利用马铃薯全粉加工废水培养解淀粉芽胞杆菌产抗菌脂肽条件优化
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摘要
为实现马铃薯全粉加工废水资源高值化利用,以马铃薯全粉加工废水为基础培养基,采用Plackett-Burman设计和Box-Behnken响应面设计,对解淀粉芽胞杆菌BacillusamyloliquefaciensPC2利用马铃薯全粉加工废水发酵产抗菌脂肽的培养基进行优化。结果表明,马铃薯全粉加工废水蛋白质、游离氨基酸、游离淀粉和还原糖含量分别为16.0、2.11、0.18和1.29mg/mL,钾、钙和镁含量分别为1.07、0.05和0.10 mg/mL,铁、锌、锰和铜含量分别为7.10、2.34、0.25和0.09μg/mL。蔗糖、谷氨酸钠和硫酸铵是影响菌株PC2发酵抗菌脂肽的3个主要因素。菌株PC2以马铃薯全粉废水为基础培养基无抗菌脂肽产生,采用响应面法优化获得了产抗菌脂肽的最佳培养基配方为:蔗糖30.2 g,谷氨酸钠3.96 g,硫酸铵6.0 g,马铃薯全粉加工废水1000 mL,所得抗菌脂肽粗提物对金黄色葡萄球菌的抑菌圈直径为21.74 mm。采用单因素试验结合响应面法,获得最适发酵条件为:接种量6.0%,装液量50 mL/250 mL,初始pH 6.8,发酵温度31℃,摇床转速170 r/min,发酵时间35 h。获得的抗菌脂肽粗提物抑菌圈直径可达22.81 mm,较优化前增加了1.08 mm,发酵液活菌数由优化前的2.6×10~8 CFU/mL提高至3.2×10~(10) CFU/mL。试验结果为马铃薯全粉加工废水资源化利用奠定了基础。
The fermentation medium based on potato powder wastewater for lipopeptides production by Bacillus amyloliquefaciens PC2 strain was optimized by Plackett-Burman design and Box-Behnken response surface methodology. The results showed that contents of total protein, total free amino acids, total free starch and total reducing sugar in wastewater were 16.00 mg/mL, 2.11 mg/mL, 0.18 mg/mL and 1.29 mg/mL, respectively. K~+, Ca~(2+), Mg~(2+), Fe~(3+), Zn~(2+), Cu~(2+) and Mn~(2+) were present at concentration of 1.07 mg/mL, 0.05 mg/mL, 0.10 mg/mL, 7.10 μg/mL, 2.34 μg/mL, 0.25 μg/mL and 0.09 μg/mL, respectively. The fermentation broth of PC2 strain based on potato powder wastewater alone did not show lipopeptides inhibitory effect. The optimal medium components were as the follows: sucrose 30.2 g, sodium glutamate 3.96 g,(NH4)2 SO4 6.0 g, potato powder wastewater 1000 mL. The inhibitory zone diameter of lipopeptides fermented by the optimal medium against Staphylococcus aureus was 21.74 mm. Moreover, the fermentation conditions were optimized by single factor horizontal and response surface methodology. The optimal fermentation conditions were 6.0% inoculation, 50 mL/250 mL medium capacity, temperature at 31 ℃, medium initial pH 6.8, rotate rate at 170 r/min and fermentation time for 35 h. The inhibitory zone diameter of the lipopeptides crude extract under the optimized conditions against S. aureus was 22.81 mm, increased by 1.08 mm compared with that before optimization, and the viable bacteria number in the fermentation broth increased from 2.6×10~8 CFU/mL to 3.2×10~(10) CFU/mL. The results suggested the optimized medium based on potato powder wastewater could be used for the low-cost production of lipopeptides by B. amyloliquefaciens PC2.
The fermentation medium based on potato powder wastewater for lipopeptides production by Bacillus amyloliquefaciens PC2 strain was optimized by Plackett-Burman design and Box-Behnken response surface methodology. The results showed that contents of total protein, total free amino acids, total free starch and total reducing sugar in wastewater were 16.00 mg/mL, 2.11 mg/mL, 0.18 mg/mL and 1.29 mg/mL, respectively. K~+, Ca~(2+), Mg~(2+), Fe~(3+), Zn~(2+), Cu~(2+) and Mn~(2+) were present at concentration of 1.07 mg/mL, 0.05 mg/mL, 0.10 mg/mL, 7.10 μg/mL, 2.34 μg/mL, 0.25 μg/mL and 0.09 μg/mL, respectively. The fermentation broth of PC2 strain based on potato powder wastewater alone did not show lipopeptides inhibitory effect. The optimal medium components were as the follows: sucrose 30.2 g, sodium glutamate 3.96 g,(NH4)2 SO4 6.0 g, potato powder wastewater 1000 mL. The inhibitory zone diameter of lipopeptides fermented by the optimal medium against Staphylococcus aureus was 21.74 mm. Moreover, the fermentation conditions were optimized by single factor horizontal and response surface methodology. The optimal fermentation conditions were 6.0% inoculation, 50 mL/250 mL medium capacity, temperature at 31 ℃, medium initial pH 6.8, rotate rate at 170 r/min and fermentation time for 35 h. The inhibitory zone diameter of the lipopeptides crude extract under the optimized conditions against S. aureus was 22.81 mm, increased by 1.08 mm compared with that before optimization, and the viable bacteria number in the fermentation broth increased from 2.6×10~8 CFU/mL to 3.2×10~(10) CFU/mL. The results suggested the optimized medium based on potato powder wastewater could be used for the low-cost production of lipopeptides by B. amyloliquefaciens PC2.
引文
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[17]申光辉,冯孟,张志清,等.一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性[J].微生物学通报,2016,43(10):2197-2206.
[18]南楠,戚向阳,袁勇军,等.枯草芽孢杆菌WL17产抗菌物质的发酵条件优化研究[J].中国食品学报,2011,11(6):77-83.
[19]刘京兰,薛雅蓉,刘常宏.内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化[J].微生物学通报,2014,41(1):75-82.
[20]卢彩鸽,董红平,张殿朋,等.解淀粉芽胞杆菌MH71摇瓶发酵培养基及发酵条件优化[J].中国生物防治学报,2015,31(3):369-377.
[21]曹建康,姜微波.果蔬采后生理生化实验指导[M].北京:中国轻工业出版社,2007,41-43,57-78.
[22]杨胜远,韦锦,郑燮茹.解淀粉芽孢杆菌抗菌物质发酵培养基的优化[J].食品科学,2015,36(11):150-156.
[23]代志凯,张翠,阮征.试验设计和优化及其在发酵培养基优化中的应用[J].微生物学通报,2010,37(6):894-903.
[24]赵朋超,权春善,金黎明,等.氮源和碳源对解淀粉芽孢杆菌Q-426抗菌脂肽合成的影响[J].中国生物工程杂志,2012,32(10):50-56.
[25]胡飞,胡本进,周子燕,等.解淀粉芽胞杆菌JDF-6液态发酵条件优化及抑菌蛋白的分析[J].中国生物防治学报,2017,33(4):561-570.
[26]关晓欢.柠檬酸废水和马铃薯淀粉废水资源化培养解淀粉芽孢杆菌及其应用[D].大连:辽宁师范大学,2013.
[27]Bai Z H,Zhou C G,Cao J X,et al.Effects of Bacillus amyloliquefaciens biofertilizer on tea yield and quality[J].Agricultural Science and Technology,2014,15(11):1883-1887.
[28]Xu S,Bai Z,Jin B,et al.Bioconversion of wastewater from sweet potato starch production to Paenibacillus polymyxa biofertilizer for tea plants[J].Scientific Reports,2014,4(1):4131.
[29]方传记,陆兆新,孙力军,等.淀粉液化芽孢杆菌抗菌脂肽发酵培养基及发酵条件的优化[J].中国农业科学,2008,41(2):533-539.
[2]刘凌,崔明学,吴娜,等.马铃薯淀粉工业废水的环境影响与资源化利用[J].食品与发酵工业,2011,37(8):131-135.
[3]周添红,赵鑫,高祥虎,等.有机化纳米粘土基絮凝吸附材料对马铃薯淀粉加工废水的资源化处理[J].环境污染与防治,2011,33(11):44-48.
[4]WANG H F.Treatment of sweet potato starch wastewater with UASB[J].Advanced Materials Research,2013,699:234-237.
[5]颜东方,贠建民.马铃薯淀粉废水生产微生物絮凝剂菌株筛选及其营养条件优化[J].农业工程学报,2013,29(3):198-206.
[6]Guo J,Zhang Y,Zhao J,et al.Characterization of a bioflocculant from potato starch wastewater and its application in sludge dewatering[J].Applied Microbiology and Biotechnology,2015,99(13):5429-5437.
[7]王宏勋,邓张双,周帅,等.刺孢小克银汉霉菌株利用马铃薯淀粉废水生产GLA油脂的研究[J].中国油脂,2007,32(8):49-51.
[8]Muniraj I K,XiaO L,Hu Z,et al.Microbial lipid production from potato processing wastewater using oleaginous filamentous fungi Aspergillus oryzae[J].Water Research,2013,47(10):3477-3483.
[9]ZemenováJ,Sykora D,MaletínskáL,et al.Lipopeptides as therapeutics:applications and in vivo quantitative analysis[J].Bioanalysis,2017,9(2):215-230.
[10]汪静杰,赵东洋,刘永贵,等.解淀粉芽孢杆菌SWB16菌株脂肽类代谢产物对球孢白僵菌的拮抗作用[J].微生物学报,2014,54(7):778-785.
[11]张荣胜,王晓宇,罗楚平,等.解淀粉芽孢杆菌Lx-11产脂肽类物质鉴定及表面活性素对水稻细菌性条斑病的防治作用[J].中国农业科学,2013,46(10):2014-2021.
[12]蔡文韬,夏菠,夏延斌,等.解淀粉芽孢杆菌发酵液处理提高辣椒采后品质[J].农业工程学报,2013,29(23):253-261.
[13]Kaewklom S,Lumlert S,Kraikul W,et al.Control of Listeria monocytogenes on sliced bologna sausage using a novel bacteriocin,amysin,produced by Bacillus amyloliquefaciens isolated from Thai shrimp paste(Kapi)[J].Food Control,2013,32(2):552-557.
[14]周广麒,马蓬勃,刘俏,等.解淀粉芽孢杆菌Q-426培养基优化及抑菌活性的预测[J].中国生物工程杂志,2013,33(11):21-26.
[15]刘宇帅,孟利强,陈静宇,等.解淀粉芽胞杆菌TF28产抗菌脂肽培养基优化[J].微生物学杂志,2017,37(30):52-58.
[16]刘文波,熊燕红,秦春秀,等.解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HAB-7的培养基优化及抑菌活性[J].中国植保导刊,2017,37(6):5-13.
[17]申光辉,冯孟,张志清,等.一株蜂粮源拮抗细菌的分离鉴定及其抑菌物质特性[J].微生物学通报,2016,43(10):2197-2206.
[18]南楠,戚向阳,袁勇军,等.枯草芽孢杆菌WL17产抗菌物质的发酵条件优化研究[J].中国食品学报,2011,11(6):77-83.
[19]刘京兰,薛雅蓉,刘常宏.内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化[J].微生物学通报,2014,41(1):75-82.
[20]卢彩鸽,董红平,张殿朋,等.解淀粉芽胞杆菌MH71摇瓶发酵培养基及发酵条件优化[J].中国生物防治学报,2015,31(3):369-377.
[21]曹建康,姜微波.果蔬采后生理生化实验指导[M].北京:中国轻工业出版社,2007,41-43,57-78.
[22]杨胜远,韦锦,郑燮茹.解淀粉芽孢杆菌抗菌物质发酵培养基的优化[J].食品科学,2015,36(11):150-156.
[23]代志凯,张翠,阮征.试验设计和优化及其在发酵培养基优化中的应用[J].微生物学通报,2010,37(6):894-903.
[24]赵朋超,权春善,金黎明,等.氮源和碳源对解淀粉芽孢杆菌Q-426抗菌脂肽合成的影响[J].中国生物工程杂志,2012,32(10):50-56.
[25]胡飞,胡本进,周子燕,等.解淀粉芽胞杆菌JDF-6液态发酵条件优化及抑菌蛋白的分析[J].中国生物防治学报,2017,33(4):561-570.
[26]关晓欢.柠檬酸废水和马铃薯淀粉废水资源化培养解淀粉芽孢杆菌及其应用[D].大连:辽宁师范大学,2013.
[27]Bai Z H,Zhou C G,Cao J X,et al.Effects of Bacillus amyloliquefaciens biofertilizer on tea yield and quality[J].Agricultural Science and Technology,2014,15(11):1883-1887.
[28]Xu S,Bai Z,Jin B,et al.Bioconversion of wastewater from sweet potato starch production to Paenibacillus polymyxa biofertilizer for tea plants[J].Scientific Reports,2014,4(1):4131.
[29]方传记,陆兆新,孙力军,等.淀粉液化芽孢杆菌抗菌脂肽发酵培养基及发酵条件的优化[J].中国农业科学,2008,41(2):533-539.