Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96018
標題: Characterization of high exopolysaccharide-producing Lactobacillus strains isolated from mustard pickles and enhancement of exopolysaccharide production by Lactobacillus pentosus SLC 13
從酸菜中分離高胞外多醣乳酸桿菌之特性與促進戊醣乳酸桿菌SLC 13胞外多醣產量之研究
作者: Jing-Yao Huang
黃靖堯
關鍵字: 乳酸菌
乳酸桿菌屬
胞外多醣
益生菌
益生質
柳丁果皮纖維
農工業副產物
Lactic acid bacteria
Lactobacillus spp.
Exopolysaccharide
Probiotics
Prebiotics
Orange peel fiber
Agro-industrial by-products
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摘要: 本研究的目的為分離和鑑定台灣酸菜中高胞外多醣(Exopolysaccharide, EPS)乳酸菌之潛在益生特性,並評估比較柳丁果皮纖維粉末(Orange peel fiber powder, OPFP)與黃白木耳發酵液粉末(Tremella flava Chen fermented powder, TFP)對戊醣乳酸桿菌SLC 13 (Lactobacillus pentosus SLC 13) EPS產量的影響,以用作未來使用植物源乳酸菌開發富含EPS發酵飲料之研究依據。從酸菜收集39株乳酸菌中,選擇四株EPS產量最大的菌株進行進一步的實驗分析。比較16S rDNA、rpoA和pheS基因序列分析,證明這些乳酸菌菌株是植物乳酸桿菌群組(Lactobacillus plantarum-group, LPG)的成員。乳酸菌NCD 2、NLD 4、SLC 13和NLD 16在模擬胃腸道條件下的存活率分別為95.83 ± 0.49%、95.07 ± 0.64%、105.84 ± 0.82%和99.65 ± 0.31%。在這四株乳酸菌中(SLC 13除外),以低劑量(1 μg/ mL)乳酸菌死菌刺激物處理巨噬細胞RAW 264.7時,沒有發現細胞毒性作用。而各種乳酸菌死菌刺激物對巨噬細胞RAW 264.7之一氧化氮產量,顯示具劑量依存性。在四株乳酸菌中,SLC 13對糞腸球菌(Enterococcus faecalis) (BCRC 12302)和小腸結腸炎耶爾辛氏菌(Yersinia enterocolitica) (BCRC 10807)具有較高的抑制活性,與其他三株乳酸菌相比,NLD 4對大腸桿菌O157:H7 (Escherichia coli O157:H7) (ATCC 43894)的抑制活性較強。在MRS培養基中添加不同濃度葡萄糖、OPFP和TFP,以研究戊醣乳酸桿菌SLC 13之EPS產量和生長的影響。研究結果發現添加6%葡萄糖、2% OPFP和2% TFP可顯著促進EPS產量(分別從0.4322 ± 0.0145至2.0302 ± 0.1572、0.4340 ± 0.0142至2.4691 ± 0.0268和0.4372 ± 0.0395至2.5582 ± 0.0873 g/ L)和乳酸菌菌數(分別從7.06 ± 0.08至8.88 ± 0.05、7.02 ± 0.01至7.67 ± 0.07和6.99 ± 0.04至7.96 ± 0.07 log 10 CFU/ mL)。而當以3% HCl預處理的2% OPFP加入培養基中,培養戊醣乳酸桿菌SLC 13可產生最大的EPS產量(2.6647 ± 0.0380 g/ L)。若以20 U聚木醣酶、20 U纖維素酶和10 U纖維素酶加10 U聚木醣酶預處理的2 % OPFP加入培養基中,可增加培養戊醣乳酸桿菌SLC 13的EPS產量(分別從2.4691 ± 0.0268至3.1488 ± 0.0410、3.1467 ± 0.0574和3.2169 ± 0.0895 g/ L)。藉由MRS培養基中加入6%葡萄糖、2% OPFP和2% TFP,研究戊醣乳酸桿菌SLC 13之EPS的抗氧化活性和戊醣乳酸桿菌SLC 13培養液的抗菌性,當培養基添加6 %葡萄糖,培養戊醣乳酸桿菌SLC 13產生的EPS (20 mg/ mL)之還原力、DPPH和ABTS自由基清除能力最高(分別為121.1548 ± 2.1861、73.7676 ± 3.1502和85.1297 ± 0.7075%)。而當培養基添加6%葡萄糖和2% OPFP分別可提高戊醣乳酸桿菌SLC 13培養液對金黃色葡萄球菌(Staphylococcus aureus) (BCRC 11863)和腸炎沙門氏菌(Salmonella enterica) (BCRC 10746)的抗菌能力。綜上所述,我們的研究結果建議,戊醣乳酸桿菌SLC 13對於益生特性應用和抗菌化合物開發為良好的候選者,並發現2% OPFP添加於培養液中可促進戊醣乳酸桿菌SLC 13之EPS產量(增為5.6892倍)、生長(單位細胞之胞外多醣產量約為0.0528 ng/ cell)和抗菌能力,而OPFP是具有經濟性的益生質,具有應用於工業的潛力。
The aim of this study was to characterize high exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) isolated from mustard pickles in Taiwan for potential probiotic applications and evaluate the effect of orange peel fiber powder (OPFP) in comparison to that of Tremella flava Chen fermented powder (TFP) on Lactobacillus pentosus SLC 13 exopolysaccharide (EPS) production for future development of EPS-rich fermented drinks by botanical LAB. Among 39 collected LAB strains from mustard pickles, four most productive EPS-producing strains were selected for further analysis. Comparative analyses of 16S rDNA, rpoA, and pheS sequences demonstrated that these strains were members of Lactobacillus plantarum-group (LPG). LAB NCD 2, NLD 4, SLC 13, and NLD 16 showed survival rates of 95.83 ± 0.49%, 95.07 ± 0.64%, 105.84 ± 0.82%, and 99.65 ± 0.31%, respectively, under simulated gastrointestinal condition. No cytotoxic effects on macrophage RAW 264.7 cells were observed when they were treated with a low dose (1 μg/mL) of stimulants extracted from the tested four LAB strains(except SLC 13). The production of nitric oxide in RAW 264.7 cells incubated with various LAB stimulants showed a dose-dependent increase. Among the four strains, SLC 13 showed higher inhibitory activity on growth of Enterococcus faecalis (BCRC 12302) and Yersinia enterocolitica (BCRC 10807). NLD 4 showed strong inhibitory activity against Escherichia coli O157:H7 (ATCC 43894) as compared with the other three strains. EPS production and growth by L. pentosus SLC 13 in MRS broth supplemented with different concentrations of glucose, OPFP, and TFP were investigated. The results of this study showed that supplementation with 6% glucose, 2% OPFP, and 2% TFP significantly promoted EPS production (from 0.4322 ± 0.0145 to 2.0302 ± 0.1572, from 0.4340 ± 0.0142 to 2.4691 ± 0.0268, and from 0.4372 ± 0.0395 to 2.5582 ± 0.0873 g/ L, respectively) and bacterial viability (from 7.06 ± 0.08 to 8.88 ± 0.05, from 7.02 ± 0.01 to 7.67 ± 0.07, and 6.99 ± 0.04 to 7.96 ± 0.07 log 10 CFU/ mL, respectively). Maximum EPS production (2.6647 ± 0.0380 g/ L) of L. pentosus SLC 13 was observed when 2% OPFP pretreated with 3 N HCl was added to the culture medium. Supplementation of 2% OPFP treated with 20 U xylanase, 20 U cellulase, and 10 U cellulase/10 U xylanase increased the EPS production (from 2.4691 ± 0.0268 to 3.1488 ± 0.0410, 3.1467 ± 0.0574, and 3.2169 ± 0.0895 g/ L, respectively) by strain L. pentosus SLC 13. The antioxidant activity of EPS and antibacterial property of the MRS broth were investigated by adding 6% glucose, 2% OPFP, and 2% TFP to the L. pentosus SLC 13 culture medium. The highest reducing power and DPPH and ABTS radical scavenging activities of EPS (20 mg/ mL) were observed when the culture medium was supplemented with 6% glucose (121.1548 ± 2.1861, 73.7676 ± 3.1502, and 85.1297 ± 0.7075%, respectively). Supplementation with 6% glucose and 2% OPFP enhanced the antibacterial property of the L. pentosus SLC 13 medium against Staphylococcus aureus (BCRC 11863) and Salmonella enterica (BCRC 10746) respectively. In summary, our results suggest that L. pentosus SLC 13 may be a good candidate for probiotic applications and for development of antibacterial compounds. This study regarding culture medium by adding 2% OPFP promotes EPS production (increased to 5.6892 times), growth (EPS yield per unit cell was about 0.0528 ng/ cell), and antibacterial property of L. pentosus SLC 13 suggest that it has potential for industrial application as an economic prebiotic.
URI: http://hdl.handle.net/11455/96018
文章公開時間: 2018-02-09
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