The EHL1/2/3 genes were identified by whole-genome sequencing of Kyokai No. 7 (K7), which is a well-known representative Japanese sake yeast Saccharomyces cerevisiae. The genes are present in K7, but not in laboratory strain S288C. Although the genes were presumed to encode epoxide hydrolase based on homology analysis, their effect on cellular metabolism in sake yeast has not yet been clarified. We constructed ehl1/2/3 mutants harboring a stop codon in each gene using the haploid yeast strain H3 as the parental strain, which was derived from K701, and investigated the physiological role and effects of the EHL1/2/3 genes on sake quality. Metabolome analysis and vitamin requirement testing revealed that the EHL1/2/3 genes are partly responsible for the synthesis of pantothenate. For fermentation profiles, ethanol production by the ehl1/2/3 mutant was comparable with that of strain H3, but succinate production was decreased in the ehl1/2/3 mutant compared to strain H3 when cultured in yeast malt (YM) medium containing 10% glucose and during sake brewing. Ethyl hexanoate and isoamyl acetate levels in the ehl1/2/3 mutant strain were decreased compared to those of strain H3 during sake brewing. Thus, the EHL1/2/3 genes did not affect ethanol production but did affect the production of organic acids and aromatic components during sake brewing.
{"title":"Physiological role of the EHL gene in sake yeast and its effects on quality of sake","authors":"Kazuko Tomonaga , Jumpei Tanaka , Keiji Kiyoshi , Takeshi Akao , Kota Watanabe , Toshimori Kadokura , Shunichi Nakayama","doi":"10.1016/j.jbiosc.2023.12.001","DOIUrl":"10.1016/j.jbiosc.2023.12.001","url":null,"abstract":"<div><p>The <em>EHL1/2/3</em> genes were identified by whole-genome sequencing of Kyokai No. 7 (K7), which is a well-known representative Japanese sake yeast <span><span>Saccharomyces cerevisiae</span></span><span>. The genes are present in K7, but not in laboratory strain S288C. Although the genes were presumed to encode epoxide hydrolase based on homology analysis, their effect on cellular metabolism in sake yeast has not yet been clarified. We constructed </span><em>ehl1/2/3</em><span> mutants harboring a stop codon in each gene using the haploid yeast strain H3 as the parental strain, which was derived from K701, and investigated the physiological role and effects of the </span><em>EHL1/2/3</em><span> genes on sake quality. Metabolome analysis and vitamin requirement testing revealed that the </span><em>EHL1/2/3</em> genes are partly responsible for the synthesis of pantothenate. For fermentation profiles, ethanol production by the <em>ehl1/2/3</em> mutant was comparable with that of strain H3, but succinate production was decreased in the <em>ehl1/2/3</em><span> mutant compared to strain H3 when cultured in yeast malt (YM) medium containing 10% glucose and during sake brewing. Ethyl hexanoate and isoamyl acetate levels in the </span><em>ehl1/2/3</em> mutant strain were decreased compared to those of strain H3 during sake brewing. Thus, the <em>EHL1/2/3</em> genes did not affect ethanol production but did affect the production of organic acids and aromatic components during sake brewing.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139502432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01DOI: 10.1016/j.jbiosc.2023.12.014
Weijun Leng , Weiwei Li , Ying Li , Hongyun Lu , Xiuting Li , Ruichang Gao
To improve the flavor profile and sensory quality of baijiu, the utilization of bioaugmented fermentation inoculated with functional microbiota normally serves as an effective method for directional regulation during the baijiu fermentation process. In this study, a systematic analysis of the succession patterns and volatile flavor compound profiles of microbial communities was carried out by high-throughput sequencing and solid-phase microextraction gas chromatography-mass spectrometry, respectively. The results demonstrated that the Saccharomyces cerevisiae YS222-related bioaugmentation clearly altered the microbial composition, particularly the assembly of bacteria, and promoted the quantity of the most volatile flavoring compounds, including alcohols, esters, and pyrazines. In addition, the correlation analysis showed that Saccharomyces and Lactobacillus in the augmented group were the main biomarkers associated with the dynamics of microbial community and greatly contributed to the brewing of sauce-flavor baijiu, which congruent with the outcomes of the enrichment analysis of integrated metabolic pathway. Thus, this work is beneficial for promoting the quality of baijiu and will serve as a useful reference for clarifying the possible mechanism of augmented fermentation on flavor development.
{"title":"Insight investigation into the response pattern of microbial assembly succession and volatile profiles during the brewing of sauce-flavor baijiu based on bioaugmentation","authors":"Weijun Leng , Weiwei Li , Ying Li , Hongyun Lu , Xiuting Li , Ruichang Gao","doi":"10.1016/j.jbiosc.2023.12.014","DOIUrl":"10.1016/j.jbiosc.2023.12.014","url":null,"abstract":"<div><p>To improve the flavor profile and sensory quality of <em>baijiu</em><span>, the utilization of bioaugmented fermentation inoculated with functional microbiota normally serves as an effective method for directional regulation during the </span><em>baijiu</em> fermentation process. In this study, a systematic analysis of the succession patterns and volatile flavor compound profiles of microbial communities was carried out by high-throughput sequencing and solid-phase microextraction gas chromatography-mass spectrometry, respectively. The results demonstrated that the <span><em>Saccharomyces</em><em> cerevisiae</em></span><span> YS222-related bioaugmentation clearly altered the microbial composition, particularly the assembly of bacteria, and promoted the quantity of the most volatile flavoring compounds, including alcohols, esters, and pyrazines. In addition, the correlation analysis showed that </span><em>Saccharomyces</em> and <span><span>Lactobacillus</span></span><span> in the augmented group were the main biomarkers associated with the dynamics of microbial community and greatly contributed to the brewing of sauce-flavor </span><em>baijiu</em>, which congruent with the outcomes of the enrichment analysis of integrated metabolic pathway. Thus, this work is beneficial for promoting the quality of <em>baijiu</em> and will serve as a useful reference for clarifying the possible mechanism of augmented fermentation on flavor development.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139557237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01DOI: 10.1016/j.jbiosc.2023.12.010
Negin Nouri, Leila Sadeghi, Arezu Marefat
Enzymes derived from microbial sources have gained increasing popularity in industrial applications over the past decades. Despite the high production cost, alkaline proteases have wide applications in industries such as tanneries, food production, and detergents. In recent years, there has been a shift towards utilizing natural carbon sources for cultivating microorganisms and extracting proteases in order to reduce production costs. This study aimed to investigate the biochemical and kinetic properties of protease enzymes obtained from Aspergillus niger cultivated in a paper waste medium and compare with the enzyme produced in a basal medium. Glucose is a more favorable carbon source compared to cellulose, so paper waste was pretreated with cellulose-degrading bacteria to convert cellulose into smaller carbohydrates. After the growth of A. niger in basal and combinational media, the enzymatic properties were compared between the extracted enzymes by using casein as substrate. The results demonstrated that A. niger could produce protease enzymes in the paper waste medium similar to the basal medium with more than 5-fold cost saving. The specific activity of the enzymes isolated from the basal and paper waste media was calculated to be 184.95 ± 10.56 U ml−1 and 169.88 ± 11.05 U ml−1, respectively. Carbon sources did not affect the optimum pH and temperature of the protease enzyme, which were found to be 8 and 37 °C, respectively. This study provides valuable insights into the production of alkaline protease from A. niger using a combinational medium (paper waste pretreated by cellulose-degrading bacteria), offering a cost-effective approach for industrial applications.
过去几十年来,从微生物中提取的酶在工业应用中越来越受欢迎。尽管生产成本较高,但碱性蛋白酶在制革、食品生产和洗涤剂等行业有着广泛的应用。近年来,人们开始转向利用天然碳源来培养微生物和提取蛋白酶,以降低生产成本。本研究旨在调查在废纸培养基中培养的黑曲霉所产生的蛋白酶的生化和动力学特性,并与在基础培养基中产生的酶进行比较。与纤维素相比,葡萄糖是更有利的碳源,因此用纤维素降解细菌对废纸进行预处理,将纤维素转化为更小的碳水化合物。黑曲霉在基础培养基和复合培养基中生长后,以酪蛋白为底物,比较了提取的酶的特性。结果表明,黑曲霉能在废纸培养基中产生类似于基础培养基的蛋白酶,成本节约 5 倍以上。经计算,从基础培养基和废纸培养基中分离出来的酶的比活力分别为 184.95 ± 10.56 U ml-1 和 169.88 ± 11.05 U ml-1。碳源并不影响蛋白酶的最适 pH 值和温度,分别为 8 和 37 °C。这项研究为利用组合培养基(经纤维素降解细菌预处理的废纸)从黑曲霉中生产碱性蛋白酶提供了宝贵的见解,为工业应用提供了一种具有成本效益的方法。
{"title":"Production of alkaline protease by Aspergillus niger in a new combinational paper waste culture medium","authors":"Negin Nouri, Leila Sadeghi, Arezu Marefat","doi":"10.1016/j.jbiosc.2023.12.010","DOIUrl":"10.1016/j.jbiosc.2023.12.010","url":null,"abstract":"<div><p><span>Enzymes derived from microbial sources have gained increasing popularity in industrial applications over the past decades. Despite the high production cost, alkaline proteases have wide applications in industries such as tanneries, food production, and detergents. In recent years, there has been a shift towards utilizing natural carbon sources for cultivating microorganisms and extracting proteases in order to reduce production costs. This study aimed to investigate the biochemical and kinetic properties of protease enzymes obtained from </span><span><span>Aspergillus niger</span></span><span> cultivated in a paper waste medium and compare with the enzyme produced in a basal medium. Glucose is a more favorable carbon source compared to cellulose, so paper waste was pretreated with cellulose-degrading bacteria to convert cellulose into smaller carbohydrates. After the growth of </span><em>A. niger</em> in basal and combinational media, the enzymatic properties were compared between the extracted enzymes by using casein as substrate. The results demonstrated that <em>A. niger</em> could produce protease enzymes in the paper waste medium similar to the basal medium with more than 5-fold cost saving. The specific activity of the enzymes isolated from the basal and paper waste media was calculated to be 184.95 ± 10.56 U ml<sup>−1</sup> and 169.88 ± 11.05 U ml<sup>−1</sup>, respectively. Carbon sources did not affect the optimum pH and temperature of the protease enzyme, which were found to be 8 and 37 °C, respectively. This study provides valuable insights into the production of alkaline protease from <em>A. niger</em> using a combinational medium (paper waste pretreated by cellulose-degrading bacteria), offering a cost-effective approach for industrial applications.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139495410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01DOI: 10.1016/j.jbiosc.2023.12.006
Wei-Ming Chai , Qiuhan Bai , Qiuxia Pan , Linjun Wang , Du Zhu
6,7-Bis-(2-methoxyethoxy)-4(3H)-quinazolinone (BMEQ) was selected from quinazolinones for its strong tyrosinase inhibitory activity (IC50 = 160 ± 6 μM). It suppressed tyrosinase activity in a competitive way and quenched the fluorescence of the enzyme through a static mechanism. The binding of BMEQ to tyrosinase increased the hydrophobicity of the latter and facilitated non-radiative energy transfer between them. The formation of BMEQ–tyrosinase complex was driven by hydrogen bonds and hydrophobic interactions, and it loosened the basic framework structure of tyrosinase, affecting the conformation of the enzyme, and leading to a decrease in tyrosinase activity. In addition, the BMEQ postponed the oxidation of phenolics and flavonoids by inhibiting polyphenol oxidase (PPO) and peroxidase (POD), which resulted in the inhibition of the browning of fresh-cut apples. This study identified a novel tyrosinase inhibitor BMEQ and verified its potential application for improving the preservation of postharvest fruits.
{"title":"6,7-Bis-(2-methoxyethoxy)-4(3H)-quinazolinone as a novel inhibitor of tyrosinase and potential anti-browning agent of fresh-cut apples","authors":"Wei-Ming Chai , Qiuhan Bai , Qiuxia Pan , Linjun Wang , Du Zhu","doi":"10.1016/j.jbiosc.2023.12.006","DOIUrl":"10.1016/j.jbiosc.2023.12.006","url":null,"abstract":"<div><p>6,7-Bis-(2-methoxyethoxy)-4(3<em>H</em><span>)-quinazolinone (BMEQ) was selected from quinazolinones for its strong tyrosinase inhibitory activity (</span><em>IC</em><sub><em>50</em></sub><span> = 160 ± 6 μM). It suppressed tyrosinase activity in a competitive way and quenched the fluorescence of the enzyme<span><span> through a static mechanism. The binding of BMEQ to tyrosinase increased the hydrophobicity<span> of the latter and facilitated non-radiative energy transfer between them. The formation of BMEQ–tyrosinase complex was driven by hydrogen bonds and hydrophobic interactions, and it loosened the basic framework structure of tyrosinase, affecting the conformation of the enzyme, and leading to a decrease in tyrosinase activity. In addition, the BMEQ postponed the </span></span>oxidation<span><span> of phenolics and flavonoids<span> by inhibiting polyphenol oxidase (PPO) and </span></span>peroxidase (POD), which resulted in the inhibition of the browning of fresh-cut apples. This study identified a novel tyrosinase inhibitor BMEQ and verified its potential application for improving the preservation of postharvest fruits.</span></span></span></p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139424878","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01DOI: 10.1016/j.jbiosc.2023.12.013
Jun Cheng , Yanmin Zhang , Yuan Tian , Lei Cao , Xuping Liu , Shiwei Miao , Liang Zhao , Qian Ye , Yan Zhou , Wen-Song Tan
Efficiently expanding Chinese hamster ovary (CHO) cells, which serve as the primary host cells for recombinant protein production, have gained increasing industrial significance. A significant hurdle in stable cell line development is the low efficiency of the target gene integrated into the host genome, implying the necessity for an effective screening and selection procedure to separate these stable cells. In this study, the genes of phenylalanine hydroxylase (PAH) and pterin 4 alpha carbinolamine dehydratase 1 (PCBD1), which are key enzymes in the tyrosine synthesis pathway, were utilized as selection markers and transduced into host cells together with the target genes. This research investigated the enrichment effect of this system and advanced further in understanding its benefits for cell line development and rCHO cell culture. A novel tyrosine-based selection system that only used PCBD1 as a selection marker was designed to promote the enrichment effect. Post 9 days of starvation, positive transductants in the cell pool approached 100%. Applied the novel tyrosine-based selection system, rCHO cells expressing E2 protein were generated and named CHO TS cells. It could continue to grow, and the yield of E2 achieved 95.95 mg/L in a tyrosine-free and chemically-defined (CD) medium. Herein, we introduced an alternative to antibiotic-based selections for the establishment of CHO cell lines and provided useful insights for the design and development of CD medium.
高效扩增的中国仓鼠卵巢(CHO)细胞是重组蛋白质生产的主要宿主细胞,在工业上的重要性与日俱增。稳定细胞系开发中的一个重要障碍是目标基因整合到宿主基因组中的效率较低,这意味着需要一种有效的筛选和选择程序来分离这些稳定细胞。本研究利用酪氨酸合成途径中的关键酶--苯丙氨酸羟化酶(PAH)和蝶呤 4 α-氨基甲醇脱水酶 1(PCBD1)基因作为选择标记,并将其与目的基因一起转导到宿主细胞中。这项研究调查了这一系统的富集效应,并进一步了解了它对细胞系开发和 rCHO 细胞培养的益处。为了促进富集效应,我们设计了一种新型的基于酪氨酸的选择系统,该系统仅使用 PCBD1 作为选择标记。饥饿 9 天后,细胞池中的阳性转导子接近 100%。应用新颖的基于酪氨酸的选择系统,产生了表达 E2 蛋白的 rCHO 细胞,并将其命名为 CHO TS 细胞。它可以继续生长,在不含酪氨酸的化学定义(CD)培养基中,E2的产量达到了95.95 mg/L。在此,我们为建立 CHO 细胞系介绍了一种基于抗生素选择的替代方法,并为 CD 培养基的设计和开发提供了有益的启示。
{"title":"Development of a novel tyrosine-based selection system for generation of recombinant Chinese hamster ovary cells","authors":"Jun Cheng , Yanmin Zhang , Yuan Tian , Lei Cao , Xuping Liu , Shiwei Miao , Liang Zhao , Qian Ye , Yan Zhou , Wen-Song Tan","doi":"10.1016/j.jbiosc.2023.12.013","DOIUrl":"10.1016/j.jbiosc.2023.12.013","url":null,"abstract":"<div><p><span><span><span>Efficiently expanding Chinese hamster ovary (CHO) cells, which serve as the primary host cells for </span>recombinant protein production, have gained increasing industrial significance. A significant hurdle in stable cell line development is the low efficiency of the target gene integrated into the host genome, implying the necessity for an effective screening and selection procedure to separate these stable cells. In this study, the genes of </span>phenylalanine hydroxylase<span><span> (PAH) and pterin 4 alpha carbinolamine dehydratase 1 (PCBD1), which are key </span>enzymes<span> in the tyrosine synthesis pathway, were utilized as selection markers and transduced into host cells together with the target genes. This research investigated the enrichment effect of this system and advanced further in understanding its benefits for cell line development and rCHO cell culture. A novel tyrosine-based selection system that only used </span></span></span><em>PCBD1</em><span> as a selection marker was designed to promote the enrichment effect. Post 9 days of starvation, positive transductants in the cell pool approached 100%. Applied the novel tyrosine-based selection system, rCHO cells expressing E2 protein were generated and named CHO TS cells. It could continue to grow, and the yield of E2 achieved 95.95 mg/L in a tyrosine-free and chemically-defined (CD) medium. Herein, we introduced an alternative to antibiotic-based selections for the establishment of CHO cell lines and provided useful insights for the design and development of CD medium.</span></p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139464591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Germline and somatic mutations cause various diseases, including cancer. Clinical applications of genome editing are keenly anticipated, since it can cure genetic diseases. Recently, we reported that a 5′-tailed duplex (TD), consisting of an approximately 80-base editor strand oligodeoxyribonucleotide and a 35-base assistant strand oligodeoxyribonucleotide, could edit a target gene on plasmid DNA and correct a single-base substitution mutation without an artificial nuclease in human cells. In this study, we assessed the ability of the TD to correct base substitution mutations located consecutively or separately, and deletion and insertion mutations. A TD with an 80-base editor strand was co-introduced into human U2OS cells with plasmid DNA bearing either a wild-type or mutated copepod green fluorescent protein (copGFP) gene. Among the mutations, three-base consecutive substitutions were efficiently repaired. The correction efficiencies of deletion mutations were similar to those of substitution mutations, and two to three times higher than those of insertion mutations. Up to three-base substitution, deletion, and insertion mutations were excellent targets for correction by TDs. These results suggested that the TDs are useful for editing disease-causing genes with small mutations.
{"title":"Correction of substitution, deletion, and insertion mutations by 5′-tailed duplexes","authors":"Hidehiko Kawai, Kento Sato, Taiki Kato, Hiroyuki Kamiya","doi":"10.1016/j.jbiosc.2023.12.011","DOIUrl":"10.1016/j.jbiosc.2023.12.011","url":null,"abstract":"<div><p><span><span><span><span>Germline and somatic mutations<span> cause various diseases, including cancer. Clinical applications of genome editing are keenly anticipated, since it can cure genetic diseases. Recently, we reported that a 5′-tailed duplex (TD), consisting of an approximately 80-base editor strand oligodeoxyribonucleotide and a 35-base assistant strand oligodeoxyribonucleotide, could edit a target gene on plasmid DNA and correct a single-base substitution mutation without an artificial </span></span>nuclease<span><span><span> in human cells. In this study, we assessed the ability of the TD to correct </span>base substitution mutations located consecutively or separately, and </span>deletion and insertion mutations. A TD with an 80-base editor strand was co-introduced into human U2OS cells with plasmid DNA bearing either a wild-type or mutated </span></span>copepod </span>green fluorescent protein (</span><em>copGFP</em>) gene. Among the mutations, three-base consecutive substitutions were efficiently repaired. The correction efficiencies of deletion mutations were similar to those of substitution mutations, and two to three times higher than those of insertion mutations. Up to three-base substitution, deletion, and insertion mutations were excellent targets for correction by TDs. These results suggested that the TDs are useful for editing disease-causing genes with small mutations.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139420836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for in vitro bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve in vitro bile analysis.
{"title":"Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery","authors":"Fumiya Tokito , Mikito Kiyofuji , Hyunjin Choi , Masaki Nishikawa , Toshiaki Takezawa , Yasuyuki Sakai","doi":"10.1016/j.jbiosc.2024.01.017","DOIUrl":"10.1016/j.jbiosc.2024.01.017","url":null,"abstract":"<div><p>Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for <em>in vitro</em> bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve <em>in vitro</em> bile analysis.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139983007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-26DOI: 10.1016/j.jbiosc.2023.12.018
Jiale Chen , Ye Lu , Li Liu , Ruoxuan Bai , Shuting Zhang , Yaqiao Hao , Fangxu Xu , Buyun Wei , Hongxin Zhao
A high-yielding microbial polysaccharide-producing strain, named RM1603, was isolated from rhizosphere soil and identified by morphological and phylogenetic analysis. The extracellular polysaccharides (EPS) were identified by thin-layer chromatography and infrared spectroscopy. The fermentation conditions were optimized by single factor experiments in shake flasks and a 5-L fermentor. The results of morphological and phylogenetic tree analysis showed that RM1603 was a strain of Aureobasidium pullulans. Its microbial polysaccharide was identified as pullulan, and the EPS production capacity reached 33.07 ± 1.03 g L−1 in shake flasks. The fermentation conditions were optimized in a 5-L fermentor, and were found to encompass an initial pH of 6.5, aeration rate of 2 vvm, rotor speed of 600 rpm, and inoculum size of 2 %. Under these conditions, the pullulan yield of RM1603 reached 62.52 ± 0.24 g L−1. Thus, this study contributes RM1603 as a new isolation with high-yielding pullulan and potential application value in biotechnology.
{"title":"Characteristic analysis and fermentation optimization of a novel Aureobasidium pullulans RM1603 with high pullulan yield","authors":"Jiale Chen , Ye Lu , Li Liu , Ruoxuan Bai , Shuting Zhang , Yaqiao Hao , Fangxu Xu , Buyun Wei , Hongxin Zhao","doi":"10.1016/j.jbiosc.2023.12.018","DOIUrl":"10.1016/j.jbiosc.2023.12.018","url":null,"abstract":"<div><p>A high-yielding microbial polysaccharide-producing strain, named RM1603, was isolated from rhizosphere soil and identified by morphological and phylogenetic analysis. The extracellular polysaccharides (EPS) were identified by thin-layer chromatography and infrared spectroscopy. The fermentation conditions were optimized by single factor experiments in shake flasks and a 5-L fermentor. The results of morphological and phylogenetic tree analysis showed that RM1603 was a strain of <em>Aureobasidium pullulans</em>. Its microbial polysaccharide was identified as pullulan, and the EPS production capacity reached 33.07 ± 1.03 g L<sup>−1</sup> in shake flasks. The fermentation conditions were optimized in a 5-L fermentor, and were found to encompass an initial pH of 6.5, aeration rate of 2 vvm, rotor speed of 600 rpm, and inoculum size of 2 %. Under these conditions, the pullulan yield of RM1603 reached 62.52 ± 0.24 g L<sup>−1</sup>. Thus, this study contributes RM1603 as a new isolation with high-yielding pullulan and potential application value in biotechnology.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139983006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-17DOI: 10.1016/j.jbiosc.2023.12.009
Jinyang Li , Ze Ding , Wenqi Dong , Weiwei Li , Yanfang Wu , Lining Zhu , Huifeng Ma , Baoguo Sun , Xiuting Li
The unique cellar fermentation process of Chinese strong-flavor Baijiu is the reason for its characteristic cellar aroma flavor. The types, abundance, community structure and metabolic activity of microorganisms in the pit mud directly affect the microbial balance in the white spirit production environment, promoting the formation of typical aromas and influencing the quality of CFSB. During the production process, the production of off-flavor in the cellar may occur. The aim of this study is to elucidate the differences in microbiota and flavor between normal pit mud and abnormal pit mud (pit mud with off-flavor). A total of 46 major volatile compounds were identified, and 24 bacterial genera and 21 fungal genera were screened. The esters, acids, and alcohols in the abnormal pit mud were lower than those in the normal pit mud, while the aldehydes were higher. 3-Methyl indole, which has been proven to be responsible for the muddy and musty flavors, was detected in both types of pit mud, and for the first time, high levels of 4-methylanisole was detected in the pit mud. The microbial composition of the two types of pit mud showed significant differences in the bacterial genera of Sporosarcina, Lactobacillus, Garciella, Anaerosalibacter, Lentimicrobium, HN–HF0106, Petrimonas, Clostridium_sensu_stricto_12 and Bacillus, and the fungal genera of Millerozyma, Penicillium, Mortierella, Monascus, Saccharomyces, Issatchenkia, Pithoascus, Pseudallescheria, and Wickerhamomyces. Additionally, we speculate that Sporosarcina is the predominant bacterial genus responsible for the imbalance of microbiota in pit mud.
{"title":"Analysis of differences in microorganisms and aroma profiles between normal and off-flavor pit mud in Chinese strong-flavor Baijiu","authors":"Jinyang Li , Ze Ding , Wenqi Dong , Weiwei Li , Yanfang Wu , Lining Zhu , Huifeng Ma , Baoguo Sun , Xiuting Li","doi":"10.1016/j.jbiosc.2023.12.009","DOIUrl":"10.1016/j.jbiosc.2023.12.009","url":null,"abstract":"<div><p>The unique cellar fermentation process of Chinese strong-flavor <em>Baijiu</em> is the reason for its characteristic cellar aroma flavor. The types, abundance, community structure and metabolic activity of microorganisms in the pit mud directly affect the microbial balance in the white spirit production environment, promoting the formation of typical aromas and influencing the quality of CFSB. During the production process, the production of off-flavor in the cellar may occur. The aim of this study is to elucidate the differences in microbiota and flavor between normal pit mud and abnormal pit mud (pit mud with off-flavor). A total of 46 major volatile compounds were identified, and 24 bacterial genera and 21 fungal genera were screened. The esters, acids, and alcohols in the abnormal pit mud were lower than those in the normal pit mud, while the aldehydes were higher. 3-Methyl indole, which has been proven to be responsible for the muddy and musty flavors, was detected in both types of pit mud, and for the first time, high levels of 4-methylanisole was detected in the pit mud. The microbial composition of the two types of pit mud showed significant differences in the bacterial genera of <em>Sporosarcina</em>, <em>Lactobacillus</em>, <em>Garciella</em>, <em>Anaerosalibacter</em>, <em>Lentimicrobium</em>, HN–HF0106, <em>Petrimonas</em>, <em>Clostridium_sensu_stricto_12</em> and <em>Bacillus</em>, and the fungal genera of <em>Millerozyma</em>, <em>Penicillium</em>, <em>Mortierella</em>, <em>Monascus</em>, <em>Saccharomyces</em>, <em>Issatchenkia</em>, <em>Pithoascus</em>, <em>Pseudallescheria</em>, and <em>Wickerhamomyces</em>. Additionally, we speculate that <em>Sporosarcina</em> is the predominant bacterial genus responsible for the imbalance of microbiota in pit mud.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139899948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fermented seasonings have pleasant flavors that stimulate our appetite. Their flavoring properties change depending on factors such as their materials and fermented conditions. Therefore, a comparative analysis of their flavor is important when evaluating their quality. However, seasonings contain high levels of various matrices such as sugars, proteins, lipids, and ethanol, making it difficult to extract aroma compounds efficiently from them. In this study, we verified a high-efficient and high-throughput volatile flavor analysis of fermented seasonings by solvent-assisted stir bar solid extraction (SA-SBSE) with reverse extraction. We applied SA-SBSE to Japanese fermented seasonings, soy sauce, miso (fermented beans), and mirin (sweet rice wine) and compared their profiles with those from other common extraction methods, headspace gas-solid-phase microextraction (HS-SPME), liquid extraction with solvent-assisted flavor evaporation (LE-SAFE), and conventional SBSE (C-SBSE). The aroma properties and profiles of extracts from SA-SBSE were close to those of the original sample, being similar to that of LE-SAFE. In addition, potent aroma compounds in each sample were extracted by SA-SBSE and LE-SAFE, which were far superior to those by C-SBSE. For quantification, SA-SBSE extracts showed a good standard curve by the standard addition method. We could quantify maltol, one of the most common potent aroma compounds in all samples, for various commercial samples by such high-throughput analysis.
{"title":"Superior high-efficiency and high-throughput volatile flavor extraction of Japanese fermented seasonings by solvent-assisted stir bar solid extraction with reverse extraction","authors":"Yoko Iijima , Azusa Miwa , Kaito Shimada , Shunsuke Horita , Yuho Kamiko , Yusuke Ito , Kikuo Sasamoto , Takeharu Nakahara , Taichi Koizumi , Nobuo Ochiai","doi":"10.1016/j.jbiosc.2024.01.013","DOIUrl":"10.1016/j.jbiosc.2024.01.013","url":null,"abstract":"<div><p>Fermented seasonings have pleasant flavors that stimulate our appetite. Their flavoring properties change depending on factors such as their materials and fermented conditions. Therefore, a comparative analysis of their flavor is important when evaluating their quality. However, seasonings contain high levels of various matrices such as sugars, proteins, lipids, and ethanol, making it difficult to extract aroma compounds efficiently from them. In this study, we verified a high-efficient and high-throughput volatile flavor analysis of fermented seasonings by solvent-assisted stir bar solid extraction (SA-SBSE) with reverse extraction. We applied SA-SBSE to Japanese fermented seasonings, soy sauce, miso (fermented beans), and mirin (sweet rice wine) and compared their profiles with those from other common extraction methods, headspace gas-solid-phase microextraction (HS-SPME), liquid extraction with solvent-assisted flavor evaporation (LE-SAFE), and conventional SBSE (C-SBSE). The aroma properties and profiles of extracts from SA-SBSE were close to those of the original sample, being similar to that of LE-SAFE. In addition, potent aroma compounds in each sample were extracted by SA-SBSE and LE-SAFE, which were far superior to those by C-SBSE. For quantification, SA-SBSE extracts showed a good standard curve by the standard addition method. We could quantify maltol, one of the most common potent aroma compounds in all samples, for various commercial samples by such high-throughput analysis.</p></div>","PeriodicalId":15199,"journal":{"name":"Journal of bioscience and bioengineering","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139898032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}