Optimization of β-Glucuronidase Activity from Lactobacillus delbrueckii Rh2 and Its Use for Biotransformation of Baicalin and Wogonoside

Seockmo Ku,Hua Zheng,Myeong Soo Park,지근억 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.2

Scutellariae radix, the root of Scutellaria baicalensis, is a traditional medicinal herb containing baicalin and wogonoside as the main flavonoids. Before absorption, these flavonoids are metabolized into their corresponding aglycones, baicalein and wogonin, by β-glucuronidase produced by the intestinal microflora. To develop a method for efficient transformation of these flavonoids, optimal conditions for the production of β-glucuronidase from Lactobacillus delbrueckii Rh2 (Rh2) were determined. Addition of 4% galactose to basal medium increased the enzyme activity of Rh2 by about 8-fold. Optimal pH and temperature for the β-glucuronidase activity of cell extract were 5.0 and 50oC, respectively, and more than 95% of the enzyme activity was maintained at 60oC for 11 h. Under optimal conditions, more than 90% of the glycones were converted into their corresponding aglycones within 3 h. These results demonstrate that the transformation of baicalin and wogonoside from S. baicalensis by Rh2 would be useful for the efficient production of wogonin and baicalein.

Enhancement of Anti-tumorigenic Polysaccharide Production, Adhesion, and Branch Formation of Bifidobacterium bifidum BGN4 by Phytic Acid

Seockmo Ku,Hyun Ju You,Geun Eog Ji 한국식품과학회 2009 Food Science and Biotechnology Vol.18 No.3

The polysaccharide (BB-pol) extracted from Bifidobacterium bifidum BGN4 showed growth inhibitory effects on several colon cancer cell lines such as HT-29 and HCT-116. To increase the yield of polysaccharide, B. bifidum BGN4 was cultured in various culture media with different compositions. When B. bifidum BGN4 was cultured in modified MRS broth containing phytic acid, the cells showed increased branch formation and enlarged morphology. The content of total carbohydrate and the ability of adhesion to intestinal epithelial cells were also increased by phytic acid. The polysaccharide obtained from the cells grown in the presence of phytic acid inhibited the proliferation of cancer cell lines such as HT-29 and MCF-7 cells but not normal colon cell line, FHC. Taken together, Bifidobacterium grown in the presence of phytic acid may confer enhanced beneficial function for the host.

Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG

( Seockmo Ku ),( Hyun Ju You ),( Myeong Soo Park ),( Geun Eog Ji ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.6

Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, wholecell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG

Ku, Seockmo,You, Hyun Ju,Park, Myeong Soo,Ji, Geun Eog The Korean Society for Microbiology and Biotechnol 2016 Journal of microbiology and biotechnology Vol.26 No.7

Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40℃, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

Review on <i>Bifidobacterium bifidum</i> BGN4: Functionality and Nutraceutical Applications as a Probiotic Microorganism

Ku, Seockmo,Park, Myeong Soo,Ji, Geun Eog,You, Hyun Ju MDPI AG 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.9

<P><I>Bifidobacterium bifidum</I> BGN4 is a probiotic strain that has been used as a major ingredient to produce nutraceutical products and as a dairy starter since 2000. The various bio-functional effects and potential for industrial application of <I>B. bifidum</I> BGN4 has been characterized and proven by in vitro (i.e., phytochemical bio-catalysis, cell adhesion and anti-carcinogenic effects on cell lines, and immunomodulatory effects on immune cells), in vivo (i.e., suppressed allergic responses in mouse model and anti-inflammatory bowel disease), and clinical studies (eczema in infants and adults with irritable bowel syndrome). Recently, the investigation of the genome sequencing was finished and this data potentially clarifies the biochemical characteristics of <I>B. bifidum</I> BGN4 that possibly illustrate its nutraceutical functionality. However, further systematic research should be continued to gain insight for academic and industrial applications so that the use of <I>B. bifidum</I> BGN4 could be expanded to result in greater benefit. This review deals with multiple studies on <I>B. bifidum</I> BGN4 to offer a greater understanding as a probiotic microorganism available in functional food ingredients. In particular, this work considers the potential for commercial application, physiological characterization and exploitation of <I>B. bifidum</I> BGN4 as a whole.</P>

Effects of ascorbic acid on α-?-arabinofuranosidase and α-?-arabinopyranosidase activities from Bifidobacterium longum RD47 and its application to whole cell bioconversion of ginsenoside

Ku, Seockmo,You, Hyun Ju,Park, Myeong Soo,Ji, Geun Eog 한국응용생명화학회 2015 Applied Biological Chemistry (Appl Biol Chem) Vol.58 No.6

Bifidobacterium longum RD47 was cultured in 24 kinds of modified MRS broths containing various ingredients to select the most promising source that induces microbial enzymes. Among the various ingredients, ascorbic acid significantly enhanced ${\alpha}$-$\small{L}$-arabinofuranosidase and ${\alpha}$-$\small{L}$-arabinopyranosidase activities in Bifidobacterium longum RD47. Addition of 2 % ascorbic acid (w/v) to MRS showed the maximum enzyme activities. Both whole cell and disrupted cell homogenates showed efficient ${\rho}$-nitrophenyl-${\beta}$-$\small{D}$-glucopyranoside and ${\rho}$-nitrophenyl-${\beta}$-$\small{D}$-glucofuranoside hydrolysis activities. The initially enhanced ${\alpha}$-$\small{L}$-arabinopyranosidase and ${\alpha}$-$\small{L}$-arabinofuranosidase activities by ascorbic acid were maintained over the cell disruption process. The optimal pH of ${\alpha}$-$\small{L}$-arabinofuranosidase and ${\alpha}$-$\small{L}$-arabinopyranosidase was 5.0 and 7.0, respectively. Both enzymes showed the maximum activities at $40.0^{\circ}C$. Under the controlled condition using Bifidobacterium longum RD47, ginsenoside Rb2, and Rc were converted to ginsenoside Rd.

Acceleration of Aglycone Isoflavone and γ-Aminobutyric Acid Production from Doenjang Using Whole-Cell Biocatalysis Accompanied by Protease Treatment

( Yincong Li ),( Seockmo Ku ),( Myeong Soo Park ),( Zhipeng Li ),( Geun Eog Ji ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.11

Recently, soybean isoflavone aglycones (i.e., daidzein and genistein) and γ-aminobutyric acid (GABA) have begun to receive considerable consumer attention owing to their potential as nutraceuticals. To produce these ingredients, multiple microorganisms and their enzymes are commonly used for catalysis in the nutraceutical industry. In this work, we introduce a novel fermentation process that uses whole-cell biocatalysis to accelerate GABA and isoflavone aglycone production in doenjang (a traditional Korean soybean paste). Microbial enzymes transform soybean isoflavone glycosides (i.e., daidzin and genistin) and monosodium glutamate into soybean isoflavone aglycones and GABA. Lactobacillus brevis GABA 100 and Aspergillus oryzae KACC 40250 significantly reduced the production time with the aid of a protease. The resulting levels of GABA and daidzein were higher, and genistein production resembled the levels in traditional doenjang fermented for over a year. Concentrations of GABA, daidzein, and genistein were measured as 7,162, 60, and 59 μg/g, respectively on the seventh day of fermentation. Our results demonstrate that the administration of whole-cell L. brevis GABA 100 and A. oryzae KACC 40250 paired with a protease treatment is an effective method to accelerate GABA, daidzein, and genistein production in doenjang.

Current Technical Approaches for the Early Detection of Foodborne Pathogens: Challenges and Opportunities

Cho, Il-Hoon,Ku, Seockmo MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.10

<P>The development of novel and high-tech solutions for rapid, accurate, and non-laborious microbial detection methods is imperative to improve the global food supply. Such solutions have begun to address the need for microbial detection that is faster and more sensitive than existing methodologies (e.g., classic culture enrichment methods). Multiple reviews report the technical functions and structures of conventional microbial detection tools. These tools, used to detect pathogens in food and food homogenates, were designed via qualitative analysis methods. The inherent disadvantage of these analytical methods is the necessity for specimen preparation, which is a time-consuming process. While some literature describes the challenges and opportunities to overcome the technical issues related to food industry legal guidelines, there is a lack of reviews of the current trials to overcome technological limitations related to sample preparation and microbial detection via nano and micro technologies. In this review, we primarily explore current analytical technologies, including metallic and magnetic nanomaterials, optics, electrochemistry, and spectroscopy. These techniques rely on the early detection of pathogens via enhanced analytical sensitivity and specificity. In order to introduce the potential combination and comparative analysis of various advanced methods, we also reference a novel sample preparation protocol that uses microbial concentration and recovery technologies. This technology has the potential to expedite the pre-enrichment step that precedes the detection process. </P>

Safety Evaluations of <i>Bifidobacterium bifidum</i> BGN4 and <i>Bifidobacterium longum</i> BORI

Kim, Min Jeong,Ku, Seockmo,Kim, Sun Young,Lee, Hyun Ha,Jin, Hui,Kang, Sini,Li, Rui,Johnston, Tony V.,Park, Myeong Soo,Ji, Geun Eog MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.5

<P>Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, <I>Bifidobacterium</I> and <I>Lactobacillus</I> species are probiotic strains that are most commonly commercially produced and actively studied. <I>Bifidobacterium bifidum</I> BGN4 and <I>Bifidobacterium longum</I> BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of <I>B. bifidum</I> BGN4 and <I>B. longum</I> BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). <I>B. bifidum</I> BGN4 and <I>B. longum</I> BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). <I>B. bifidum</I> BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of <I>B. bifidum</I> BGN4 was not transferred via conjugation with <I>L. acidophilus</I> ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of <I>B. bifidum</I> BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of <I>B. bifidum</I> BGN4. Tetracycline-resistant genes are prevalent among <I>B. longum</I> strains; <I>B. longum</I> BORI has a <I>tet</I>(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with <I>L. fermentum</I> AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of <I>B. bifidum</I> BGN4 and <I>B. longum</I> BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.</P>

Optimization of the optical transparency of bones by PACT-based passive tissue clearing

Jin Byung-Ho,Woo Jiwon,Lee Mirae,Ku Seockmo,Moon Hyung Seok,Ryu Seung Jun,Hyun Young-Min,Park Jeong-Yoon,Kuh Sung Uk,Cho Yong Eun 생화학분자생물학회 2023 Experimental and molecular medicine Vol.55 No.-

Recent developments in tissue clearing methods such as the passive clearing technique (PACT) have allowed three-dimensional analysis of biological structures in whole, intact tissues, thereby providing a greater understanding of spatial relationships and biological circuits. Nonetheless, the issues that remain in maintaining structural integrity and preventing tissue expansion/shrinkage with rapid clearing still inhibit the wide application of these techniques in hard bone tissues, such as femurs and tibias. Here, we present an optimized PACT-based bone-clearing method, Bone-mPACT+, that protects biological structures. Bone-mPACT+ and four different decalcifying procedures were tested for their ability to improve bone tissue clearing efficiency without sacrificing optical transparency; they rendered nearly all types of bone tissues transparent. Both mouse and rat bones were nearly transparent after the clearing process. We also present a further modification, the Bone-mPACT+ Advance protocol, which is specifically optimized for processing the largest and hardest rat bones for easy clearing and imaging using established tissue clearing methods.