Z., Hu, X., 2010. High hydrostatic pressure inactivation of total aerobic bacteria, lactic acid bacteria, yeasts in sour Chinese cabbage. Int. J. Food Microbiol. 142, 180–184. Li, Z., Li, H., Yang, X., Huang, Z., Liu, C., Cao, B., 2013. Characterization of Se(IV) removal from aqueous solution by Aspergillus sp. J2. Chem. Eng. J. 220, 67–71. Lv, X.C., Huang, X.L., Zhang, W., Rao, P.F., Ni, L., 2013. Yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing, revealed by culturedependent and culture-independent methods. Food Control 34, 183–190. Madoroba, E., Steenkamp, E.T., Theron, J., Scheirlinck, I., Cloete, T.E., Huys, G., 2011. Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food. Syst. Appl. Microbiol. 34, 227–234. Miambi, E., Guyot, J., Ampe, F., 2003. Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods. Int. J. Food Microbiol. 82, 111–120.
14
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
Miguel, M.G.D.C.P., Cardoso, P.G., Lago, L.D.A., Schwan, R.F., 2010. Diversity of bacteria present in milk kefir grains using culture-dependent and culture-independent methods. Food Res. Int. 43, 1523–1528. Minervini, F., Lattanzi, A., De Angelis, M., Di Cagno, R., Gobbetti, M., 2012. Influence of artisan bakery- or laboratory-propagated sourdoughs on the diversity of lactic acid bacterium and yeast microbiotas. Appl. Environ. Microbiol. 78, 5328–5340. Mukisa, I.M., Porcellato, D., Byaruhanga, Y.B., Muyanja, C.M., Rudi, K., Langsrud, T., Narvhus, J.A., 2012. The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods. Int. J. Food Microbiol. 160, 1–10. Nakatsu, C.H., Torsvik, V., Øvreås, L., 2000. Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci. Soc. Am. J. 64, 1382–1388. Ongol, M.P., Asano, K., 2009. Main microorganisms involved in the fermentation of Ugandan ghee. Int. J. Food Microbiol. 133, 286–291. Pepe, O., Villani, F., Coppola, S., 2001. Differential viable count of mixed starter cultures of lactic acid bacteria in doughs by using modified Chalmers medium. Microbiol. Res. 155, 351–354. Plessas, S., Alexopoulos, A., Mantzourani, I., Koutinas, A., Voidarou, C., Stavropoulou, E., Bezirtzoglou, E., 2011. Application of novel starter cultures for sourdough bread production. Anaerobe 17, 486–489. Quero, G.M., Fusco, V., Cocconcelli, P.S., Owczarek, L., Borcakli, M., Fontana, C., Skapska, S., Jasinska, U.T., Ozturk, T., Morea, M., 2014. Microbiological, physico-chemical, nutritional and sensory characterization of traditional Matsoni: selection and use of autochthonous multiple strain cultures to extend its shelf-life. Food Microbiol. 38, 179–191.
Reale, A., Di Renzo, T., Succi, M., Tremonte, P., Coppola, R., Sorrentino, E., 2011. Identification of lactobacilli isolated in traditional ripe wheat sourdoughs by using molecular methods. World J. Microbiol. Biotechnol. 27, 237–244. Settanni, L., Ventimiglia, G., Alfonzo, A., Corona, O., Miceli, A., Moschetti, G., 2013. An integrated technological approach to the selection of lactic acid bacteria of flour origin for sourdough production. Food Res. Int. 54, 1569–1578. Terzaghi, B.E., Sandine, W.E., 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 341. Tu, R., Wu, H., Lock, Y., Ju, C., 2010. Evaluation of microbial dynamics during the ripening of a traditional Taiwanese naturally fermented ham. Food Microbiol. 27, 460–467. Ventimiglia, G., Alfonzo, A., Galluzzo, P., Corona, O., Francesca, N., Caracappa, S., Moschetti, G., Settanni, L., 2015. Codominance of Lactobacillus plantarum and obligate heterofermentative lactic acid bacteria during sourdough fermentation. Food Microbiol. 51, 57–68. Zhang, J., Liu, W., Sun, Z., Bao, Q., Wang, F., Yu, J., Chen, W., Zhang, H., 2011. Diversity of lactic acid bacteria and yeasts in traditional sourdoughs collected from western region in Inner Mongolia of China. Food Control 22, 767–774. Zheng, X.W., Yan, Z., Han, B.Z., Zwietering, M.H., Samson, R.A., Boekhout, T., Nout, M.J.R., 2012. Complex microbiota of a Chinese “Fen” liquor fermentation starter (FenDaqu), revealed by culture-dependent and culture-independent methods. Food Microbiol. 31, 293–300.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Microbiological characterization of traditional dough fermentation starter (Jiaozi) for steamed bread making by culture-dependent and culture-independent methods Zhijian Li a, Haifeng Li b,⁎, Ke Bian a,⁎ a b
College of Food Science and Technology, Henan University of Technology, Zhengzhou 450052, China College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China
a r t i c l e
i n f o
Article history: Received 6 April 2016 Received in revised form 17 June 2016 Accepted 21 June 2016 Available online 23 June 2016 Keywords: Jiaozi PCR-DGGE Yeast Bacteria Dough fermentation starter
a b s t r a c t In this study, the microbial composition of two types of Jiaozi (a dough fermentation starter in making steamed bread) was investigated using both culture-dependent and culture-independent (PCR-DGGE) methods. The numbers of the cultivable bacteria on MRS at 30 °C and yeast on YPD at 28 °C in the maize flour Jiaozi (MFJ) were 9.21 ± 0.16 Log CFU/g and 9.18 ± 0.05 Log CFU/g, respectively, which were higher than that in the rice flour Jiaozi (RFJ) (P b 0.05). A total of 140 bacteria and 124 yeasts were isolated and identified on the basis of the sequences of their 16S rRNA gene and ITS region. The culture-dependent method showed that Acetobacter tropicalis and Enterococcus durans were the predominant bacteria strains in MFJ, and accounted for 45.7% and 25.7% of the bacteria, and Lactobacillus plantarum and Pediococcus pentosaceus represented 12.8% and 8.6%. In the RFJ sample, the most prominent isolate was P. pentosaceus (38.6%), followed by L. plantarum (24.3%), A. tropicalis (22.8%), and E. durans (5.7%). P. pentosaceus and L. plantarum were also detected in both starters by PCR-DGGE, while some bacteria species such as A. tropicalis and E. durans, recovered as pure cultures, were not detected by direct PCR-DGGE. On the other hand, Lactobacillus brevis, Weissella sp. and Lactobacillus alimentarius detected by PCR-DGGE were not recovered in any of the media and conditions used. In the MFJ sample, the isolated main yeast species were identified as Wickerhamomyces anomalus (67.2%), Saccharomyces cerevisiae (27.9%) and Torulaspora delbrueckii (4.9%). In addition to S. cerevisiae (42.9%), W. anomalus (27.0%) and T. delbrueckii (7.9%), Saccharomycopsis fibuligera was also identified as the predominant isolate in RFJ samples and accounted for 22.2%. PCR-DGGE also indicated the presence of W. anomalus and S. cerevisiae in both MFJ and RFJ starters and S. fibuligera was also detected in RFJ, but T. delbrueckii was not detected in both samples. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Steamed bread is a traditional fermented staple food largely consumed in China (Li et al., 2015a). Although instant active dry yeast is widely used for dough fermentation in making steamed bread, traditional Jiaozi is still applied in many areas due to the unique flavour and taste of the product made with it (Li et al., 2014). Jiaozi is different from traditional sourdough because non-wheat flours were used as the main material for its preparation. In particular, Chinese Qu, which is a traditional starter for Chinese liquor production, is sometimes added in the preparation of Jiaozi (Li et al., 2014; Zheng et al., 2012). Based on the raw materials used, Jiaozi can be mainly categorized into maize flour Jiaozi (MFJ) and rice flour Jiaozi (RFJ). MFJ is naturally produced using maize flour, muskmelon and wheat flour as raw materials, while RFJ is manufactured by rice flour, rice wine and Qu. Although partly
⁎ Corresponding authors. E-mail address: hfl[email protected] (H. Li).
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.06.024 0168-1605/© 2016 Elsevier B.V. All rights reserved.
mechanized, the production process of Jiaozi is still based on empirical knowledge and relies on back-slopping and spontaneous fermentation. Therefore, the product quality and stability are usually variable due to the uncontrolled non-sterile fermentation conditions as well as occasional change of environmental conditions, such as temperature and water content of fermentation. Application of defined starter cultures is one of the approaches that could be used to improve indigenous technologies and ensure starter quality consistence (Mukisa et al., 2012). A prerequisite for the commercial production of Jiaozi from appropriate starter cultures is a thorough insight of the microbial composition behind it. Furthermore, having more information about the microbial structure existing in the starter is also important to produce product with high quality (Li et al., 2015b; Plessas et al., 2011). Although Jiaozi has been manufactured for hundreds of years, only few studies have been performed to characterize it from the microbiological point of view. Earlier microbiological work only studied RFJ using conventional culture-dependent techniques (Li et al., 2014). Several LAB and yeasts have been isolated from Jiaozi (Li et al., 2014); however, the study does not include
10
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
quantitative data of the strains isolated, even though this step is important to understand the ecological significance of these microorganisms. Furthermore, to date, no study investigated the microbial populations in Jiaozi by molecular method. In recent years, studies have shown that culture-dependent methods may lead to the underestimation of microbial diversity and failure to isolate the dominant flora in some cases. Therefore, complementing culture-dependent methods with culture-independent methods has attracted a lot of attention, and is recommended as a strategy to reduce bias and generate a better view of microbial diversity (Iacumin et al., 2009; Ongol and Asano, 2009). Various studies have shown that polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE) is one reliable and reproducible technique to study the ecology of traditional dough fermentation starters, such as sourdoughs (Cocolin et al., 2013). In this study, the diversity of the representative microbial populations associated with two types of Jiaozi was investigated and compared using a combination of culture-dependent and cultureindependent (PCR-DGGE) methods. This study is the first to extensively investigate the bacterial and yeast diversity in the Jiaozi, and is relevant for future development of effective defined starter cultures. 2. Materials and methods
applicable. Data were analyzed using SPSS software (SPSS 19.0, SPSS Inc., Chicago, USA).
2.3. DNA extraction from pure culture and PCR amplification Genomic DNA of bacteria and fungi was extracted by Bacteria and Yeast Genomic DNA Purification Kit (Tiangen, Beijing, China), respectively, according to the manufacturer's instructions. The 16S rRNA gene was amplified using forward primer P0 (5′-GAG AGT TTG ATC CTG GCT CAG-3′) and reverse primer P6 (5′-CTA CGG CTA CCT TGT TAC-3′) (Quero et al., 2014). The ITS regions were amplified by forward primer ITS4 (5′-TCC TCC GCT GAC TAA TAT GC-3′) and reverse primer ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) (Li et al., 2013). Amplifications were performed in a MasterCycler Gradient thermal cycler (Eppendorf, Germany). The PCR mixture (25 μL) contained 2.5 μL of 10× PCR buffer, 10 ng of template DNA, 0.5 μL of each primer (10 μM), 0.5 μL of dNTP mixture (10 mM each) and 2.5 U of Taq polymerase (Sangon Biotech Co. Ltd., Shanghai, China). Template DNA was denatured for 5 min at 94 °C, 30 cycles of 94 °C for 30 s, 54 °C for 45 s, 72 °C for 2 min and a final extension of 72 °C for 5 min. The same PCR amplification conditions were used for both bacterial and fungal pairs of primers. After purification, the PCR products were sent to a commercial facility for sequencing (Sangon Biotech Co. Ltd., Shanghai, China). Sequences were aligned with those in GeneBank with the BLAST program to determine the closest known relatives.
2.1. Sample collection Two kinds of Jiaozi final products (2.0 kg of each sample) were collected from a local market. The RFJ made from rice wine, Chinese Qu and rice flour was obtained from Nanyang, Henan, China. The MFJ made from maize flour, muskmelon and wheat flour was obtained from Shangqiu, Henan, China. The dimensions of RFJ and MFJ blocks were about 8 × 5 × 3 cm and 3 × 3 × 3 cm, respectively. Jiaozi blocks (500 g) were randomly selected and ground to powder in an alcoholdisinfected grinder; the powder was kept in plastic jars at 4 °C. 2.2. Microbiological analysis Samples of Jiaozi (10 g each) were homogenized with 90 mL sterile PPS (peptone physiological salt) solution containing 0.1% peptone and 0.85% NaCl. Appropriate serial dilutions were prepared using the same diluent. Bacteria were enumerated on plate count agar (PCA), de Man– Rogosa–Sharpe agar (MRS), medium 17 agar (M17) and modified Chalmers agar (MC) with 0.1% (w/v) natamycin to prevent yeast growth (Li et al., 2010; Pepe et al., 2001; Terzaghi and Sandine, 1975). Plates were incubated at 30 °C and 42 °C under aerobic or anaerobic conditions for 3 days. Anaerobic conditions were obtained via incubation in anaerobic jars (AnaeroPack Rectangular Jar™ Mitsubishi Gas Chemical Co., Tokyo, Japan) containing an O2 absorbing and CO2 releasing pack (AnaeroPack ™, Mitsubishi Gas Chemical Co., Tokyo, Japan) (Ongol and Asano, 2009). Fungi were enumerated on three different media, namely, Czapek– Dox agar (CDA), yeast peptone dextrose agar (YPD) and Rose Bengal agar (RBA), to which 100 mg/L chloramphenicol was added (Li et al., 2010, 2013, 2014). The diluted suspension (0.2 mL) was added to make spread plates which were incubated at 28 °C for 2 days. From plates with 20–200 colonies, the representative colonies of yeasts and bacteria were randomly selected according to their macromorphological aspects and isolated in proportion to their frequencies. The results reported in this article are the average values ± S.D. The significant differences between two treatments with different media were detected using one-way analysis of variance (ANOVA) by the Tukey's range test (P b 0.05). Student's t-test was used to compare the means of microorganism number between two Jiaozi samples wherever
2.4. PCR-DGGE analyses DNA from Jiaozi samples was extracted as previously described (Lv et al., 2013). The V3 region of the 16S rRNA gene of the total bacteria was amplified using the universal primers: GC-338F (CGCCCGGGGCGCGCCCCGGGGCGGGGCGGGGGCGCGGGGGG CCT ACG GGA GGC AGC AG) and 518R (ATT ACC GCG GCT GCT GG) (Nakatsu et al., 2000). The Eucarya-specific 18S rRNA genes were amplified by one set of universal primers: NS1 (GTA GTC ATA TGC TTG TCT C) and GCfung (CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCC CAT TCC CCG TTA CCC GTT G) (Hoshino and Morimoto, 2008). PCR reactions were carried out using a PTC220 thermal cycler (Bio-Rad, USA). The reaction mixture (50 μL) contained 5 μL of 10× PCR buffer, 50 ng of template DNA, 1 μL of each primer (10 μM), 3.2 μL of dNTP (2.5 mM) and 2.5 U of Taq polymerase. The amplification program was 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 55 °C for 45 s and 72 °C for 1 min and a final extension at 72 °C for 10 min. The sizes and quantities of the PCR products were determined using 1.5% agarose gel electrophoresis. The gradient gel preparation and electrophoresis were performed using the Bio-Rad Dcode Universal Mutation Detection System according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). The PCR products were separated on an 8% polyacrylamide gel with a 30%–55% urea–formamide linear denaturing gradient for bacteria DGGE analysis and 20%–45% denaturing gradient for fungi DGGE analysis. The 100% denaturing solution consisted of 40% (v/v) formamide deionized and 7.0 M urea. Electrophoresis was conducted in 1× TAE buffer at a constant voltage of 150 V at 60 °C for 4 h for bacteria and at a constant voltage of 50 V at 60 °C for 20 h for fungi. Following electrophoresis, the gels were stained by silver staining (Zhang et al., 2011). The gel was scanned with “Quantity One” software using a calibrated imaging Gel-Doc2000 (Bio-Rad, CA, USA). Stained bands were excised and recycled using Poly-Gel DNA Extraction Kit of OMEGA and re-amplified with the primer without the GC clamp under the same conditions as described above. The product was cloned into pGEM-T easy vector and the inserts of the positive clones were sequenced. Sequencing of DNA was carried out by Sangon Biotech Co. Ltd., Shanghai, China and analysis of sequence data was performed using BLAST program as describe above.
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
3. Results and discussion
Table 2 Identities of bacteria and yeast isolates from two Jiaozi samples on MRS, M17, MC, YPD, RBA and CDA.
3.1. Enumeration of bacteria and yeast on selective media Traditionally, culture media are only partially selective and exclude parts of the microbial community. In order to find out more kinds of bacteria from Jiaozi, enumeration of total bacteria from the two Jiaozi samples was carried out on four different types of media (PCA, MRS, M17 and MC) at 30 °C or 42 °C under aerobic or anaerobic cultivation. The results of the microbiological analysis are shown in Table 1. The total aerobic bacteria count in MFJ was about 9.20 Log CFU/g on PCA, MRS and M17 media at 30 °C, which was higher than that on MC medium (P b 0.05). The bacteria number decreased to 5.94–6.70 Log CFU/g at 42 °C on these three media. The total anaerobic bacteria in MFJ were counted about 8.48–8.86 Log CFU/g on these three media. In RFJ sample, aerobic, anaerobic and thermophilic bacteria counts were comparable on MRS, which were higher than that on M17 and MC. In general, bacterial counts in the RFJ were lower than that in the MFJ on each of the four media used at 30 °C (P b 0.05). In addition, the high level of thermophilic bacteria in RFJ on MRS and M17 media at 42 °C makes this starter different from MFJ (P b 0.05). In this study, three mycological media (YPD, RBA and CDA) were used to understand the diverse yeast biota of Jiaozi. The yeast counts were present at levels of 8.83–9.18 Log CFU/g on three media in MFJ, which were about ten times higher than that in RFJ. The data obtained by traditional microbiological counting showed that the number of bacteria was comparable to the number of yeasts in each Jiaozi sample, especially in MFJ. This high level of yeast number in Jiaozi makes this kind of starter different from other types of traditional starters, such as sourdough and starter prepared by fed batch fermentation of which the yeast/LAB ratio is generally 1:100 or 1:10 (Iacumin et al., 2009; Li et al., 2014).
3.2. Identification of isolates A total of 140 bacterial isolates were randomly selected and identified from the two Jiaozi samples. The identities of the isolated strains are presented in Table 2. The result indicated that LAB and acetic acid bacteria (AAB) were the dominant bacterial groups in Jiaozi. Acetobacter tropicalis, Enterococcus durans, Lactobacillus plantarum and Pediococcus pentosaceus were found in both samples, whereas Acetobacter orleanensis, Acetobacter cerevisiae and Escherichia coli were only observed in MFJ and Enterococcus faecalis, Bacillus cereus and Staphylococcus warneri were only found in RFJ samples. Table 1 Microbiological analysis of the two Jiaozi samples. Microbial groups
Media and incubation conditions
MFJ (Log CFU/g)
RFJ (Log CFU/g)
Bacteria PCA (30 °C) MRS (30 °C) MRS (anaerobic conditions, 30
9.20 ± 0.02a 9.21 ± 0.16a 8.86 ± 0.03b
8.95 ± 0.04a 8.37 ± 0.06b 8.31 ± 0.11b
°C) MRS (42 °C) M17 (30 °C) M17 (anaerobic conditions, 30
6.69 ± 0.04c 9.20 ± 0.01a 8.58 ± 0.02d
8.46 ± 0.06b 7.85 ± 0.02cd 8.08 ± 0.12ef
5.94 ± 0.01e 8.62 ± 0.04d 8.48 ± 0.03d
7.68 ± 0.06c 6.69 ± 0.04g 7.82 ± 0.03cd
6.70 ± 0.06c 9.18 ± 0.05a 8.83 ± 0.02b 8.84 ± 0.03b
6.09 ± 0.05h 7.91 ± 0.05de 8.12 ± 0.04f 7.43 ± 0.04i
°C) M17 (42 °C) MC (30 °C) MC (anaerobic conditions, 30 ° C) MC (42 °C) Yeasts YPD (28 °C) RBA (28 °C) CDA (28 °C)
11
Means with different superscript letters within the same column are significantly different (P b 0.05).
Microbial groups
Species identification
Bacteria Acetobacter tropicalis Enterococcus durans Lactobacillus plantarum Pediococcus pentosaceus Acetobacter orleanensis Acetobacter cerevisiae Enterococcus faecium Bacillus cereus Escherichia coli Staphylococcus warneri Yeasts Wickerhamomyces anomalus Saccharomyces cerevisiae Torulaspora delbrueckii Saccharomycopsis fibuligera
MFJ
RFJ
(% of the 70 isolates) 45.7 25.7 12.8 8.6 2.9 2.9
(% of the 70 isolates) 22.8 5.7 24.3 38.6
1.4 2.9 1.4 (% of the 61 isolates) 67.2 27.9 4.9
4.3 (% of the 63 isolates) 27.0 42.9 7.9 22.2
In MFJ, the most commonly isolated species were A. tropicalis and E. durans constituting 45.7% and 25.7% of the total bacterial colonies, respectively (Table 2), and L. plantarum and P. pentosaceus represented 12.8% and 8.6%, respectively. P. pentosaceus, L. plantarum and A. tropicalis represented 38.6%, 24.3% and 22.8%, respectively, of the total number of isolates from RFJ. The isolates identified as E. coli in MFJ and B. cereus, E. faecium and S. warneri in RFJ constituted a minor proportion of the total number of isolates. B. cereus could originate from Qu, which was a material used for making RFJ, because it was revealed that Bacillus sp. was an abundant bacterial species in that ingredient (Zheng et al., 2012). The presence of E. coli in MFJ, E. faecium and S. warneri in RFJ was probably caused by contamination from the raw material or environment during Jiaozi manufacturing process. It was interesting to find a similarity of the predominant bacteria species between the two different kinds of Jiaozi samples from different regions, but their percentages in each Jiaozi were completely different. Although it has been reported that LAB play an important role in traditional dough fermentation starters (Alfonzo et al., 2013; Zhang et al., 2011), there is limited report indicating that AAB could be of importance in dough starter (Minervini et al., 2012). This report highlights the involvement of AAB in traditional dough fermentation starters. Yeast species composition in Jiaozi was also analyzed by the culturedependent analysis and the isolation percentages of the yeast species are shown in Table 2. Wickerhamomyces anomalus was the most commonly isolated species in sample MFJ (67.2%), followed by Saccharomyces cerevisiae (27.9%). S. cerevisiae, W. anomalus and Saccharomycopsis fibuligera were the three most abundant yeast species in RFJ, constituting 42.9%, 27.0% and 22.2% of the total isolates, respectively. Torulaspora delbrueckii was identified in both Jiaozi samples, but it only accounted for a small proportion. The analysis indicated that the dominant yeasts were in a different pattern and their ratio also varied between two types of Jiaozi. The key difference between the two samples can be attributed to S. fibuligera, which was only isolated in the RFJ sample. The variations could probably be influenced by differences in yeast flora of the raw materials and the production technologies used by the different producers. Various authors have shown that S. cerevisiae and W. anomalus are prevalent yeasts typically associated with sourdoughs (Garofalo et al., 2011; Daniel et al., 2011; Iacumin et al., 2009). The presence of S. fibuligera observed in RFJ could be related to the use of Qu for RFJ making (Lv et al., 2013; Zheng et al., 2012). These results indicate that non-
12
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
Saccharomyces yeasts are part of the microbial community in Jiaozi and could be adjunct cultures for dough fermentation. 3.3. PCR-DGGE analysis The microbial diversity of two types of Jiaozi was further studied using culture-independent technique (PCR-DGGE). The results of the diversity of the bacteria and fungi associated with two Jiaozi are reported in Fig. 1 and Table 3. DGGE profiles also displayed some similarities of dominant bacteria flora in two Jiaozi. Sequencing and identification of
Fig. 1. Bacterial (A) and fungal (B) PCR-DGGE profiles of MFJ and RFJ.
the 20 DNA bands revealed that more microbial species existed in RFJ than that in MFJ. Besides the P. pentosaceus, L. plantarum, Lactobacillus brevis and Lactobacillus alimentarius in both samples (Table 3), Lactobacillus farciminis, Weissella sp. and Bacillus sp. were also identified in RFJ. The results showed that direct PCR-DGGE of total community DNA yielded a different description of bacteria composition in the starter compared with culturing results. Although L. plantarum and P. pentosaceus were identified by both methods, L. brevis, L. farciminis, Weissella sp. and L. alimentarius were not recovered by cultivation techniques. L. brevis and W. cibaria are commonly associated with sourdoughs and L. plantarum particularly codominated with these LAB in sourdough production (Settanni et al., 2013; Ventimiglia et al., 2015). The possible explanation for the failure to isolate them might be due to their scarcity to be selected from the plates or due to the selectiveness of the culture media and culturing condition. In contrast, it was especially surprising that the other two cultivable species, A. tropicalis and E. durans that had been identified as the dominant species in MFJ and RFJ by culture-dependent analysis, were not detected by the PCRDGGE. The discrepancy could be attributed to the efficiency of DNA extraction or the selective PCR amplification (Madoroba et al., 2011; Mukisa et al., 2012; Reale et al., 2011). It has been reported that DNA extraction yields can vary significantly, even for closely related strains (Miambi et al., 2003). In addition, some community members may affect the detection of certain species due to competition during PCR (Miambi et al., 2003). Discrepancies between culturing and PCR-DGGE results have also been observed for sourdough (Iacumin et al., 2009; Zhang et al., 2011). This highlights different limitations of culturing and PCR-DGGE in studying microbial diversity. Therefore, a combination of both methods is indeed useful for studying microbial diversity of traditional starters. Bands 2, 17 and 20 were identified as a chloroplast DNA of plant and band 18 could not be identified using the GenBank database. The problematic multiple banding pattern observed for L. plantarum, L. brevis and P. pentosaceus could be related to the presence of multicopies of the 16S rRNA gene for these microorganisms as reported in previous studies (Chao et al., 2013; Miguel et al., 2010). Combining the results of culture-dependent and culture-independent analysis, it can be found that the Jiaozi has a broader microbial diversity and higher numbers of microorganisms. LAB strains are mainly responsible for the dough acidification (Li et al., 2015b). They contributed to the flavour development of steamed bread due to the formation of organic acids and other flavour compounds (Li et al., 2014). The results of sequencing of the fungal DGGE bands are reported in Table 3. The fungal results revealed by culture-independent method were almost the same as that of culture-dependent method. S. cerevisiae and W. anomalus were identified by both methods and were the dominant species in two Jiaozi samples. S. fibuligera observed in RFJ by PCRDGGE further confirmed the results observed by culture method. Conversely, T. delbrueckii was not detected by PCR-DGGE in both samples. Possible reasons might be that a small fraction of its DNA was extracted which was lower than the detection limit of PCR-DGGE (Chao et al., 2013; Mukisa et al., 2012). On the other hand, large amounts of DNA from other yeast species had the potential to interfere with the specific binding of the primers to the template of this species (Chen et al., 2008; Tu et al., 2010). The presence of Mucor indicus was probably caused by contamination from the raw material or environment (Zheng et al., 2012). Several bands derived from a single species may be due to the amplification of the interspecific or intraspecific heterogeneous nature of the ITS region targeted (Madoroba et al., 2011), as the case for the bacteria detected in this study. The simple and similar diversity of dominant yeasts in two types of Jiaozi indicates a long-term natural selection process of the yeasts originating from ancient times. It is highly likely that a constant recycling of inoculum, as with back-slopping, results in the dominance of these yeast species. These results are in agreement with the generally accepted concept that traditional fermentation products are dominated by a
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
13
Table 3 Identification of the bands obtained by PCR-DGGE. Microbial groups
Band(s)a
Closest relative
Accession number
Identity (%)
Sample
AB889691 KM352501.1 HG798506 KF668014 KC753454 KC753459 KF746906 NR_044701 HG670306.1 GU301189 AP014692.1
99 100 100 100 100 99 99 99 100 99 100
RFJ MFJ MFJ and RFJ RFJ MFJ and RFJ MFJ and RFJ RFJ MFJ and RFJ MFJ RFJ MFJ and RFJ
NR132222 KJ659884 KP119822 AY054699
99 99 99 100
MFJ and RFJ MFJ and RFJ RFJ MFJ and RFJ
Bacteria 1 2 3, 4, 5, 7, 8, 9 6 10, 19 11, 12, 14, 16 13 15 17 18 20
Lactobacillus farciminis Triticum turgidum subsp. durum cultivar Langdon chloroplast Lactobacillus plantarum Weissella sp. FB12 Lactobacillus brevis Pediococcus pentosaceus Bacillus sp. M-237-24 Lactobacillus alimentarius Triticum aestivum uncultured bacterium Vigna radiata var. sublobata chloroplast
Yeasts 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 12, 16 11, 13 14, 15 a
Saccharomyces cerevisiae Wickerhamomyces anomalus Saccharomycopsis fibuligera Mucor indicus SDM-13
Bands are numbered as indicated on the DGGE gels shown in Fig. 1.
few microbial species that are selected during the course of fermentation as a result of physiological and metabolic adaptation to the food matrix (Madoroba et al., 2011). S. cerevisiae is the most effective ethanol producer in the fermentation of cereals and alcoholic beverages (Lv et al., 2013). It also plays a leading role in the fermentation of wheat and maize dough and is normally reported as being predominant such that it forms the basis for a variety of different foods (Greppi et al., 2013; Iacumin et al., 2009). This could explain the predominance of S. cerevisiae in both Jiaozi, especially in RFJ, which is normally produced from rice wine. S. cerevisiae is positively affected by the presence of L. plantarum, which produced much more CO2 than when in association with Lactobacillus sanfranciscensis (Iacumin et al., 2009). W. anomalus was observed in Jiaozi as well by both detection approaches. This yeast species is common in cereal fermentations (wheat dough and Qu) and in combination with LAB, has been associated with the production of flavour and ethanol (Iacumin et al., 2009; Zheng et al., 2012). S. fibuligera, another abundant yeast species isolated in RFJ in this study, can secrete a large amount of α-amylase, which may facilitate the conversion of starch into fermentable carbohydrates, thus generating a suitable substrate for dough fermentation (Lv et al., 2013; Zheng et al., 2012). T. delbrueckii isolated in this study showed high activity of maltase (data not shown). Although it was detected in low percentage by culture dependent method in both samples, this yeast could play an important role to provide sufficient carbon source in the dough fermentation. In recent years, the significance of non-Saccharomyces yeasts in winemaking has attracted the interest of winemaking researchers (Lv et al., 2013). Nonetheless, the function of non-Saccharomyces yeasts in Jiaozi is still poorly understood. The functionality of the yeast biota needs further investigation. The results in this study reveal that the distribution profiles of microorganisms in Jiaozi products have a common pattern comprising of above predominant species only if special raw materials was used. The results enrich our knowledge of dough starter-related microorganisms, and could help in narrowing down the isolates that can be evaluated for future studies to generate effective defined starter cultures. Furthermore, the present study highlights a combined use of culture-dependent and -independent approaches for detection of microbial communities, especially bacteria within complex matrices. Acknowledgements This research was supported by the Fundamental Research Funds for the Henan Provincial Colleges and Universities in Henan University of Technology (2015QNJH01 and 2014YWQQ18), Special Fund for Agro-
scientific Research in the Public Interest (grant no. 201303070), National Natural Science Foundation of China (31400103) and Science and Technology Project of Henan Province (132300410423). References Alfonzo, A., Ventimiglia, G., Corona, O., Di Gerlando, R., Gaglio, R., Francesca, N., Moschetti, G., Settanni, L., 2013. Diversity and technological potential of lactic acid bacteria of wheat flours. Food Microbiol. 36, 343–354. Chao, S.H., Huang, H.Y., Kang, Y.H., Watanabe, K., Tsai, Y.C., 2013. The diversity of lactic acid bacteria in a traditional Taiwanese millet alcoholic beverage during fermentation. LWT Food Sci. Technol. 51, 135–142. Chen, H., Wang, S., Chen, M., 2008. Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods. Food Microbiol. 25, 492–501. Cocolin, L., Alessandria, V., Dolci, P., Gorra, R., Rantsiou, K., 2013. Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation. Int. J. Food Microbiol. 167, 29–43. Daniel, H.M., Moons, M.C., Huret, S., Vrancken, G., De Vuyst, L., 2011. Wickerhamomyces anomalus in the sourdough microbial ecosystem. Antonie Van Leeuwenhoek 99, 63–73. Garofalo, C., Aquilanti, L., Clementi, F., 2011. The biodiversity of the microbiota of traditional Italian sourdoughs. In: Almeida, M.T. (Ed.), Wheat: Genetics, Crops and Food Production. Nova Science Publishers, Inc., New York, pp. 366–392. Greppi, A., Rantisou, K., Padonou, W., Hounhouigan, J., Jespersen, L., Jakobsen, M., Cocolin, L., 2013. Yeast dynamics during spontaneous fermentation of mawè and tchoukoutou, two traditional products from Benin. Int. J. Food Microbiol. 165, 200–207. Hoshino, Y.T., Morimoto, S., 2008. Comparison of 18S rDNA primers for estimating fungal diversity in agricultural soils using polymerase chain reaction-denaturing gradient gel electrophoresis. Soil Sci. Plant Nutr. 54, 701–710. Iacumin, L., Cecchini, F., Manzano, M., Osualdini, M., Boscolo, D., Orlic, S., Comi, G., 2009. Description of the microflora of sourdoughs by culture-dependent and culture-independent methods. Food Microbiol. 26, 128–135. Li, Z., Li, H., Deng, C., Liu, C., 2014. Effect of mixed strain starter culture on rheological properties of wheat dough and quality of steamed bread. J. Texture Stud. 45, 180–186. Li, Z., Deng, C., Li, H., Liu, C., Bian, K., 2015a. Characteristics of remixed fermentation dough and its influence on the quality of steamed bread. Food Chem. 179, 257–262. Li, Z., Li, H., Deng, C., Bian, K., Liu, C., 2015b. Effect of Lactobacillus plantarum DM616 on dough fermentation and Chinese steamed bread quality. J. Food Process. Preserv. 39, 30–37. Li, L., Feng, L., Yi, J., Hua, C., Chen, F., Liao, X., Wang, Z., Hu, X., 2010. High hydrostatic pressure inactivation of total aerobic bacteria, lactic acid bacteria, yeasts in sour Chinese cabbage. Int. J. Food Microbiol. 142, 180–184. Li, Z., Li, H., Yang, X., Huang, Z., Liu, C., Cao, B., 2013. Characterization of Se(IV) removal from aqueous solution by Aspergillus sp. J2. Chem. Eng. J. 220, 67–71. Lv, X.C., Huang, X.L., Zhang, W., Rao, P.F., Ni, L., 2013. Yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing, revealed by culturedependent and culture-independent methods. Food Control 34, 183–190. Madoroba, E., Steenkamp, E.T., Theron, J., Scheirlinck, I., Cloete, T.E., Huys, G., 2011. Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food. Syst. Appl. Microbiol. 34, 227–234. Miambi, E., Guyot, J., Ampe, F., 2003. Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods. Int. J. Food Microbiol. 82, 111–120.
14
Z. Li et al. / International Journal of Food Microbiology 234 (2016) 9–14
Miguel, M.G.D.C.P., Cardoso, P.G., Lago, L.D.A., Schwan, R.F., 2010. Diversity of bacteria present in milk kefir grains using culture-dependent and culture-independent methods. Food Res. Int. 43, 1523–1528. Minervini, F., Lattanzi, A., De Angelis, M., Di Cagno, R., Gobbetti, M., 2012. Influence of artisan bakery- or laboratory-propagated sourdoughs on the diversity of lactic acid bacterium and yeast microbiotas. Appl. Environ. Microbiol. 78, 5328–5340. Mukisa, I.M., Porcellato, D., Byaruhanga, Y.B., Muyanja, C.M., Rudi, K., Langsrud, T., Narvhus, J.A., 2012. The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods. Int. J. Food Microbiol. 160, 1–10. Nakatsu, C.H., Torsvik, V., Øvreås, L., 2000. Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci. Soc. Am. J. 64, 1382–1388. Ongol, M.P., Asano, K., 2009. Main microorganisms involved in the fermentation of Ugandan ghee. Int. J. Food Microbiol. 133, 286–291. Pepe, O., Villani, F., Coppola, S., 2001. Differential viable count of mixed starter cultures of lactic acid bacteria in doughs by using modified Chalmers medium. Microbiol. Res. 155, 351–354. Plessas, S., Alexopoulos, A., Mantzourani, I., Koutinas, A., Voidarou, C., Stavropoulou, E., Bezirtzoglou, E., 2011. Application of novel starter cultures for sourdough bread production. Anaerobe 17, 486–489. Quero, G.M., Fusco, V., Cocconcelli, P.S., Owczarek, L., Borcakli, M., Fontana, C., Skapska, S., Jasinska, U.T., Ozturk, T., Morea, M., 2014. Microbiological, physico-chemical, nutritional and sensory characterization of traditional Matsoni: selection and use of autochthonous multiple strain cultures to extend its shelf-life. Food Microbiol. 38, 179–191.
Reale, A., Di Renzo, T., Succi, M., Tremonte, P., Coppola, R., Sorrentino, E., 2011. Identification of lactobacilli isolated in traditional ripe wheat sourdoughs by using molecular methods. World J. Microbiol. Biotechnol. 27, 237–244. Settanni, L., Ventimiglia, G., Alfonzo, A., Corona, O., Miceli, A., Moschetti, G., 2013. An integrated technological approach to the selection of lactic acid bacteria of flour origin for sourdough production. Food Res. Int. 54, 1569–1578. Terzaghi, B.E., Sandine, W.E., 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 341. Tu, R., Wu, H., Lock, Y., Ju, C., 2010. Evaluation of microbial dynamics during the ripening of a traditional Taiwanese naturally fermented ham. Food Microbiol. 27, 460–467. Ventimiglia, G., Alfonzo, A., Galluzzo, P., Corona, O., Francesca, N., Caracappa, S., Moschetti, G., Settanni, L., 2015. Codominance of Lactobacillus plantarum and obligate heterofermentative lactic acid bacteria during sourdough fermentation. Food Microbiol. 51, 57–68. Zhang, J., Liu, W., Sun, Z., Bao, Q., Wang, F., Yu, J., Chen, W., Zhang, H., 2011. Diversity of lactic acid bacteria and yeasts in traditional sourdoughs collected from western region in Inner Mongolia of China. Food Control 22, 767–774. Zheng, X.W., Yan, Z., Han, B.Z., Zwietering, M.H., Samson, R.A., Boekhout, T., Nout, M.J.R., 2012. Complex microbiota of a Chinese “Fen” liquor fermentation starter (FenDaqu), revealed by culture-dependent and culture-independent methods. Food Microbiol. 31, 293–300.