Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Summary

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

Abstract

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

Introduction

Bacillus licheniformis is a species of Bacillus in the family Firmicutes, which is widely distributed in various environments such as water, soil, and animal intestines¹. B. licheniformis has a short and stout rod-like structure and moves individually². The colony is nearly round and dull, with a central bulge and neat edges of grayish white³. It has strong growth and reproduction ability and can absorb and utilize nutrients from various carbon sources, such as monosaccharides, polysaccharides, ketose, and organic acids⁴. In the later sta....

Protocol

All experimental procedures were documented and approved by the Ethics Committee of Nanjing Tech University. The feces were derived from Taihu sows about 2 years old (see Table of Materials), which were raised on professional and standard pig farms.

1. Preparation of media

1. Luria-Bertani (LB) liquid medium: Add 10 g of NaCl, 5 g of yeast extract powder, and 10 g of tryptone into a conical bottle, add distilled water to 1 L, stir, and dissolve with a.......

Discussion

B. licheniformis grows rapidly with simple culture conditions and fast sugar consumption, and the mature fermentation technology is helpful in saving industrial production costs¹³. The wide application of B. licheniformis and its secretions, bacitracin, has determined its promising market value. In agriculture, B. licheniformis is employed as a biofertilizer to improve plant growth and nutrient uptake by enhancing soil fertility, promoting root development, and aiding in.......

Materials

Name	Company	Catalog Number	Comments
2 × Phanta Flash Master Mix	Nanjing Vazyme Biotechnology Co., Ltd., Nanjing,China	P252-01
2kb DNA Marker	Beijing Trans Biotechnology Co., Ltd., Beijing, China	BM121-01
Acetonitrile	Shanghai Aladdin Biochemical Technology Co.,Ltd., Shanghai, China	A104443
Agar powder	Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China	A800730
Agarose	Shanghai Aladdin Biochemical Technology Co.,Ltd., Shanghai, China	A104062
Ammonium sulfate ((NH4)2SO4)	Sinopharm Chemical Reagent Co., Ltd., Shanghai, China	10002917
Autoclave sterilizer	Zealway Instrument Inc., Xiamen, China	GI36DWS
Bacillus biochemical identification strip	Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China	HBIG14
Bacitracin	Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China	B65740
Bacteria DNA Extraction Kit	Tiangen Biochemical Technology Co., Ltd., Beijing, China	DP209
Breathable sealing film	Beijing Leiborun Biotechnology Co., Ltd.	BS-QM-01A
Butanol	Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China	B433378
C18 (5 μm, 4.6 × 250 mm) HPLC column	Rizhao Kepuno New Material Co., Ltd., Rizhao, China	C1805-462510
Calcium carbonate (CaCO3)	Sinopharm Chemical Reagent Co., Ltd., Shanghai, China	10005717
Centrifuge	New Brunswick Scientific Co., Inc., UK	5452
Chromatographic tank	Nanjing Tenghui Experimental Technology Co., Ltd., Nanjing, China	P-1
Conical bottle	Sichuan Shubo  Co., Ltd., Chengdu, China	18012
Constant temperature incubator	Taist Instrument Co., Ltd., Tianjin, China	GH4500
Dipotassium phosphate (K2HPO4)	Xilong Chemical Co., Ltd., Guangdong, China	XL0015
EDTA-2Na	Shanghai Aladdin Biochemical Technology Co.,Ltd., Shanghai, China	E397526
Electronic balance	Mettler Toledo International Co., Ltd.	FA2104
Ethyl alcohol	Shanghai Aladdin Biochemical Technology Co.,Ltd., Shanghai, China	E130059
Gel Midi Purification Kit	Tiangen Biochemical Technology Co., Ltd., Beijing, China	DP302
Glass rod	Chengdu Yibang Kexi Instrument Co., Ltd.	1294
Glucose	Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China	D823520
Gram 's staining solution kit	Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China	HB8278
High performance liquid chromatograph	Agilent Technologies, Inc., California, America	1260
Horizontal electrophoresis apparatus	Beijing Liuyi Biotechnology Co., Ltd., Beijing, China	DYCP-31DN BIOMATE
Inoculation ring	Shanghai Muchen Biotechnology Co., Ltd., Shanghai, China	3171026
Magnetic stirrer	Wiggens GmbH Co., Ltd., Germany	WH220 PLUS
Methyl alcohol	Shanghai Aladdin Biochemical Technology Co.,Ltd., Shanghai, China	M116115
Microcentrifuge tube	Shanghai Muchen Biotechnology Co., Ltd., Shanghai, China	1351171
Micrococcus luteus	Bena Culture Collection, Suzhou, China	BNCC102589
Microporous filter membrane	Nantong Suri Experimental Equipment Co., Ltd.	PES0.22
Ninhydrin	Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China	N105629
Optical microscope	Optical Instrument Factory, Shanghai, China	DYS-108
Pig feces	Nanjing Quanfu Pig Farm, Nanjing, China
Polymerase chain reaction (PCR) Amplifier	Suzhou Dongsheng Xingye Scientific Instrument Co., Ltd., Suzhou, China	ETC811
Professional sequencing company	General Biology (Anhui) Co., Ltd., Anhui, China
Pyridine	Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China	P111516
Shaker	Taicang Qiangle Experimental Equipment Co., Ltd.,Taicang, China	HYL-C
Silica gel GF254 thin layer plate	Yantai Huayang New Material Co., Ltd., Yantai, China	HPT-HSGF5025023
Sodium chloride (NaCl)	Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China	S805275
Sodium citrate	Sinopharm Chemical Reagent Co., Ltd., Shanghai, China	C39197100001
Soluble starch	Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China	S817547
Thermostat water bath	Shanghai Heheng Instrument Equipment Co., Ltd., Shanghai, China	DK-8D
Tryptone	Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China	T139519
Ultra GelRed	Nanjing Vazyme Biotechnology Co., Ltd., Nanjing,China	GR501-01
Ultra pure water instrument	Merck KGaA Co., Ltd., Germany	Milli Direct-Q8
Ultrasonic cleaner	Jiangsu Huaguan Electric Appliance Group Co., Ltd., Jiangsu, China	SB-100DT
Vernier caliper	Sanfeng Company, Japan	N20P
Yeast extract powder	Vicbio Biotechnology Co., Ltd., Beijing, China	LP0021