蓝藻酸解产物成分及其饲用安全性研究
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摘要
为了利用太湖蓝藻的蛋白质资源,开发新型饲料产品,本试验采用盐酸水解法研究蓝藻酸解产物中氨基酸组成及残留的微囊藻毒素和重金属含量,并进行动物急性毒理试验,研究其饲用安全性。结果表明:蓝藻经盐酸水解14 h后,酸解产物中氨基酸种类达17种,氨基酸总量达52.63%,其中畜禽必需氨基酸总量为30.25%,微囊藻毒素-LR残留量为1.33μg/kg。蓝藻酸解18 h产物中微囊藻毒素-LR残留量为0.90μg/kg(低于饮用水限量标准1.0μg/L),且微囊藻毒素-LR和微囊藻毒素-RR去除率均高于99.99%。蓝藻酸解14 h产物中重金属砷(As)、铅(Pb)、汞(Hg)、铬(Cr)的含量均远低于相关国家标准的限量。小鼠急性毒性试验、小鼠骨髓嗜多染红细胞微核试验和小鼠精子畸形试验测试的蓝藻酸解产物的饲用安全性结果表明:蓝藻酸解物的小鼠半致死剂量(LD50)>9.09 g/kg BW,属于实际无毒的毒性级别;微核试验和致畸试验检测结果均显示为阴性,蓝藻酸解产物无致突变性和无精子致畸性。由此得出,蓝藻酸解14 h产物的氨基酸总量超50%且畜禽易缺乏而常添加的赖氨酸、蛋氨酸、苏氨酸含量较高;蓝藻经酸解14 h以上,其产物中微囊藻毒素残留量接近饮用水限量标准,急性毒理评价为无毒、无致突变性、无精子致畸性。
In order to utilize the protein resource of cyanobacteria from Taihu lake and develop new feed product,the amino acid composition,residual microcystin and heavy metal contents in cyanobacterial acid hydrolysate were analyzed by hydrochloric acid hydrolysis method. Meanwhile,the feeding safety of cyanobacterial acid hydrolysate was also studied by using animal acute toxicity test. The results showed as follows: after hydrolysis by hydrochloric acid for 14 h,a total of 17 kinds of amino acids were found in the cyanobacterial acid hydrolysate,and the total amino acid content reached to 52.63%,the livestock and poultry essential amino acid content was 30.25%,the microcystin-LR residual amount was 1.33 μg/kg. After hydrolysis by hydrochloric acid for 18 h,microcystin-LR residual amount in cyanobacterial acid hydrolysate was 0.90 μg/kg,which was lower than the standard limit of drinking water( 1.0 μg/L),and the removal ratios of microcystin-LR and microcystin-RR were all higher than 99.99%. The contents of heavy metals [arsenic( As),plumbum( Pb),mercury( Hg) and chromium( Cr) ]in the cyanobacterial acid hydrolysate was in conformity with relative national standards after hydrolysis by hydrochloric acid for 14 h.The feeding safety of cyanobacterial acid hydrolysate was tested by mouse acute toxicity test,mouse bone marrow cell micronucleus test and mouse sperm deformity test. The results showed that the half lethal dose( LD50) of cyanobacterial acid hydrolysate for mice was >9.09 g/kg BW,which belonged to actually non-toxic level. The results of micronucleus test and teratogenic test were negative,and the cyanobacterial acid hydrolysate showed no mutagenicity and no sperm teratogenicity. The results indicate that the total amino acid content exceeded 50% of cyanobacterial acid hydrolysate( cyanobacterial is hydrolyzed by hydrochloric acid for 14 h),and it contains lysine,methionine and threonine which are easily deficient and needed to be added for livestock and poultry. The residual amounts of microcystins in products are close to the standard limit of drinking water after acid hydrolysis for more than 14 h,and the acute toxicological evaluation shows that cyanobacterial acid hydrolysate is non-toxic,non-mutagenic and azoospermic teratogenicity.
In order to utilize the protein resource of cyanobacteria from Taihu lake and develop new feed product,the amino acid composition,residual microcystin and heavy metal contents in cyanobacterial acid hydrolysate were analyzed by hydrochloric acid hydrolysis method. Meanwhile,the feeding safety of cyanobacterial acid hydrolysate was also studied by using animal acute toxicity test. The results showed as follows: after hydrolysis by hydrochloric acid for 14 h,a total of 17 kinds of amino acids were found in the cyanobacterial acid hydrolysate,and the total amino acid content reached to 52.63%,the livestock and poultry essential amino acid content was 30.25%,the microcystin-LR residual amount was 1.33 μg/kg. After hydrolysis by hydrochloric acid for 18 h,microcystin-LR residual amount in cyanobacterial acid hydrolysate was 0.90 μg/kg,which was lower than the standard limit of drinking water( 1.0 μg/L),and the removal ratios of microcystin-LR and microcystin-RR were all higher than 99.99%. The contents of heavy metals [arsenic( As),plumbum( Pb),mercury( Hg) and chromium( Cr) ]in the cyanobacterial acid hydrolysate was in conformity with relative national standards after hydrolysis by hydrochloric acid for 14 h.The feeding safety of cyanobacterial acid hydrolysate was tested by mouse acute toxicity test,mouse bone marrow cell micronucleus test and mouse sperm deformity test. The results showed that the half lethal dose( LD50) of cyanobacterial acid hydrolysate for mice was >9.09 g/kg BW,which belonged to actually non-toxic level. The results of micronucleus test and teratogenic test were negative,and the cyanobacterial acid hydrolysate showed no mutagenicity and no sperm teratogenicity. The results indicate that the total amino acid content exceeded 50% of cyanobacterial acid hydrolysate( cyanobacterial is hydrolyzed by hydrochloric acid for 14 h),and it contains lysine,methionine and threonine which are easily deficient and needed to be added for livestock and poultry. The residual amounts of microcystins in products are close to the standard limit of drinking water after acid hydrolysis for more than 14 h,and the acute toxicological evaluation shows that cyanobacterial acid hydrolysate is non-toxic,non-mutagenic and azoospermic teratogenicity.
引文
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[12]周利英,戴璐,倪小波,等.微波消解ICP-AES法同时测定饲料中8种重金属元素[J].饲料工业,2018,39(1):46-48.
[13]中华人民共和国卫生部,中国国家标准化管理委员会.GB 15193.3-2003急性毒性试验[S].北京:中国标准出版社,2004:581-596.
[14]ZHOU W S,ZHANG X Z,XIE P,et al.The suppression of hematopoiesis function in BALB/C mice induced by prolonged exposure of microcystin-LR[J].Toxicology Letters,2013,219(2):194-201.
[15]WANG J Z,SUN F F,TANG S S,et al.Mutagenicity and teratogenicity studies of vitacoxib in rats and mice[J].Toxicology Reports,2018,5:827-831.
[16]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB/T 5916-2004产蛋后备鸡、产蛋鸡、肉用仔鸡配合饲料[S].北京:中国标准出版社,2004:478-485.
[17]UNGER N,HOLZGRABE U.Stability and assessment of amino acids in parenteral nutrition solutions[J].Journal of Pharmaceutical and Biomedical Analysis,2018,147:125-139.
[18]VAN NGUYEN M,JORDAL A E O,ESPE M,et al.Feed intake and brain neuropeptide Y(NPY)and cholecystokinin(CCK)gene expression in juvenile cobia fed plant-based protein diets with different lysine to arginine ratios[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2013,165(3):328-337.
[19]吴幸强,龚艳,肖邦定,等.微囊藻毒素在滇池鱼体内的积累水平及分布特征[J].水生生物学报,2010,34(2):388-393.
[2]刘刚,屠春宝,毕相东,等.碱法热处理对蓝藻厌氧发酵生物转化及微囊藻毒素降解效果的影响[J].农业资源与环境学报,2016,33(6):547-553.
[3]陈婷,韩士群,周庆.巢湖藻-草-泥有机肥的重金属安全性评价[J].江苏农业科学,2017,45(18):251-254.
[4]钱玉婷,常志州,王世梅,等.水华蓝藻酸解制备复合氨基酸液的研究[J].江苏农业学报,2008,24(5):706-710.
[5]HAN S Q,ZHOU Q,XU Y D,et al.Valuable ingredients and feed toxicity evaluation of Microcystis aeruginosa acidolysis product in mice[J].Experimental Biology and Medicine,2015,240(10):1333-1339.
[6]WOOLBRIGHT B L,WILLIAMS C D,NI H M,et al.Microcystin-LR induced liver injury in mice and in primary human hepatocytes is caused by oncotic necrosis[J].Toxicon,2017,125:99-109.
[7]CHEN L,GIESY J P,XIE P.The dose makes the poison[J].Science of The Total Environment,2018,621:649-653.
[8]NOVAK M,HERCOG K,ZEGURA B.Assessment of the mutagenic and genotoxic activity of cyanobacterial toxin beta-N-methyl-amino-L-alanine in Salmonella typhimurium[J].Toxicon,2016,118:134-140.
[9]DEZ-QUIJADA L,PUERTO M,GUTIRREZ-PRAENAD,et al Microcystin-RR:occurrence,content in water and food and toxicological studies.A review[J].Environmental Research,2019,168:457-489.
[10]国家质量技术监督局.GB/T 18246-2000饲料中氨基酸的测定[S].北京:中国标准出版社,2001:712-715.
[11]中华人民共和国卫生部,中国国家标准化管理委员会.GB 5749-2006生活饮用水卫生标准[S].北京:中国标准出版社,2007:21-25.
[12]周利英,戴璐,倪小波,等.微波消解ICP-AES法同时测定饲料中8种重金属元素[J].饲料工业,2018,39(1):46-48.
[13]中华人民共和国卫生部,中国国家标准化管理委员会.GB 15193.3-2003急性毒性试验[S].北京:中国标准出版社,2004:581-596.
[14]ZHOU W S,ZHANG X Z,XIE P,et al.The suppression of hematopoiesis function in BALB/C mice induced by prolonged exposure of microcystin-LR[J].Toxicology Letters,2013,219(2):194-201.
[15]WANG J Z,SUN F F,TANG S S,et al.Mutagenicity and teratogenicity studies of vitacoxib in rats and mice[J].Toxicology Reports,2018,5:827-831.
[16]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB/T 5916-2004产蛋后备鸡、产蛋鸡、肉用仔鸡配合饲料[S].北京:中国标准出版社,2004:478-485.
[17]UNGER N,HOLZGRABE U.Stability and assessment of amino acids in parenteral nutrition solutions[J].Journal of Pharmaceutical and Biomedical Analysis,2018,147:125-139.
[18]VAN NGUYEN M,JORDAL A E O,ESPE M,et al.Feed intake and brain neuropeptide Y(NPY)and cholecystokinin(CCK)gene expression in juvenile cobia fed plant-based protein diets with different lysine to arginine ratios[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2013,165(3):328-337.
[19]吴幸强,龚艳,肖邦定,等.微囊藻毒素在滇池鱼体内的积累水平及分布特征[J].水生生物学报,2010,34(2):388-393.
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