菌酶联合法提取松墨天牛甲壳素
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摘要
甲壳素(chitin)是一种重要的可再生资源。松墨天牛(Monochamus alternatus)是松属植物的重要蛀干害虫,其壳质化程度高,是甲壳素的优质资源。以松墨天牛为原料,探讨"菌酶联合法"提取昆虫甲壳素的可行性和应用前景。结果表明:1)联合发酵最佳条件是8%松墨天牛粉末、8%葡萄糖液体培养基,接种11%的凝结芽孢杆菌,恒温摇床温度为50℃,转速为100r/min,同时加入蛋白酶K和凝结芽孢杆菌发酵5d后得到发酵产物,最终甲壳素得率为32.51%,产物中甲壳素纯度为72.78%,灰分残留量占甲壳素产品的1.42%,蛋白质残留量占甲壳素产品的25.74%。2)红外光谱鉴定结果表明,通过菌酶联合法制取的甲壳素相比于传统酸碱法提取的甲壳素品质好,具有良好的应用前景。
Chitin is an important renewable resource.Monochamus alternatus,is an key pest to genus Pinus.Compared to other insects,it has a higher degree of chitinization and a higher content of chitin,it is a highquality of chitin resource.In view of this,this paper usesd M.alternatus as the raw material to explore the feasibility and application prospect of the extraction of chitin by the"bacterium-protease combined method".The main findings were as follows:1)The optimum conditions for combined fermentation were 8% M.alternifolius powder,8%glucose liquid medium,inoculation of 11%Bacillus coagulans,constant temperature shaker at 50℃,rotation speed at 100 r/min,and simultaneous addition of proteinase K After fermentation with B.coagulans for 5 days.After being identified as chitin,the final yield of chitin was 32.51%,the purity of the chitin in the product was 72.78%,the residual ash content was 1.42% of the chitin product,and the protein residue was 25.74% of the chitin product.2)The infrared spectrum identification results indicated that the chitin prepared by the bacterium-protease combined method had higher quality than the traditional acid-alkali method extraction,and thus had more advantages and application prospects.
Chitin is an important renewable resource.Monochamus alternatus,is an key pest to genus Pinus.Compared to other insects,it has a higher degree of chitinization and a higher content of chitin,it is a highquality of chitin resource.In view of this,this paper usesd M.alternatus as the raw material to explore the feasibility and application prospect of the extraction of chitin by the"bacterium-protease combined method".The main findings were as follows:1)The optimum conditions for combined fermentation were 8% M.alternifolius powder,8%glucose liquid medium,inoculation of 11%Bacillus coagulans,constant temperature shaker at 50℃,rotation speed at 100 r/min,and simultaneous addition of proteinase K After fermentation with B.coagulans for 5 days.After being identified as chitin,the final yield of chitin was 32.51%,the purity of the chitin in the product was 72.78%,the residual ash content was 1.42% of the chitin product,and the protein residue was 25.74% of the chitin product.2)The infrared spectrum identification results indicated that the chitin prepared by the bacterium-protease combined method had higher quality than the traditional acid-alkali method extraction,and thus had more advantages and application prospects.
引文
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[29]王少梅,陈少莲,崔奕波.用氯仿-甲醇抽提法测定鱼体脂肪含量的研究[J].水生生物学报,1993(2):193-19WANG S M,CHEN S L,CUI Y B.On the procedures of chlooroform-methanol extraction for the determination of lipid content of fish samples[J].Journal of Hydrobiology,1993(2):193-196.(in Chinese)
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[31]刘高强,王晓玲.昆虫壳聚糖在食品工业中的开发应用[J].食品科技,2004(3):92-94.
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[34]倪天庆,胡思源.甲壳素及其衍生物壳聚糖的药理作用和临床应用研究进展[J].现代药物与临床,2012,27(3):313-316.
[35]梁玉佳.南极磷虾虾壳中甲壳素的制取与应用[D].大连:大连工业大学,2013.
[36]蒋小姝,莫海涛,苏海佳,等.甲壳素及壳聚糖在农业领域方面的应用[J].中国农学通报,2013,29(6):170-174.JIANG X T,MO H T,SU H J,et al.The application of chitin and chitosan in agriculture[J].Chinese Agricultural Science Bulletin,2013,29(6):170-174.(in Chinese)
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[38]赵黎明,夏文水.虾蟹壳生产甲壳素废碱液的成分分析[J].食品工业科技,2009,30(8):75-77.ZHAO L M,XIA W S.Component analysis of alkali wastewater from chitin processing[J].Food Industry Science and Technology,2009,30(8):75-77.(in Chinese)
[39]杨慧智.利用虾蟹壳制甲壳素大有可为[J].中国水产,1984(7):26-27.
[40]陈宇,马小军,谢红国,等.去除甲壳素/壳聚糖中蛋白质的研究进展[J].现代化工,2012(Supp.2):59-62.CHEN Y,MA X J,XIE H G,et al.Progress in removal protein from chitin/chitosan[J].Modern Chemical Industry,2012(Supp.2):59-62.(in Chinese)
[2]孙绪兵,杜京城,由耀辉.纳米甲壳素的制备、改性及应用研究进展[J].高分子通报,2016(8):71-80.
[3]陈耀华.人类第6生命要素---甲壳质(几丁质、几丁聚糖)种子(续三)[J].锦州医学院学报,2000,21(1):65-68.
[4]刘婉.甲壳素纤维及其应用[J].纺织科技进展,2015(03):4-7.
[5]侯佰立.、壳聚糖改良工艺的研究[D].广州:广东海洋大学,2011.
[6]刘高强,刘卫星,魏美才,等.虫类甲壳素/壳聚糖的研究及其开发前景[J].西北林学院学报,2005,20(4):143-146.LIU G Q,LIU W X,WEI M C,et al.Development situation and perspectives on chitin and chitosan from insects[J].Journal of Northwest Forestry University,2005,20(4):143-146.(in Chinese)
[7]刘高强,刘卫星,魏美才,等.从害虫马尾松毛虫中提取甲壳素的初步研究[J].西北林学院学报,2007,22(2):138-141.LIU G Q,LIU W X,WEI M C,et al.A preliminary study on the extraction of chitin from pupa of Dendrolimus punctatus[J].Journal of Northwest Forestry University,2007,22(2):138-141.(in Chinese)
[8]蒋晓宇,刘高强,魏美才.松毛虫作为新食品工业原料的研究进展[J].食品科技,2009,34(3):111-114.JIANG X Y,LIU G Q,WEI M C.Research advances in pine caterpillar(Dendrolimus spp.)as a new resource of food industry[J].Food Science and Technology,2009,34(3):111-114.(in Chinese)
[9]朱巽,旷建军,林仲桂.南岳松墨天牛生物学特性研究[J].西北林学院学报,2006,21(5):113-115.ZHU X,KUANG J J,LIN Z G.Bionomics of Monochamus alternatus qccurring in Nanyue Mountains[J].Journal of Northwest Forestry University,2006,21(5):113-115.(in Chinese)
[10]郝德君,张永慧,戴华国,等.松墨天牛对寄主树木的产卵选择[J].昆虫学报,2005,48(3):460-464.HAO D J,ZHANG Y H,DAI H G,et al.Oviposition preference of Monochamus alternatus Hope(Coleoptera:Cerambycidae)to host plants[J].Acta Entomologies,2005,48(3):460-464.(in Chinese)
[11]宋红敏,徐汝梅.松墨天牛的全球潜在分布区分析[J].昆虫知识,2006,43(4):535-539.SONG H M,XU R M.Global potential geographical distribution of Monochamus alternatus[J].Entomological Knowledge,2006,43(4):535-539.(in Chinese)
[12]朱诚棋,王博,沈婧,等.松墨天牛综合防治进展[J].中国植保导刊,2017,37(2):19-24.ZHU C Q,WANG B,SHEN J,et al.Research progress on integrated controlling techniques against Monochamus alternatus[J].China Plant Protection Guide,2017,37(2):19-24.(in Chinese)
[13]展茂魁.马尾松蛀干害虫种群动态与松材线虫病的关系及松褐天牛天敌研究[D].北京:中国林业科学研究院,2014.
[14]杨建霞,郝德君,周曙东,等.寄主植物对松墨天牛的营养效应及对体内酯酶与羧酸酯酶活性的影响[J].林业科学,2009,45(1):97-101.YANG J X,HAO D J,ZHOU S D,et al.Effects of plant hosts on the nutrient indices and activity of the esterase and carboxylesterase of Monochamus alternatus[J].Forestry Science and Technology,2009,45(1):97-101.(in Chinese)
[15]李彦艳,张闪闪,任国栋.甲壳动物、昆虫、真菌中甲壳素的提取进展[J].食品研究与开发,2015,36(7):122-126.LI Y Y,ZHANG S S,REN G D.The progress of chitin extracted from crustaceans,insects and fungi[J].Food Research and Development,2015,36(7):122-126.(in Chinese)
[16]李晓丹,赖鹤鋆,林婉琪,等.潮汕地区虾蟹壳提取甲壳素的工艺优化[J].湖北农业科学,2015,54(3):668-670.
[17]胡瑞.酶解法制备特定重均分子量及窄分布壳寡糖的工艺研究[D].武汉:武汉理工大学,2012.
[18]严丽平.微波法制备壳聚糖的试验研究[D].上海:东华大学,2004.
[19]张其清,张瑗,王临钊,等.一种壳聚糖的提取和制备方法[P].2017-06-13.
[20]段元斐,何忠诚,庄桂东,等.甲壳素提取新工艺的研究[J].食品工业,2007(3):7-8.
[21]杜敬河.甲壳素酶产生菌的筛选及酶法制备甲壳低聚糖的研究[D].广州:华南农业大学,2016.
[22]ARBIA W,ARBIA L,ADOUR L,et al.Chitin extraction from crustacean shells using biological methods-a review[J].Food Technology and Biotechnology,2013(51):12.
[23]CIRA L A,HUERTA S,HALL G M,et al.Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery[J].Process Biochemistry,2002(37):359-1366.
[24]FLORES-ALBINO B,ARIAS L,GMEZ J,et al.Chitin and L(+)-lactic acid production from crab(Callinectes bellicosus)wastes by fermentation of Lactobacillus sp.B2using sugar cane molasses as carbon source[J].Bioprocess and Biosystems Engineering,2012(35):1193-1200.
[25]GHORBEL-BELLAAJ O,HAJJI S,YOUNES I,et al.Optimization of chitin extraction from shrimp waste with Bacillus pumilus A1using response surface methodology[J].International Journal of Biological Macromolecules,2013(61):243-250.
[26]GHORBEL-BELLAAJ O,HMIDET N,JELLOULI K,et al.Shrimp waste fermentation with seudomonas aeruginosa A2:optimization of chitin extraction conditions through plackettburman and response surface methodology approaches[J].International Journal of Biological Acromolecules,2011(48):596-602.
[27]莫姣娇,孙强.昆虫蛋白质含量的测定方法[J].安徽农学通报,2007,13(11):40-41.
[28]涂荣秀,马珺,陈志兵,等.饲用昆虫常规营养成分指标的测定[J].上海畜牧兽医通讯,2006(6):49-50.
[29]王少梅,陈少莲,崔奕波.用氯仿-甲醇抽提法测定鱼体脂肪含量的研究[J].水生生物学报,1993(2):193-19WANG S M,CHEN S L,CUI Y B.On the procedures of chlooroform-methanol extraction for the determination of lipid content of fish samples[J].Journal of Hydrobiology,1993(2):193-196.(in Chinese)
[30]郭宝华.四种昆虫甲壳素的提取及昆虫壳聚糖和羧甲基壳聚糖的制备[D].北京:中国林业科学研究院,2005.
[31]刘高强,王晓玲.昆虫壳聚糖在食品工业中的开发应用[J].食品科技,2004(3):92-94.
[32]雷朝亮,钟昌珍,宗良炳,等.蝇蛆几丁糖保健功能的评价[J].华中农业大学学报,1998,17(2):22-26.
[33]何玲,于波,王慧丽,等.甲壳素/壳聚糖生物抗菌敷料在皮肤组织工程中的应用[J].中国组织工程研究与临床康复,2010,14(38):7149-7152.
[34]倪天庆,胡思源.甲壳素及其衍生物壳聚糖的药理作用和临床应用研究进展[J].现代药物与临床,2012,27(3):313-316.
[35]梁玉佳.南极磷虾虾壳中甲壳素的制取与应用[D].大连:大连工业大学,2013.
[36]蒋小姝,莫海涛,苏海佳,等.甲壳素及壳聚糖在农业领域方面的应用[J].中国农学通报,2013,29(6):170-174.JIANG X T,MO H T,SU H J,et al.The application of chitin and chitosan in agriculture[J].Chinese Agricultural Science Bulletin,2013,29(6):170-174.(in Chinese)
[37]黄立新,谢林明,崔毅华.蛹壳聚糖对真丝织物的抗菌防皱整理[J].丝绸,2003(4):36-37.HUANG L X,XIE L M,CUI Y H.Anti-bacterial and creaseresist finishing of silk fabrics with chrysalis chitosan[J].Silk,2003(4):36-37.(in Chinese)
[38]赵黎明,夏文水.虾蟹壳生产甲壳素废碱液的成分分析[J].食品工业科技,2009,30(8):75-77.ZHAO L M,XIA W S.Component analysis of alkali wastewater from chitin processing[J].Food Industry Science and Technology,2009,30(8):75-77.(in Chinese)
[39]杨慧智.利用虾蟹壳制甲壳素大有可为[J].中国水产,1984(7):26-27.
[40]陈宇,马小军,谢红国,等.去除甲壳素/壳聚糖中蛋白质的研究进展[J].现代化工,2012(Supp.2):59-62.CHEN Y,MA X J,XIE H G,et al.Progress in removal protein from chitin/chitosan[J].Modern Chemical Industry,2012(Supp.2):59-62.(in Chinese)