Publications by authors named "Zhihao Tu"

9 Publications

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Analysis of bacterial flora of indigo fermentation fluids utilizing composted indigo leaves (sukumo) and indigo extracted from plants (Ryukyu-ai and Indian indigo).

J Biosci Bioeng 2021 Jun 11. Epub 2021 Jun 11.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan; Laboratory of Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan. Electronic address:

Indigo is a fabric dye that requires reduction by microbial activity or chemical reagents to render it soluble in water. Sources of indigo for fermentation are primarily divided into composted indigo-containing plants and indigo extracted from plants. To elucidate the factors responsible for bacterial diversity, and for sustaining reduced state of indigo in different preparations, this study assessed fermentation-derived fluids using composted plant leaves, sukumo, and extracted indigo (Ryukyu-ai paste, and Indian indigo cake) prepared using different procedures. Regardless of the indigo source, obligate anaerobic bacteria, including the families Proteinivoraceae and Tissierellaceae, predominate (16.9-46.1%), suggesting their high affinity for this fermentation ecosystem (hyperalkaline and low redox potential). Moreover, bacterial communities in sukumo fermentations are more diverse than those from indigo extracts with the diversity tending to increase based on the fermentation period. Our results further suggest that the microbiota composition in sukumo fermentation is associated with the various bacterial nutrients derived from sukumo, including seed microorganisms. In addition, the debris derived from sukumo can reduce the pH stress experienced by the microorganisms. Further, regardless of 5.4 years difference in the fermentation age, the bacterial flora in two Ryukyu-ai batches exhibit similar features with low microbial diversities. The uniformity of the nutrient, along with the simple, yet strong, bacterial network in Ryukyu-ai fluids may be responsible for the stable bacterial flora composition. Taken together, these results indicate that the microbiota in indigo fermentation is highly influenced by the seed culture, the nutrient derived from raw materials, and the fermentation conditions.
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http://dx.doi.org/10.1016/j.jbiosc.2021.05.004DOI Listing
June 2021

Driver-passenger communicative stress and psychological distress among Chinese bus drivers: the mediating effect of job burnout.

BMC Public Health 2021 03 20;21(1):547. Epub 2021 Mar 20.

Department of Nautical psychology, Faculty of Psychology, The Second Military Medical University, Shanghai, China.

Background: This study aimed to investigate the relationship between driver-passenger communicative stress and psychological distress among bus drivers, as well as whether job burnout mediates the effect of driver-passenger communicative stress on psychological distress.

Methods: A questionnaire consisting of a 12-item version of the General Health Questionnaire (GHQ-12), a one-item driver-passenger communicative stress scale, the Maslach Burnout Inventory-General Survey (MBI-GS), as well as sociodemographic and work factors, was distributed to 310 bus drivers in Shanghai, of which 307 completed it (99.0% response rate). A parallel multiple mediation model with bootstrap approach, was calculated to test the mediating effect.

Results: Driver-passenger communicative stress, emotional exhaustion and cynicism were positively associated with psychological distress. Communicative stress was significantly positively linked with two of the three dimensions of burnout (emotional exhaustion and cynicism) and dependent variable. Emotional exhaustion and cynicism were positively associated with the dependent variable. The results indicate that emotional exhaustion and cynicism partially mediated the effect of communicative stress on psychological health, and that 60.0% of this effect can be explained by mediating effects, in which emotional exhaustion and cynicism weighed 63.2% and 36.8%, respectively.

Conclusions: Communicative stress had effects on psychological distress among Chinese bus drivers, and job burnout was a mediator in this relationship.
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http://dx.doi.org/10.1186/s12889-021-10618-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980616PMC
March 2021

Characterization of the microbiota in long- and short-term natural indigo fermentation.

J Ind Microbiol Biotechnol 2019 Dec 20;46(12):1657-1667. Epub 2019 Aug 20.

Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.

The duration for which the indigo-reducing state maintenance in indigo natural fermentation in batch dependent. The microbiota was analyzed in two batches of sukumo fermentation fluids that lasted for different durations (Batch 1: less than 2 months; Batch 2: nearly 1 year) to understand the mechanisms underlying the sustainability and deterioration of this natural fermentation process. The transformation of the microbiota suggested that the deterioration of the fermentation fluid is associated with the relative abundance of Alcaligenaceae. Principal coordinates analysis (PCoA) showed that the microbial community maintained a very stable state in only the long-term Batch 2. Therefore, entry of the microbiota into a stable state under alkaline anaerobic condition is an important factor for maintenance of indigo fermentation for long duration. This is the first report on the total transformation of the microbiota for investigation of long-term maintenance mechanisms and to address the problem of deterioration in indigo fermentation.
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http://dx.doi.org/10.1007/s10295-019-02223-0DOI Listing
December 2019

Analysis of the microbiota involved in the early changes associated with indigo reduction in the natural fermentation of indigo.

World J Microbiol Biotechnol 2019 Jul 26;35(8):123. Epub 2019 Jul 26.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo, 062-8517, Japan.

Constituents of the seed microbiota and initial changes in the microbiota in fermentations are important in fermentation progression. To identify the origin of indigo-reducing bacteria and understand the initial changes in the microbiota that occur concomitantly with the initiation of indigo reduction during indigo fermentation, we analysed the initial changes in the microbiota. The proportions of the reported indigo-reducing taxa Alkalibacterium, Amphibacillus and Polygonibacillus increased to 24.0% on the 5th day, to 15.2% on the 7th day and to 42.8% at 4.5 months, and the relative abundances of these taxa were 0.048%, 0.14% and 0.02%, respectively, in sukumo (composted Japanese indigo plant material used for fermentation). In the early phase of the microbiota transition, two substantial changes were observed. The first change may be attributed to the substantial environmental changes caused by the introduction of heated wood ash extract (pH ≥ 10.5, temperature ≥ 60 °C). This change increased the proportions of Alkalibacterium and the family Bacillaceae. The second change in microbiota might be initiated by the consumption of oxygen by aerobic microorganisms until the 5th day followed by an increase in the abundance of the obligate anaerobe Anaerobranca and the aerotolerant Amphibacillus and a decrease in the abundance of Bacillaceae. This experiment demonstrated that the 0.048% Alkalibacterium in the original material was augmented to 23.6% of the microbiological community within 5 days. This means that using the appropriate material and performing appropriate pretreatment and adjustment of fermentation conditions are important to increase the abundance of the taxa that reduce indigo.
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http://dx.doi.org/10.1007/s11274-019-2699-5DOI Listing
July 2019

Microbial Diversity in the Edible Gall on White Bamboo Formed by the Interaction between Ustilago esculenta and Zizania latifolia.

Curr Microbiol 2019 Jul 24;76(7):824-834. Epub 2019 Apr 24.

Department of Food Science and Human Wellness, Rakuno Gakuen University, Midorimachi 582, Bunkyodai, Ebetsu-shi, Hokkaido, 069-8501, Japan.

An edible gall is formed between the third and fourth nodes beneath the apical meristem near the base of Zizania latifolia shoots. This gall is harbored by and interacts with the smut fungus Ustilago esculenta. The gall is also a valuable vegetable called "white bamboo," jiaobai or gausun in China and makomotake in Japan. Five samples of the galls harvested at different stages of swelling were used to isolate microorganisms by culturing. Isolated fungal and bacterial colonies were identified by DNA sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, respectively. Several strains of U. esculenta as well as 6 other species of fungi and 10 species of bacteria were isolated. The microbiome was also evaluated by simple and outlined DNA profiling with automated rRNA intergenic spacer analysis (ARISA), and the amount of DNA of U. esculenta was determined by qPCR. At least 16 species of fungi and 40 species of bacteria were confirmed by ARISA of the overall sample. Interestingly, the greatest bacterial diversity, i.e., 18 species, was observed in the most mature sample, whereas the fungal diversity observed in this sample, i.e., 4 species, was rather poor. Based on qPCR, U. esculenta occurred in samples from all stages; however, the abundance of U. esculenta exhibited unique U-shaped relationships with growth. These results may explain why the interaction between U. esculenta and Z. latifolia also influences the unique microbial diversity observed throughout the growth stages of the swollen shoot, although the limited sample size does not allow conclusive findings.
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http://dx.doi.org/10.1007/s00284-019-01693-wDOI Listing
July 2019

Microbial Communities Associated With Indigo Fermentation That Thrive in Anaerobic Alkaline Environments.

Front Microbiol 2018 18;9:2196. Epub 2018 Sep 18.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Sapporo, Japan.

Indigo fermentation, which depends on the indigo-reducing action of microorganisms, has traditionally been performed to dye textiles blue in Asia as well as in Europe. This fermentation process is carried out by naturally occurring microbial communities and occurs under alkaline, anaerobic conditions. Therefore, there is uncertainty regarding the fermentation process, and many unknown microorganisms thrive in this unique fermentation environment. Until recently, there was limited information available on bacteria associated with this fermentation process. Indigo reduction normally occurs from 4 days to 2 weeks after initiation of fermentation. However, the changes in the microbiota that occur during the transition to an indigo-reducing state have not been elucidated. Here, the structural changes in the bacterial community were estimated by PCR-based methods. On the second day of fermentation, a large change in the redox potential occurred. On the fourth day, distinct substitution of the genus with the aerotolerant genus was observed, corresponding to marked changes in indigo reduction. Under open-air conditions, indigo reduction during the fermentation process continued for 6 months on average. The microbiota, including indigo-reducing bacteria, was continuously replaced with other microbial communities that consisted of other types of indigo-reducing bacteria. A stable state consisting mainly of the genus was also observed in a long-term fermentation sample. The stability of the microbiota, proportion of indigo-reducing microorganisms, and appropriate diversity and microbiota within the fluid may play key factors in the maintenance of a reducing state during long-term indigo fermentation. Although more than 10 species of indigo-reducing bacteria were identified, the reduction mechanism of indigo particle is riddle. It can be predicted that the mechanism involves electrons, as byproducts of metabolism, being discarded by analogs mechanisms reported in bacterial extracellular solid Fe reduction under alkaline anaerobic condition.
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http://dx.doi.org/10.3389/fmicb.2018.02196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153312PMC
September 2018

Bacillus fermenti sp. nov., an indigo-reducing obligate alkaliphile isolated from indigo fermentation liquor for dyeing.

Int J Syst Evol Microbiol 2018 Apr 16;68(4):1123-1129. Epub 2018 Feb 16.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8571, Japan.

The indigo-reducing, facultatively anaerobic and obligately alkaliphilic strains Bf-1, Bf-2 and Bf-4 were isolated from an indigo fermentation liquor used for dyeing, which uses sukumo [composted Polygonum indigo (Polygonum tinctorium Lour.) leaves] as a basic ingredient and was obtained from a craft centre in Date City, Hokkaido, Japan. The 16S rRNA gene sequence analyses indicated that the closest neighbours of strain Bf-1 are Bacillus maritimus DSM 100413 (98.3 % 16S rRNA gene sequence similarity), Bacillus persicus DSM 25386 (98.2 %) and Bacillus rigiliprofundi LMG 28275 (97.7 %). The 16S rRNA gene sequence of strain Bf-1 was almost identical to the sequences of strains Bf-2 and Bf-4 (99.9 %). Cells of strain Bf-1 stained Gram-positive and formed straight rods that achieved motility through a pair of subpolar flagella. Strain Bf-1 grew at temperatures of between 15 and 45 °C with optimum growth at 33‒40 °C. The strain grew in the pH range of pH 8‒12, with optimum growth at pH 10. The isoprenoid quinone detected was menaquinone-7 (MK-7), and the DNA G+C content was 41.7 %. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C15 : 0 and iso-C16 : 0. Phylogenetically related neighbours, although demonstrating high 16S rRNA gene sequence similarity (>97.6 %) with strain Bf-1, exhibited less than 9 % relatedness in DNA-DNA hybridization experiments. Based on evidence from this polyphasic study, the isolates represent a novel species, for which the name Bacillus fermenti sp. nov. is proposed. The type strain of this species is Bf-1 (=JCM 31807=NCIMB 15079).
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http://dx.doi.org/10.1099/ijsem.0.002636DOI Listing
April 2018

Rapid and reliable species identification of wild mushrooms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

Anal Chim Acta 2016 Aug 4;934:163-9. Epub 2016 Jun 4.

Department of Food Science and Human Wellness, Rakuno Gakuen University, Midorimachi 582, Bunkyodai, Ebetsu-shi, Hokkaido, 069-8501, Japan. Electronic address:

Mushrooms are a favourite natural food in many countries. However, some wild species cause food poisoning, sometimes lethal, due to misidentification caused by confusing fruiting bodies similar to those of edible species. The morphological inspection of mycelia, spores and fruiting bodies have been traditionally used for the identification of mushrooms. More recently, DNA sequencing analysis has been successfully applied to mushrooms and to many other species. This study focuses on a simpler and more rapid methodology for the identification of wild mushrooms via protein profiling based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). A preliminary study using 6 commercially available cultivated mushrooms suggested that a more reproducible spectrum was obtained from a portion of the cap than from the stem of a fruiting body by the extraction of proteins with a formic acid-acetonitrile mixture (1 + 1). We used 157 wild mushroom-fruiting bodies collected in the centre of Hokkaido from June to November 2014. Sequencing analysis of a portion of the ribosomal RNA gene provided 134 identifications of mushrooms by genus or species, however 23 samples containing 10 unknown species that had lower concordance rate of the nucleotide sequences in a BLAST search (less than 97%) and 13 samples that had unidentifiable poor or mixed sequencing signals remained unknown. MALDI-TOF MS analysis yielded a reproducible spectrum (frequency of matching score ≥ 2.0 was ≥6 spectra from 12 spectra measurements) for 114 of 157 samples. Profiling scores that matched each other within the database gave correct species identification (with scores of ≥2.0) for 110 samples (96%). An in-house prepared database was constructed from 106 independent species, except for overlapping identifications. We used 48 wild mushrooms that were collected in autumn 2015 to validate the in-house database. As a result, 21 mushrooms were identified at the species level with scores ≥2.0 and 5 mushrooms at the genus level with scores ≥1.7, although the signals of 2 mushrooms were insufficient for analysis. The remaining 20 samples were recognized as "unreliable identification" with scores <1.7. Subsequent DNA analysis confirmed that the correct species or genus identifications were achieved by MALDI-TOF MS for the 26 former samples, whereas the 18 mushrooms with poorly matched scores were species that were not included in the database. Thus, the proposed MALDI-TOF MS coupled with our database could be a powerful tool for the rapid and reliable identification of mushrooms; however, continuous updating of the database is necessary to enrich it with more abundant species.
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http://dx.doi.org/10.1016/j.aca.2016.05.056DOI Listing
August 2016

Identification of Mushroom Species by Automated rRNA Intergenic Spacer Analysis (ARISA) and Its Application to a Suspected Case of Food Poisoning with Tricholoma ustale.

Shokuhin Eiseigaku Zasshi 2016 ;57(2):37-45

Department of Food Science and Human Wellness, Rakuno Gakuen Uninversity.

Automated rRNA intergenic spacer analysis (ARISA), a method of microbiome analysis, was evaluated for species identification of mushrooms based on the specific fragment sizes. We used 51 wild mushroom-fruiting bodies collected in the centre of Hokkaido and two cultivated mushrooms. Samples were hot-air-dried and DNA were extracted by a beads beating procedure. Sequencing analysis of portions of the rRNA gene (rDNA) provided 33 identifications of mushrooms by genus or species. The results of ARISA identification based on the combination of the fragment sizes corresponding to two inter spacer regions (ITS2 and ITS1) of rDNA within±0.1% accuracy showed that 27 out of the 33 species had specific fragment sizes differentiated from other species. The remaining 6 species formed 3 pairs that showed overlapping fragment sizes. In addition, within-species polymorphisms were observed as 1 bp differences among 32 samples of 13 species. ARISA was applied to investigate a case of suspected food poisoning in which the mushroom was thought to be a toxic Kakishimeji. The morphological identification of the mushroom was ambiguous since the remaining sample lacked a part of the fruiting body. Further, yeast colonies had grown on the surface of the fruiting body during storage. The ARISA fragment size of the mushroom showed 7 bp difference from that of the candidate toxic mushroom. Although ARISA could be a useful tools for estimation of mushroom species, especially in case where the fruiting bodies have deteriorated or been processed, further studies are necessary for reliable identification. For example, it may be necessary to adopt more informative genes which could provide clearer species-specific polymorphisms than the ITS regions.
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http://dx.doi.org/10.3358/shokueishi.57.37DOI Listing
March 2017