Isolation， Identification， and Whole-genome Sequencing Analysis of Murine-Derived Limosilactobacillus reuteri

doi:10.13560/j.cnki.biotech.bull.1985.2025-0964

Abstract

Abstract:

Objective Limosilactobacillus reuteri is a facultative anaerobic lactic acid bacterium commonly found in the intestines of vertebrates and mammals. This bacterium possesses various probiotic functions, such as cholesterol reduction, inhibition of pathogenic bacteria growth, and enhancement of immune function, making the isolation of strains highly applicable to the food fermentation industry crucial. Method In this study, MRS-CaCO₃ medium was used to isolate and cultivate L. reuteri from mouse feces. Preliminary identification was conducted through morphological observation, Gram staining, and 16S rRNA sequence analysis. The identified strains were subjected to growth performance, stress resistance, and antibacterial efficacy tests. Whole-genome sequencing and annotation were performed to explore the probiotic mechanisms and safety of the strain at the genetic level. Result The isolated L. reuteri GF304 had no hemolytic activity and demonstrated promising growth performance. It showed strong tolerances to acid and bile salts, with high survival rates in artificial gastrointestinal fluids. Additionally, it effectively inhibited the growth of Escherichia coli and Staphylococcus aureus, indicating its probiotic properties. Whole-genome sequencing analysis revealed that the genome of L. reuteri GF304 contained no virulence or antibiotic resistance genes. Instead, it harbored stress resistance and probiotic genes related to heat shock tolerance, cold shock tolerance, acid resistance, bile salt tolerance, adhesion, antioxidant capacity, and organic acid synthesis. The most prevalent CAZy classifications for strain GF304 were GH and GT. Secondary metabolite analysis revealed that strain GF304 possesses Type Ⅲ Polyketide Synthase biosynthetic gene clusters and RiPP biosynthetic gene clusters. Conclusion Based on its secure genomic profile (absence of virulence and antibiotic resistance genes) and the abundance of probiotic-associated gene clusters, combined with its excellent growth, stress tolerance, and antibacterial properties, L. reuteri GF304 demonstrates significant potential for development as a probiotic preparation.

Key words: Limosilactobacillus reuteri, isolation and identification, probiotic properties, whole-genome sequencing

GAO Fei, ZHANG Yu-xi, MU Di, CHEN Zheng, CHEN Hong-yan. Isolation， Identification， and Whole-genome Sequencing Analysis of Murine-Derived Limosilactobacillus reuteri[J]. Biotechnology Bulletin, 2026, 42(7): 1-13.

Figures/Tables 10

References 39

[1]	Afrc RF. Probiotics in man and animals [J]. J Appl Bacteriol, 1989, 66(5): 365-378.
[2]	Lata P, Savitri. Probiotics and human health [J]. Res J Biotech, 2023, 18(7): 173-180.
[3]	Cristofori F, Dargenio VN, Dargenio C, et al. Anti-inflammatory and immunomodulatory effects of probiotics in gut inflammation: a door to the body [J]. Front Immunol, 2021, 12: 578386.
[4]	程雨, 谢坤, 姜艳平, 等. 兔源罗伊乳杆菌的分离鉴定及益生效果评价 [J]. 中国兽医学报, 2024, 44(10): 2136-2144, 2293.
	Cheng Y, Xie K, Jiang YP, et al. Isolation and identification of rabbit-derived Lactobacillus reuteri and evaluation of its probiotic function [J]. Chin J Vet Sci, 2024, 44(10): 2136-2144, 2293.
[5]	Falcinelli S, Rodiles A, Hatef A, et al. Influence of probiotics administration on gut microbiota core: a review on the effects on appetite control, glucose, and lipid metabolism [J]. J Clin Gastroenterol, 2018, 52(): S50-S56.
[6]	徐乐, 陈诗宇, 王上, 等. 茶花鸡源罗伊特氏黏液乳杆菌CHF7-2的益生特性及全基因组测序分析 [J]. 微生物学报, 2025, 65(3): 1197-1218.
	Xu L, Chen SY, Wang S, et al. Probiotic characterization and whole genome sequencing of Limosilactobacillus reuteri CHF7-2 from Chahua chicken [J]. Acta Microbiol Sin, 2025, 65(3): 1197-1218.
[7]	国家卫生健康委员会. 解读关于《可用于食品的菌种名单》和《可用于婴幼儿食品的菌种名单》更新的公告(2022年第4号) [J]. 饮料工业, 2022, 25(5): 3-4.
	National Health Commission of the People’s Republic of China. Interpretation of the announcement on updating the list of bacteria used in food and the list of bacteria used in infant food (No.4, 2022) [J]. Beverage Ind, 2022, 25(5): 3-4.
[8]	Abuqwider J, Altamimi M, Mauriello G. Limosilactobacillus reuteri in health and disease [J]. Microorganisms, 2022, 10(3): 522.
[9]	张晓莉, 杨勇, 王光强, 等. 基于全基因组和比较基因组分析罗伊氏粘液乳杆菌的益生特性 [J]. 中国食品学报, 2025, 25(8): 8-20.
	Zhang XL, Yang Y, Wang GQ, et al. Probiotic properties of Limosilactobacillus reuteri based on whole-genome and comparative genome analysis [J]. J Chin Inst Food Sci Technol, 2025, 25(8): 8-20.
[10]	张媛媛, 赵梦迪, 李悦垚, 等. 犬源罗伊氏乳杆菌LRA7对比格犬短链脂肪酸含量、肠道菌群及代谢组的影响 [J]. 动物营养学报, 2025, 37(4): 2648-2660.
	Zhang YY, Zhao MD, Li YY, et al. Effects of canine-derived Lactobacillus reuteri LRA7 on short-chain fatty acid contents, intestinal flora and metabolome in beagle dogs [J]. Chin J Anim Nutr, 2025, 37(4): 2648-2660.
[11]	刘春艳. 猪源罗伊氏乳杆菌对断奶仔猪肠道屏障功能和细胞外基质的影响 [D]. 南宁: 广西大学, 2023.
	Liu CY. Effects of Lactobacillus reuteri from pigs on intestinal barrier function and extracellular matrix of weaned piglets [D]. Nanning: Guangxi University, 2023.
[12]	杨巍巍, 彭子维, 谢昊炅, 等. 罗伊氏黏液乳杆菌的分离鉴定及对瘤胃体外发酵参数的影响 [J]. 饲料工业, 2025, 46(15): 143-150.
	Yang WW, Peng ZW, Xie HJ, et al. Isolation and identification of Limosilactobacillus reuteri and its effects on rumen fermentation parameters in vitro [J]. Feed Ind, 2025, 46(15): 143-150.
[13]	王志刚, 徐伟慧, 张迎, 等. 一种谷氨酸浓缩母液的常温脱盐以及资源化利用方法及其装置: CN115872482B [P]. 2023-08-11.
	Wang ZG, Xu WH, Zhang Y, et al. A method and apparatus for room temperature desalination and resource utilization of glutamic acid concentrate mother liquor: CN115872482B [P]. 2023-08-11.
[14]	农业农村部办公厅关于印发《直接饲喂微生物和发酵制品生产菌株鉴定及其安全性评价指南》的通知(农办牧[2021]43号) [J]. 中华人民共和国农业农村部公报, 2021(11): 97-111.
	Circular of the general office of the ministry of agriculture and rural affairs on printing and distributing the guidelines on identification and safety evaluation of direct-fed microbials and fermented-food-derived bacterial strains [J]. Gaz Minist Agric Rural Aff People’s Repub China, 2021(11): 97-111.
[15]	Zhou WQ, Gao S, Zheng J, et al. Identification of an Aerococcus urinaeequi isolate by whole genome sequencing and average nucleotide identity analysis [J]. J Glob Antimicrob Resist, 2022, 29: 353-359.
[16]	李兰溪. 植物乳杆菌LPJZ-658对成犬血液指标、消化吸收及肠道菌群的影响 [D]. 长春: 吉林农业大学, 2024.
	Li LX. Effects of Lactiplantibacillus plantarum LPJZ-658 on blood indexes, digestion and metabolism, and intestinal microbiota in dogs [D].Changchun: Jilin Agricultural University, 2024.
[17]	仇聪蕊, 舒祥力, 张云飞, 等. 一株猪源罗伊氏黏液乳杆菌的分离鉴定及其益生特性研究 [J]. 中国兽医科学, 2025, 55(9): 1216-1226.
	Qiu CR, Shu XL, Zhang YF, et al. Isolation, identification and probiotic characteristics analysis of Limosilactobacillus reuteri from porcine [J]. Chin Vet Sci, 2025, 55(9): 1216-1226.
[18]	林龙镇, 邹卫玲, 李安章, 等. 产酸、耐酸乳酸菌的分离鉴定及益生特性 [J]. 华南农业大学学报, 2018, 39(2): 95-102.
	Lin LZ, Zou WL, Li AZ, et al. Isolation, identification and probiotic characteristics of acidproducing and acid-resistant Lactobacillus strains [J]. J South China Agric Univ, 2018, 39(2): 95-102.
[19]	Wu JJ, Zhou QY, Liu DM, et al. Evaluation of the safety and probiotic properties of Lactobacillus gasseri LGZ1029 based on whole genome analysis [J]. LWT, 2023, 184: 114759.
[20]	Yogeswara IBA, Maneerat S, Haltrich D. Glutamate decarboxylase from lactic acid bacteria-a key enzyme in GABA synthesis [J]. Microorganisms, 2020, 8(12): 1923.
[21]	Yang H, He MW, Wu CD. Cross protection of lactic acid bacteria during environmental stresses: Stress responses and underlying mechanisms [J]. LWT, 2021, 144: 111203.
[22]	Chintakovid N, Singkhamanan K, Yaikhan T, et al. Probiogenomic analysis of Lactiplantibacillus plantarum SPS109: a potential GABA-producing and cholesterol-lowering probiotic strain [J]. Heliyon, 2024, 10(13): e33823.
[23]	Kompramool S, Singkhamanan K, Pomwised R, et al. Genomic insights into Pediococcus pentosaceus ENM104: a probiotic with potential antimicrobial and cholesterol-reducing properties [J]. Antibiotics, 2024, 13(9): 813.
[24]	Bäumler AJ, Sperandio V. Interactions between the microbiota and pathogenic bacteria in the gut [J]. Nature, 2016, 535(7610): 85-93.
[25]	李彩玉, 刘东慧, 权衡, 等. 益生菌细菌素防御病毒和细菌感染的机制研究 [J]. 中国兽医科学, 2024, 54(6): 808-815.
	Li CY, Liu DH, Quan H, et al. Mechanism study of probiotics bacteriocins defense against viral and bacterial infections [J]. Chin Vet Sci, 2024, 54(6): 808-815.
[26]	Ginsburg I. Role of lipoteichoic acid in infection and inflammation [J]. Lancet Infect Dis, 2002, 2(3): 171-179.
[27]	Averill-bates DA. The antioxidant glutathione [M]//Antioxidants. Amsterdam: Elsevier, 2023: 109-141.
[28]	Li XM, Zhang BX, Yan CX, et al. A fast and specific fluorescent probe for thioredoxin reductase that works via disulphide bond cleavage [J]. Nat Commun, 2019, 10: 2745.
[29]	Suwannasom N, Kao I, Pruß A, et al. Riboflavin: the health benefits of a forgotten natural vitamin [J]. Int J Mol Sci, 2020, 21(3): 950.
[30]	Martin-Gallausiaux C, Marinelli L, Blottière HM, et al. SCFA: mechanisms and functional importance in the gut [J]. Proc Nutr Soc, 2021, 80(1): 37-49.
[31]	Smythe P, Efthimiou G. In silico genomic and metabolic atlas of Limosilactobacillus reuteri DSM 20016: an insight into human health [J]. Microorganisms, 2022, 10(7): 1341.
[32]	Douillard FP, Ribbera A, Kant R, et al. Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG [J]. PLoS Genet, 2013, 9(8): e1003683.
[33]	苏世友, 滕超, 张维, 等. 细菌Ⅲ型聚酮合酶研究进展 [J]. 中国农业科技导报, 2013, 15(6): 119-129.
	Su SY, Teng C, Zhang W, et al. Progress on type Ⅲ polyketide synthase from bacteria [J]. J Agric Sci Technol, 2013, 15(6): 119-129.
[34]	Benjdia A, Balty C, Berteau O. Radical SAM enzymes in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) [J]. Front Chem, 2017, 5: 87.
[35]	汪金秀, 张琪, 丁伟, 等. 核糖体肽生物合成中的典型翻译后修饰研究 [J]. 生物技术通报, 2020, 36(10): 215-225.
	Wang JX, Zhang Q, Ding W, et al. Classic post-translational modification in ribosomally synthesized and post-translationally modified peptides biosynthesis [J]. Biotechnol Bull, 2020, 36(10): 215-225.
[36]	Bonelli RR, Schneider T, Sahl HG, et al. Insights into in vivo activities of lantibiotics from gallidermin and epidermin mode-of-action studies [J]. Antimicrob Agents Chemother, 2006, 50(4): 1449-1457.
[37]	柳少燕, 陈捷胤, 李蕾, 等. 拮抗菌与病原菌碳水化合物酶类比较分析 [J]. 基因组学与应用生物学, 2013, 32(1): 97-104.
	Liu SY, Chen JY, Li L, et al. Comparative analysis of the carbohydrate-active enzymes between antagonistic microorganism and plant pathogen [J]. Genom Appl Biol, 2013, 32(1): 97-104.
[38]	Oehme DP, Shafee T, Downton MT, et al. Differences in protein structural regions that impact functional specificity in GT2 family β-glucan synthases [J]. PLoS One, 2019, 14(10): e0224442.
[39]	Inglin RC, Meile L, Stevens MJA. Clustering of pan- and core-genome of Lactobacillus provides novel evolutionary insights for differentiation [J]. BMC Genom, 2018, 19(1): 284.

菌株	人工胃液			人工肠液
	活菌数（×10⁶ CFU·mL^-1）		存活率（%）	活菌数（×10⁵ CFU·mL^-1）			存活率/%
	0 h	2 h	2 h/0 h	0 h	2 h	4 h	2 h/0 h	4 h/0 h
GF304	3.26±0.16	2.73±0.43	83.38±0.09	4.75±0.67	4.00±0.85	2.72±0.70	83.66±0.07	57.80±0.14

菌株	人工胃液			人工肠液
	活菌数（×10⁶ CFU·mL^-1）		存活率（%）	活菌数（×10⁵ CFU·mL^-1）			存活率/%
	0 h	2 h	2 h/0 h	0 h	2 h	4 h	2 h/0 h	4 h/0 h
GF304	3.26±0.16	2.73±0.43	83.38±0.09	4.75±0.67	4.00±0.85	2.72±0.70	83.66±0.07	57.80±0.14

Gene name	Gene product	Gene ID
Temperature
groES	Co-chaperone GroES	gene0399
groEL	Chaperonin GroEL	gene0400
smpB	SsrA-binding protein SmpB	gene0450
hrcA	Heat-inducible transcriptional repressor HrcA	gene0768
dnaK	Molecular chaperone DnaK	gene0770
dnaJ	Molecular chaperone DnaJ	gene0771
cspA	Cold shock domain-containing protein，cold-shock protein	gene0676/gene1783
pH
atpB	F0F1 ATP synthase subunit A	gene0512
atpE	F0F1 ATP synthase subunit C	gene0513
atpF	F0F1 ATP synthase subunit B	gene0514
atpH	ATP synthase F1 subunit delta	gene0515
atpA	F0F1 ATP synthase subunit alpha	gene0516
atpG	F0F1 ATP synthase subunit gamma	gene0517
atpD	F0F1 ATP synthase subunit beta	gene0518
atpC	F0F1 ATP synthase subunit epsilon	gene0519
-	Alkaline shock response membrane anchor protein AmaP	gene0966
arcA	Arginine deiminase	gene0496
arcC	Carbamate kinase	gene0480
argR	Arginine repressor/ArgR family transcriptional regulator	gene0497/gene1329
argG	Argininosuccinate synthase	gene0802
argH	Argininosuccinate lyase	gene0803
gadB	Glutamate decarboxylase	gene0539
gadC	Gamma-aminobutyrate antiporte/glutamate/gamma-aminobutyrate family transporter YjeM	gene0542/gene0540/gene1458/gene1618
nhaC	Na⁺/H⁺ antiporter NhaC family protein/Na⁺/H⁺ antiporter NhaC	gene0171/gene2094
Bile salt resistance
cbh	Choloylglycine hydrolase	gene0801
ppaC	Manganese-dependent inorganic pyrophosphatase	gene0959
Production of adhesion molecules
ltaS	LTA synthase family protein	gene1935/gene2098
Oxidative resistance
trxA	Thioredoxin	gene0582/gene2088
trxB	Thioredoxin-disulfide reductase	gene0423
gshA	Glutamate--cysteine ligase	gene0072/gene1632
pepN	M1 family metallopeptidase	gene2145
nfrA1	NADPH-dependent oxidoreductase	gene1863
Riboflavin biosynthesis
ribD	Bifunctional diaminohydroxyphosphoribosylaminopyrimidine Deaminase/5-amino-6-（5-phosphoribosylamino）uracil reductase RibD	gene0993
ribE	riboflavin synthase	gene0994
ribBA	Bifunctional 3，4-dihydroxy-2-butanone-4-phosphate synthase/GTP cyclohydrolase Ⅱ	gene0995
ribH	6，7-dimethyl-8-ribityllumazine synthase	gene0996
ribF	Riboflavin biosynthesis protein RibF	gene0766
ribT	Reductase	gene0825
Organic acid biosynthesis
ackA	Acetate kinase	gene0604

Gene name	Gene product	Gene ID
Temperature
groES	Co-chaperone GroES	gene0399
groEL	Chaperonin GroEL	gene0400
smpB	SsrA-binding protein SmpB	gene0450
hrcA	Heat-inducible transcriptional repressor HrcA	gene0768
dnaK	Molecular chaperone DnaK	gene0770
dnaJ	Molecular chaperone DnaJ	gene0771
cspA	Cold shock domain-containing protein，cold-shock protein	gene0676/gene1783
pH
atpB	F0F1 ATP synthase subunit A	gene0512
atpE	F0F1 ATP synthase subunit C	gene0513
atpF	F0F1 ATP synthase subunit B	gene0514
atpH	ATP synthase F1 subunit delta	gene0515
atpA	F0F1 ATP synthase subunit alpha	gene0516
atpG	F0F1 ATP synthase subunit gamma	gene0517
atpD	F0F1 ATP synthase subunit beta	gene0518
atpC	F0F1 ATP synthase subunit epsilon	gene0519
-	Alkaline shock response membrane anchor protein AmaP	gene0966
arcA	Arginine deiminase	gene0496
arcC	Carbamate kinase	gene0480
argR	Arginine repressor/ArgR family transcriptional regulator	gene0497/gene1329
argG	Argininosuccinate synthase	gene0802
argH	Argininosuccinate lyase	gene0803
gadB	Glutamate decarboxylase	gene0539
gadC	Gamma-aminobutyrate antiporte/glutamate/gamma-aminobutyrate family transporter YjeM	gene0542/gene0540/gene1458/gene1618
nhaC	Na⁺/H⁺ antiporter NhaC family protein/Na⁺/H⁺ antiporter NhaC	gene0171/gene2094
Bile salt resistance
cbh	Choloylglycine hydrolase	gene0801
ppaC	Manganese-dependent inorganic pyrophosphatase	gene0959
Production of adhesion molecules
ltaS	LTA synthase family protein	gene1935/gene2098
Oxidative resistance
trxA	Thioredoxin	gene0582/gene2088
trxB	Thioredoxin-disulfide reductase	gene0423
gshA	Glutamate--cysteine ligase	gene0072/gene1632
pepN	M1 family metallopeptidase	gene2145
nfrA1	NADPH-dependent oxidoreductase	gene1863
Riboflavin biosynthesis
ribD	Bifunctional diaminohydroxyphosphoribosylaminopyrimidine Deaminase/5-amino-6-（5-phosphoribosylamino）uracil reductase RibD	gene0993
ribE	riboflavin synthase	gene0994
ribBA	Bifunctional 3，4-dihydroxy-2-butanone-4-phosphate synthase/GTP cyclohydrolase Ⅱ	gene0995
ribH	6，7-dimethyl-8-ribityllumazine synthase	gene0996
ribF	Riboflavin biosynthesis protein RibF	gene0766
ribT	Reductase	gene0825
Organic acid biosynthesis
ackA	Acetate kinase	gene0604