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

Published: May 31, 2024

doi:

¹College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University

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 stage of growth and development, B. licheniformis can exist in the form of dormant spores and produce antibacterial substances such as bacitracin, lichenysin, and surfactin. It can also resist nutritional deficiency and extreme external environment⁵. There is no obvious codon preference, and the efficient secretion system determines the heterologous protein secretion of B. licheniformis, which is twice that of Bacillus subtilis⁶. It is often used to produce enzyme preparations such as protease, amylase, and cellulase⁷. Because of its lack of endogenous toxins, it is certified as a food-safe strain and listed on the QPS by EFSA⁸. Therefore, there are several potential uses, including bioactive compound production, which have a wide range of applications in aquaculture, agriculture, food, biomedicine, and pharmaceutical industries. Also, B. licheniformis is an important component of animal intestinal flora, which can promote animals to improve production performance, improve intestinal flora balance, and prevent diseases. The entire genome of B. licheniformis ATCC14580 was analyzed in 2004, and the background information of transcription translation, protein folding, and secretion mechanism has been gradually understood⁹. This genetic information makes it conducive for genetic modification at the molecular level, contributing to facilitating the large-scale production of B. licheniformis.

Bacitracin is a dodecacyclic peptide antibiotic produced by non-ribosomal peptide synthetase by secondary metabolism in B. subtilis and B. licheniformis. Bacitracin is a mixture composed of various components such as bacitracin A, B, and C, where one or two amino acids differ between each component; among these, bacitracin A has the strongest biological activity¹⁰. Bacitracin can inhibit gram-positive bacteria such as Staphylococcus and Micrococcus luteus and some gram-negative bacteria by inhibiting cell wall formation and interacting with membrane-binding proteins¹¹. Meanwhile, bacitracin is safe and stable, not easy to produce drug resistance, and can be compatible with other antibacterial drugs¹². Therefore, bacitracin is used in medical and veterinary practice. In addition, because of its fast elimination rate and low absorption rate, it can also be used as an additive for animal feed¹³.

B. licheniformis can colonize the intestine and improve the gastrointestinal microenvironment. The adhesion and reproduction ability and related physiological functions of Bacillus from different sources in the gastrointestinal tract of different animals are different. Pig-derived B. licheniformis is more conducive to colonization in the intestines of pigs and other livestock. There is a close relationship between the relative abundance of intestinal probiotics and the health status of host¹⁴. Dietary supplementation with B. licheniformis mix in weaned piglets improves the intestinal ecosystem by changing microbiota composition and metabolic activity, and also effects the intestinal mucosa¹⁵. Animal feces can reflect the type and quantity of animal intestinal flora. This protocol describes the isolation and purification of bacitracin-producing Bacillus spp. from healthy pig feces. The feces are derived from Taihu sows that are not fed with compound feed and have excellent production performance in pig farms. The isolates were identified as B. licheniformis based on their morphological characteristics, physicochemical properties, and biochemical identification.

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 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…

Representative Results

In this experiment, 48 strains of Bacillus were isolated from fresh feces of healthy pigs, numbered from 1001 to 1048. Among them, 15 strains had antibacterial activity against M. luteus. From the 15 strains, the titers of bacitracin were measured by high-performance liquid chromatography, as shown in Table 1. Among them, B. licheniformis No. 1026 had the highest bacitracin titer, 456.35 ± 21.75 U/mL, so No. 1026 was selected for subsequent experiments. <p class="jove_…

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study was supported by the National Key Research and Development Program of China (No. 2022YFC2104800), and the Six Talent Peaks Project in Jiangsu Province (No. 2019-NY-058).

Materials

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

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