Effects of cellulase-producing bacteria on bacterial community structure and diversity during fermentation of Chinese liquor grains
2017; Wiley; Volume: 123; Issue: 1 Linguagem: Inglês
10.1002/jib.390
ISSN2050-0416
AutoresJian-Hua Guo, Lihua Sun, X. L. Liu,
Tópico(s)Probiotics and Fermented Foods
ResumoJournal of the Institute of BrewingVolume 123, Issue 1 p. 130-137 Research articleFree Access Effects of cellulase-producing bacteria on bacterial community structure and diversity during fermentation of Chinese liquor grains J. H. Guo, Corresponding Author J. H. Guo gjh19771123@163.com College of Food and Biological Engineering, Qiqihar University, Qiqihar, 161006 People's Republic of ChinaCorrespondence to: J. H. Guo, College of Food and Biological Engineering, Qiqihar University, Qiqihar 161006, People's Republic of China. E-mail: gjh19771123@163.comSearch for more papers by this authorL. H. Sun, L. H. Sun Biological Engineering Department, Liaoning Economic Management Cadre Institute Shenyang, 110122 People's Republic of ChinaSearch for more papers by this authorX. L. Liu, X. L. Liu College of Food and Biological Engineering, Qiqihar University, Qiqihar, 161006 People's Republic of ChinaSearch for more papers by this author J. H. Guo, Corresponding Author J. H. Guo gjh19771123@163.com College of Food and Biological Engineering, Qiqihar University, Qiqihar, 161006 People's Republic of ChinaCorrespondence to: J. H. Guo, College of Food and Biological Engineering, Qiqihar University, Qiqihar 161006, People's Republic of China. E-mail: gjh19771123@163.comSearch for more papers by this authorL. H. Sun, L. H. Sun Biological Engineering Department, Liaoning Economic Management Cadre Institute Shenyang, 110122 People's Republic of ChinaSearch for more papers by this authorX. L. Liu, X. L. Liu College of Food and Biological Engineering, Qiqihar University, Qiqihar, 161006 People's Republic of ChinaSearch for more papers by this author First published: 15 March 2017 https://doi.org/10.1002/jib.390Citations: 5AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract To study role of cellulase-producing bacteria on bacterial community structure during fermentation of Chinese liquor grains, a cellulase-producing strain called DM-4 was added to the grains at different levels. The bacterial community structure and diversity were then studied using a high-throughput sequencing method. Results showed that the bacterial community structure exhibited varied characteristics at different levels of grain fermentation, which amply illustrated that adding cellulase-producing bacteria to fermenting grains had significant effect on the bacterial community structure. Diversity analysis indicated that abundance and diversity of bacterial community increased significantly by adding 104–106 cfu/g DM-4 cellulase-producing bacteria. Also when 0–106 cfu/g of DM-4 cellulase-producing bacteria was added, there was significant correlation between the dose of cellulase-producing bacteria added and bacterial community diversity. The study thus concluded that bacterial community diversity and uniformity increased by adding cellulase-producing bacteria during fermentation. Copyright © 2017 The Institute of Brewing & Distilling Introduction Microorganisms play a dominant role in Chinese liquor fermentation process, and the species and structure of the microbial community in fermenting grains have a significant effect on the type and quantity of flavouring substances. Adding microorganisms with special properties to liquor grains could change the original bacterial community's structure and the proportion of different microorganisms during the fermentation process, thereby changing the bacterial species and quantity of flavouring substances. Based on the paper by Gao et al., ethyl caproate content in liquor increased with addition of a fermentation liquid containing caproic acid-producing bacteria to the fermenting grains 1. According to Liu et al., adding lactate-decomposing bacteria to fermenting grains increased ethyl acetate content and the distillation yield, while reducing lactic acid and ethyl lactate content in liquor 2. There were several cellulosic materials that played a limited role in fermenting liquor grains. A lot of nutritive substances were wrapped up in cellulosic materials and degraded incompletely during fermentation, resulting in low utilization and high residue content. This also affected biochemical reactions within the system. Many researchers have already used cellulase or cellulase-producing bacteria in liquor making to improve utilization ratio of raw materials, liquor yield and quality. 3. Bacillus producing flavouring substances are important in the liquor fermentation process 4. Bacillus licheniformis could produce tetramethylpyrazine, phenethyl alcohol and advanced fatty acid esters, which impart a unique flavour to the finished products and also have added health benefits. Liu et al. added four strains of B. licheniformis known for producing large amounts of amylase to fermenting grains 5. Results showed that ethyl lactate content decreased and ethyl acetate content increased in liquor. Bacillus subtilis could synthesize acetoin and amyl alcohol as well as other components, thus imparting a richer flavour to liquor 6. Based on the paper by Shi et al., adding three strains of thermophilic B. subtilis to fermenting grains increased pyrazine content in liquor, while concentrations of tetramethylpyrazine increased by 3.70, 2.67 and 4.99 times, respectively 7. Bo et al. isolated a strain of Bacillus subtilis that could produce n-propyl alcohol from Daqu and fermented grains, and added it to fermenting grains after culture amplification 8. Results showed that content of n-propyl alcohol in liquor increased by 3.72%, thus proving that Bacillus subtilis has a positive effect on n-propyl alcohol quantity in liquor. The microbial community's structure and diversity in a system can be detected using high-throughput sequencing technology 9. Acosta-Martínez et al. studied the effects of different modes of tillage and land usage patterns on soil bacterial diversity, and the effects of different cropping patterns on soil bacterial communities in semi-arid regions through 16SrDNA sequencing analysis, using 454 high-throughput sequencing technology 10, 11. Yang et al. studied the difference in salivary microbial composition of healthy people and patients with dental caries using 454 high-throughput sequencing technology 12. Costello and Knight evaluated the human bacterial community's biogeography using high-throughput sequencing technology, thus establishing a baseline related to disease 13. Researching the change in rules of microbial community structure in fermented grains was beneficial in exploring liquor fermentation characteristics. Hou et al. studied fungal community structure and variational regularity of Luzhou-flavoured fermented grains using PCR-DGGE technique 14. Results showed that the similarity index of fungus DGGE map of fermenting grains was very low at different fermentation periods, while the difference in fungal community varied greatly at different fermentation stages. Shao et al. studied yeast community structure in Maotai liquor fermentation and its effect on imparting flavour to liquor using PCR-DGGE Technology 15. The results showed that yeast community diversity varied greatly during Maotai liquor fermentation, and these changes had a significant effect on liquor yield and quality. Zhao et al. analysed microbes at different levels of grain fermentation in a cylinder, using a high-throughput sequencing method. They found that anaerobic or facultative aerobic bacteria were the dominant microorganisms in the center of the cylinder, like Lactobacillaceae, Streptococcaceae, etc. 16. Also the outer wall of the cylinder mostly had aerobic bacteria, such as Bacillaceae. Zheng et al. researched the relationship between protein expression and microbial community diversity in 30- and 300-year pit muds, by proteomic and high-throughput analytic technique 17. In this paper, a cellulase-producing strain DM-4 identified as B. subtilis was added to Chinese liquor fermenting grains at different levels, and the effect of cellulase-producing strain DM-4 on bacterial community structure and diversity of Chinese liquor fermenting grains was discussed based on the high-throughput sequencing results. Materials and methods Treatment of fermenting grains Fermenting grains were supplied by Heilongjiang Beidacang Group Co. Ltd. A cellulase-producing strain DM-4 was incubated for 24 h in a stirred tank bioreactor containing 10 L medium (glucose, 20 g/L; peptone, 5 g/L; yeast extract, 3 g/L; wort, 3 g/L; pH 5.0). The cells were separated from the culture medium by centrifugation at 7000 g, and four suspensions were made, at cell concentrations of 104, 106, 108 and 1010 cfu/mL, respectively with sterile water. Each suspension (150 mL) was then added to 15 kg of fermenting grains, so that they contained 102, 104, 106 and 108 cfu/g cellulase-producing bacteria, respectively (groups 1–4). Sterile water (150 mL) was added to the control group (group 0). The fermenting grains were then transferred to wooden boxes. The wooden boxes' dimensions were 600 × 350 × 150 mm each, and were sealed with a preservative film, before placing in a liquor factory brewing workshop for anaerobic fermentation. The fermenting grains in the fermented pool at the liquor factory were also used as control group (group P). Determining bacterial community structure and diversity of fermenting grains by high-throughput sequencing method Aliquots of 5 g of fermenting grains were taken at 6 day intervals and the total bacterial DNA was extracted with an Ez Up post genomic DNA extraction kit. The bacterial 16S ribosomal DNA (rDNA) was amplified by a polymerase chain reaction (PCR) using the primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) 18. PCR products were analysed by a high-throughput sequencing method, using a MiSeq PE300 Illumina sequencing platform from Shanghai Majorbio Bio-Pharm Technology Co. Ltd. Sequencing results were optimized and OTU (operational taxonomic units) were generated based on a sequence similarity level of 97%. Based on the OTU results, the number of sequences of each OTU and the sequence in the SILVA database were statistically analysed, and the sequences' taxonomic information was obtained. On this basis, the abundance index (Ace/Chao), diversity index (Shannon/Simpson) and coverage index (Coverage) of the bacterial community in fermenting grain samples were calculated, using Mothur software 19. The correlation was analysed by Pearson's correlation analysis and calculated by SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) 20. Results and discussion Results of high-throughput sequencing test showed that each fermenting grain sample had more than 30,000 optimized sequences. Based on sequence similarity of 97%, 95 OTU (were obtained. The sequence's taxonomic information indicated that the bacteria detected by high-throughput sequencing method included five phyla, 14 classes, 27 orders (two species with no rank), 46 families (three species with no rank), 70 genera (four species with no rank) and 23 species that were uncultured. Based on genus, the figures of bacterial community structure from different fermenting grains during fermentation were established with 30 dominant genera, as indicated in Figs 1-6. Figure 1Open in figure viewerPowerPoint Bacterial communities' species structure on day 1 of fermentation in fermenting grains. Figure 2Open in figure viewerPowerPoint Bacterial communities' species structure on day 6 of fermentation in fermenting grains. Figure 3Open in figure viewerPowerPoint Bacterial communities' species structure on day 12 of fermentation in fermenting grains. Figure 4Open in figure viewerPowerPoint Bacterial communities' species structure on the 18th day of fermentation in fermenting grains. Figure 5Open in figure viewerPowerPoint Bacterial communities' species structure on day 24 of fermentation in fermenting grains. Figure 6Open in figure viewerPowerPoint Bacterial communities' species structure on day 30 of fermentation in fermenting grains. From the above figures, it can be seen that bacterial community structure in different fermenting grains has different characteristics that change with fermentation time. At the beginning of fermentation (Fig. 1), the rank order of top 30 bacterial genus in the fermenting grains were as follows: Lactococcus, Bacillus, Lactobacillus, Pseudomonas, Solibacillus, Kroppenstedtia, Streptococcus, Lysinibacillus, Neisseria, Arthrobacter, Acinetobacter, Pseudochrobactrum, Escherichia-Shigella, Saccharopolyspora, Enterobacteriaceae (no rank), Actinopolyspora, Thermoactinomyces, Brevibacterium, Geobacillus, Leuconostoc, Paenibacillus, Marinilabiaceae uncultured, Brochothrix, mitochondria_norank, Ewingella, Cupriavidus, Stenotrophomonas, Psychrobacter, Acetobacter and Ignatzschineri. Among them, the total percentage of Lactococcus, Bacillus, Lactobacillus and Pseudomonas was >50% in each fermenting grain, and the aforementioned bacteria provided an absolute advantage. With addition of cellulase-producing bacteria, the percentage of absolutely dominant bacteria decreased, with the lowest in group 3. As the DM-4 cellulase-producing strain was the Bacillus strain, its percentage in the fermenting grains increased with addition of cellulase-producing bacteria, increasing to 56.84% in group 4. On day 6 of fermentation (Fig. 2), the bacterial community in each of the fermenting grains had changed and percentage of Thermoactinomyces and Pedobacter increased in each of them. The percentages of Pseudochrobactrum, Marinilabiaceae (uncultured) and Neisseria significantly increased in groups 1–3, where cellulase-producing bacteria were added. In group 4, the percentage of Bacillus sp. significantly decreased, but the percentage of Lactobacillus significantly increased. On day 12 of fermentation (Fig. 3), the percentages of Lactococcus, Bacillus sp., Lactobacillus and Pseudomonas significantly decreased in each fermenting grain, especially in group 3, while others such as Lysinibacillus increased. The percentage of Acinetobacter was very low in group P, but increased significantly in group 0. Bacterial numbers showed relatively uniform distribution in groups 2 and 3. On day 18 of fermentation (Fig. 4), the total percentages of Lactococcus, Bacillus, Lactobacillus and Pseudomonas increased, especially in group 0. On day 24 of fermentation (Fig. 5), the percentage of dominant bacteria decreased, while the percentage of Streptococcus and Thermoactinomyces increased significantly, and the percentage of Pseudomonas was much lower. The bacterial species distribution tended to be uniform in all kinds of fermenting grains, especially in group 3. Towards the end of the fermentation process (Fig. 6), the percentage of Lactococcus and Lactobacillus increased tremendously in groups P, 0 and 1. In groups 2–4, only the Lactobacillus percentage increased a little, but the percentage of Bacillus increased significantly. Hence, Lactococcus and Lactobacillus growth can be inhibited by increasing number of Bacillus sp. in fermenting grains. The diversity indices of bacterial community in different grains during fermentation were calculated according to OTU, as shown in Tables 1-6. The results showed that coverage indices of fermenting grain sequencing were >0.999, indicating the species and structure of bacterial community in the fermenting grains. Abundance indices (Ace/Chao) and Shannon indices of bacterial community in the fermenting grains decreased initially during fermentation, increasing again and then finally decreasing, while the Simpson index showed the opposite trend. At the time of fermentation, the indices of bacterial community in the fermenting grains were very different, compared with group 0. Incremental addition of cellulase-producing strain DM-4 resulted in a larger difference. The difference was larger in group 3 as compared with group 4. In order to compare total difference in diversity indices among fermenting grains, average values of bacterial community diversity index in each fermenting grain were calculated, during the fermentation process, as shown in Fig. 7. As seen in this figure, abundance indices (Ace/Chao) and diversity indices (Shannon/Simpson) of groups 2 and 3 had greater differences compared with groups 0 and P, while group 1 had no significant difference when compared with groups 0 and P. Compared with groups 0 and P, the Shannon index in group 4 decreased significantly, while the Simpson index increased. Table 1. Bacterial communities' diversity indices of fermenting grains on day 0 of fermentation Types of fermented grains Ace Chao Shannon Simpson Coverage P# group 94.56 92.50 3.0953 0.1248 0.999871 0# group 89.60 90.75 2.9472 0.1069 0.999800 1# group 91.52 90.50 2.9648 0.1301 0.999869 2# group 96.18 94.50 3.3202 0.0926 0.999867 3# group 96.59 96.33 3.4201 0.0685 0.999836 4# group 93.93 93.14 2.2573 0.3008 0.999808 Table 2. Bacterial communities' diversity indices of fermenting grains on day 6 of fermentation Types of fermented grains Ace Chao Shannon Simpson Coverage P# group 82.48 82.00 2.9154 0.1234 0.999867 0# group 91.99 82.86 2.8533 0.1261 0.999637 1# group 89.77 87.00 2.9088 0.1145 0.999636 2# group 87.36 84.60 3.0714 0.0917 0.999734 3# group 93.25 94.25 3.2893 0.0607 0.999668 4# group 80.59 71.00 2.6074 0.1567 0.999736 Table 3. Bacterial communities' diversity indices of fermenting grains on day 12 of fermentation Types of fermented grains Ace Chao Shannon Simpson Coverage P# group 93.05 91.43 3.3172 0.0753 0.999902 0# group 98.96 96.86 3.1998 0.0738 0.999643 1# group 99.53 99.00 3.3314 0.0721 0.999706 2# group 105.40 112.50 3.5638 0.0590 0.999672 3# group 120.28 100.00 3.7112 0.0543 0.999776 4# group 102.08 135.50 3.2319 0.0870 0.999535 Table 4. Bacterial communities' diversity indices of fermenting grains on day 18 of fermentation Types of fermented grains Ace Chao Shannon Simpson Coverage P# group 98.48 93.86 3.2086 0.0876 0.999634 0# group 97.44 89.12 3.0388 0.0962 0.999634 1# group 100.93 93.00 3.1708 0.0908 0.999703 2# group 117.35 89.60 3.2851 0.0838 0.999739 3# group 126.44 103.00 3.4550 0.0758 0.999679 4# group 94.74 96.50 3.0037 0.1170 0.999672 Table 5. Bacterial communities' diversity indices of fermenting grains on day 24 of fermentation Types of fermented grains Ace Chao Shannon Simpson Coverage P# group 127.57 102.00 3.4968 0.0437 0.999486 0# group 99.72 94.63 3.3343 0.0562 0.99967 1# group 121.34 99.43 3.4637 0.0500 0.999613 2# group 125.54 123.00 3.5779 0.0424 0.999623 3# group 191.13 127.00 3.7467 0.0397 0.999718 4# group 115.69 112.50 3.4642 0.0596 0.999673 Table 6. Bacterial communities' diversity indices of fermenting grains on day 30 of fermentation Types of fermented grains Ace Chao Shannon Simpson Coverage P# group 109.12 100.50 3.0488 0.0908 0.999506 0# group 94.09 91.00 2.9085 0.0979 0.999668 1# group 106.18 97.00 3.0054 0.0953 0.999636 2# group 122.84 142.00 3.2486 0.0774 0.999637 3# group 128.61 106.00 3.3492 0.0758 0.999711 4# group 98.36 94.43 3.1443 0.0862 0.999625 The diversity index of group 0 was used as a benchmark to calculate ratio of other groups accordingly. The significant differences tests of diversity indices between other groups and group 0 are shown in Table 7 and 8. As seen in these tables, the abundance indices (Chao) of groups 2 and 3 show significant differences when compared with group 0 (p < 0.05), with an extremely significant level of difference in group 3 (p < 0.01). The diversity indices (Shannon) of groups 2 and 3 show significant differences from group 0 (p < 0.01). The dominance index of group 4 shows a significant difference from group 0 (p < 0.05). Therefore, appropriate addition of cellulase-producing strain of DM-4 (104–106 cfu/g) to liquor grains, significantly improves the quantity and diversity of bacterial community in the fermenting grains. Figure 7Open in figure viewerPowerPoint Average diversity indices of bacterial communities during fermentation in different fermenting grains. Table 7. Significance difference test of richness indices (Ace/Chao) between 0# group and others LSD (0# group) Mean difference Standard error Significance Ace Chao Ace Chao Ace Chao P# group 0.0569919 0.0313848 0.0943212 0.0730192 0.550 0.670 1# group 0.0640289 0.0382496 0.0943212 0.0730192 0.502 0.604 2# group 0.1428329 0.1815694* 0.0943212 0.0730192 0.140 0.019 3# group 0.3147199** 0.1490026* 0.0943212 0.0730192 0.002 0.049 4# group 0.0222914 0.0985787 0.0943212 0.0730192 0.815 0.187 * The mean difference is significant at the 0.05 level. ** The mean difference is significant at the 0.01 level. Table 8. Significance difference test of diversity indices(Shannon/Simpson) between 0# group and others LSD (0# group) Mean difference Standard error Significance Shannon Simpson Shannon Simpson Shannon Simpson P# group 0.0435927 −0.0363300 0.0277792 0.1740962 0.127 0.836 1# group 0.0303523 −0.0151686 0.0277792 0.1740962 0.283 0.931 2# group 0.0979666** −0.1984931 0.0277792 0.1740962 0.001 0.263 3# group 0.1475421** −0.3122452 0.0277792 0.1740962 0.000 0.083 4# group 0.0336258 0.3987626* 0.0277792 0.1740962 0.236 0.029 * The mean difference is significant at the 0.05 level. ** The mean difference is significant at the 0.01 level. As per the results of the high-throughput sequencing method, the addition of cellulase-producing bacteria changed the diversity of bacterial community in the fermenting grains. Also, the degree of change varied with additions at different levels. In order to research the relationship between the diversity of bacterial community and quantity of cellulase-producing strain to be added, the correlation coefficients between them were calculated, as shown in Tables 9 and 10. Table 9. Correlation coefficients between addition level of the cellulase-producing bacteria and bacterial communities' diversity indices of fermenting grains Shannon index Simpson index Addition level 0.117 0.272 * Correlation is significant at the 0.05 level (two-tailed). ** Correlation is significant at the 0.01 level (two-tailed). Table 10. Correlation coefficients between addition level (≤106 cfu/g) of the cellulase-producing bacteria and bacterial communities diversity indices of fermenting grains Shannon index Simpson index Addition level 0.933* −0.946** * Correlation is significant at the 0.05 level (two-tailed). ** Correlation is significant at the 0.01 level (two-tailed). As seen in Tables 9 and 10, when the quantity of appropriate bacterial strain added was 0–108 cfu/g, there was no obvious correlation between the quantity of cellulase-producing bacteria and the diversity index of the bacterial community (Table 9). However, when the quantity added was 0–106 cfu/g, there was a significantly positive correlation between the Shannon index and the quantity of cellulase-producing strain (p < 0.05), and also a significantly negative correlation between the Simpson index and the quantity of cellulase-producing strain (p < 0.01; Table 10). Therefore, when the quantity of cellulase-producing strain added was 0–106 cfu/g, the diversity index significantly increased, but the dominance index decreased, thus, increasing uniformity of bacterial community in fermenting grains when increasing quantities were added. The above discussion indicates that addition of cellulase-producing strain DM-4 could change bacterial community structure and diversity index of fermenting grains during fermentation. When the quantity of cellulase-producing strain DM-4 added was 106 cfu/g, the Shannon index of bacterial community in fermenting grains increased significantly. Simultaneously, the Simpson index decreased significantly, which ensured a more uniform distribution of bacterial species in the fermenting grains. Bacterial species are an important factor for imparting flavour to liquor. Increasing bacterial community numbers and distribution uniformity made fermenting grains richer and more uniform. Therefore, fermenting grains' properties were enhanced with coordinated proportions of flavouring substances in liquor. 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