-
摘要:
目的 评估普通拟杆菌(Bacteroides vulgatus)Bv46的生物特性和益生潜能。 方法 模拟胃肠道的酸性和胆盐环境,评估普通拟杆菌Bv46耐逆性特点;通过溶血、药敏和明胶酶定性实验评估其安全性;基于其自聚集、疏水性和对HT-29细胞的黏附情况评估其黏附能力;利用琼脂扩散、色谱和吸光度测定,评估Bv46对致病菌的拮抗、产酸和抗氧化功能。 结果 分离自健康人群的普通拟杆菌Bv46在模拟胃液和0.3%胆盐中的存活率分别为59.55%和63.76%;无溶血现象和明胶酶活性,除对氨苄西林和青霉素G耐药外,对其他所检测抗生素均敏感;Bv46具有中等自聚集能力、高疏水性和对HT-29细胞的中等黏附性;对致病性大肠埃希菌和鼠伤寒沙门菌的生长有抑制作用;此外,Bv46可以产生短链脂肪酸,并且具有抗氧化作用。 结论 普通拟杆菌Bv46是潜在的益生菌。 Abstract:Objective To evaluate probiotic potential of Bacteroides vulgatus Bv46. Methods The Bv46 strain was tested on their tolerance to artificial gastric juices and bile salts. The Bv46 was assessed the hemolytic activity, antimicrobial susceptibility and gelatinase activity. Moreover, the probiotic properties of Bv46 were evaluated about auto-aggregation, hydrophobicity, adhesion to HT-29 cells, antimicrobial activity against pathogens, acid production and antioxidant properties. Results It was 59.55% and 63.76% to the survival rates of Bv46 isolated from healthy people in simulated gastric juice and 0.3% bile salts respectively. Bv46 showed no haemolytic and gelatinase activities, and were only resistant to ampicillin and penicillin G. Bv46 showed medium auto-aggregation ability, but high hydrophobicity and medium adhesion to HT-29 cells. Moreover, Bv46 inhibited the growth of pathogenic Escherichia coli and Salmonella typhimurium. Furthermore, Bv46 produced short-chain fatty acids, and had antioxidant effects. Conclusion The Bv46 is a potential probiotic. -
Key words:
- Bacteroides vulgatus /
- Adhesion /
- Probiotic /
- Short chain fatty acids
-
图 1 普通拟杆菌Bv46溶血和明胶酶活性检测结果
注:A. 普通拟杆菌Bv46溶血实验阴性;B. 普通拟杆菌Bv46明胶酶实验阴性;C. 金黄色葡萄球菌(ATCC 25923)作为明胶实验阳性对照[9]
Figure 1. Haemolytic and gelatinase activity of Bacteroides vulgatus Bv46
表 1 普通拟杆菌Bv46对酸和0.3%胆盐耐受性
Table 1. Acid and 0.3% bile salt resistance of Bacteroides vulgatus Bv46
菌株名称 酸耐受性a
(存活率,%)0.3%胆盐耐受性
(存活率,%)普通拟杆菌Bv46 59.55±0.18 63.76±0.85 注:a. pH值3.0的含有3 g/L胃蛋白酶的BHI液体培养基 表 2 用 E-test试纸条检测普通拟杆菌Bv46的抗生素敏感性
Table 2. Antibiotic susceptibility test of Bacteroides vulgatus Bv46 by E-test strips
抗生素名称 普通拟杆菌Bv46 氨苄西林 耐药 青霉素G 耐药 阿莫西林–克拉维酸 敏感 头孢曲松 敏感 美罗培南 敏感 四环素 敏感 克林霉素 敏感 氯霉素 敏感 甲硝唑 敏感 表 3 普通拟杆菌Bv46自聚集、疏水性和DPPH自由基清除率结果
Table 3. The self-aggregation, hydrophobicity and DPPH free radical scavenging rate of Bacteroides vulgatus Bv46
菌株名称 自聚集
(%)疏水性
(%)DPPH自由基
清除率(%)普通拟杆菌Bv46 38.53±1.32 69.27±1.56 81.43±2.01 植物乳杆菌HT121 73.32±1.45 64.70±1.78 98.87±1.64 表 4 普通拟杆菌Bv46对致病菌的抑制作用
Table 4. Antimicrobial activity against pathogens of Bacteroides vulgatus Bv46
致病菌名称 大肠埃希菌
(CICC 24186)大肠埃希菌
(ATCC 43895)鼠伤寒沙门菌
(ATCC 14028)金黄色葡萄球菌
(ATCC 25923)李斯特菌
( ATCCBAA-697)抑菌情况 ++ ++ ++ − − 注:符号与直径的对应关系:−. 无抑菌圈;+. 2~6 mm抑菌圈;++. 7~11 mm抑菌圈 表 5 普通拟杆菌Bv46培养上清液的pH监测及短链脂肪酸含量测定
Table 5. pH value and short chain fatty acids in the culture supernatents of Bacteroides vulgatus Bv46
上清液pH监测 上清中短链脂肪酸含量测定(μg/mL) 0 h pH 24h pH 乙酸 丙酸 异丁酸 丁酸 7.40±0.03 5.88±0.09 1486.21 173.75 6.07 0.95 -
[1] Wexler AG, Goodman AL. An insider's perspective: Bacteroides as a window into the microbiome[J]. Nat Microbiol, 2017, 2(5): 17026. DOI: 10.1038/nmicrobiol.2017.26. [2] Waidmann M, Bechtold O, Frick JS, et al. Bacteroides vulgatus protects against escherichia coli-induced colitis in gnotobiotic interleukin-2-deficient mice[J]. Gastroenterology, 2003, 125(1): 162–177. DOI: 10.1016/S0016−5085(03)00672−3. [3] Li SJ, Wang C, Zhang CC, et al. Evaluation of the effects of different Bacteroides vulgatus strains against DSS-induced colitis[J]. J Immunol Res, 2021, 2021: 9117805. DOI: 10.1155/2021/9117805. [4] Santacroce L, Charitos IA, Bottalico L. A successful history: probiotics and their potential as antimicrobials[J]. Expert Rev Anti Infect Ther, 2019, 17(8): 635–645. DOI: 10.1080/14787210.2019.1645597. [5] Chua JCL, Hale JDF, Silcock P, et al. Bacterial survival and adhesion for formulating new oral probiotic foods[J]. Crit Rev Food Sci, 2020, 60(17): 2926–2937. DOI: 10.1080/10408398.2019.1669528. [6] Angmo K, Kumari A, Savitri N, et al. Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh[J]. Lwt-Food Sci Technol, 2016, 66: 428–435. DOI: 10.1016/j.lwt.2015.10.057. [7] Humphries R, Bobenchik AM, Hindler JA, et al. Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing, M100, 31st Edition[J]. J Clin Microbiol, 2021, 59(12): JCM0021321. DOI: 10.1128/JCM.00213−21. [8] Kondrotiene K, Lauciene L, Andruleviciute V, et al. Safety assessment and preliminary in vitro evaluation of probiotic potential of Lactococcus lactis strains naturally present in raw and fermented milk[J]. Curr Microbiol, 2020, 77(10): 3013–3023. DOI: 10.1007/s00284−020−02119−8. [9] Ribeiro SC, Coelho MC, Todorov SD, et al. Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese[J]. J Appl Microbiol, 2014, 116(3): 573–585. DOI: 10.1111/jam.12388. [10] del Re B, Sgorbati B, Miglioli M, et al. Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum[J]. Lett Appl Microbiol, 2000, 31(6): 438–442. DOI: 10.1046/j.1365−2672.2000.00845. [11] Kos B, Susković J, Vuković S, et al. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92[J]. J Appl Microbiol, 2003, 94(6): 981–987. DOI: 10.1046/j.1365−2672.2003.01915.x. [12] Li XP, Xiao YC, Song LQ, et al. Effect of Lactobacillus plantarum HT121 on serum lipid profile, gut microbiota, and liver transcriptome and metabolomics in a high-cholesterol diet–induced hypercholesterolemia rat model[J]. Nutrition, 2020, 79-80: 110966. DOI: 10.1016/j.nut.2020.110966. [13] Sui Y, Liu JT, Liu YX, et al. In vitro probiotic characterization of Lactobacillus strains from fermented tangerine vinegar and their cholesterol degradation activity[J]. Food Biosci, 2021, 39: 100843. DOI: 10.1016/j.fbio.2020.100843. [14] Liu LY, Zhang L, Zhou HJ, et al. Antimicrobial resistance and molecular characterization of Citrobacter spp. causing extraintestinal infections[J]. Front Cell Infect Microbiol, 2021, 11: 737636. DOI: 10.3389/fcimb.2021.737636. [15] Abe Sato ST, Marques JM, da Luz de freitas A, et al. Isolation and genetic identification of endophytic lactic acid bacteria from the Amazonian açai fruits: probiotics features of selected strains and their potential to inhibit pathogens[J]. Front Microbiol, 2021, 11: 610524. DOI: 10.3389/fmicb.2020.610524. [16] Patrignani F, Parolin C, D'alessandro M, et al. Evaluation of the fate of Lactobacillus crispatus BC4, carried in Squacquerone cheese, throughout the simulator of the human intestinal microbial ecosystem (SHIME)[J]. Food Res Int, 2020, 137: 109580. DOI: 10.1016/j.foodres.2020.109580. [17] Bian X, Evivie SE, Muhammad Z, et al. In vitro assessment of the antimicrobial potentials of Lactobacillus helveticus strains isolated from traditional cheese in Sinkiang China against food-borne pathogens[J]. Food Funct, 2016, 7(2): 789–797. DOI: 10.1039/c5fo01041a. [18] Shivangi S, Devi PB, Ragul K, et al. Probiotic potential of Bacillus strains isolated from an acidic fermented food Idli[J]. Probiotics Antimicrob Proteins, 2020, 12(4): 1502–1513. DOI: 10.1007/s12602−020−09650. [19] Botes M, Loos B, Van Reenen CA, et al. Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments[J]. Arch Microbiol, 2008, 190(5): 573–584. DOI: 10.1007/s00203−008−0408−0. [20] Duary RK, Rajput YS, Batish VK, et al. Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells[J]. Indian J Med Res, 2011, 134(5): 664–671. DOI: 10.4103/0971−5916.90992. [21] Monteagudo-Mera A, Rastall RA, Gibson GR, et al. Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health[J]. Appl Microbiol Biotechnol, 2019, 103(16): 6463–6472. DOI: 10.1007/s00253−019−09978−7. [22] Vinolo MAR, Rodrigues HG, Nachbar RT, et al. Regulation of inflammation by short chain fatty acids[J]. Nutrients, 2011, 3(10): 858–876. DOI: 10.3390/nu3100858. [23] De Almeida Júnior WLG, Ferrari ÍDS, De Souza JV, et al. Characterization and evaluation of lactic acid bacteria isolated from goat milk[J]. Food Control, 2015, 53: 96–103. DOI: 10.1016/j.foodcont.2015.01.013. [24] Ebrahimi M, Sadeghi A, Rahimi D, et al. Postbiotic and anti-aflatoxigenic capabilities of Lactobacillus kunkeei as the potential probiotic LAB isolated from the natural honey[J]. Probiotics Antimicro, 2021, 13(2): 343–355. DOI: 10.1007/s12602−020−09697. [25] Zhang L, Cui YJ, Yang YY, et al. The virulence factor GroEL promotes gelatinase secretion from cells in the osteoblast lineage: implication for direct crosstalk between bacteria and adult cells[J]. Arch Oral Biol, 2021, 122: 104991. DOI: 10.1016/j.archoralbio.2020.104991. [26] Sóki J, Wybo I, Hajdú E, et al. A Europe-wide assessment of antibiotic resistance rates in Bacteroides and Parabacteroides isolates from intestinal microbiota of healthy subjects[J]. Anaerobe, 2020, 62: 102182. DOI: 10.1016/j.anaerobe.2020.102182. [27] Arokiyaraj S, Hairul Islam VI, Bharanidharan R, et al. Antibacterial, anti-inflammatory and probiotic potential of Enterococcus hirae isolated from the rumen of Bos primigenius[J]. World J Microbiol Biotechnol, 2014, 30(7): 2111–2118. DOI: 10.1007/s11274−014−1625−0. [28] Han Q, Kong BH, Chen Q, et al. In vitro comparison of probiotic properties of lactic acid bacteria isolated from Harbin dry sausages and selected probiotics[J]. J Func Foods, 2017, 32: 391–400. DOI: 10.1016/j.jff.2017.03.020. [29] Soltan-Dallal M, Mojarrad M, Baghbani F, et al. Effects of probiotic Lactobacillus acidophilus and Lactobacillus casei on colorectal tumor cells activity (CaCo-2)[J]. Arch Iran Med, 2015, 18(3): 167–172. [30] Ragul K, Syiem I, Sundar K, et al. Characterization of probiotic potential of Bacillus species isolated from a traditional brine pickle[J]. J Food Sci Technol, 2017, 54(13): 4473–4483. DOI: 10.1007/s13197−017−2928−6. [31] Hojjati M, Behabahani BA, Falah F. Aggregation, adherence, anti-adhesion and antagonistic activity properties relating to surface charge of probiotic Lactobacillus brevis gp104 against Staphylococcus aureus[J]. Microb Pathog, 2020, 147: 104420. DOI: 10.1016/j.micpath.2020.104420. [32] Caggia C, De Angelis M, Pitino I, et al. Probiotic features of Lactobacillus strains isolated from ragusano and pecorino siciliano cheeses[J]. Food Microbiol, 2015, 50: 109–117. DOI: 10.1016/j.fm.2015.03.010. [33] Tejero-Sariñena S, Barlow J, Costabile A, et al. In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: evidence for the effects of organic acids[J]. Anaerobe, 2012, 18(5): 530–538. DOI: 10.1016/j.anaerobe.2012.08.004. [34] Hong L, Kim WS, Lee SM, et al. Pullulan nanoparticles as prebiotics enhance the antibacterial properties of Lactobacillus plantarum through the induction of mild stress in probiotics[J]. Front Microbiol, 2019, 10: 142. DOI: 10.3389/fmicb.2019.00142. [35] Jang HJ, Lee NK, Paik HD. Lactobacillus brevis Probiotic characterization of KU15153 showing antimicrobial and antioxidant effect isolated from kimchi[J]. Food Sci Biotechnol, 2019, 28(5): 1521–1528. DOI: 10.1007/s10068−019−00576. [36] Fukuda S, Toh H, Hase K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate[J]. Nature, 2011, 469(7331): 543–547. DOI: 10.1038/nature09646. [37] Arpaia N, Campbell C, Fan XY, et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation[J]. Nature, 2013, 504(7480): 451–455. DOI: 10.1038/nature12726. [38] Furusawa Y, Obata Y, Fukuda S, et al. Erratum: Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells[J]. Nature, 2014, 506(7487): 254. DOI: 10.1038/nature13041. -