基于宏基因组测序对中国北方地区生乳中嗜冷菌的群落结构研究

doi:10.11843/j.issn.0366-6964.2024.08.037

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (8): 3659-3668.doi: 10.11843/j.issn.0366-6964.2024.08.037

基于宏基因组测序对中国北方地区生乳中嗜冷菌的群落结构研究

许文君¹^,²^,³(), 郑楠²^,³, 王加启²^,³, 孟璐²^,³^,*()

1. 青岛农业大学食品科学与工程学院，青岛 266109
2. 中国农业科学院北京畜牧兽医研究所农业农村部奶及奶制品质量安全控制重点实验室，北京 100193
3. 中国农业科学院北京畜牧兽医研究所农业农村部奶产品质量安全风险评估实验室(北京)，北京 100193

收稿日期:2023-11-15 出版日期:2024-08-23 发布日期:2024-08-28
通讯作者: 孟璐 E-mail:xubj2022@163.com;menglu@caas.cn
作者简介:许文君(1996-)，女，山东滨州人，硕士生，主要从事牛奶微生物研究，E-mail: xubj2022@163.com
基金资助:
国家重点研发计划(2022YFD1301003);农产品质量安全风险评估(GJFP20220304);国家奶牛产业技术体系(CARS36);中国农业科学院科技创新工程(ASTIP-IAS12)

Study on the Community Structure of Psychrophilic Bacteria in Raw Milk from North Regions of China based on Metagenomic Sequencing

Wenjun XU¹^,²^,³(), Nan ZHENG²^,³, Jiaqi WANG²^,³, Lu MENG²^,³^,*()

1. College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
2. Key Laboratory of Quality and Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
3. Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs (Beijing), Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2023-11-15 Online:2024-08-23 Published:2024-08-28
Contact: Lu MENG E-mail:xubj2022@163.com;menglu@caas.cn

摘要/Abstract

摘要：

本研究旨在利用宏基因组测序探究中国北方地区生乳中嗜冷菌群落结构的多样性。在冬季从中国内蒙古地区和北京地区的14个牧场共采集14份生乳样品，分离纯化可培养嗜冷菌和提取生乳中微生物总DNA，并利用16S rDNA测序和宏基因组测序结合的方法对生乳中嗜冷菌进行分析。结果发现，北京地区和内蒙古地区生乳中嗜冷菌的群落结构无显著性差异。两个地区生乳中优势嗜冷菌属均是假单胞杆菌属、寡养单胞菌属和短波单胞杆菌属。全局和概述图谱、氨基酸代谢和碳水化合物代谢是最主要的功能代谢途径，且嗜冷菌中含有多种抗生素抗性基因。综上，本研究利用传统培养和宏基因组测序准确测定了生乳中嗜冷菌群落的物种和功能信息，有助于了解生乳中嗜冷菌的生命活动及为嗜冷菌风险评估提供基础信息，促进中国北京及内蒙古牧场开展防控。

关键词: 生乳, 嗜冷菌, 宏基因组测序, 地区, 结构

Abstract:

The aim of this study was to investigate the community structure diversity of psychrophilic bacteria in raw milk in northern China by metagenomic sequencing. A total of 14 raw milk samples were collected from 14 pastures in Inner Mongolia and Beijing, China in winter. The culturable psychrophilic bacteria were isolated and purified, total microbial DNA in raw milk was extracted as well. Moreover, the psychrophilic bacteria in raw milk were analyzed by 16S rDNA sequencing and metagenomic sequencing, respectively. The results showed that no significant difference was found in community structure of psychrophilic bacteria in raw milk between Beijing and Inner Mongolia. Pseudomonas, Stenotrophomonas and Brevundimonas were the dominant psychrophiles bacteria in raw milk from the two regions. The global and overview profiles, amino acid metabolism and carbohydrate metabolism were the most important functional metabolic pathways, and psychrophilic bacteria contained a variety of antibiotic resistance genes. In conclusion, this study used traditional culture and metagenomic sequencing to accurately determine the species and functional information of the psychrophilic community in raw milk, which is helpful to understand the life activities of psychrophilic bacteria in raw milk and provide basic information for risk assessment of psychrophilic bacteria. Furthermore, it lays down foundational knowledge pivotal for the effective prevention and management of pasture-related issues in Beijing and Inner Mongolia, China.

Key words: raw milk, psychrophilic bacteria, metagenomic sequencing, region, structure

中图分类号:

TS252.1

许文君, 郑楠, 王加启, 孟璐. 基于宏基因组测序对中国北方地区生乳中嗜冷菌的群落结构研究[J]. 畜牧兽医学报, 2024, 55(8): 3659-3668.

Wenjun XU, Nan ZHENG, Jiaqi WANG, Lu MENG. Study on the Community Structure of Psychrophilic Bacteria in Raw Milk from North Regions of China based on Metagenomic Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3659-3668.

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参考文献 30

1	YUAN L , SADIQ F A , BURMOLLE M , et al. Insights into psychrotrophic bacteria in raw milk: a review[J]. J Food Prot, 2019, 82 (7): 1148- 1159. doi: 10.4315/0362-028X.JFP-19-032
2	DOGAN B , BOOR K J . Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants[J]. Appl Environ Microbiol, 2003, 69 (1): 130- 138. doi: 10.1128/AEM.69.1.130-138.2003
3	JIN S S , WANG Y Z , ZHAO X H . Cold-adaptive mechanism of psychrophilic bacteria in food and its application[J]. Microb Pathog, 2022, 169, 105652. doi: 10.1016/j.micpath.2022.105652
4	QUIGLEY L , O'SULLIVAN O , STANTON C , et al. The complex microbiota of raw milk[J]. FEMS Microbiol Rev, 2013, 37 (5): 664- 698. doi: 10.1111/1574-6976.12030
5	KIM I S , HUR Y K , KIM E J , et al. Comparative analysis of the microbial communities in raw milk produced in different regions of Korea[J]. Asian-Australas J Anim Sci, 2017, 30 (11): 1643- 1650. doi: 10.5713/ajas.17.0689
6	YUAN L , SADIQ F A , LIU T J , et al. Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes[J]. J Zhejiang Univ Sci B, 2018, 19 (8): 630- 642. doi: 10.1631/jzus.B1700352
7	SAMARŽIJA D , ZAMBERLIN Š , POGAČIĆ T . Psychrotrophic bacteria and milk and dairy products quality[J]. Mljekarstvo, 2012, 62 (2): 77- 95.
8	MARTIN N H , ORRES-FRENZEL P , WIEDMANN M . Invited review: Controlling dairy product spoilage to reduce food loss and waste[J]. J Dairy Sci, 2021, 104 (2): 1251- 1261. doi: 10.3168/jds.2020-19130
9	OUAMBA A J K , GAGNON M , LAPOINTE G , et al. Graduate Student Literature Review: Farm management practices: Potential microbial sources that determine the microbiota of raw bovine milk[J]. J Dairy Sci, 2022, 105 (9): 7276- 7287. doi: 10.3168/jds.2021-21758
10	YAP M , GLEESON D , O'TOOLE P W , et al. Seasonality and geography have a greater influence than the use of chlorine-based cleaning agents on the microbiota of bulk tank raw milk[J]. Appl Environ Microbiol, 2021, 87 (22): e01081- 21.
11	GUO X C , YU Z J , ZHAO F Y , et al. Both sampling seasonality and geographic origin contribute significantly to variations in raw milk microbiota, but sampling seasonality is the more determining factor[J]. J Dairy Sci, 2021, 104 (10): 10609- 10627. doi: 10.3168/jds.2021-20480
12	LIANG L J , WANG P , ZHAO X M , et al. Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China[J]. J Dairy Sci, 2022, 105 (7): 5669- 5684. doi: 10.3168/jds.2021-21445
13	FRANZOSA E A , HSU T , SIROTA-MADI A , et al. Sequencing and beyond: integrating molecular 'omics' for microbial community profiling[J]. Nat Rev Microbiol, 2015, 13 (6): 360- 372. doi: 10.1038/nrmicro3451
14	WANG J C , ZHENG Y , XI X X , et al. Application of PacBio Single Molecule Real-Time (SMRT) sequencing in bacterial source tracking analysis during milk powder production[J]. Food Control, 2018, 93, 226- 234. doi: 10.1016/j.foodcont.2018.05.044
15	CHEN S F , ZHOU Y Q , CHEN Y R , et al. fastp: an ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics, 2018, 34 (17): i884- i890. doi: 10.1093/bioinformatics/bty560
16	LI H , DURBIN R . Fast and accurate short read alignment with Burrows-Wheeler transform[J]. Bioinformatics, 2009, 25 (14): 1754- 1760. doi: 10.1093/bioinformatics/btp324
17	REN Y , YU G , SHI C P , et al. Majorbio Cloud: A one-stop, comprehensive bioinformatic platform for multiomics analyses[J]. iMeta, 2022, 1 (2): e12. doi: 10.1002/imt2.12
18	ZHANG D , LI S Q , PALMER J , et al. The relationship between numbers of Pseudomonas bacteria in milk used to manufacture UHT milk and the effect on product quality[J]. Int Dairy J, 2020, 105, 104687. doi: 10.1016/j.idairyj.2020.104687
19	ZHANG D , PALMER J , TEH K H , et al. Identification and selection of heat-stable protease and lipase-producing psychrotrophic bacteria from fresh and chilled raw milk during up to five days storage[J]. LWT, 2020, 134, 110165. doi: 10.1016/j.lwt.2020.110165
20	YANG X Y , GUO X J , LIU W P , et al. The complex community structures and seasonal variations of psychrotrophic bacteria in raw milk in Heilongjiang Province, China[J]. LWT, 2020, 134, 110218. doi: 10.1016/j.lwt.2020.110218
21	DU B Y , MENG L , LIU H M , et al. Single molecule real-time sequencing and traditional cultivation techniques reveal complex community structures and regional variations of psychrotrophic bacteria in raw milk[J]. Front Microbiol, 2022, 13, 853263. doi: 10.3389/fmicb.2022.853263
22	NARVHUS J A , BÆKKELUND O N , TIDEMANN E M , et al. Isolates of Pseudomonas spp. from cold-stored raw milk show variation in proteolytic and lipolytic properties[J]. Int Dairy J, 2021, 123, 105049. doi: 10.1016/j.idairyj.2021.105049
23	RUEGG P L . A 100-Year Review: Mastitis detection, management, and prevention[J]. J Dairy Sci, 2017, 100 (12): 10381- 10397.
24	DU B Y , MENG L , LIU H M , et al. Impacts of milking and housing environment on milk microbiota[J]. Animals (Basel), 2020, 10 (12): 2339.
25	JIANG Y , YU P , LIU X M , et al. Shifts in diversity and function of bacterial community during manufacture of rushan[J]. J Dairy Sci, 2021, 104 (12): 12375- 12393.
26	胡海燕, 刘慧敏, 孟璐, 等. 宏基因组学在微生物抗生素抗性基因检测中的应用[J]. 微生物学通报, 2019, 46 (11): 3110- 3123.
	HU H Y , LIU H M , MENG L , et al. Application of metagenomics in the detection of microbial antibiotic resistance genes[J]. Microbiology China, 2019, 46 (11): 3110- 3123.
27	TÓTH A G , CSABAI I , KRIKÓ E , et al. Antimicrobial resistance genes in raw milk for human consumption[J]. Sci Rep, 2020, 10 (1): 7464.
28	MENG L , LIU H M , LAN T , et al. Antibiotic resistance patterns of Pseudomonas spp. isolated from raw milk revealed by whole genome sequencing[J]. Front Microbiol, 2020, 11, 1005.
29	LIU J X , ZHU Y T , JAY-RUSSELL M , et al. Reservoirs of antimicrobial resistance genes in retail raw milk[J]. Microbiome, 2020, 8 (1): 99.
30	PATEL K , AMARESAN N . Antimicrobials compounds from extreme environment Rhizosphere organisms for plant growth[J]. Int J Curr Microbiol App Sci, 2014, 3 (7): 651- 664.

引物 Primer	序列 Sequence	参考文献 Reference
27F	AGAGTTTGATCMTGGCTCAG	[14]
1492R	GGTTACCTTGTTACGACTT

属 Genera	种 Species	总数(株) Total number (strains)	北京地区 Beijing	内蒙古地区 Inner Mongolia
Acinetobacter	albensis	1	0	1
	sp. TAC-1	1	0	1
Arthrobacter	alpinus	1	1	0
Carnobacterium	maltaromaticum	1	1	0
Chryseobacterium	balustinum	1	0	1
	indoltheticum	2	1	1
	sp. Sin57	2	2	0
	sp. WW-RP1	1	0	1
	vrystaatense	1	0	1
	yeoncheonense	1	1	0
Flavobacterium	sp. F-340	1	0	1
Glutamicibacter	bergerei	1	0	1
Lactococcus	carnosus	1	1	0
Paeniglutamicibacter	antarcticus	1	0	1
Pseudomonas	azotoformans	1	1	0
	cedrina	1	0	1
	fluorescens	3	1	2
	gessardii	3	2	1
	mandelii	1	0	1
	paralactis	1	1	0
	poae	1	1	0
	rhodesiae	1	0	1
	sp. HW08	2	2	0
Psychrobacter	faecalis	1	1	0
	pulmonis	5	2	3
	sp. S06	2	0	2
	sp. SOD-1301	1	0	1
Serratia	liquefaciens	4	2	2
	grimesii	2	1	1

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基于宏基因组测序对中国北方地区生乳中嗜冷菌的群落结构研究

Study on the Community Structure of Psychrophilic Bacteria in Raw Milk from North Regions of China based on Metagenomic Sequencing

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