Microsoft Word tb 苏本宪sy.doc

Size: px

Start display at page:

Download "Microsoft Word tb 苏本宪sy.doc"

征耄扈
7 years ago
Views:

1 微生物学通报 Microbiology China 研究报告 Jan. 20, 2015, 42(1): by Institute of Microbiology, CAS DOI: /j.microbiol.china 乳杆菌代谢产物对化脓性链球菌的抑制作用 * 苏本宪艾静朱德全孟祥晨 ( ) 摘要目的分析乳杆菌代谢产物对化脓性链球菌的抑制作用方法基于双层平板打孔法, 通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用 ; 然后分别采用高效液相色谱法和 4- 氨酰安替比林法检测乳杆菌代谢产物中的有机酸和 H 2 O 2 含量 ; 最后, 检测乳酸乙酸和 H 2 O 2 对化脓性链球菌的最小抑菌浓度 (MIC) 最小杀菌浓度(MBC) 结果对化脓性链球菌的抑菌效果以植物乳杆菌 KLDS 最好, 副干酪乳杆菌 KLDS 次之, 瑞士乳杆菌 KLDS 抑菌效果最差 ; 乳酸和乙酸产量 KLDS1.0667>KLDS > KLDS1.0203;H 2 O 2 产量 KLDS1.0203>KLDS1.0667>KLDS 在抑菌试验中, 乳杆菌的发酵上清液经去除 H 2 O 2 处理后抑菌圈直径都减小 ; 将发酵上清液的 ph 调至 7.0 后均检测不到抑菌圈结果表明, 乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和 H 2 O 2, 其中乳酸是产生抑菌作用的最主要物质乳酸乙酸和 H 2 O 2 对化脓性链球菌的最小抑菌浓度 (MIC) 分别为和 g/l, 对化脓性链球菌的最小杀菌浓度 (MBC) 分别为和 g/l 结论乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用, 主要抑菌物质为有机酸和 H 2 O 2 关键词乳杆菌, 化脓性链球菌, 抑菌, 有机酸,H 2 O 2 Inhibition of Streptococcus pyogenes by lactobacilli metabolites SU Ben-Xian AI Jing ZHU De-Quan MENG Xiang-Chen * (Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin, Heilongjiang , China) Abstract: [Objective] Inhibition of lactobacilli metabolites on Streptococcus pyogenes was analyzed. [Methods] Three lactobacilli strains was grown in MRS broth overnight, then the culture supernatants were collected and evaluated for inhibitory activity. Inhibition was determined by measuring inhibition zone diameters with double plate punching method. Then HPLC and 4-Aminoantipyrine were used to determine the concentration of organic acids and H 2 O 2 respectively in lactobacilli metabolites. Finally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of lactic acid, acetic acid and H 2 O 2 on S. pyogenes were determined. [Results] Lactobacillus plantarum KLDS had the strongest inhibitory effect among the three strains, followed by L. paracasei KLDS and L. helveticus KLDS L. plantarum KLDS had the highest production of lactic acid and acetic acid, followed by * 通讯作者 :Tel: ; :xchmeng@163.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 118 微生物学通报 Microbiol. China 2015, Vol.42, No.1 KLDS and KLDS However, L. helveticus KLDS had the highest production of H 2 O 2, followed by KLDS and KLDS The diameters of inhibition zone all decreased after elimination of H 2 O 2 in lactobacilli supernatants, and no obvious inhibition zones were detected when the ph of lactobacilli supernatants was adjusted to 7.0. The results indicated that organic acids and H 2 O 2 were the main inhibiting substances in lactobacilli metabolites, further lactic acid was the most important inhibiting substance. MIC of lactic acid, acetic acid and H 2 O 2 on Streptococcus pyogenes were 1.28, 0.64 and g/l, respectively. MBC of lactic acid, acetic acid and H 2 O 2 on Streptococcus pyogenes were 5.12, 2.56 and g/l, respectively. [Conclusion] The metabolites of lactobacilli could inhibit the growth of Streptococcus pyogenes, and the organic acids and H 2 O 2 were the main inhibiting substances. Keywords: Lactobacilli, Streptococcus pyogenes, Inhibition, Organic acid, H 2 O 2 (Lactic acid bacteria LAB) [1-2] (Lactobacillus) [3-4] [5-7] ( ) ( ) [5-6] [8] Streptococcus pyogenes (Group A streptococcus GAS) [8-9] 90% [10] 1 材料与方法 1.1 材料和设备材料 :(Lactobacillus plantarum) KLDS (Lactobacillus paracasei) KLDS (Lactobacillus helveticus) KLDS Streptococcus pyogenes (Group A streptococcus GAS) ATCC 试剂和仪器 : Sigma 30% H 2 O 2 4- MRS (De man rogosa and sharpe medium) BHI (Brian heart infusion) BHIA (Brian heart infusion agar) Eppondorf UV-2401PC WATERS2695 GL-21M

3 : 119 Aminex HPX-87H HPLC 1.2 方法菌株活化与培养 : 0.2 ml 10 ml MRS [2] 37 C 24 h C ATCC BHIA 37 C 48 h 10 ml BHI 37 C 24 h C 乳杆菌代谢产物对化脓性链球菌的抑制作用 :(1) ph 1% 150 ml MRS 37 C 24 h g 4 C 15 min [3,6] 20 ml ph (2) 1) 20 ml 0.22 μm A 2) 20 ml 3 mol/l NaOH ph μm B 3) 20 ml 80 C 3 h H 2 O [4] μm C 4) 20 ml 80 C 3 h H 2 O 2 3 mol/l NaOH ph μm D 5) 20 ml 80 C 3 h H 2 O 2 3 mol/l NaOH ph ml 8 ml 4 C g 4 C 15 min 2 ml [11] 0.22 μm E CFU/mL (Colony-forming units CFU CFU/mL ) [12-13] 1% ml 10 ml H + 浓度对抑菌作用的影响 : 1 mol/l HCl MRS ph [14-15] 乳杆菌在培养过程中抑菌活性主要有机酸及 H 2 O 2 含量的测定 : 1% MRS 37 C 36 h 4 h (10 ml) g 4 C 15 min 0.22 μm 20 C (1) (2) [16-17] Aminex HPX-87H HPLC 65 C5 mmol/l 0.5 ml/min10 μl (3) H 2 O 2 H 2 O 2 Marty-Teysset [18-19] H 2 O 2 MRS 5 15 min H 2 O 乳酸乙酸和 H 2 O 2 最小抑菌浓度 (MIC) 和最小杀菌浓度 (MBC) 的测定 :(1) (MIC) H 2 O 2 (Minimum inhibition concentration MIC) 1 BHI 3.2 ml

4 120 微生物学通报 Microbiol. China 2015, Vol.42, No.1 2 ml ml 1.28 g/l ( ) 2 ml 2 2 ml 0 2 ml BHI 2 ml 10 6 CFU/mL 37 C 11 h OD ( 610 nm) OD OD (MIC) [20] (2) (MBC) MIC 2 ml BHI 2 ml 10 6 CFU/mL 37 C 11 h (%)=(a b)/a 100 a( ) ( CFU/mL) b ( ) (Minimal bactericidal concentration MBC) 90% (MBC) [20-21] 数据处理与分析 : 3 SPSS 结果与分析 2.1 乳杆菌代谢产物对化脓性链球菌的抑菌作用乳杆菌代谢产物经不同处理后的抑菌作用 : 37 C 24 h 1 1 KLDS KLDS KLDS ph KLDS KLDS KLDS ph H + H 2 O C ( 122 C C) 80 C 3 H 2 O 2 H 2 O 2 ph 7.0 H 2 O 2 ph H 2 O 2 表 1 乳杆菌发酵上清液的 ph 测定及经不同处理后对化脓性链球菌的抑菌效果 Table 1 Determination of ph of lactobacilli supernatant and its inhibition effect on Streptococcus pyogenes after different treatment Strains ph ph of lactobacilli supernatant Inhibition zone diameters (mm) A B C D E KLDS ± ± ± ±0.39 KLDS ± ± ± ±0.34 KLDS ± ± ± ± ±0.2 mm. A B H 2 O 2 C ph 7.0 D H 2 O 2 ph 7.0 E H 2 O 2 ph Note: Diameter of double plate punching hole was 7.0±0.2 mm. A: Supernatant with no treatment; B: Supernatant with H 2 O 2 elimination; C: Supernatant with ph adjusted; D: Supernatant with H 2 O 2 elimination and ph adjustment; E: Supernatant of 10 fold concentration after H 2 O 2 elimination and ph adjustment. : Undetected inhibition zone.

5 : 121 H 2 O 2 H 2 O 上清液中 H + 浓度对抑菌作用的影响 : ( 24 h) ph 3 ph MRS ph H + ph MRS ph 3.5 MRS (8.46 mm) KLDS ph mm H + ph H 乳杆菌在培养过程中发酵上清液的抑菌活性 36 h 1 4 h 8 h KLDS KLDS KLDS h KLDS KLDS h 20 h 图 1 乳杆菌培养过程中发酵上清液的抑菌活性 Figure 1 Inhibition activity of supernatant in lactobacilli cultivation 7.0±0.2 mm. Note: Diameter of double plate punching hole was 7.0±0.2 mm. KLDS h 24 h h KLDS KLDS (P>0.05) KLDS KLDS KLDS KLDS (P<0.05) 2.3 乳杆菌代谢产物中主要抑菌物质的分析乳杆菌在培养过程中发酵上清液中有机酸含量分析 : (HPLC) y=71 707x R 2 = min 36 h 2A h KLDS KLDS h 24 h KLDS h 20 h (HPLC) y=71 906x R 2 = min 36 h 2B h 32 h MRS MRS h KLDS KLDS KLDS (P>0.05) KLDS KLDS KLDS (P<0.05) KLDS KLDS KLDS KLDS KLDS (P<0.05)

6 122 微生物学通报 Microbiol. China 2015, Vol.42, No.1 图 2 乳杆菌发酵上清液中乳酸 (A) 和乙酸 (B) 含量随培养时间的变化趋势图 Figure 2 Change of lactic acid (A) and acetic acid (B) concentration in lactobacilli supernatant 2B MRS 3.6 g/l. Note: Acetate concentration in MRS was 3.6 g/l in Figure 2B 乳杆菌在培养过程中发酵上清液中 H 2 O 2 含量的测定 : H 2 O g/ml H 2 O 2 y= x R 2 = h H 2 O h H 2 O h 32 h 8 h H 2 O 2 H 2 O h H 2 O h H 2 O 2 KLDS H 2 O 2 KLDS H 2 O 2 KLDS H 2 O 2 KLDS KLDS (P<0.05) 2.2 H 2 O 乳酸乙酸和 H 2 O 2 最小抑菌浓度 (MIC) 和最小杀菌浓度 (MBC) 的测定 MIC 的测定 : H 2 O 2 OD 4 H 2 O 2 MIC g/l MIC H 2 O 2 < < H 2 O 2 > > MBC 的测定 : MBC 2 H 2 O 2 MBC g/l MBC H 2 O 2 < < H 2 O 2 > > 图 3 乳杆菌发酵上清液中 H 2 O 2 含量的变化趋势图 Figure 3 Change of H 2 O 2 concentration in lactobacilli supernatant

7 : 123 图 4 乳酸乙酸 (A) 和 H 2 O 2 (B) 对化脓性链球菌的 MIC Figure 4 MIC of lactic acid, acetic acid (A) and H 2 O 2 (B) on Streptococcus pyogenes OD H 2 O 2 MIC. Note: The inflexion point of the curve obtained by plotting organic acid and H 2 O 2 concentration vs optical density was MIC of them to index strain. 表 2 乳酸乙酸和 H 2 O 2 对化脓性链球菌的 MBC 测定结果 Table 2 MBC of lactic acid, acetic acid and H 2 O 2 on Streptococcus pyogenes MBC (g/l) Inhibiting substances Sterilizing rate (%) Lactic acid Acetic acid H 2 O 结论 3 KLDS1.0667>KLDS > KLDS KLDS1.0667>KLDS >KLDS H 2 O 2 KLDS1.0203>KLDS1.0667> KLDS H 2 O 2 H 2 O 2 H 2 O 2 > > H 2 O 2 H 2 O 2 参考文献 [1] Zhang Y. The isolation, identification and genetic diversity analysis of lactobacilli of two sources[d]. Harbin: Masterʼs Thesis of Northeast Agricultural University, 2008: 1-59 (in Chinese). [D]. :, 2008: 1-59 [2] Song TS, Kim YG, Kim KH, et al. In vitro evaluation of probiotic lactic acid bacteria isolated from dairy and non-dairy environments[j]. Food Science and Biotechnology, 2010, 19(1): [3] Cui YW, Meng XC. Isolation and identification of several Lactobacilli[J]. Journal of Northeast Agricultural University, 2008, 39(6): (in Chinese),. [J]., 2008, 39(6): [4] Wu J, Mang L, Wang YL, et al. Screening and identification of lactobacillus producing antimicrobial substance from Koumiss[J]. Journal of Inner Mongolia Agricultural University, 2011, 32(1): (in Chinese),,,. [J]., 2011, 32(1): [5] Xiong J, Han RN, Zhang ZH, et al. Screening of lactic acid bacteria strains with high antibacterial activity from Douchi and it s research of bacteriostatic[j]. Chinese Journal of Microecology, 2011, 23(6): (in Chinese),,,. [J]., 2011, 23(6): [6] Mezaini A, Chihib NE, Bouras AD, et al. Antibacterial activity of some lactic acid bacteria isolated from an Algerian dairy product[j]. Journal of Environmental and Public Health, 2007: 1-6 [7] González L, Sandoval H, Sacristán N, et al. Identification of lactic acid bacteria isolated from Genestoso cheese throughout ripening and study of their antimicrobial activity[j]. Food Control, 2007, 18: [8] Maudsdotter L, Jonsson H, Roos S, et al. Lactobacilli reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid that degrades the toxic component lipoteichoic acid[j]. Antimicrobial Agents and Chemotherapy, 2011, 55(4): [9] Cunningham MW. Pathogenesis of group A streptococcal infections[j]. Clinical Microbiology Reviews, 2000, 13(3):

8 124 微生物学通报 Microbiol. China 2015, Vol.42, No [10] Wang CQ, Wang Y, Wang XH, et al. Recovery rates and antimicrobial susceptibility of Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis collected from children with respiratory tract infection in Children s Hospital of Fuden University from 2000 to 2006[J]. Chinese Journal of Evidence Based Pediatrics, 2007, 2(3): (in Chinese),,, [J]., 2007, 2(3): [11] Gao YR, Jia SR, Gao Q, et al. A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage[j]. Food Control, 2010, 21: [12] Wang H, Gong HS, Meng XC. Partial characteristics of the bacteriocin produced by Lactobacillus brevis[j]. Microbiology China, 2011, 38(7): (in Chinese),,. [J]., 2011, 38(7): [13] Oh SJ, Kim SH, Ko Y, et al. Effect of bacteriocin produced by Lactococcus sp. HY 449 on skin-inflammatory bacteria[j]. Food and Chemical Toxicology, 2006, 44: [14] Ito A, Sato Y, Kudo S, et al. The screening of hydrogen peroxide-producing lactic acid bacteria and their application to inactivating psychrotrophic food-borne pathogens[j]. Current Microbiology, 2003, 47: [15] Li PL, Zhang C, Jiang HH. Analysis of characteristics of the bacteriocin produced by a Lactobacillus plantarum[j]. China Brewing, 1999(5): (in Chinese),,. [J]., 1999(5): [16] Lü ZL, Lin X, Deng WH, et al. Content determination of malic acid and lactic acid in cider by HPLC[J]. Fine and Specialty Chemicals, 2012, 20(1): 9-13 (in Chinese),,,. HPLC [J]., 2012, 20(1): 9-13 [17] Bianchini A. Factors affecting production and stability of antifungal compounds of Lactobacillus plantarum, and effects of the antifungal compounds on growth and aflatoxin production by Aspergillus spp.[d]. Nebraska: Faculty of the Graduate College at the University of Nebraska, 2010: [18] Batdorj B, Trinetta V, Dalgalarrondo M, et al. Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens[j]. Journal of Applied Microbiology, 2006, 103: [19] Marty-Teysset C, De La Torre F, Garel JR. Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress[j]. Applied and Environmental Microbiology, 2000, 66(1): [20] Qiao ZH, Cheng YQ, Lu ZH, et al. Antibacterial and bactericide activity of lactic acid against three strains of food-borne pathogenic bacteria[j]. Food Science and Technology, 2008, 10: (in Chinese),,,. [J]., 2008, 10: [21] Otero MC, Nader-Macías ME. Inhibition of Staphylococcus aureus by H 2 O 2 -producing Lactobacillus gasseri isolated from the vaginal tract of cattle[j]. Animal Reproduction Science, 2006, 96: 35-46

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇