乳酸菌富硒能力及其体外生物活性评估
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摘要
从13株乳酸菌中筛选出耐硒较好的菌株,对其富硒能力及生物活性进行研究。通过测定OD值及菌体显色观察,确定菌株的最适耐硒质量浓度;通过3,3’-二氨基联苯胺(3,3’-DAB)比色法测定菌株富硒量;对富硒菌株在抗氧化活性、胆盐耐受以及体外抗菌活性的变化进行探究;通过电镜扫描结合EDX能谱分析,观察富硒乳酸菌细胞表面结构,分析其化学成分。试验结果表明:13株中有8株菌具有较好的耐硒能力;WY1,WY2适宜的富硒质量浓度为60μg/mL,WY5,WY8,WY9,WY10,WY12富硒质量浓度为80μg/mL,WY3富硒质量浓度为100μg/mL;WY12的富硒率最高,为79.19%±3.01%;其它依次为WY3(78.72%±1.07%)、WY10(73.42%±1.61%)、WY9(67.65%±3.24%)、WY1(64.27%±3.31%)、WY8(61.49%±4.49%)、WY2(56.29%±3.01%)和WY5(34.98%±1.46%);富硒菌在1,1-二苯基-2-三硝基苯肼(DPPH)、2,2’-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)(ABTS)自由基清除率以及还原力上均有显著提高,胆盐耐受力也有所增强;此外,富硒菌株对于大肠杆菌以及鼠伤寒沙门氏菌具有较强的抑制作用。电镜扫描结合EDX分析证实,富硒菌菌体表面吸附有纳米硒微粒。结论:乳酸菌能够富集并转化无机硒,且富硒菌株体外生物活性有一定提高。
Thirteen lactic acid bacteria(LAB) strains were used in the present study and their selenium(Se)enrichment abilities were evaluated. Subsequently, several bioactivity indexes of these strains(with or without Se) were tested. The suitable mass concentration of inorganic Se for each strain was determined based on the OD value and cell color change. The accumulation amount of Se by LAB was measured through the 3, 3'-diaminobenzidine(3, 3'-DAB)method. A primary study was carried out to evaluate the antioxidant activity, bile tolerance and antibacterial activity of Se-enriched LAB. Cell morphologies and chemical element components analysis of the Se-enriched LAB was finished through a scanning electron microscope(SEM) equipped with energy dispersion X-ray spectrometry. The results indicated that eight among all these strains have the ability to tolerate and absorb the inorganic Se. The suitable Se mass concentration for WY1 and WY2 was 60 μg/mL, for WY5, WY8, WY9, WY10 and WY12 was 80 μg/mL, for WY3 was 100 μg/mL. Especially, the ratio of Se enrichment of WY12 was the highest(79.19%±3.01%), in order, WY3(78.72% ±1.07%), WY10(73.42% ± 1.61%), WY9(67.65%±3.24%), WY1(64.27% ± 3.31%), WY8(61.49% ± 4.49%), WY2(56.29% ± 3.01%) and WY5(34.98% ± 1.46%), respectively. Antioxidant activities test showed that Se-enriched strains exhibited improved ability in scavenging DPPH, ABTS free radical rates, as well as had a stronger reducing activity.0.1% bile salts tolerance test showed that the bile salt tolerance of Se-enriched strains was improved. In addition, the antibacterial activities showed that Se-enriched strains possess stronger inhibitory effect against Escherichia coli and Salmonella typhimurium. The observation and analyzation of Se-enriched LAB cell pellets demonstrated the existence of elemental or Nano-Se. In summary, LAB have the ability to absorb inorganic Se and transform them into organic,elemental or Nano forms. The bioactivities of Se-enriched LAB have some improvement(compared with that of the no-Se strains) in vitro.
Thirteen lactic acid bacteria(LAB) strains were used in the present study and their selenium(Se)enrichment abilities were evaluated. Subsequently, several bioactivity indexes of these strains(with or without Se) were tested. The suitable mass concentration of inorganic Se for each strain was determined based on the OD value and cell color change. The accumulation amount of Se by LAB was measured through the 3, 3'-diaminobenzidine(3, 3'-DAB)method. A primary study was carried out to evaluate the antioxidant activity, bile tolerance and antibacterial activity of Se-enriched LAB. Cell morphologies and chemical element components analysis of the Se-enriched LAB was finished through a scanning electron microscope(SEM) equipped with energy dispersion X-ray spectrometry. The results indicated that eight among all these strains have the ability to tolerate and absorb the inorganic Se. The suitable Se mass concentration for WY1 and WY2 was 60 μg/mL, for WY5, WY8, WY9, WY10 and WY12 was 80 μg/mL, for WY3 was 100 μg/mL. Especially, the ratio of Se enrichment of WY12 was the highest(79.19%±3.01%), in order, WY3(78.72% ±1.07%), WY10(73.42% ± 1.61%), WY9(67.65%±3.24%), WY1(64.27% ± 3.31%), WY8(61.49% ± 4.49%), WY2(56.29% ± 3.01%) and WY5(34.98% ± 1.46%), respectively. Antioxidant activities test showed that Se-enriched strains exhibited improved ability in scavenging DPPH, ABTS free radical rates, as well as had a stronger reducing activity.0.1% bile salts tolerance test showed that the bile salt tolerance of Se-enriched strains was improved. In addition, the antibacterial activities showed that Se-enriched strains possess stronger inhibitory effect against Escherichia coli and Salmonella typhimurium. The observation and analyzation of Se-enriched LAB cell pellets demonstrated the existence of elemental or Nano-Se. In summary, LAB have the ability to absorb inorganic Se and transform them into organic,elemental or Nano forms. The bioactivities of Se-enriched LAB have some improvement(compared with that of the no-Se strains) in vitro.
引文
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[16] MOGNA L, NICOLA S, PANE M, et al. Selenium and zinc internalized by Lactobacillus buchneri Lb26(DSM 16341)and Bifidobacterium lactis Bb1(DSM17850):improved bioavailability using a new biological approach[J]. Journal of Clinical Gastroenterology, 2012, 46S(9):S41-S45.
[17] BENKO I, NAGY G, TANCZOS B, et al. Subacute toxicity of nano-selenium compared to other selenium species in mice[J]. Environmental Toxicology and Chemistry, 2012, 31(12):2812-2820.
[18] YAZDI M H, MAHDAVI M, SETAYESH N, et al.Selenium nanoparticle-enriched Lactobacillus brevis causes more efficient immune responses in vivo and reduces the liver metastasis in metastatic form of mouse breast cancer[J]. DARU Journal of Pharma ceutical Sciences, 2013, 21(1):1.
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[22]贾姗姗,朱连勤,朱风华,等. 3,3’-二氨基联苯胺分光光度法检测硒[J].畜牧与兽医, 2012, 44(6):74-76.
[23] DENG Y, MAN C, FAN Y, et al. Preparation of elemental selenium-enriched fermented milk by newly isolated Lactobacillus brevis from kefir grains[J].International Dairy Journal, 2015, 44:31-36.
[24] SUHAJDA A, HEGOCZKI J, JANZSO B, et al.Preparation of selenium yeasts I. Preparation of sele nium-enriched Saccharomyces cerevisiae[J]. Journal of Trace Elements in Medicine and Biology, 2000,14(1):43-47.
[25] LIN M Y, CHANG F J. Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708and Lactobacillus acidophilus ATCC 4356[J]. Digestive Diseases and Sciences, 2000, 45(8):1617-1622.
[26] WOOTTON-BEARD P C, MORAN A, RYAN L.Stability of the total antioxidant capacity and total polyphenol content of 23 commercially available vegetable juices before and after in vitro digestion measured by FRAP, DPPH, ABTS and Folin-Ciocalteu methods[J]. Food Research International, 2011,44(1):217-224.
[27] LIN M Y, YEN C L. Antioxidative ability of lactic acid bacteria[J]. Journal of Agricultural and Food Chemistry, 1999, 47(4):1460-1466.
[28]黄玉军,梁文星,刘冬,等.长寿人群肠道具有降胆固醇特性益生菌的筛选[J].中国乳品工业, 2013,41(12):13-16.
[29]王津,杨燕萍,王叶琛,等.母猪粪便中乳酸菌菌株体外益生特性初步研究[J].中国饲料, 2014(14):20-24.
[30]宋照军,王树宁,潘润淑,等.富硒乳酸菌的分离,筛选,驯化及富硒研究[J].中国酿造, 2004, 23(11):4-6.
[31]靳志强,张博文,李平兰.动物双歧杆菌01耐硒性能及富硒条件的研究[J].食品科学, 2009, 30(15):184-187.
[32] XIA S K, CHEN L, LIANG J Q. Enriched selenium and its effects on growth and biochemical composition in Lactobacillus bulgaricus[J]. Journal of Agricultural and Food Chemistry, 2007, 55(6):2413-2417.
[33]黄秀锦.富硒乳酸菌发酵条件的研究[J].中国调味品, 2009, 34(12):54-60.
[34] SHEN Q, ZHANG B, XU R, et al. Antioxidant activity in vitro of the selenium-contained protein from the Se-enriched Bifidobacterium animalis 01[J].Anaerobe, 2010, 16(4):380-386.
[35] PE譙AS E, MARTINEZ-VILLALUENGA C, FRIAS J, et al. Se improves indole glucosinolate hydrolysis products content, Se-methylselenocysteine content,antioxidant capacity and potential anti-inflammatory properties of sauerkraut[J]. Food Chemistry, 2012,132(2):907-914.
[36] CHEN L, PAN D D, ZHOU J, et al. Protective effect of selenium-enriched Lactobacillus on CCl4-induced liver injury in mice and its possible mechanisms[J]. World Journal of Gastroenterology, 2005,11(37):5795.
[37] REN Z, ZHAO Z, WANG Y, et al. Preparation of selenium/zinc-enriched probiotics and their effect on blood selenium and zinc concentrations, antioxidant capacities, and intestinal microflora in canine[J]. Biological Trace Element Research, 2011, 141(1/2/3):170-183.
[38] GUO Y, PAN D, LI H, et al. Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis[J].Food Chemistry, 2013, 138(1):84-89.
[39]黄玉军,刘冬,赵兰凤,等. 6株人源乳酸菌体外抗氧化活性的比较[J].现代食品科技, 2013, 29(7):1518-1522.
[40] REID G, BURTON J. Use of Lactobacillus to prevent infection by pathogenic bacteria[J]. Microbes and Infection, 2002, 4(3):319-324.
[41] YANG J, HUANG K, QIN S, et al. Antibacterial action of selenium-enriched probiotics against pathogenic Escherichia coli[J]. Digestive Diseases and Sciences, 2009, 54(2):246-254.
[2] MATER D D G, BRETIGNY L, FIRMESSE O, et al. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal transit of healthy volunteers consuming yogurt[J].FEMS Microbiology Letters, 2005, 250(2):185-187.
[3] NAVARRO-ALARCON M, LOPEZ-MARTINEZ M C. Essentiality of selenium in the human body:relationship with different diseases[J]. Science of the Total Environment, 2000, 249(1):347-371.
[4] RAYMAN M P. The importance of selenium to human health[J]. The Lancet, 2000, 356(9225):233-241.
[5] KIM Y S, MILNER J. Molecular targets for selenium in cancer prevention[J]. Nutrition and Cancer, 2001,40(1):50-54.
[6] ZHU Z, JIANG W, GANTHER H E, et al. Activity of Se-allylselenocysteine in the presence of methionineγ-lyase on cell growth, DNA integrity,apoptosis, and cell-cycle regulatory molecules[J].Molecular Carcinogenesis, 2000, 29(4):191-197.
[7] ZHANG J, WANG H, YAN X, et al. Comparison of short-term toxicity between Nano-Se and selenite in mice[J]. Life Sciences, 2005, 76(10):1099-1109.
[8] NUTTALL K L. Evaluating selenium poisoning[J].Annals of Clinical&Laboratory Science, 2006, 36(4):409-420.
[9] SARATHCHANDRA S U, WATKINSON J H. Oxidation of elemental selenium to selenite by Bacillus megaterium[J]. Science, 1981, 211(4482):600-601.
[10] KESSI J, HANSELMANN K W. Similarities between the abiotic reduction of selenite with glutathione and the dissimilatory reaction mediated by Rhodospirillum rubrum and Escherichia coli[J]. Journal of Biological Chemistry, 2004, 279(49):50662-50669.
[11] NELSON D C, CASEY W H, SISON J D, et al.Selenium uptake by sulfur-accumulating bacteria[J].Geochimica et Cosmochimica Acta, 1996, 60(18):3531-3539.
[12] GARBISU C, ISHII T, LEIGHTON T, et al. Bacterial reduction of selenite to elemental selenium[J].Chemical Geology, 1996, 132(1):199-204.
[13] DUMONT E, VANHAECKE F, CORNELIS R. Selenium speciation from food source to metabolites:a critical review[J]. Analytical and Bioanalytical Chemistry, 2006, 385(7):1304-1323.
[14] ANDREONI V, LUISCHI M M, CAVALCA L, et al. Selenite tolerance and accumulation in the Lactobacillus species[J]. Annals of Microbiology, 2000,50(1):77-88.
[15] GALANO E, MANGIAPANE E, BIANGA J, et al.Privileged incorporation of selenium as selenocysteine in Lactobacillus reuteri proteins demonstrated by selenium-specific imaging and proteomics[J]. Molecular&Cellular Proteomics, 2013, 12(8):2196-2204.
[16] MOGNA L, NICOLA S, PANE M, et al. Selenium and zinc internalized by Lactobacillus buchneri Lb26(DSM 16341)and Bifidobacterium lactis Bb1(DSM17850):improved bioavailability using a new biological approach[J]. Journal of Clinical Gastroenterology, 2012, 46S(9):S41-S45.
[17] BENKO I, NAGY G, TANCZOS B, et al. Subacute toxicity of nano-selenium compared to other selenium species in mice[J]. Environmental Toxicology and Chemistry, 2012, 31(12):2812-2820.
[18] YAZDI M H, MAHDAVI M, SETAYESH N, et al.Selenium nanoparticle-enriched Lactobacillus brevis causes more efficient immune responses in vivo and reduces the liver metastasis in metastatic form of mouse breast cancer[J]. DARU Journal of Pharma ceutical Sciences, 2013, 21(1):1.
[19] CALOMME M R, BRANDEN K, BERGHE D A.Selenium and Lactobacillus species[J]. Journal of Applied Bacteriology, 1995, 79(3):331-340.
[20] SAINI K, TOMAR S K, SANGWAN V, et al. Evaluation of Lactobacilli from human sources for uptake and accumulation of selenium[J]. Biological Trace Element Research, 2014, 160(3):433-436.
[21]侯少范,王五一.微量硒的测定方法简介[J].分析化学, 1980, 8(2):183-187.
[22]贾姗姗,朱连勤,朱风华,等. 3,3’-二氨基联苯胺分光光度法检测硒[J].畜牧与兽医, 2012, 44(6):74-76.
[23] DENG Y, MAN C, FAN Y, et al. Preparation of elemental selenium-enriched fermented milk by newly isolated Lactobacillus brevis from kefir grains[J].International Dairy Journal, 2015, 44:31-36.
[24] SUHAJDA A, HEGOCZKI J, JANZSO B, et al.Preparation of selenium yeasts I. Preparation of sele nium-enriched Saccharomyces cerevisiae[J]. Journal of Trace Elements in Medicine and Biology, 2000,14(1):43-47.
[25] LIN M Y, CHANG F J. Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708and Lactobacillus acidophilus ATCC 4356[J]. Digestive Diseases and Sciences, 2000, 45(8):1617-1622.
[26] WOOTTON-BEARD P C, MORAN A, RYAN L.Stability of the total antioxidant capacity and total polyphenol content of 23 commercially available vegetable juices before and after in vitro digestion measured by FRAP, DPPH, ABTS and Folin-Ciocalteu methods[J]. Food Research International, 2011,44(1):217-224.
[27] LIN M Y, YEN C L. Antioxidative ability of lactic acid bacteria[J]. Journal of Agricultural and Food Chemistry, 1999, 47(4):1460-1466.
[28]黄玉军,梁文星,刘冬,等.长寿人群肠道具有降胆固醇特性益生菌的筛选[J].中国乳品工业, 2013,41(12):13-16.
[29]王津,杨燕萍,王叶琛,等.母猪粪便中乳酸菌菌株体外益生特性初步研究[J].中国饲料, 2014(14):20-24.
[30]宋照军,王树宁,潘润淑,等.富硒乳酸菌的分离,筛选,驯化及富硒研究[J].中国酿造, 2004, 23(11):4-6.
[31]靳志强,张博文,李平兰.动物双歧杆菌01耐硒性能及富硒条件的研究[J].食品科学, 2009, 30(15):184-187.
[32] XIA S K, CHEN L, LIANG J Q. Enriched selenium and its effects on growth and biochemical composition in Lactobacillus bulgaricus[J]. Journal of Agricultural and Food Chemistry, 2007, 55(6):2413-2417.
[33]黄秀锦.富硒乳酸菌发酵条件的研究[J].中国调味品, 2009, 34(12):54-60.
[34] SHEN Q, ZHANG B, XU R, et al. Antioxidant activity in vitro of the selenium-contained protein from the Se-enriched Bifidobacterium animalis 01[J].Anaerobe, 2010, 16(4):380-386.
[35] PE譙AS E, MARTINEZ-VILLALUENGA C, FRIAS J, et al. Se improves indole glucosinolate hydrolysis products content, Se-methylselenocysteine content,antioxidant capacity and potential anti-inflammatory properties of sauerkraut[J]. Food Chemistry, 2012,132(2):907-914.
[36] CHEN L, PAN D D, ZHOU J, et al. Protective effect of selenium-enriched Lactobacillus on CCl4-induced liver injury in mice and its possible mechanisms[J]. World Journal of Gastroenterology, 2005,11(37):5795.
[37] REN Z, ZHAO Z, WANG Y, et al. Preparation of selenium/zinc-enriched probiotics and their effect on blood selenium and zinc concentrations, antioxidant capacities, and intestinal microflora in canine[J]. Biological Trace Element Research, 2011, 141(1/2/3):170-183.
[38] GUO Y, PAN D, LI H, et al. Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis[J].Food Chemistry, 2013, 138(1):84-89.
[39]黄玉军,刘冬,赵兰凤,等. 6株人源乳酸菌体外抗氧化活性的比较[J].现代食品科技, 2013, 29(7):1518-1522.
[40] REID G, BURTON J. Use of Lactobacillus to prevent infection by pathogenic bacteria[J]. Microbes and Infection, 2002, 4(3):319-324.
[41] YANG J, HUANG K, QIN S, et al. Antibacterial action of selenium-enriched probiotics against pathogenic Escherichia coli[J]. Digestive Diseases and Sciences, 2009, 54(2):246-254.