鱼池中二甲基异莰醇和土臭味素的含量、来源及产生影响因素的研究
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摘要
目前在池塘中养殖的水产品有许多带有不同程度的土腥异味,降低水产品的食用口感和食用价值,并危及养殖效益。现有研究表明,水产品土腥异味主要由浮游藻类和放线菌分泌的土腥异味物质土臭味素(Geosmin)与二甲基异莰醇(MIB)引起。为研究我国精养鱼池中Geosmin与MIB存在现状、主要来源及产生的主要限制因素,筛选有效的消除异味技术,提高水产品质量安全水平,本文开展了相关试验研究工作,取得如下结论。
1.采用顶空固相微萃取-气质联用色谱法(HSPME-GC-MS)可有效测定水体中Geosmin与MIB含量,滤除浮游藻类措施对Geosmin测定结果有显著影响,而对MIB测定结果基本没有影响。建立了针对淡水鱼的感官评价方法,采用感官评价方法可以将鱼的土腥异味程度划分为5个级别:正常、轻微、明显、较重和极重,依据鱼体土腥异味程度可推测鱼体中MIB大致含量,人们对鱼肉中MIB的异味阈值在500 ng/kg。
2.北京地区淡水鱼池中Geosmin是主要的土腥异味物质成份,普遍存在,含量为1.22～35.58 ng/L,MIB在部分鱼池中被检出,含量1.39～6.00 ng/L。经过分析表明:鱼池中Geosmin含量与浮游藻类总生物量正相关,浮游藻类中的颗粒直链藻Melosira granulata和条纹小环藻Cyclotella striata是北京地区夏秋季节淡水精养鱼池中能够产生Geosmin的主要藻类。Geosmin和MIB含量均与放线菌无关。天津地区寡盐水养鱼池水体中普遍存在Geosmin和MIB,其中MIB是鱼池中的主要异味物质成份,浓度0.33～5302.70ng/L,Geosmin浓度相对较低,0.29～12.10ng/L。天津地区鱼池中MIB含量与放线菌正相关。池水盐度对MIB浓度及放线菌数量有重要影响。
3.经高氏1号培养基分离培养,北京地区鱼池水中放线菌生物量(1～27)×10~6个/L,天津鱼池水中放线菌生物量(0.01～1.50)×10~6个/L,两地放线菌优势种均为链霉菌Streptomyces。北京鱼池中放线菌生物量明显高于天津鱼池,但数学分析显示北京鱼池中较高生物量的放线菌与异味物质(Geosmin和MIB)无关,而天津鱼池中相对较低生物量的放线菌与异味物质MIB有关。这可能是由于天津鱼池是寡盐水(盐度1～5),盐度促进放线菌产生异味物质,尤其是MIB。
4.本文采用16SrRNA方法,结合菌落形态特征研究了天津鱼池中放线菌的种类组成,并构建系统发育树关系图。在鱼池中共分离出常见、优势放线菌种4属9种。其中链霉菌是优势种,链霉菌、类诺卡氏菌Nocardioides、小单胞菌Micromonospora是天津鱼池放线菌常见种。鱼池中链霉菌生物量(0.007～0.575)×10~6个/L;类诺卡氏菌生物量(0.0033～0.07)×10~6个/L;小单胞菌生物量(0.00133～0.4776)×10~6个/L。放线菌总生物量(0.01～1.50)×10~6个/L。
5.对由天津鱼池中分离到的6株链霉菌的产味能力进行测定,发现不同种类链霉菌产生异味物质能力差异极大,产MIB能力由高到低依次为:Sp2(5570.0ng/ml或2×10~6ng/g)、Sp3(776.0ng/ml或8.5×10~4ng/g)、Sp5(72.4ng/ml或2.6×10~4ng/g)、Sp4(19.8ng/ml或7.1×10~3ng/g)、Sp1(14.3ng/ml或5.8×10~3ng/g)、Sp6(2.6ng/ml或7.6×10~2ng/g)。Sp2等链霉菌是中国天津寡盐水鱼池土腥异味主要产源。
6.经过形态及培养特征观察以及分子生物学分析,确定放线菌SP2是早期链霉菌Streptomyces praecox。该菌产生MIB的量显著高于产生Geosmin的量。MIB和Geosmin主要存在于Streptomyces praecox细胞外。温度和盐度是影响Streptomycespraecox产生MIB和Geosmin的重要因素,25℃～30℃是Streptomyces praecox产生两种异味物质的“突变区域”;在培养盐度2和1时,MIB和Geosmin的产生量最大。NH_4~+、pH及其交互作用对Streptomyces praecox产生MIB有显著影响;NH_4~+、PO_4~(3-)和pH,以及任二者间的交互作用对Streptomyces praecox产生Geosmin有极显著影响。栅藻Scenedesmus obliquus胞外产物能促进Streptomyces praecox产生MIB和Geosmin。
7.本文试验了各种消除异味的技术方法,发现采用清水、流水、活性炭消除鱼的异味均有一定效果。用流水处理15 h和用活性炭吸附法处理12 h,均可有效地去除鲤Cyprinuscarpio的异味。施用光合细菌,可降低或消除鲂、草鱼和淡水白鲨的异味,但作用时间较长。
Many aquatic products in ponds have different degrees of earthy-musty odor,which decreases their taste and edible value and threatens the cultivation efficiency.Existing research has demonstrated that earthy-musty odor,are caused by geosmin and MIB excreted by planktonic algae and actinomycetes.This study was conducted to evaluate the present condition,the primary source and the major limiting factor of geosmin and MIB in intensive cultivation ponds.This information was then used to select an effective technology for the elimination of earthy-musty odor and to improve the level of quality and safety of aquatic products.The results of this study were as follows.
1.The head space solid-phase microextraction-gas chromatography-mass spectrophotometer(HSPME-GC-MS) method can be used to effectively measure the content of geosmin and MIB in water.Filtering algae had a significant impact on the levels of geosmin,but little effect on the concentrations of MIB.In addition,the results of this study enabled establishment of a sensory evaluation method for freshwater fish.In this method,the earthy-musty odor of fish was divided into five levels:normal,mild, clear,heavy and very heavy.The MIB content in the fish could be estimated based on the level of earthy-musty odor.The human olfact value for MIB in fish meat was 500 ng/kg.
2.Geosmin was found to be the primary element responsible for the earthy-musty odor of fish in freshwater ponds in Beijing.Furthermore,geosmin was present in almost all ponds,as indicated by concentrations of 1.22～35.58 ng/L.MIB was present in some fishponds,with average concentrations of 1.39-6.00 ng/L being observed.Additionally, the geosmin content in fishponds was found to be related to the gross biomass of algae in the ponds.Cyclotella striata and Melosira granulata were found to be the primary algae responsible for the production of geosmin in freshwater fishponds in summer and autumn in Beijing.The MIB and geosmin content were not related to actinomycetes.Geosmin and MIB were found to be widespread in saline water ponds in Tianjin.In these ponds, MIB was the main factor responsible for off-flavor and was present at concentrations of 0.33-5302.70 ng/L.However,the concentration of geosmin in Tianjin was relatively low,as indicated by observed values of 0.29-12.10 ng/L.The MIB content was positively correlated with actinomycetes in fishponds in Tianjin.Water salinity was found to have an important impact on the concentration of actinomycetes and MIB.
3.When separately cultivated using Agar No.1,the biomass of actinomycetes in fishponds in Beijing was found to be(1-27)×10~6/L,while that of actinomycetes in fishponds in Tianjin was(0.01-1.50)×10~6/L.The dominant species of actinomycetes in both areas was Streptomyces.The biomass of actinomycetes in fishponds in Beijing was higher that of fishponds in Tianjin.However,mathematical analysis revealed that the higher biomass of actinomycetes in the fishponds in Beijing was not related to the concentrations of earthy-musty odor elements(geosmin and MIB),while the relatively low biomass of actinomycetes in fishponds in Tianjin was related to the MIB.This may have been due to the presence of saline water ponds in Tianjin(salinity 1 to 5),because salinity may cause actinomycetes to produce odorous substances such as MIB.
4.In this paper,sequencing of the 16SrRNA combined with evaluation of the colony morphology was used to identify the species of actinomycetes in the fishponds in Tianjin, and a phylogenetic tree diagram was constructed.Four genera and 9 species of common and dominant actinomycetes were identified,with Streptomyces being the dominant genus.The most common genera in Tianjin were Streptomyces,Nocardioides,and Micromonospora,which were present in concentrations of(0.007-0.575)×10~6/L, (0.0033-0.07)×10~6/L;and(0.00133-0.4776)×10~6/L,respectively.The total biomass of actinomycetes was(0.01-1.50)×10~6 / L.
5.Six types of Streptomyces were observed in fishponds in Tianjin.Furthermore,the earthy-musty odor production among Streptomyces was found to differ greatly. Specifically,the ability of the various species of Streptomyces found in fishponds in Tianjin to produce MIB were as follows:Sp2(5570.0 ng/ml or 2×10~6 ng/g),Sp3(776.0 ng/ml or 8.5×10~4 ng/g),Sp5(72.4 ng/ml or 2.6×10~4 ng/g),Sp4(19.8 ng/ml or 7.1×103 ng/g),Sp1(14.3 ng/ml or 5.8×103 ng/g),Sp6(2.6 ng/ml or 7.6×102 ng/g).Sp2 and other types of Streptomyces were found to be the primary cause of off-flavor in saline water ponds in Tianjin,China.
6.After observation of the morphological and cultivation features and molecular analysis,Actinomycete SP2 was identified as Streptomyces praecox.This species was found to produce much more MIB than geosmin,and both of these compounds were found to primarily exist outside of the cells.Temperature and salinity were also important factors that affected the production of MIB and geosmin by Streptomyces praecox. Specifically,25℃-30℃was found to be the mutation region for Streptomyces praecox, at which they produced earthy-musty odor substances.Additionally,Streptomyces praecox produced the greatest amounts of MIB and geosmin when cultivated at salinities of 2 and 1,respectively.NH_4~+,pH and their interactions had a significant impact on the ability of Streptomyces praecox to produce MIB,while NH_4~+,PO_4~(3-),and pH,and any interaction between these compounds had a significant effect on its production of geosmin.Extracellular metabolites produced by Scenedesmus obliquus may accelerate the production of MIB and Streptomyces praecox to produce MIB and geosmin.
7.In this study,a variety of techniques for reducing earthy-musty odor were tested. The results revealed that the use of clear water,flowing water and active carbon can eliminate earthy-musty odor of fish to some extent.For example,15 h of treatment with flowing water and the use of active carbon adsorption treatment for 12 h were found to effectively remove earthy-musty odor associated with Cyprinus carpio.In addition,the application of photosynthetic bacteria was also found to reduce or eliminate earthy-musty odor of John Dory,grass carp and fresh water white sharks,but this required a longer treatment than the aforementioned treatments.
1.采用顶空固相微萃取-气质联用色谱法(HSPME-GC-MS)可有效测定水体中Geosmin与MIB含量,滤除浮游藻类措施对Geosmin测定结果有显著影响,而对MIB测定结果基本没有影响。建立了针对淡水鱼的感官评价方法,采用感官评价方法可以将鱼的土腥异味程度划分为5个级别:正常、轻微、明显、较重和极重,依据鱼体土腥异味程度可推测鱼体中MIB大致含量,人们对鱼肉中MIB的异味阈值在500 ng/kg。
2.北京地区淡水鱼池中Geosmin是主要的土腥异味物质成份,普遍存在,含量为1.22～35.58 ng/L,MIB在部分鱼池中被检出,含量1.39～6.00 ng/L。经过分析表明:鱼池中Geosmin含量与浮游藻类总生物量正相关,浮游藻类中的颗粒直链藻Melosira granulata和条纹小环藻Cyclotella striata是北京地区夏秋季节淡水精养鱼池中能够产生Geosmin的主要藻类。Geosmin和MIB含量均与放线菌无关。天津地区寡盐水养鱼池水体中普遍存在Geosmin和MIB,其中MIB是鱼池中的主要异味物质成份,浓度0.33～5302.70ng/L,Geosmin浓度相对较低,0.29～12.10ng/L。天津地区鱼池中MIB含量与放线菌正相关。池水盐度对MIB浓度及放线菌数量有重要影响。
3.经高氏1号培养基分离培养,北京地区鱼池水中放线菌生物量(1～27)×10~6个/L,天津鱼池水中放线菌生物量(0.01～1.50)×10~6个/L,两地放线菌优势种均为链霉菌Streptomyces。北京鱼池中放线菌生物量明显高于天津鱼池,但数学分析显示北京鱼池中较高生物量的放线菌与异味物质(Geosmin和MIB)无关,而天津鱼池中相对较低生物量的放线菌与异味物质MIB有关。这可能是由于天津鱼池是寡盐水(盐度1～5),盐度促进放线菌产生异味物质,尤其是MIB。
4.本文采用16SrRNA方法,结合菌落形态特征研究了天津鱼池中放线菌的种类组成,并构建系统发育树关系图。在鱼池中共分离出常见、优势放线菌种4属9种。其中链霉菌是优势种,链霉菌、类诺卡氏菌Nocardioides、小单胞菌Micromonospora是天津鱼池放线菌常见种。鱼池中链霉菌生物量(0.007～0.575)×10~6个/L;类诺卡氏菌生物量(0.0033～0.07)×10~6个/L;小单胞菌生物量(0.00133～0.4776)×10~6个/L。放线菌总生物量(0.01～1.50)×10~6个/L。
5.对由天津鱼池中分离到的6株链霉菌的产味能力进行测定,发现不同种类链霉菌产生异味物质能力差异极大,产MIB能力由高到低依次为:Sp2(5570.0ng/ml或2×10~6ng/g)、Sp3(776.0ng/ml或8.5×10~4ng/g)、Sp5(72.4ng/ml或2.6×10~4ng/g)、Sp4(19.8ng/ml或7.1×10~3ng/g)、Sp1(14.3ng/ml或5.8×10~3ng/g)、Sp6(2.6ng/ml或7.6×10~2ng/g)。Sp2等链霉菌是中国天津寡盐水鱼池土腥异味主要产源。
6.经过形态及培养特征观察以及分子生物学分析,确定放线菌SP2是早期链霉菌Streptomyces praecox。该菌产生MIB的量显著高于产生Geosmin的量。MIB和Geosmin主要存在于Streptomyces praecox细胞外。温度和盐度是影响Streptomycespraecox产生MIB和Geosmin的重要因素,25℃～30℃是Streptomyces praecox产生两种异味物质的“突变区域”;在培养盐度2和1时,MIB和Geosmin的产生量最大。NH_4~+、pH及其交互作用对Streptomyces praecox产生MIB有显著影响;NH_4~+、PO_4~(3-)和pH,以及任二者间的交互作用对Streptomyces praecox产生Geosmin有极显著影响。栅藻Scenedesmus obliquus胞外产物能促进Streptomyces praecox产生MIB和Geosmin。
7.本文试验了各种消除异味的技术方法,发现采用清水、流水、活性炭消除鱼的异味均有一定效果。用流水处理15 h和用活性炭吸附法处理12 h,均可有效地去除鲤Cyprinuscarpio的异味。施用光合细菌,可降低或消除鲂、草鱼和淡水白鲨的异味,但作用时间较长。
Many aquatic products in ponds have different degrees of earthy-musty odor,which decreases their taste and edible value and threatens the cultivation efficiency.Existing research has demonstrated that earthy-musty odor,are caused by geosmin and MIB excreted by planktonic algae and actinomycetes.This study was conducted to evaluate the present condition,the primary source and the major limiting factor of geosmin and MIB in intensive cultivation ponds.This information was then used to select an effective technology for the elimination of earthy-musty odor and to improve the level of quality and safety of aquatic products.The results of this study were as follows.
1.The head space solid-phase microextraction-gas chromatography-mass spectrophotometer(HSPME-GC-MS) method can be used to effectively measure the content of geosmin and MIB in water.Filtering algae had a significant impact on the levels of geosmin,but little effect on the concentrations of MIB.In addition,the results of this study enabled establishment of a sensory evaluation method for freshwater fish.In this method,the earthy-musty odor of fish was divided into five levels:normal,mild, clear,heavy and very heavy.The MIB content in the fish could be estimated based on the level of earthy-musty odor.The human olfact value for MIB in fish meat was 500 ng/kg.
2.Geosmin was found to be the primary element responsible for the earthy-musty odor of fish in freshwater ponds in Beijing.Furthermore,geosmin was present in almost all ponds,as indicated by concentrations of 1.22～35.58 ng/L.MIB was present in some fishponds,with average concentrations of 1.39-6.00 ng/L being observed.Additionally, the geosmin content in fishponds was found to be related to the gross biomass of algae in the ponds.Cyclotella striata and Melosira granulata were found to be the primary algae responsible for the production of geosmin in freshwater fishponds in summer and autumn in Beijing.The MIB and geosmin content were not related to actinomycetes.Geosmin and MIB were found to be widespread in saline water ponds in Tianjin.In these ponds, MIB was the main factor responsible for off-flavor and was present at concentrations of 0.33-5302.70 ng/L.However,the concentration of geosmin in Tianjin was relatively low,as indicated by observed values of 0.29-12.10 ng/L.The MIB content was positively correlated with actinomycetes in fishponds in Tianjin.Water salinity was found to have an important impact on the concentration of actinomycetes and MIB.
3.When separately cultivated using Agar No.1,the biomass of actinomycetes in fishponds in Beijing was found to be(1-27)×10~6/L,while that of actinomycetes in fishponds in Tianjin was(0.01-1.50)×10~6/L.The dominant species of actinomycetes in both areas was Streptomyces.The biomass of actinomycetes in fishponds in Beijing was higher that of fishponds in Tianjin.However,mathematical analysis revealed that the higher biomass of actinomycetes in the fishponds in Beijing was not related to the concentrations of earthy-musty odor elements(geosmin and MIB),while the relatively low biomass of actinomycetes in fishponds in Tianjin was related to the MIB.This may have been due to the presence of saline water ponds in Tianjin(salinity 1 to 5),because salinity may cause actinomycetes to produce odorous substances such as MIB.
4.In this paper,sequencing of the 16SrRNA combined with evaluation of the colony morphology was used to identify the species of actinomycetes in the fishponds in Tianjin, and a phylogenetic tree diagram was constructed.Four genera and 9 species of common and dominant actinomycetes were identified,with Streptomyces being the dominant genus.The most common genera in Tianjin were Streptomyces,Nocardioides,and Micromonospora,which were present in concentrations of(0.007-0.575)×10~6/L, (0.0033-0.07)×10~6/L;and(0.00133-0.4776)×10~6/L,respectively.The total biomass of actinomycetes was(0.01-1.50)×10~6 / L.
5.Six types of Streptomyces were observed in fishponds in Tianjin.Furthermore,the earthy-musty odor production among Streptomyces was found to differ greatly. Specifically,the ability of the various species of Streptomyces found in fishponds in Tianjin to produce MIB were as follows:Sp2(5570.0 ng/ml or 2×10~6 ng/g),Sp3(776.0 ng/ml or 8.5×10~4 ng/g),Sp5(72.4 ng/ml or 2.6×10~4 ng/g),Sp4(19.8 ng/ml or 7.1×103 ng/g),Sp1(14.3 ng/ml or 5.8×103 ng/g),Sp6(2.6 ng/ml or 7.6×102 ng/g).Sp2 and other types of Streptomyces were found to be the primary cause of off-flavor in saline water ponds in Tianjin,China.
6.After observation of the morphological and cultivation features and molecular analysis,Actinomycete SP2 was identified as Streptomyces praecox.This species was found to produce much more MIB than geosmin,and both of these compounds were found to primarily exist outside of the cells.Temperature and salinity were also important factors that affected the production of MIB and geosmin by Streptomyces praecox. Specifically,25℃-30℃was found to be the mutation region for Streptomyces praecox, at which they produced earthy-musty odor substances.Additionally,Streptomyces praecox produced the greatest amounts of MIB and geosmin when cultivated at salinities of 2 and 1,respectively.NH_4~+,pH and their interactions had a significant impact on the ability of Streptomyces praecox to produce MIB,while NH_4~+,PO_4~(3-),and pH,and any interaction between these compounds had a significant effect on its production of geosmin.Extracellular metabolites produced by Scenedesmus obliquus may accelerate the production of MIB and Streptomyces praecox to produce MIB and geosmin.
7.In this study,a variety of techniques for reducing earthy-musty odor were tested. The results revealed that the use of clear water,flowing water and active carbon can eliminate earthy-musty odor of fish to some extent.For example,15 h of treatment with flowing water and the use of active carbon adsorption treatment for 12 h were found to effectively remove earthy-musty odor associated with Cyprinus carpio.In addition,the application of photosynthetic bacteria was also found to reduce or eliminate earthy-musty odor of John Dory,grass carp and fresh water white sharks,but this required a longer treatment than the aforementioned treatments.
引文
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