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,

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....

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