植物乳杆菌的筛选、鉴定及其抑菌物质研究
|
摘要
采用溶钙圈法和琼脂扩散法从传统发酵食品中筛选具有抑菌作用的菌株,通过形态学观察、生理生化反应和16S rDNA分析进行菌株鉴定,经过酶敏感性反应、排酸实验和HPLC检测对所产抑菌物质进行初步分析,采用琼脂扩散法对抑菌物质进行抑菌谱研究。结果显示,从东北酸菜中筛选到一株具有较强抑菌作用的植物乳杆菌(Lactobacillus plantarum)CNQ3,该菌株在MRS培养基中发酵24 h,活菌数为6.5×10~(11) CFU/mL,抑菌圈直径为19.50 mm。L.plantarum CNQ3所产抑菌物质表现为有机酸,HPLC检测其主要为乳酸和醋酸,二者含量分别为11.23和5.58 mg/mL,同时含有0.083 mg/mL的苯乳酸。该抑菌物质的抑菌谱广,对受试的8株食源性致病菌和腐败菌具有抑菌作用,其中对单核增生李斯特菌(Listeria monocytogenes)的抑菌效果最好,抑菌圈直径23.30 mm,对藤黄微球菌(Micrococcus luteus)的抑菌圈直径最小,为18.96 mm。本研究为筛选具有抑菌活性的乳酸菌提供参考价值。
The strain was isolated and screened from the traditional fermented food samples with the methods of dissolving calcium ring and agar diffusion inhibition circle. The strain identification was studied by physiological characterization observation,biochemical test and 16 S rDNA sequencing analysis. The analysis of antibacterial substances was carried out by enzyme sensitivity reaction,acid elimination test and HPLC detection. The antibacterial spectrum was investigated by the method of agar diffusion inhibition circle. The results showed that the strain DB was isolated from the northeast sauerkraut sample,and it was identified as Lactobacillus plantarum CNQ3.After fermentation for 24 h in MRS,the viable count of L. plantarum CNQ3 was 6.5×10~(11) CFU/mL and the diameter of inhibitory zone against Staphylococcus aureus was 19.50 mm. The antibacterial fermentation broth of L. plantarum CNQ3 was primarily determined as organic acids. The acid compounds were analyzed by HPLC and they were mainly presented as lactic acid and acetic acid,in which the contents were 11.23 mg/mL and 5.58 mg/mL,respectively,moreover,they also contained phenyllactic acid for 0.083 mg/mL. The active compounds showed a wider antibacterial spectrum with the tested foodborne pathogens and spoilage bacteria,in which,they exhibited the best antagonistic activity against Listeria monocytogenes,and the inhibitory zone diameter was 23.30 mm,while,the antibacterial effect was weak on Micrococcus luteus,which with an inhibitory zone diameter of 18.96 mm. This study provided a certain reference value for screening of LAB with antibacterial activity.
The strain was isolated and screened from the traditional fermented food samples with the methods of dissolving calcium ring and agar diffusion inhibition circle. The strain identification was studied by physiological characterization observation,biochemical test and 16 S rDNA sequencing analysis. The analysis of antibacterial substances was carried out by enzyme sensitivity reaction,acid elimination test and HPLC detection. The antibacterial spectrum was investigated by the method of agar diffusion inhibition circle. The results showed that the strain DB was isolated from the northeast sauerkraut sample,and it was identified as Lactobacillus plantarum CNQ3.After fermentation for 24 h in MRS,the viable count of L. plantarum CNQ3 was 6.5×10~(11) CFU/mL and the diameter of inhibitory zone against Staphylococcus aureus was 19.50 mm. The antibacterial fermentation broth of L. plantarum CNQ3 was primarily determined as organic acids. The acid compounds were analyzed by HPLC and they were mainly presented as lactic acid and acetic acid,in which the contents were 11.23 mg/mL and 5.58 mg/mL,respectively,moreover,they also contained phenyllactic acid for 0.083 mg/mL. The active compounds showed a wider antibacterial spectrum with the tested foodborne pathogens and spoilage bacteria,in which,they exhibited the best antagonistic activity against Listeria monocytogenes,and the inhibitory zone diameter was 23.30 mm,while,the antibacterial effect was weak on Micrococcus luteus,which with an inhibitory zone diameter of 18.96 mm. This study provided a certain reference value for screening of LAB with antibacterial activity.
引文
[1]Prakash O,Nimonkar Y,Shouche Y S.Practice and prospects of microbial preservation[J].Fems Microbiology Letters,2013,339(1):1-9.
[2]Rai M,Pandit R,Gaikwad S,et al.Antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food[J].Journal of Food Science and Technology,2016,53(9):3381-3394.
[3]Olaoye O A,Idowu O A.Features and functional properties of lactic acid bacteria used as biological preservatives of meat processing:A review article[J].Journal of Agricultural Technology,2010,6(3):449-460.
[4]Anyogu A,Awamaria B,Sutherland J P,et al.Molecular characterisation and antimicrobial activity of bacteria associated with submerged lactic acid cassava fermentation[J].Food Control,2014,39:119-127.
[5]Gemechu T.Review on lactic acid bacteria function in milk fermentation and preservation[J].African Journal of Food Science,2015,9(4):170-175.
[6]Waters D M,Mauch A,Coffey A,et al.Lactic acid bacteria as a cell factory for the delivery of functional biomolecules and ingredients in cereal-based beverages:A review[J].Critical Reviews in Food Science and Nutrition,2015,55(4):503-520.
[7]Arena M P,Silvain A,Normanno G,et al.Use of Lactobacillus plantarum strains as a bio-control strategy against food-borne pathogenic microorganisms[J].Frontiers in Microbiology,2016,7:464.
[8]Lavermicocca P,Valerio F,Evidente A,et al.Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B[J].Applied and Environmental Microbiology,2000,66(9):4084-4090.
[9]Corsetti A,Valmorri S.Lactobacillus spp.:Lactobacillus plantarum,in:Fuquay J W,Fox P F,McSweeney P L H(Eds).Encyclopedia of dairy sciences(Second edition)[M].New York:Academic Press,2011:111-118.
[10]Zhang N,Liu J,Li J,et al.Characteristics and application in food preservatives of Lactobacillus plantarum TK9 isolated from naturally fermented congee[J].International Journal of Food Engineering,2016,12(4):377-384.
[11]Guimaraes A,Santiago A,Teixeira J A,et al.Anti-aflatoxigenic effect of organic acids produced by Lactobacillus plantarum[J].International Journal of Food Microbiology,2018,264:31-38.
[12]Tran V,Thu,Foo H L,et al.Inhibitory activity and organic acid concentrations of metabolite combinations produced by various strains of Lactobacillus plantarum[J].African Journal of Biotechnology,2011,10(8):1359-1363.
[13]Chanos P,Mygind T.Co-culture-inducible bacteriocin production in lactic acid bacteria[J].Applied Microbiology and Biotechnology,2016,100(10):4297-4308.
[14]Cortés-Zavaleta O,López-Malo A,Hernández-Mendoza A,et al.Antifungal activity of lactobacilli and its relationship with 3-phenyllactic acid production[J].International Journal of Food Microbiology,2014,173(3):30-35.
[15]Russo P,Arena M P,Fiocco D,et al.Lactobacillus plantarum with broad antifungal activity:A promising approach to increase safety and shelf-life of cereal-based products[J].International Journal of Food Microbiology,2017,247(17):48-54.
[16]Paucean A,Vodnar D C,Socaci S A,et al.Carbohydrate metabolic conversions to lactic acid and volatile derivatives,as influenced by Lactobacillus plantarum ATCC 8014 and Lactobacillus casei ATCC 393 efficiency during in vitro and sourdough fermentation[J].European Food Research and Technology,2013,237(5):679-689.
[17]Chen R,Chen W,Chen H,et al.Comparative evaluation of the antioxidant capacities,organic acids,and volatiles of papaya juices fermented by Lactobacillus acidophilus and Lactobacillus plantarum[J].Journal of Food Quality,2018,2018:1-12.
[18]Laref N,Bettache G.Antifungal activity of newly isolates of lactic acid bacteria[J].Innovative Romanian Food Biotechnology,2013,13:80-88.
[19]Li X,Ning Y,Liu D,et al.Metabolic mechanism of phenyllactic acid naturally occurring in Chinese pickles[J].Food Chemistry,2015,186:265-270.
[20]Rather I A,Seo B J,Kumar V J R,et al.Biopreservative potential of Lactobacillus plantarum YML007 and efficacy as a replacement for chemical preservatives in animal feed[J].Food Science and Biotechnology,2014,23(1):195-200.
[21]何艳霞.抑真菌乳酸菌的筛选及其在蒸蛋糕中的应用[D].无锡:江南大学,2017.
[22]Zhang Z H,Wu S X,Yin J Z,et al.A high phenyllactic acid producing Lactobacillus plantarum strain isolated from ‘Douchi’-A traditional fermented soybean food in Yunnan Province of China[J].Annals of Surgery,2011,90(2):1-5.
[23]Li X,Jiang B,Pan B.Biotransformation of phenylpyruvic acid to phenyllactic acid by growing and resting cells of a Lactobacillus sp[J].Biotechnology letters,2007,29(4):593-597.
[24]Chaudhari S S,Gokhale D V.Phenyllactic acid:A potential antimicrobial compound in lactic acid bacteria[J].Journal of Bacteriology & Mycology Open Access,2016,2(5):37.
[25]Neetoo H,Mahomoodally F.Use of antimicrobial films and edible coatings incorporating chemical and biological preservatives to control growth of Listeria monocytogenes on cold smoked salmon[J].BioMed Research International,2014,2014:534915.
[26]凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999.
[27]赵圣明,曹雪珂,赵岩岩,等.抑制芽孢杆菌乳酸菌的筛选鉴定及其抗菌物质的分离纯化[J].食品工业科技,2018,11(39):144-150,165.
[28]Coelho L F,Lima C J B De,Rodovalho C M,et al.Lacticacid production by new Lactobacillus plantarum LMISM6 grown in molasses:Optimization of medium composition[J].Brazilian Journal of Chemical Engineering,2011,28(1):27-36.
[29]Zhao S,Han J,Bie X,et al.Purification and characterization of plantaricin JLA-9:A novel bacteriocin against Bacillus spp.produced by Lactobacillus plantarum JLA-9 from Suan-Tsai,a traditional chinese fermented cabbage[J].Journal of Agricultural and Food Chemistry,2016,64(13):2754-2764.
[30]黄国昌,顾斌涛,黄朝,等.苯乳酸生产菌的筛选及鉴定[J].食品与发酵工业,2018,44(3):97-100.
[31]熊涛,宋苏华,黄涛,等.植物乳杆菌 NCU116 抑菌性能的研究[J].食品与发酵工业,2012,38(6):97-101.
[32]李强坤,柳陈坚,张园莲,等.植物乳杆菌对食源性病原菌作用的研究[J].食品科学,2016,37(11):18-23.
[33]Prema P,Smila D,Palavesam A,et al.Production and characterization of an antifungal compound(3-phenyllactic acid)produced by Lactobacillus plantarum strain[J].Food and Bioprocess Technology,2010,3(3):379-386.
[34]Dieuleveux V,Lemarinier S,Guéguen M.Antimicrobial spectrum and target site of d-3-phenyllactic acid[J].International Journal of Food Microbiology,1998,40(3):177-183.
[35]Ning Y,Yan A,Yang K,et al.Antibacterial activity of phenyllactic acid against Listeria monocytogenes and Escherichia coli by dual mechanisms[J].Food Chemistry,2017,228:533-540.
[2]Rai M,Pandit R,Gaikwad S,et al.Antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food[J].Journal of Food Science and Technology,2016,53(9):3381-3394.
[3]Olaoye O A,Idowu O A.Features and functional properties of lactic acid bacteria used as biological preservatives of meat processing:A review article[J].Journal of Agricultural Technology,2010,6(3):449-460.
[4]Anyogu A,Awamaria B,Sutherland J P,et al.Molecular characterisation and antimicrobial activity of bacteria associated with submerged lactic acid cassava fermentation[J].Food Control,2014,39:119-127.
[5]Gemechu T.Review on lactic acid bacteria function in milk fermentation and preservation[J].African Journal of Food Science,2015,9(4):170-175.
[6]Waters D M,Mauch A,Coffey A,et al.Lactic acid bacteria as a cell factory for the delivery of functional biomolecules and ingredients in cereal-based beverages:A review[J].Critical Reviews in Food Science and Nutrition,2015,55(4):503-520.
[7]Arena M P,Silvain A,Normanno G,et al.Use of Lactobacillus plantarum strains as a bio-control strategy against food-borne pathogenic microorganisms[J].Frontiers in Microbiology,2016,7:464.
[8]Lavermicocca P,Valerio F,Evidente A,et al.Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B[J].Applied and Environmental Microbiology,2000,66(9):4084-4090.
[9]Corsetti A,Valmorri S.Lactobacillus spp.:Lactobacillus plantarum,in:Fuquay J W,Fox P F,McSweeney P L H(Eds).Encyclopedia of dairy sciences(Second edition)[M].New York:Academic Press,2011:111-118.
[10]Zhang N,Liu J,Li J,et al.Characteristics and application in food preservatives of Lactobacillus plantarum TK9 isolated from naturally fermented congee[J].International Journal of Food Engineering,2016,12(4):377-384.
[11]Guimaraes A,Santiago A,Teixeira J A,et al.Anti-aflatoxigenic effect of organic acids produced by Lactobacillus plantarum[J].International Journal of Food Microbiology,2018,264:31-38.
[12]Tran V,Thu,Foo H L,et al.Inhibitory activity and organic acid concentrations of metabolite combinations produced by various strains of Lactobacillus plantarum[J].African Journal of Biotechnology,2011,10(8):1359-1363.
[13]Chanos P,Mygind T.Co-culture-inducible bacteriocin production in lactic acid bacteria[J].Applied Microbiology and Biotechnology,2016,100(10):4297-4308.
[14]Cortés-Zavaleta O,López-Malo A,Hernández-Mendoza A,et al.Antifungal activity of lactobacilli and its relationship with 3-phenyllactic acid production[J].International Journal of Food Microbiology,2014,173(3):30-35.
[15]Russo P,Arena M P,Fiocco D,et al.Lactobacillus plantarum with broad antifungal activity:A promising approach to increase safety and shelf-life of cereal-based products[J].International Journal of Food Microbiology,2017,247(17):48-54.
[16]Paucean A,Vodnar D C,Socaci S A,et al.Carbohydrate metabolic conversions to lactic acid and volatile derivatives,as influenced by Lactobacillus plantarum ATCC 8014 and Lactobacillus casei ATCC 393 efficiency during in vitro and sourdough fermentation[J].European Food Research and Technology,2013,237(5):679-689.
[17]Chen R,Chen W,Chen H,et al.Comparative evaluation of the antioxidant capacities,organic acids,and volatiles of papaya juices fermented by Lactobacillus acidophilus and Lactobacillus plantarum[J].Journal of Food Quality,2018,2018:1-12.
[18]Laref N,Bettache G.Antifungal activity of newly isolates of lactic acid bacteria[J].Innovative Romanian Food Biotechnology,2013,13:80-88.
[19]Li X,Ning Y,Liu D,et al.Metabolic mechanism of phenyllactic acid naturally occurring in Chinese pickles[J].Food Chemistry,2015,186:265-270.
[20]Rather I A,Seo B J,Kumar V J R,et al.Biopreservative potential of Lactobacillus plantarum YML007 and efficacy as a replacement for chemical preservatives in animal feed[J].Food Science and Biotechnology,2014,23(1):195-200.
[21]何艳霞.抑真菌乳酸菌的筛选及其在蒸蛋糕中的应用[D].无锡:江南大学,2017.
[22]Zhang Z H,Wu S X,Yin J Z,et al.A high phenyllactic acid producing Lactobacillus plantarum strain isolated from ‘Douchi’-A traditional fermented soybean food in Yunnan Province of China[J].Annals of Surgery,2011,90(2):1-5.
[23]Li X,Jiang B,Pan B.Biotransformation of phenylpyruvic acid to phenyllactic acid by growing and resting cells of a Lactobacillus sp[J].Biotechnology letters,2007,29(4):593-597.
[24]Chaudhari S S,Gokhale D V.Phenyllactic acid:A potential antimicrobial compound in lactic acid bacteria[J].Journal of Bacteriology & Mycology Open Access,2016,2(5):37.
[25]Neetoo H,Mahomoodally F.Use of antimicrobial films and edible coatings incorporating chemical and biological preservatives to control growth of Listeria monocytogenes on cold smoked salmon[J].BioMed Research International,2014,2014:534915.
[26]凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999.
[27]赵圣明,曹雪珂,赵岩岩,等.抑制芽孢杆菌乳酸菌的筛选鉴定及其抗菌物质的分离纯化[J].食品工业科技,2018,11(39):144-150,165.
[28]Coelho L F,Lima C J B De,Rodovalho C M,et al.Lacticacid production by new Lactobacillus plantarum LMISM6 grown in molasses:Optimization of medium composition[J].Brazilian Journal of Chemical Engineering,2011,28(1):27-36.
[29]Zhao S,Han J,Bie X,et al.Purification and characterization of plantaricin JLA-9:A novel bacteriocin against Bacillus spp.produced by Lactobacillus plantarum JLA-9 from Suan-Tsai,a traditional chinese fermented cabbage[J].Journal of Agricultural and Food Chemistry,2016,64(13):2754-2764.
[30]黄国昌,顾斌涛,黄朝,等.苯乳酸生产菌的筛选及鉴定[J].食品与发酵工业,2018,44(3):97-100.
[31]熊涛,宋苏华,黄涛,等.植物乳杆菌 NCU116 抑菌性能的研究[J].食品与发酵工业,2012,38(6):97-101.
[32]李强坤,柳陈坚,张园莲,等.植物乳杆菌对食源性病原菌作用的研究[J].食品科学,2016,37(11):18-23.
[33]Prema P,Smila D,Palavesam A,et al.Production and characterization of an antifungal compound(3-phenyllactic acid)produced by Lactobacillus plantarum strain[J].Food and Bioprocess Technology,2010,3(3):379-386.
[34]Dieuleveux V,Lemarinier S,Guéguen M.Antimicrobial spectrum and target site of d-3-phenyllactic acid[J].International Journal of Food Microbiology,1998,40(3):177-183.
[35]Ning Y,Yan A,Yang K,et al.Antibacterial activity of phenyllactic acid against Listeria monocytogenes and Escherichia coli by dual mechanisms[J].Food Chemistry,2017,228:533-540.