Name: Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria (Video) | JoVE Journal
Uploaded: 2025-02-28T12:20:00.000+00:00
Duration: 6 min 38 s
Description: Learn to screen and isolate C-glycoside-cleaving intestinal bacteria using a low carbon source medium protocol. This method identifies active bacterial strains for microbiology and biotechnology research.

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5 minutes

The work was supported by the National Natural Science Foundation of China 82374134.

The experiments conducted adhered to local, national, and international biosafety containment regulations appropriate to the specific biosafety hazards associated with each strain. Fecal samples were collected from healthy volunteers who had not taken any drugs for at least one week. Details of the reagents and equipment used are provided in the Table of Materials.
1. Construction of human intestinal bacterial transformation model&#160;&#160;in vitro
<ol>
	<li>Preparation of General Anaerobic Medium (GAM)25
	<ol>
		<li>Preparation of Solution A
		<ol>
			<li>Combine 10.0 g of tryptone, 10.0 g of proteose peptone, 3.0 g of soya peptone, 13.5 g of digestive serum powder, 1.2 g of liver extract powder, 2.2 g of beef extract, and 5.0 g of yeast extract in a 500 mL Erlenmeyer flask.</li>
			<li>Add 500 mL of distilled water and stir under heating until the substances are dissolved completely.</li>
		</ol>
		</li>
		<li>Preparation of Solution B
		<ol>
			<li>Prepare Solution B following the same procedure as Solution A, except combine 3.0 g of glucose, 5.0 g of soluble starch, 2.5 g of dipotassium hydrogen phosphate, and 3.0 g of sodium chloride in a 250 mL Erlenmeyer flask.</li>
		</ol>
		</li>
		<li>Preparation of Solution C
		<ol>
			<li>Prepare Solution C following the same procedure as Solutions A and B, except combine 0.3 g of L-cysteine hydrochloride and 0.3 g of sodium thioglycolate in a 100 mL Erlenmeyer flask.</li>
			<li>Mix all three solutions in a 1000 mL Erlenmeyer flask, adjust the pH to 7.2 using a 10% sodium hydroxide solution, and add distilled water to make up a final volume of 1000 mL.</li>
			<li>Distribute the medium into 250 mL Erlenmeyer flasks and autoclave at 121 &#730;C for 30 min. Cool to room temperature and store at 4 &#730;C.</li>
		</ol>
		</li>
		<li>Preparation of solid general anaerobic medium
		<ol>
			<li>Add 1%-2% agar to the GAM liquid medium and heat until completely dissolved. Distribute into 250 mL Erlenmeyer flasks and autoclave at 121 &#730;C for 30 min.</li>
		</ol>
		</li>
		<li>Preparation of low-carbon source medium
		<ol>
			<li>Prepare the low-carbon source medium using the same procedure as GAM, but omit yeast extract, glucose, and soluble starch.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of Mixed Human Intestinal Flora
	<ol>
		<li>Collect 1-2 g of fresh human feces in a disposable sterile stool collection tube filled with nitrogen gas. Seal the tube promptly. Add 5 mL of GAM medium, seal, and culture at 37 &#730;C for 24 h in a sterile anaerobic incubator. 
		NOTE: Use an oxygen meter to monitor the anaerobic incubator&#39;s oxygen levels, and replace nitrogen gas as necessary to maintain anaerobic conditions.</li>
	</ol>
	</li>
	<li>Activation of mixed human intestinal flora
	<ol>
		<li>After 24 h, transfer 0.5 mL of the human intestinal bacterial suspension to a new microcentrifuge tube containing 4.5 mL of fresh GAM medium.</li>
		<li>Seal and incubate at 37 &#730;C for 24 h under anaerobic conditions to obtain active mixed human intestinal flora.</li>
	</ol>
	</li>
	<li>Preparation of reference solutions
	<ol>
		<li>Weigh 5 mg each of orientin and luteolin, and dissolve them separately in 1 mL of DMSO to prepare orientin and luteolin reference solutions at a concentration of 5 mg/mL.</li>
	</ol>
	</li>
	<li>Screening of active intestinal flora capable of cleaving C-glycosides
	<ol>
		<li>Mix 260 &#181;L of activated intestinal flora with 6 mL of fresh low-carbon source medium containing 40 &#181;L of orientin reference solution, and incubate at 37 &#730;C.</li>
		<li>Include a control group (6 mL of low-carbon source medium, 40 &#181;L of orientin reference solution, and 260 &#181;L of normal saline) and a blank group (6 mL of low-carbon source medium, 40 &#181;L of normal saline, and 260 &#181;L of activated intestinal flora). 
		NOTE: Perform all tests in triplicate in an anaerobic incubator.</li>
	</ol>
	</li>
	<li>Pretreatment of transformation solutions
	<ol>
		<li>After 24 h and 48 h, pipette 1 mL of the reaction solutions into 1.5 mL microcentrifuge tubes and centrifuge at 13,400 x g for 15 min at room temperature to remove bacteria.</li>
		<li>Add 200 &#181;L of the supernatant to 600 &#181;L of methanol, mix, and centrifuge at 13,400 x g for 15 min at room temperature to remove proteins.</li>
	</ol>
	</li>
	<li>Detection of transformation products
	<ol>
		<li>Filter the supernatant using a 0.22 &#181;m microporous membrane and analyze the filtrate via high performance liquid chromatography (HPLC). Conduct HPLC analysis on an instrument equipped with a C18 column (4.6 mm &#215; 250 mm, 5 &#181;m).</li>
		<li>Maintain the column oven temperature at 40 &#730;C, with a flow rate of 1 mL/min. Use acetonitrile as mobile phase A and 0.1% formic acid in purified water as mobile phase B. 
		NOTE: Employ a linear gradient system as follows: 5%-55% A at 0-20 min, 55%-95% A at 20-25 min, 95%-5% A at 25-30 min, and 5% A at 30-32 min.</li>
	</ol>
	</li>
</ol>
2. Isolation of single strains from human intestinal bacterial flora
<ol>
	<li>Activation of mixed human intestinal bacterial flora
	<ol>
		<li>Activate the mixed human intestinal bacterial flora capable of cleaving orientin, as described in step 1.3, to obtain the activated human intestinal bacterial flora solution.</li>
	</ol>
	</li>
	<li>Preparation of solid GAM plates
	<ol>
		<li>Dissolve the prepared solid GAM medium by heating, and pour it into disposable sterile petri dishes in an anaerobic incubator. Allow the medium to solidify to prepare solid GAM plates.</li>
	</ol>
	</li>
	<li>Preliminary screening of single strains from human intestinal bacterial flora
	<ol>
		<li>Pipette 100 &#181;L of the activated bacterial solution into a 1.5 mL microcentrifuge tube containing 1 mL of normal saline.</li>
		<li>Inoculate an appropriate volume of the bacterial solution onto a petri dish using a disposable inoculation ring. Seal the dish and incubate it in an anaerobic incubator.</li>
	</ol>
	</li>
	<li>Observation of colony morphology
	<ol>
		<li>After 48 h of incubation at 37 &#730;C, observe the size, morphology, and color of colonies on the plate.</li>
	</ol>
	</li>
	<li>Selection of individual colonies to obtain single strains
	<ol>
		<li>Select distinct single bacterial colonies and culture them in 10 mL microcentrifuge tubes containing 4.5 mL of fresh GAM medium. Incubate under anaerobic conditions at 37 &#730;C for 24 h to obtain single strains.</li>
	</ol>
	</li>
	<li>Verification of single strain activity
	<ol>
		<li>Verify the activity of single strains as described in steps 1.5, 1.6, and 1.7. Perform activity verification using high performance liquid chromatography (HPLC) and plate marking25.</li>
	</ol>
	</li>
	<li>Purification of single strains from human intestinal bacterial flora
	<ol>
		<li>Purify the active single strain as described in step 2.1. Verify its activity through transformation experiments with orientin, following the procedures outlined in steps 1.5, 1.6, and 1.7.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors declare no conflicts of interest.

Standard operating procedure&#160;(SOP) for screening human intestinal bacteria capable of cleaving C-glycosides were established. Using these procedures, a pure active strain was successfully obtained, and its deglycosylation property was confirmed through transformation tests. The SOP consists of the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium.
The most important aspect of the screening process is the u...

C-glycosides are a group of compounds characterized by the direct linkage of glycosyl groups to aglycones through C-C bonds1. In nature, orientin, vitexin, puerarin, and their derivatives are commonly identified as C-glycosides2. These compounds are frequently found in medicinal plants such as Trollius chinensis3 and in animals such as Styela plicata4. Studies have demonstrated that these compounds provide health benefits and exhibit various bioactivities, including antibacterial5,6,7, anti-inflammatory8,9,10,11,12, antiviral3,12,13, antioxidant14,15,16, and antineoplastic activities17.
Due to the linkage of glycosyl groups to the skeleton through C-C bonds, these compounds exhibit high stability and resistance to acidic and enzymatic hydrolysis18, leading to poor absorbability and low bioavailability19. This limitation affects the development and utilization of C-glycosides. However, drugs containing C-glycosides, often administered orally, are deglycosylated by specific enzymes, producing more potent bioactive aglycones20,21,22,23. While host&#39;s own enzymes are unable to deglycosylate C-glycosides, intestinal bacteria have been reported to metabolize certain types. For instance, some intestinal bacteria convert mangiferin into norathyriol, which exhibits greater potency as an antidiabetic or antineoplastic agent24. Despite these findings, only a limited number of bacteria capable of cleaving C-glycosides have been characterized, and the underlying mechanisms remain poorly understood. Efficient and standardized screening of these bacteria will enhance understanding of their functions and accelerate the development of C-glycosides.
The screening method has been developed and refined over time into a mature system. The core approaches include enrichment, activity-oriented screening, and activity validation in low-carbon source media. This method facilitates the isolation of pure target strains, as well as the identification of species, genomic data, and traits of target microorganisms. Using this system, intestinal bacteria capable of cleaving C-glycosides can be effectively screened and isolated.

<table><tbody><tr><td>Acetonitrile</td><td>Thermo Fisher Scientific</td><td>F2408R205</td><td>HPLC</td></tr><tr><td>Anaerobic Incubator</td><td>Shanghai CIMO Medical Instrument Manufacturing Co., LTD</td><td></td><td>YQX- II</td></tr><tr><td>Beef Extract</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-009</td><td>BR</td></tr><tr><td>Digestive Serum Powder</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-087</td><td>BR</td></tr><tr><td>Dipotassium Hydrogen Phosphate</td><td>Beijing Chemical Works</td><td>M26298</td><td>AR</td></tr><tr><td>Disposable Sterile Stool Collection Tube</td><td>Lang Fu Co., LTD</td><td></td><td>5 mL</td></tr><tr><td>Distilled Water</td><td>Department of Biopharmaceutical, Beijing University of Traditional Chinese Medicine</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich Corporation</td><td>WXBD2861V</td><td>AR</td></tr><tr><td>EP Tube</td><td>Beijing Biodee Biotechnology Co., LTD</td><td></td><td>10 mL/1.5 mL</td></tr><tr><td>Eppendorf Centrifuge</td><td>Eppendorf AG</td><td></td><td>5418</td></tr><tr><td>Glucose</td><td>Beijing Chemical Works</td><td>GC205003</td><td>AR</td></tr><tr><td>High Performance Liquid Chromatograph</td><td>Shimadzu Corporation</td><td></td><td>LC-20</td></tr><tr><td>High-pressure Steam Sterilizer</td><td>Sanyo Denki Shanghai Co., LTD</td><td></td><td>MLS-3780</td></tr><tr><td>Innoval C&lt;sub&gt;18 &lt;/sub&gt;Chromatographic Column</td><td>Agela Technologies Co., LTD</td><td></td><td>4.6 mm &amp;times; 250 mm, 5 &amp;micro;m</td></tr><tr><td>L-cysteine Hydrochloride</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>BGASY01</td><td>BR</td></tr><tr><td>Liver Extract Powder</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-085</td><td>BR</td></tr><tr><td>Luteolin</td><td>National Institutes for Food and Drug Control</td><td></td><td>&gt;98%</td></tr><tr><td>Magnetic Stirrer</td><td>Ika Werke Co., LTD</td><td></td><td>RCT basic</td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific</td><td>20240901312</td><td>AR</td></tr><tr><td>Millipore Filter Membrane</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td></td><td>0.22 &amp;micro;L &amp;times; 50 mm</td></tr><tr><td>Orientin</td><td>Yishiming (Beijing) Biotechnology Co., LTD</td><td>19120601</td><td>&gt;98%</td></tr><tr><td>Peptone</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>1685787</td><td>BR</td></tr><tr><td>Petri Dish</td><td>Beijing Biodee Biotechnology Co., LTD</td><td></td><td>150 mm</td></tr><tr><td>Sodium Chloride</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>BN20008</td><td>AR</td></tr><tr><td>Sodium Thioglycolate</td><td>Shanghai Jianglai Biotechnology Co., LTD</td><td>J031S219019</td><td>AR</td></tr><tr><td>Soluble Starch</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>S9765</td><td>BR</td></tr><tr><td>Soya Peptone</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>2147955</td><td>BR</td></tr><tr><td>Tryptone</td><td>Agela Technologies Co., LTD</td><td>1685787</td><td>BR</td></tr><tr><td>Ultrasonic Cleaner</td><td>Kun Shan Ultrasonic Instruments Co., LTD</td><td></td><td>KQ-500DE</td></tr><tr><td>Yeast Extract</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-014</td><td>BR</td></tr></tbody></table>

screening and isolation of c-glycoside-cleaving intestinal bacteria

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

Fecal samples from ten healthy volunteers were screened using transformation experiments, resulting in one sample demonstrating activity in deglycosylating orientin. This finding confirmed the feasibility of screening for active samples. The active sample was isolated using the plate marking method. Based on the morphology and characteristics of the colonies, approximately 18 single bacterial colonies with yellow or white coloration, varied shapes, and uneven edges were selected for further analysis. Among these, only on...

This study establishes a set of standard operating procedures (SOPs) for efficiently screening and isolating intestinal bacteria capable of cleaving C-glycosides.

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

screening-and-isolation-of-c-glycoside-cleaving-intestinal-bacteria

Research

JoVE Journal

Biochemistry

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Isolation and Culture of Clostridioides difficile Bacteria from Larval Zebrafish Intestine

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Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model

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Isolating Enteric Glial Cells From the Submucosa and Lamina Propria of a Mouse

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Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

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Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

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Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

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Recent studies highlight the roles of human gut microbiota enzymes in metabolizing C-glycosides. Here, we present a comprehensive methodology integrating analytical chemistry techniques (LC-MS/MS and NMR) with structural biology approaches to systematically characterize the structural and enzymatic reaction chains of these C-glycoside-metabolizing enzymes.

Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota

Learn to screen and isolate C-glycoside-cleaving intestinal bacteria using a low carbon source medium protocol. This method identifies active bacterial strains for microbiology and biotechnology research.

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

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