辣椒炭疽病菌拮抗内生菌筛选及其对辣椒采后保鲜效果的研究
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摘要
从辣椒果实中分离、筛选出了一株对辣椒炭疽病菌具有拮抗作用的内生细菌P5,通过菌体、菌落形态观察、生理生化测定及16S r DNA序列分析,鉴定P5为枯草芽孢杆菌。以辣椒炭疽病作为指示菌,测定其发酵滤液理化性质。用发酵滤液处理辣椒进行采后保鲜研究,测定品质指标:质量损失率、腐烂率、可滴定酸含量、Vc含量、总糖含量、叶绿素含量等品质指标。结果表明,发酵滤液热稳定性良好,在p H 8.0以下,40~100℃范围内,抑菌活性稳定无变化,121℃处理30 min活性有所下降,为对照组的85%;对p H稳定,在室温下,p H 2.0~12范围内,抑菌活性不发生变化;对紫外线稳定,经紫外光照射12 h,其活性仍为对照的89.87%。发酵滤液处理过的辣椒保鲜效果明显优于无菌水对照组,显著降低了辣椒失重率和腐烂率,并有效抑制了果实中可滴定酸、Vc、总糖、叶绿素等含量的减少,对辣椒有显著的保鲜效果。
An antagonistic endophytic bacterium strain(P5) against Colletotrichum capsici was isolated from capsicum fruits, and identified as Bacillus subtilis based on observations of bacterial cell and colony morphologies, physiological and biochemical measurements, and 16 S r DNA sequence analysis. The physical and chemical properties of the crude fermentation extract were determined with Colletotrichum capsici as the indicative bacterium. In addition, the fresh-keeping effects of the crude fermentation extract of P5 on postharvest capsicum were studied by measuring the weight loss rate, rate of decay, titratable acidity, and the contents of vitamin C, total carbohydrates, and chlorophyll, along with other quality indices of capsicum. The results indicated that the crude fermentation extract had good thermal stability, and the antibacterial activity was stable, showing no changes in an environment with a p H value <8.0 and a temperature range from 40~100 ℃. The antibacterial activity decreased to 85% of that of the control group after treatment at 121 ℃ for 30 min. No changes in the antibacterial activity were observed from p H 2.0 to p H 12.0 under room temperature, showing good stability to p H fluctuations. The crude fermentation extract was stable to ultraviolet(UV) light and could maintain 89.87% of the initial activities under UV irradiation for 12 h. The crude fermentation extract treatment group exhibited better fresh-keeping effect on postharvest capsicum compared with the sterile water treatment group, and the fermentation extract could effectively reduce the weight loss rate and rate of decay. The treatment inhibited the reductions in titratable acidity and the contents of vitamin C, total carbohydrates, and chlorophyll, showing an apparent fresh-keeping effect on capsicum.
An antagonistic endophytic bacterium strain(P5) against Colletotrichum capsici was isolated from capsicum fruits, and identified as Bacillus subtilis based on observations of bacterial cell and colony morphologies, physiological and biochemical measurements, and 16 S r DNA sequence analysis. The physical and chemical properties of the crude fermentation extract were determined with Colletotrichum capsici as the indicative bacterium. In addition, the fresh-keeping effects of the crude fermentation extract of P5 on postharvest capsicum were studied by measuring the weight loss rate, rate of decay, titratable acidity, and the contents of vitamin C, total carbohydrates, and chlorophyll, along with other quality indices of capsicum. The results indicated that the crude fermentation extract had good thermal stability, and the antibacterial activity was stable, showing no changes in an environment with a p H value <8.0 and a temperature range from 40~100 ℃. The antibacterial activity decreased to 85% of that of the control group after treatment at 121 ℃ for 30 min. No changes in the antibacterial activity were observed from p H 2.0 to p H 12.0 under room temperature, showing good stability to p H fluctuations. The crude fermentation extract was stable to ultraviolet(UV) light and could maintain 89.87% of the initial activities under UV irradiation for 12 h. The crude fermentation extract treatment group exhibited better fresh-keeping effect on postharvest capsicum compared with the sterile water treatment group, and the fermentation extract could effectively reduce the weight loss rate and rate of decay. The treatment inhibited the reductions in titratable acidity and the contents of vitamin C, total carbohydrates, and chlorophyll, showing an apparent fresh-keeping effect on capsicum.
引文
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[13]高芬,吴元华,穆凌霄,等.一株拮抗链霉菌的室内抑菌活性及发酵液理化性质测定[J].中国植物病理学会2007年学术年会论文集,2007GAO Feng,WU Yuan-hua,MU Ling-xiao,et al.Inhibition effect of antagonistic micioorganism against alternaria alternate and the Physical and chemical properties of the fermented broth[J]Proceedings of the Annual Meeting of Chinese Society for Plant Pathology,2007
[14]靳敏,夏玉宇.食品技术检验[M].北京:化学工业出版社,2003
[15]李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社,2000
[16]食品分析与感官评定[M].农业出版社,2002
[17]赵奇,杨玉珍,郭运宏,等.油用牡丹丹皮提取液对青椒的保鲜效应[J].食品工业科技,2015,36(2):339-342ZHAO Qi,YANG Yu-zhen,GUO Yun-hong,WANG Guo-xia,et al.Preservation effects of cortex oil peony extracts on green pepper[J]Science and Technology of Food Industry,2015,36(2):339-342
[18]Yu G Y,Sinclair J B,Hartman G L,et al.Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J].Soil Biology and Biochemistry,2002,34(7):955-963
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[22]Bernal G,Illanes A,Ciampi L.Isolation and partial purification of a metabolite from a mutant strain of Bacillus sp.with antibiotic activity against plant pathogenic agents[J].Electronic Journal of Biotechnology,2002,5(1):7-8
[23]Tsuge K,Ano T,Shoda M.Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8[J].Archives of Microbiology,1996,165(4):243-251
[24]Paik S H,Chakicherla A,Hansen J N.Identification and characterization of the structural and transporter genes for,and the chemical and biological properties of,sublancin 168,a novel lantibiotic produced by Bacillus subtilis 168[J].Journal of Biological Chemistry,1998,273(36):23134-23142
[25]Hansen J N.Antibiotics synthesized by posttranslational modification[J].Annual Reviews in Microbiology,1993,47(1):535-564
[26]何红,邱思鑫,蔡学清,等.辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定[J].微生物学报,2004,44(1):13-18HE Hong,QIU Si-Xin,CAI Xue-qing,et al.Colonization in plants and identification of endophytic bacteria BS-1and BS-2 from capsicum annuum[J].Acta Microbiologica Sinica,2004,44(1):13-18
[27]孔建,王文夕,赵白鸽.枯草芽孢杆菌B-903菌株抗菌物质的研究[J].微生物学报,1992,32(6):445-449KONG Jian,WAN Wen-xi,Zh AO Bai-ge,etal.Study on characteristic of antifungu substance fromb Bacillus subtilis B-903 strain[J]Acta Microbiologica Sinica.1992,32(6):445-449
[28]别小妹,吕凤霞,陆兆新,等.枯草芽孢杆菌fmb R抗菌物质稳定性研究[J].食品科学,2006,27(6):104-108BIE Xiao-mei,LU Feng-xia,LU Zhao-xin,et al.Study on stability of antimicrobial substance produced by bacillussubtilis fmb R[J]Food Scicence.2006,27(6):104-108
[29]Nakayama S,Takahashi S,Hirai M,et al.Isolation of new variants of surfactin by a recombinant Bacillus subtilis[J].Applied Microbiology and Biotechnology,1997,48(1):80-82
[2]庞学群,张昭其,黄雪梅.果蔬采后病害的生物防治(综述)[J].热带亚热带植物学报,2002,10(2):186-192PANG Xue-qun,ZHANG Zhao-qi,ZHUANG Xue-mei.Biological control of postharvest diseases of fruits and vegetables[J].Journal of Tropical and Subtropical Botany2002,10(2):186-192
[3]Assis J S.Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit[J].Postharvest Biology and Technology,2001,23(1):33-39
[4]吴士云,孙力军,周声,等.内生菌EJS防治油桃采后病害的研究[J].食品科技,2007,32(6):239-242WU Shi-yun,SUN Li-jun,ZHOU Sheng,et al.Study on the inhibition of endophytic strain EJS on penicilliosis of nectarine after harvest[J].Food Science and Technology,2007,32(6):239-242
[5]孙力军,陆兆新,刘俊,等.一株产胞外多糖植物内生菌EJS-3菌株的分离和鉴定[J].食品科学,2006,27(7):65-68SUN Li-jun,LU Zhao-xin,LIU Jun,et al.Isolation and identification of an endophytic strain EJS-3 producing exopolysaccharide[J].Food Science,2006,27(7):65-68
[6]刘西莉,马安捷,林吉柏,等.精甲霜灵与外消旋体甲霜灵对掘氏疫霉菌的抑菌活性比较[J].农药学学报,2003,5(3):45-49LIU Xi-li,MA An-jie,LIN Ji-bai,et al.The comparison of inhibitory action between stereoisomers of metalaxyl[J].Chinese Journal of Pesticide Science,2003,5(3):45-49
[7]刘杰凤,韩寒冰,张进凤,等.茄类内生菌的分离及拮抗细菌的筛选[J].农业科学与技术:英文版,2009,10(1):35-38LIU Jie-feng,HAN Han-bing,ZHANG Jing-feng,et al.Isolation of endophyte and screening for antagonistic bacteria in solanaceae[J].Journal of Anhui Agri.Sci,2009,10(1):35-38
[8]童志丹,易有金,杨建奎,等.番茄采后早疫病菌拮抗菌的筛选及发酵条件优化研究[J].安徽农业科学,2011,39(27):16645-16647TONG Zhi-dan,YI You-jin,YANG Jian-kun,et al.Screening of antagonistic bacteria for alternaria solani and optimizing of its fermentation[J].Journal of Anhui Agri.Sci,2011,39(27):16645-16647
[9]常见细菌系统鉴定手册[M].科学出版社,2001
[10]童志丹.番茄采后早疫病菌拮抗微生物的筛选,鉴定及发酵条件优化[D].湖南农业大学,2011 TONG Zhi-dan.Sereening,identification and fermentation conditions optimization of antagonistic strains on tomato postharvest diseases caused by alternaria solani[D].Hunan Agricultural University,2011
[11]何红,蔡学清,洪永聪,等.辣椒内生细菌的分离及拮抗菌的筛选[J].中国生物防治,2002,18(4):171-175HE Hong,CAI Xue-qing,HONG Yong-cong,et al.Selection of endophytic antifungal bacteria from capsicum[J].Chinese Journal of Biological Control,2002,18(4):171-175
[12]王金爱,许文涛,梁志宏,等.一株生防菌LX-07的鉴定及其拮抗物的理化特性[J].食品工业科技,2011,3:88-90WANG Jin-ai,XU Wen-tao,LIANG Zhi-bong,et al.Identification of a bio-control bacteria LX-07 and characterizations of its antifungal substance[J].Science and Technology of Food Industry,2011,3:88-90
[13]高芬,吴元华,穆凌霄,等.一株拮抗链霉菌的室内抑菌活性及发酵液理化性质测定[J].中国植物病理学会2007年学术年会论文集,2007GAO Feng,WU Yuan-hua,MU Ling-xiao,et al.Inhibition effect of antagonistic micioorganism against alternaria alternate and the Physical and chemical properties of the fermented broth[J]Proceedings of the Annual Meeting of Chinese Society for Plant Pathology,2007
[14]靳敏,夏玉宇.食品技术检验[M].北京:化学工业出版社,2003
[15]李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社,2000
[16]食品分析与感官评定[M].农业出版社,2002
[17]赵奇,杨玉珍,郭运宏,等.油用牡丹丹皮提取液对青椒的保鲜效应[J].食品工业科技,2015,36(2):339-342ZHAO Qi,YANG Yu-zhen,GUO Yun-hong,WANG Guo-xia,et al.Preservation effects of cortex oil peony extracts on green pepper[J]Science and Technology of Food Industry,2015,36(2):339-342
[18]Yu G Y,Sinclair J B,Hartman G L,et al.Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani[J].Soil Biology and Biochemistry,2002,34(7):955-963
[19]Gond S K,Bergen M,Torres M,et al.Endophytic Bacillus spp.produce antifungal lipopeptides and induce host defence gene expression in maize[J].Microbiological Research,2014
[20]Pérez-García A,Romero D,De Vicente A.Plant protection and growth stimulation by microorganisms:biotechnological applications of Bacilli in agriculture[J].Current Opinion in Biotechnology,2011,22(2):187-193
[21]Nakayama S,Takahashi S,Hirai M,et al.Isolation of new variants of surfactin by a recombinant Bacillus subtilis[J].Applied Microbiology and Biotechnology,1997,48(1):80-82
[22]Bernal G,Illanes A,Ciampi L.Isolation and partial purification of a metabolite from a mutant strain of Bacillus sp.with antibiotic activity against plant pathogenic agents[J].Electronic Journal of Biotechnology,2002,5(1):7-8
[23]Tsuge K,Ano T,Shoda M.Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8[J].Archives of Microbiology,1996,165(4):243-251
[24]Paik S H,Chakicherla A,Hansen J N.Identification and characterization of the structural and transporter genes for,and the chemical and biological properties of,sublancin 168,a novel lantibiotic produced by Bacillus subtilis 168[J].Journal of Biological Chemistry,1998,273(36):23134-23142
[25]Hansen J N.Antibiotics synthesized by posttranslational modification[J].Annual Reviews in Microbiology,1993,47(1):535-564
[26]何红,邱思鑫,蔡学清,等.辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定[J].微生物学报,2004,44(1):13-18HE Hong,QIU Si-Xin,CAI Xue-qing,et al.Colonization in plants and identification of endophytic bacteria BS-1and BS-2 from capsicum annuum[J].Acta Microbiologica Sinica,2004,44(1):13-18
[27]孔建,王文夕,赵白鸽.枯草芽孢杆菌B-903菌株抗菌物质的研究[J].微生物学报,1992,32(6):445-449KONG Jian,WAN Wen-xi,Zh AO Bai-ge,etal.Study on characteristic of antifungu substance fromb Bacillus subtilis B-903 strain[J]Acta Microbiologica Sinica.1992,32(6):445-449
[28]别小妹,吕凤霞,陆兆新,等.枯草芽孢杆菌fmb R抗菌物质稳定性研究[J].食品科学,2006,27(6):104-108BIE Xiao-mei,LU Feng-xia,LU Zhao-xin,et al.Study on stability of antimicrobial substance produced by bacillussubtilis fmb R[J]Food Scicence.2006,27(6):104-108
[29]Nakayama S,Takahashi S,Hirai M,et al.Isolation of new variants of surfactin by a recombinant Bacillus subtilis[J].Applied Microbiology and Biotechnology,1997,48(1):80-82