Frontiers in Life Science, 2015 Vol. 8, No. 3, 264–270, http://dx.doi.org/10.1080/21553769.2015.1041188 Cultivable bacterial diversity and amylase production in two typical light-ﬂavor Daqus of Chinese spirits a , b b b∗ c a a Zuming Li , Lin Chen , Zhihui Bai , Deliang Wang , Liping Gao and Bodi Hui a b College of Arts and Sciences,Beijing Union University, Beijing 100191, PR China; Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China; China National Research Institute of Food and Fermentation Industries, Beijing 100027, PR China (Received 19 October 2014; accepted 12 April 2015 ) Culture-dependent methods and molecular techniques were used to simultaneously investigate the cultivable bacterial diversity and amylase production in two typical light-ﬂavor Daqus of Chinese spirits. Eight bacteria were identiﬁed from Niulanshan Daqu (Bacillus licheniformis, Bacillus subtilis, Bacillus cereus, Bacillus sonorensis, Streptomyces albus, Bacillus atrophaeus, Bacillus tequilensis and Bacillus megaterium) and eight from Hongxing Daqu (B. licheniformis, B. subtilis, B. cereus, Bacillus thuringiensis, Bacillus altitudinis, Bacillus pumilus, Geobacillus stearothermophilus and Bacil- lus amyloliquefaciens). All of the bacterial isolates from the Hongxing Daqu could produce extracellular α-amylase with a maximum yield of 25.3 U/ml by B. cereus H17, whereas B. licheniformis H55 could produce a maximum glucoamylase yield of 41.6 U/ml. Some of the bacterial isolates from the Niulanshan Daqu could also produce extracellular α-amylase and glucoamylase. The maximum yield of 27.6 U/ml α-amylase was achieved by B. subtilis N3, and the maximum yield of 58.1 U/ml glucoamylase was achieved by B. cereus N25. Bacillus licheniformis, B. subtilis and B. cereus were not only the dominant bacteria, but also possessed high α-amylase and glucoamylase activities, which may play very important roles during fermentation. Keywords: Chinese spirits; Daqu; bacterial diversity; 16S rRNA gene; α-amylase; glucoamylase Introduction Traditional microbiological methods, such as isolation Chinese spirits are commonly known as ‘Chinese liquor’ and enumeration in selective media, are eﬀective in qual- and are very popular in China. There are three main types itative and quantitative analysis (Shi et al. 2009; Zheng of Chinese spirit: sauce-ﬂavor liquor, intense-ﬂavor liquor et al. 2012). Sequencing analysis of the 16S rRNA gene and light-ﬂavor liquor (Li et al. 2013). Chinese spirits are has been used for rapid bacterial identiﬁcation (Jeyaram typically obtained from cereals such as sorghum and wheat et al. 2010). In this study, reports on an investigation of the by complex fermentation processes using natural mixed cultivable bacterial diversity and amylase production from culture starters (i.e. ‘Daqu’). Daqu, which is made from two typical light-ﬂavor Daqus in Chinese spirits. raw wheat, barley and/or peas, is associated with the degra- dation of starch and the production of alcohol, and is also Materials and methods the determinative factor for liquor ﬂavoring (Liu et al. The two Daqus were obtained from two major Chinese 2012). Daqu contains microbes and enzymes, and both glu- spirit factories in Beijing, which are well known in China. coamylase and α-amylase are two important amylases for The sampling data are shown in Table 1. Three parallel starch hydrolysis. In the brewing industry, the hydrolyzates samples were collected from the top, middle and bottom are used as nutrients in microbial fermentation for ethanol in the store room. After being milled and mixed, samples production after enzymatic hydrolysis. However, amylase were transferred to sterile bags, sealed and stored at 4°C production of bacteria from Daqus is poorly understood. for further use. During fermentation, the degradation of biopolymers such Ten grams of Daqu powder was homogenized in 90 ml as starch and the formation of aromatic compounds are of a sterile 0.9% NaCl solution in a rocking incubator for usually performed by microbes (Wang et al. 2008). There- 30 min at 180 rpm. Samples (1.0 ml) of the homogenate fore, it is important to check the exact composition of the were serially diluted 10-fold in a sterile 0.9% NaCl solu- natural product’s microﬂora. However, very little is known tion, from which aliquots (0.1 ml) were plated on beef about the speciﬁc microbes associated with Daqus, despite the fact that the subject has been studied by microbiologists extract peptone agar plates with the following components (Wang et al. 2008; Zheng et al. 2012). (w/v): 0.5% beef extract, 1.0% peptone and 0.5% NaCl. *Corresponding author. Email: firstname.lastname@example.org © 2015 Taylor & Francis Frontiers in Life Science 265 Table 1. Samples of two typical light-ﬂavor Daqus of Chinese The isolates from the Daqus were diluted in a sterile spirits. 0.9% NaCl solution and then plated on to starch agar plates containing 2.0% soluble starch, 1.0% peptone and 0.5% Highest temperature NaCl. Inoculated plates were incubated for 1–3 days at Name of inside the Daqu Name of 37°C to obtain colonial growth. The incubated plates were Sample no. Daqu pile (°C) factory stained with iodine solution, and the colonies with clear zones formed by the hydrolysis of starch were evaluated as N Niulanshan 50 Beijing Niulan- amylase producers. The components (w/v) of the medium shan Liquor Co. Ltd used for liquid inoculum culture were as follows: 0.3% H Hongxing 50 Beijing Hongxing beef extract, 1.0% peptone and 0.5% NaCl, pH 7.2. The Liquor Co. Ltd medium was sterilized by autoclaving at 121°C for 20 min. Medium (20 ml) in 150 ml Erlenmeyer ﬂasks was inocu- lated and cultivated at 37°C by shaking at 180 rpm for 24 h. Inoculated plates were incubated at 37°C for 24–48 h for The medium in a 500 ml Erlenmeyer ﬂask contained (w/v): isolation and screening on the basis of diﬀerent colony soluble starch 2.0%, beef extract 0.5%, peptone 1.0% and morphologies (diameter, shape, color, surface and spores). NaCl 0.5%, pH 7.2. After sterilization, the medium (100 The genomic DNAs of the isolates were extracted, ml) was inoculated with 6 ml of a 24-h-old inoculum cul- ampliﬁed and sequenced according to the methods ture, and cultivated at 37°C by shaking at 180 rpm for 48 h, described by Li et al. (2012). The 16S rDNA sequence data then centrifuged at 8000 rpm at 4°C for 15 min to remove of the isolates reported here have been submitted to Gen- the cells and debris. The supernatant was designated as the Bank nucleotide sequence databases with the accession crude enzyme for future enzyme activity assays (Qureshi numbers shown in Tables 2 and 3. et al. 2015). Table 2. Bacterial diversity and amylase production in Niulanshan Daqu. Related Activity of GenBank Closest relative (NCBI Activity of glucoamylase Strain no. sequence accession no.) Identity (%) HC value α-amylase (U/ml) (U/ml) N1 KC441799 B. licheniformis BaDB24 99.9 1.5 5.9 12.1 (JX237858) N3 KC441800 B. subtilis FS106 (KC179631) 100 2.1 27.6 18.5 N5 KC441815 B. sonorensis HT1 99.9 – – – (JN013186) N7 KC441801 Streptomyces albus NRRL 98.3 – – – B-2365 (DQ026669) N10 KC441802 B. licheniformis DQgbc4 100 – – – (GQ470399) N12 KC441803 B. subtilis CRRI-HN-3 99.2 – – – (JQ695930.1) N15 KC441804 B. atrophaeus Aj080319IA- 99.9 1.9 11.7 19.2 12 (HQ727972) N16 KC441805 B. licheniformis LZBL-9 99.9 – – – (JX847115) N18 KC441806 B. licheniformis VPS50.2 99.9 1.3 0.5 3.7 (HE993550) N20 KC441807 B. tequilensis SH41 100 2.4 14.6 25.9 (KC172031) N21 KC441814 B. licheniformis 2J-1 99.7 – – – (FJ493045) N25 KC441808 B. cereus DZ4 (HQ143564) 99.4 1.7 15.5 58.1 N29 KC441809 B. megaterium HNYM34 99.9 – – – (JN999852) N33 KC441810 B. licheniformis CICC 10334 97.8 1.3 5.8 23.1 (GQ375243) N34 KC441811 B. cereus XX2010 100 1.2 1.7 5.6 (JX993816) N38 KC441812 B. subtilis IMAU80212 99.5 – – – (GU125629) Note: NCBI = National Center for Biotechnology Information; HC = hydrolysis capacity; B. = Bacillus;– = the value of the parameter was not detected. 266 Z. Li et al. Table 3. Bacterial diversity and amylase production in Hongxing Daqu. Related Activity of GenBank Closest relative (NCBI Activity of glucoamylase Strain no. sequence accession no.) Identity (%) HC value α-amylase (U/ml) (U/ml) H3 KC441784 B. cereus HN-Beihezhu1 98.5 1.4 6.6 21.6 (JQ917438) H12 KC441785 B. subtilis LXB3 (GQ861468) 99.9 1.7 10.3 – H17 KC441786 B. cereus DZ4 (HQ143564) 99.4 2.2 25.3 3.6 H23 KC441787 B. thuringiensis E101 97.8 1.2 1.4 – (JX051371) H32 KC441788 B. subtilis QD399 99.8 1.6 9.4 – (EF488090) H34 KC441789 B. altitudinis JF31 99.9 1.3 3.7 – (KC172006) H37 KC441790 B. licheniformis 2J-1 99.9 1.2 7.6 3.3 (FJ493045) H39 KC441791 B. pumilus NJM4 99.7 1.8 10.4 – (AB701293) H44 KC441798 B. licheniformis CICC10334 97.8 1.9 16.3 0.33 (GQ375243) H45 KC441792 Geobacillus stearother- 99.9 1.5 0.9 – mophilus DDKRC4 (JN641292) H46 KC441793 B. cereus NJU110 (FJ449630) 97.5 1.3 6.1 3.9 H49 KC441794 B. amyloliquefaciens St08 99.1 1.9 10.9 6.3 (JN700078) H54 KC441797 B. subtilis FS106 (KC179631) 100 1.8 4.1 2.2 H55 KC441795 B. licheniformis HT18 98.8 2.1 12.3 41.6 (JN013203) H56 KC441796 B. cereus CP1 (JX544748) 98.4 1.4 6.3 3.9 Note: NCBI = National Center for Biotechnology Information; HC = hydrolysis capacity; B. = Bacillus;– = the value of the parameter was not detected. For α-amylase assays, the starch–iodine method was 66.7% of the bacteria isolated from the Niulanshan and used (Shankar et al. 2009). One unit of α-amylase activity Hongxing Daqus, with approximately 37.5% and 20.0% of was deﬁned as the amount of enzyme that hydrolyzed 1 mg the sequences corresponding to B. licheniformis. In addi- of starch per hour under the assay conditions. Glucoamy- tion, B. sonorensis, Streptomyces albus, B. atrophaeus, B. lase activity was determined by measuring released reduc- tequilensis and B. megaterium were observed in the Niu- ing sugars using the dinitrosalicylic acid method (Kumar lanshan Daqu, while B. thuringiensis, B. altitudinis, B. & Satyanarayana 2009). One unit of glucoamylase activ- pumilus, Geobacillus stearothermophilus and B. amyloliq- ity was deﬁned as the amount of enzyme that liberated 1 uefaciens were found in the Hongxing Daqu (Tabassum mg of reducing sugar as glucose per hour under the assay et al. 2014). conditions. To understand the phylogenetic position of these strains from the two typical light-ﬂavor Daqus of Chinese spirits, phylogenetic trees were constructed based on comparison Results and discussion of the 16S rDNA sequences of the strains from the two typical light-ﬂavor Daqus and those of reference bacteria In total, 16 and 15 bacterial strains were randomly (Figures 1 and 2). selected and identiﬁed by sequencing the 16S rDNA from Several factors, including the regional climate, water the Niulanshan and Hongxing Daqus, respectively. The used in production and microorganisms in the air jointly sequences have been submitted to the GenBank database inﬂuenced the microorganism composition in Daqus. Nev- with accession numbers (Tables 2 and 3). Eight species ertheless, temperature was an important technological were encountered in each of the Niulanshan and Hongx- parameter, which would aﬀect the growth and death of ing Daqus. The results are presented in Tables 2 and 3. microorganisms and ﬁnally determine the microorganism The Bacillus genus was dominant in the two light-ﬂavor community structure in Daqus (Kumar & Satyanarayana style Daqus. This observation is consistent with that of 2009; Zheng et al. 2012). Both Niulanshan Daqu and previous studies (Kumar & Satyanarayana 2009; Zheng Hongxing Daqu are representatives of the light-ﬂavor et al. 2012). In particular, B. licheniformis, B. subtilis and Daqus. During its manufacture by solid-state fermentation, B. cereus together represented approximately 68.8% and Frontiers in Life Science 267 Figure 1. Phylogenetic relationships among bacterial 16S rDNA sequences in the Daqu of Niulanshan liquor and with previously reported sequences. The number on each branch indicates the percentage of 1000 replicates that are included in the branch. Sequences determined in this study are shown in bold, and GenBank accession numbers are shown in parentheses for all of the related sequences. The scale bar of 0.02 represents a 2% nucleotide substitution rate according to the Jukes–Cantor evolutionary distance (Surhio et al. 2014). the temperature in the center of the Daqu blocks during Shanxi Province (Kumar & Satyanarayana 2009; Zheng fermentation does not exceed 50°C (Table 1). Most bac- et al. 2012). The present results are in agreement with teria tolerate these temperatures and, therefore, a wide these ﬁndings. Bacillus licheniformis, a facultative anaer- range of bacterial species was observed in the two light- obe, is a bacterium that can grow in adverse ecological ﬂavor Daqus (Figures 1 and 2). Although both the Niu- niches (Shi et al. 2009). It has been found that B. pumilus lanshan and Hongxing Daqus are light-ﬂavor Daqus, the exists in Hongxing Daqu and Fen Daqu. However, B. community structures of bacteria were obviously diﬀerent cereus was not the dominant species in Fen Daqu and was between them. Bacillus species were the most frequently found only in the outer part of Fen Daqu. In addition, isolated bacteria from the two light-ﬂavor Daqus. Bacillus Brevibacterium sp., Enterococcus faecalis, Lactobacillus bacteria have a better ability than other bacteria to sur- plantarum, Pediococcus pentosaceus, Salmonella enterica, vive under conditions of low moisture content and high Leuconostoc citreum, Micrococcus luteus, Pseudomonas temperature (Wang et al. 2008). Bacillus licheniformis, aeruginosa and Escherichia coli were encountered in Fen B. subtilis and B. cereus were dominant in the Daqus Daqu (Zheng et al. 2012); nevertheless, none of them (Figures 1 and 2). Other species, such as B. atrophaeus, was observed in either Niulanshan Daqu or Hongxing B. sonorensis, B. megaterium, Streptomyces albus and Daqu. Therefore, the bacterial community structures in Bacillus tequilensis, were observed only in the Niulanshan both Niulanshan and Hongxing Daqus were obviously Daqu, whereas B. amyloliquefaciens, B. pumilus, B. altitu- diﬀerent from those in Fen Daqu. Many intrinsic and dinis, B. thuringiensis and Geobacillus stearothermophilus extrinsic factors of Daqu production inﬂuence the richness were all observed in the Hongxing Daqu. In compari- and structure of the microbial community, including the son, both B. licheniformis and B. subtilis, which were raw material variety, moisture content, temperature con- detected by a culture-dependent method, were predomi- trol, geographical location, climate and water (Wang et al. nant in Fen Daqu, which is a light-ﬂavor Daqu from the 2011). 268 Z. Li et al. Figure 2. Phylogenetic relationships among bacterial 16S rDNA sequences in the Daqu of Hongxing liquor and with previously reported sequences. The number on each branch indicates the number out of 1000 replicates that are included in the branch. Sequences determined in this study are shown in bold, and GenBank accession numbers are shown in parentheses for all of the related sequences. The scale bar of 0.02 represents a 2% nucleotide substitution rate according to the Jukes–Cantor evolutionary distance. Following staining with iodine solution, the isolates by B. cereus N25 (Table 2). Bacillus licheniformis, B. from the two Daqus that exhibited visible clear zones subtilis and B. cereus were the dominant producers of around the colonies on agar plates with starch as the sole both α-amylase and glucoamylase. Of the total enzyme, carbon source were selected as good producers of amy- the activity of α-amylase and glucoamylase produced by lase (Tables 2 and 3). All 15 bacterial isolates from the these bacteria accounted for 14.6% and 23.4%, 33.1% and Hongxing Daqu could produce extracellular α-amylase at 11.1%, and 20.6% and 38.3%, respectively. In addition, 37°C after 48 h of incubation, with a maximum yield of B. atrophaeus and B. tequilensis could produce α-amylase 25.3 U/ml by B. cereus H17, whereas B. licheniformis H55 and glucoamylase (Khaskheli et al. 2015). could produce a maximum glucoamylase yield of 41.6 The variety of bacteria found in the Daqus produced U/ml among the nine bacterial isolates that could produce complicated enzyme systems that converted the Daqus glucoamylase at 37°C after 48 h of incubation (Table 3). into unique glycation and fermented substances, which Of the total enzyme, the activity of α-amylase and glu- are useful for subsequent reactions leading to ﬂavor pro- coamylase produced by B. licheniformis, B. subtilis and duction (Shi et al. 2009). Amylases are one of the most B. cereus accounted for 27.5% and 52.2%, 18.1% and important enzymes and are of great signiﬁcance in today’s 2.5%, and 33.7% and 38.0%, respectively. Furthermore, B. biotechnology industry. They have several diﬀerent indus- amyloliquefaciens could produce glucoamylase. trial applications, including foods, pharmaceuticals, deter- In total, eight and eight bacterial isolates from the Niu- gents and animal feed. The variety of bacteria found in lanshan Daqu could produce extracellular α-amylase and the two Daqus could produce amylase, but the compo- glucoamylase, respectively. The maximum yield of 27.6 sitions of bacteria producing amylase obviously diﬀered U/ml α-amylase was achieved by B. subtilis N3 and the between them, although they all belonged to light-ﬂavor maximum yield of 58.1 U/ml glucoamylase was achieved Daqus (Tables 2 and 3). As the dominant bacterial species Frontiers in Life Science 269 in the two typical Daqus, B. licheniformis and B. subtilis the bacterial community structures and characteristics of also possessed high α-amylase and glucoamylase activi- amylase production were obviously diﬀerent. However, B. ties, which enabled them to utilize raw starch as a carbon licheniformis, B. subtilis and B. cereus were not only the source and helped them to play a very important role during dominant bacteria but also possessed high α-amylase and fermentation. Of the total enzyme from the Hongxing and glucoamylase activities that enabled them to utilize raw Niulanshan Daqus, the activity of α-amylase secreted by starch as a carbon source, and which may help them to play B. licheniformis and B. subtilis accounted for 27.5% and a very important role during fermentation. Further inves- 18.1%, and 14.6% and 33.1%, respectively. In contrast, tigation needs to be carried out to obtain more detailed the total activity of glucoamylase accounted for 52.2% information on the speciﬁc function of each individual and 2.5%, and 23.4% and 11.1%. Moreover, B. cereus microorganism and its contribution to the ﬁnal formation was a chief bacterial species that produced amylase in the of the unique aroma in Chinese spirits. Hongxing and Niulanshan Daqus, with the total activity of α-amylase and glucoamylase produced by B. cereus Acknowledgements accounting for 33.7% and 38.0%, and 20.6% and 38.3%, The authors are thankful to those who sent the Daqu samples for respectively. this research. Comparison of the activities of α-amylase and glu- coamylase in the Hongxing Daqu with those in the Niulan- Funding shan Daqu indicated that the predominant bacterial genus and species secreting both α-amylase and glucoamylase This research was ﬁnancially supported by the National Key Technology R&D Program of China [no. 2012BAK17B11] were similar, including the Bacillus genus and species of and Beijing Municipal Commission of Education and Sub- B. licheniformis, B. subtilis and B. cereus. Bacillus species sidy for Outstanding People of Beijing [no. KM201311417007; are important sources of amylases and proteases. The PXM2013_014209_07_000082; 2011A005022000004]. hydrolytic capabilities of these microorganisms can result in a precursor-rich environment, which is useful for sub- Disclosure statement sequent reactions leading to ﬂavor production (Wang et al. No potential conﬂict of interest was reported by the authors. 2008). 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Frontiers in Life Science
– Taylor & Francis
Published: Jul 3, 2015
Keywords: Chinese spirits; Daqu; bacterial diversity; 16S rRNA gene; α-amylase; glucoamylase