Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

Summary

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

Abstract

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.

Introduction

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 antibacterial^5,6,7, anti-inflammatory^8,9,10,11,12, antiviral^3,12,13, antioxidant^14,15,16, 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 aglycones^20,21,22,23. While host'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.

Protocol

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  in vitro

1. Preparation of General Anaerobic Medium (GAM)²⁵
   1. Preparation of Solution A
      1. 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.
      2. Add 500 mL of distilled water and stir under heating until the substances are dissolved completely.
   2. Preparation of Solution B
      1. 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.
   3. Preparation of Solution C
      1. 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.
      2. 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.
      3. Distribute the medium into 250 mL Erlenmeyer flasks and autoclave at 121 ˚C for 30 min. Cool to room temperature and store at 4 ˚C.
   4. Preparation of solid general anaerobic medium
      1. Add 1%-2% agar to the GAM liquid medium and heat until completely dissolved. Distribute into 250 mL Erlenmeyer flasks and autoclave at 121 ˚C for 30 min.
   5. Preparation of low-carbon source medium
      1. Prepare the low-carbon source medium using the same procedure as GAM, but omit yeast extract, glucose, and soluble starch.
2. Preparation of Mixed Human Intestinal Flora
   1. 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 ˚C for 24 h in a sterile anaerobic incubator.
      NOTE: Use an oxygen meter to monitor the anaerobic incubator's oxygen levels, and replace nitrogen gas as necessary to maintain anaerobic conditions.
3. Activation of mixed human intestinal flora
   1. 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.
   2. Seal and incubate at 37 ˚C for 24 h under anaerobic conditions to obtain active mixed human intestinal flora.
4. Preparation of reference solutions
   1. 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.
5. Screening of active intestinal flora capable of cleaving C-glycosides
   1. Mix 260 µL of activated intestinal flora with 6 mL of fresh low-carbon source medium containing 40 µL of orientin reference solution, and incubate at 37 ˚C.
   2. Include a control group (6 mL of low-carbon source medium, 40 µL of orientin reference solution, and 260 µL of normal saline) and a blank group (6 mL of low-carbon source medium, 40 µL of normal saline, and 260 µL of activated intestinal flora).
      NOTE: Perform all tests in triplicate in an anaerobic incubator.
6. Pretreatment of transformation solutions
   1. 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.
   2. Add 200 µL of the supernatant to 600 µL of methanol, mix, and centrifuge at 13,400 x g for 15 min at room temperature to remove proteins.
7. Detection of transformation products
   1. Filter the supernatant using a 0.22 µm 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 × 250 mm, 5 µm).
   2. Maintain the column oven temperature at 40 ˚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.

2. Isolation of single strains from human intestinal bacterial flora

1. Activation of mixed human intestinal bacterial flora
   1. 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.
2. Preparation of solid GAM plates
   1. 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.
3. Preliminary screening of single strains from human intestinal bacterial flora
   1. Pipette 100 µL of the activated bacterial solution into a 1.5 mL microcentrifuge tube containing 1 mL of normal saline.
   2. 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.
4. Observation of colony morphology
   1. After 48 h of incubation at 37 ˚C, observe the size, morphology, and color of colonies on the plate.
5. Selection of individual colonies to obtain single strains
   1. 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 ˚C for 24 h to obtain single strains.
6. Verification of single strain activity
   1. 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.
7. Purification of single strains from human intestinal bacterial flora
   1. 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.

Results

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...

Discussion

Standard operating procedure (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...

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	Thermo Fisher Scientific	F2408R205	HPLC
Anaerobic Incubator	Shanghai CIMO Medical Instrument Manufacturing Co., LTD		YQX- II
Beef Extract	Beijing Abxing Biotechnology Co., LTD	01-009	BR
Digestive Serum Powder	Beijing Abxing Biotechnology Co., LTD	01-087	BR
Dipotassium Hydrogen Phosphate	Beijing Chemical Works	M26298	AR
Disposable Sterile Stool Collection Tube	Lang Fu Co., LTD		5 mL
Distilled Water	Department of Biopharmaceutical, Beijing University of Traditional Chinese Medicine
DMSO	Sigma-Aldrich Corporation	WXBD2861V	AR
EP Tube	Beijing Biodee Biotechnology Co., LTD		10 mL/1.5 mL
Eppendorf Centrifuge	Eppendorf AG		5418
Glucose	Beijing Chemical Works	GC205003	AR
High Performance Liquid Chromatograph	Shimadzu Corporation		LC-20
High-pressure Steam Sterilizer	Sanyo Denki Shanghai Co., LTD		MLS-3780
Innoval C18 Chromatographic Column	Agela Technologies Co., LTD		4.6 mm × 250 mm, 5 µm
L-cysteine Hydrochloride	Beijing Abxing Biotechnology Co., LTD	BGASY01	BR
Liver Extract Powder	Beijing Abxing Biotechnology Co., LTD	01-085	BR
Luteolin	National Institutes for Food and Drug Control		>98%
Magnetic Stirrer	Ika Werke Co., LTD		RCT basic
Methanol	Thermo Fisher Scientific	20240901312	AR
Millipore Filter Membrane	Sangon Biotech (Shanghai) Co., Ltd.		0.22 µL × 50 mm
Orientin	Yishiming (Beijing) Biotechnology Co., LTD	19120601	>98%
Peptone	Beijing Abxing Biotechnology Co., LTD	1685787	BR
Petri Dish	Beijing Biodee Biotechnology Co., LTD		150 mm
Sodium Chloride	Beijing Abxing Biotechnology Co., LTD	BN20008	AR
Sodium Thioglycolate	Shanghai Jianglai Biotechnology Co., LTD	J031S219019	AR
Soluble Starch	Beijing Abxing Biotechnology Co., LTD	S9765	BR
Soya Peptone	Beijing Abxing Biotechnology Co., LTD	2147955	BR
Tryptone	Agela Technologies Co., LTD	1685787	BR
Ultrasonic Cleaner	Kun Shan Ultrasonic Instruments Co., LTD		KQ-500DE
Yeast Extract	Beijing Abxing Biotechnology Co., LTD	01-014	BR