CN108850779B - PEG-ACS/M-siRNA纳米复合物及其应用和降低鱼粉贮藏中组胺含量的方法 - Google Patents
PEG-ACS/M-siRNA纳米复合物及其应用和降低鱼粉贮藏中组胺含量的方法 Download PDFInfo
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Abstract
本发明公开了一种PEG‑ACS/M‑siRNA纳米复合物及其应用和降低鱼粉贮藏过程中组胺含量的方法:(1)根据鱼粉中主要的组胺产生菌摩氏摩根菌(Morganella morganii subsp.morganii KT)(CP004345)组氨酸脱羧酶基因MU9_RS17900(Gene ID:14672734)序列设计并制备小干扰核糖核酸(siRNA);(2)以经PEG化之后的精氨酸修饰的壳聚糖为载体,得到稳定的PEG‑ACS/M‑siRNA纳米复合物;(3)将PEG‑ACS/M‑siRNA纳米复合物按一定的比例加入鱼粉中。该方法对鱼粉贮藏过程组胺含量抑制效果显著,可降低鱼粉中组胺含量的49%~53%,对饲料工业鱼粉中生物胺的控制具有重要意义。
Description
技术领域
本发明属于饲料工业领域,具体涉及一种PEG-ACS/M-siRNA纳米复合物的制备方法及在饲用鱼粉贮藏中的应用方法,旨在降低鱼粉贮藏中危害物组胺的含量,提升鱼粉的饲用安全。
背景技术
鱼粉是以全鱼或鱼的下脚料为原料,经蒸煮、压榨、脱水、脱脂、干燥、粉碎等工序加工而成的粉状物。鱼粉作为优质的动物蛋白质饲料原料,其粗蛋白含量高达55%以上,同时富含多种必需氨基酸、必需脂肪酸、矿物质、维生素等。除此以外,鱼粉中还含有大量的能促进动物生长的“未知生长因子”,在饲料生产中具有重要地位。然而在贮藏过程中,鱼粉中丰富的组氨酸在组氨酸脱羧酶的作用下会发生脱羧反应而形成对动物毒害作用很强的组胺。研究表明,摩氏摩根菌的组氨酸脱羧酶活性高,是鱼粉中形成组胺的主要腐败菌种。因此,抑制摩氏摩根菌组氨酸脱羧酶的表达是抑制鱼粉中组胺形成的有效途径。
组胺作为一种生物胺,可通过与细胞膜上的受体作用而发生组胺中毒。组胺的衍生物称为肌胃糜烂素,它的致病能力是组胺的100倍。据报道,2.2mg/kg肌胃糜烂素可刺激家禽的胃酸分泌造成胃部疾病,鱼肉肝脏受损等。目前降低鱼粉中组胺的主要方法有辐照法、改善加工和贮藏条件等,这些方法存在着操作要求高,成本高,效果不明显等缺点,难以在生产中应用和推广。因此,一种高效、简单、实用、安全的方法降低鱼粉中组胺含量对饲料工业有着重要的意义。
RNA干扰技术是通过具有同源性的双链RNA(dsRNA)诱导序列特异的目标基因的沉默,阻断其基因活性。根据碱基互补配对原则,小干扰RNA(siRNA)有着很高的特异性,只对靶基因起干扰作用,对其他序列不起作用。目前siRNA的非病毒载体主要为阳离子脂质体和阳离子高分子,如聚乙烯亚胺和壳聚糖等。但脂质体和聚乙烯亚胺均具有较高的细胞毒性和体内毒性,而壳聚糖作为载体时又具有效率低下、靶向性不强等缺点。
本发明选用改性之后的壳聚糖作为传递siRNA的载体构建的PEG-ACS/M-siRNA纳米复合物,根据碱基互补配对原则,与鱼粉中主要的组胺产生菌摩氏摩根菌组氨酸脱羧酶基因的mRNA特异性结合,从而达到使组氨酸脱羧酶沉默的目的,进而抑制了鱼粉中组胺的合成,有效地延缓了鱼粉贮藏过程中的腐败进程,具有广泛的应用前景。
发明内容
本发明的目的在于提供一种PEG-ACS/M-siRNA纳米复合物的制备方法及应用策略。通过抑制鱼粉中主要组胺产生菌摩氏摩根菌组氨酸脱羧酶基因的表达,从而降低鱼粉在贮藏过程中组胺的含量,旨在保障鱼粉的品质和安全,提供了一种高效、易操作的组胺含量降低方法。
本发明的技术方案如下:
本发明提供一种PEG-ACS/M-siRNA纳米复合物,根据鱼粉中主要的组胺产生菌摩氏摩根菌(Morganella morganii subsp.morganii KT)(CP004345)组氨酸脱羧酶基因(MU9_RS17900Gene ID:14672734)序列(SEQ NO:1)设计M-siRNA,以PEG化精氨酸修饰的壳聚糖为载体,制得PEG-ACS/M-siRNA纳米复合物。
进一步,所述M-siRNA为双链,其序列选自以下序列:
(1)正义链:5'-GGAUCGUAUUUCUGUUGAAAU-3',
反义链:5'-UUCAACAGAAAUACGAUCCAC-3';
(2)正义链:5'-GUAUUUCUGUUGAAAUCGACU-3',
反义链:5'-UCGAUUUCAACAGAAAUACGA-3';
(3)正义链:5'-GUAUCAAACGUGAAGAUUACU-3',
反义链:5'-UAAUCUUCACGUUUGAUACCG-3'。
进一步,所述PEG化精氨酸修饰的壳聚糖采用以下步骤制备:
精氨酸修饰壳聚糖的制备:壳聚糖溶于TEMED/HCl缓冲溶液中,然后向该溶液中加入1-乙基-3(3-二甲胺丙基)碳二亚胺、N-羟基-丁二酰亚胺偶联剂,搅拌均匀后,再加入壳聚糖氨基摩尔量50~100%的精氨酸,在磁力搅拌下连续室温反应6~10h,然后经脱盐水透析和冷冻干燥处理得到精氨酸修饰的壳聚糖;
PEG-ACS的制备:取精氨酸修饰的壳聚糖,加入PEG-SPA,室温下反应,使用截留分子量为14000的透析袋透析,去掉未反应的PEG,得到PEG化的精氨酸修饰的壳聚糖。
进一步,步骤(1)中1-乙基-3(3-二甲胺丙基)碳二亚胺与壳聚糖等摩尔量,N-羟基-丁二酰亚胺偶联剂与壳聚糖等摩尔量。
进一步,PEG-ACS/M-siRNA复合物的制备包括以下步骤:
(a)siRNA溶液的制备:用DEPC水溶解siRNA,制备浓度为18~25μM的siRNA溶液;
(b)将PEG-ACS溶解于NaAc/HAc缓冲溶液中,与siRNA溶液分别置于50~55℃恒温水浴加热10~15min,然后将两种溶液等体积混合,迅速在涡旋混合器上混合30~40s,得PEG-ACS/M-siRNA纳米粒。
PEG-ACS/M-siRNA纳米复合物的粒径为150~250nm。
进一步,PEG-ACS/M-siRNA纳米复合物的具体制备方法为:
(1)根据鱼粉中主要的组胺产生菌摩氏摩根菌组氨酸脱羧酶基因MU9_RS17900靶序列:5'-GTGGATCGTATTTCTGTTGAAAT-3',设计M-siRNA序列。
(2)制备M-siRNA的序列:
正义链:5'-GGAUCGUAUUUCUGUUGAAAU-3',
反义链:5'-UUCAACAGAAAUACGAUCCAC-3'。
(3)精氨酸修饰的壳聚糖(ACS)的制备:将0.5~1.5g壳聚糖,溶于pH 4.5~5.0的N,N,N’,N’-四甲基乙二胺(TEMED)/盐酸缓冲溶液中,然后向该溶液中加入与壳聚糖氨基等摩尔量的1-乙基-3(3-二甲胺丙基)碳二亚胺(EDC)和N-羟基-丁二酰亚胺(NHS)作为偶联剂,搅拌均匀后,再加入壳聚糖氨基摩尔量50~100%的精氨酸,在磁力搅拌下室温反应6~10h,经脱盐水透析和冷冻干燥处理得到精氨酸修饰的壳聚糖。
(4)PEG化精氨酸修饰的壳聚糖(PEG-ACS)的制备:取2.5mL,10mg/mL精氨酸修饰的壳聚糖,加入25mg的PEG-SPA,室温下反应4~5h。使用截留分子量为14000的透析袋透析,去掉未反应的PEG。即得PEG化精氨酸修饰的壳聚糖,冷冻干燥备用。
(5)siRNA溶液的制备:用DEPC水溶解siRNA,制备浓度为18~25μM的siRNA溶液,-20℃保存备用。siRNA反复冻融不得超过5次。
(6)PEG-ACS/M-siRNA纳米复合物的制备:采用复凝聚的方法制备PEG-ACS/M-siRNA纳米复合物。PEG-ACS溶解于NaAc/HAc缓冲溶液(pH 5.5)中,至PEG-ACS的质量分数为0.02%。将PEG-ACS溶液和siRNA溶液分别置于50~55℃恒温水浴加热10~15min,然后将其等体积混合。迅速在涡旋混合器上混合30~40s,即得PEG-ACS/M-siRNA纳米粒。
(7)通过动态光散射法测得纳米复合物的粒径在150~250nm之间。将制备的复合物于-20℃冷冻3~6h后冷冻干燥20~26h得到冻干粉备用。
使用时,将PEG-ACS/M-siRNA冻干粉按一定比例加入至鱼粉中。
优选地,步骤(3)中所述的壳聚糖分子量为5x 104D,脱乙酰度为82%。
优选地,步骤(3)中所述的偶联剂的量为壳聚糖氨基等摩尔量。
优选地,步骤(3)中所述的精氨酸的量为壳聚糖氨基摩尔量的50%。
优选地,步骤(4)中所述的ACS和PEG-SPA质量比为1:1。
优选地,步骤(4)中所述的透析袋的分子量为14000。
优选地,步骤(6)中所述的水浴温度为50~55℃,时间为10~15min。
优选地,步骤(6)中所述的PEG-ACS溶液与siRNA溶液为等体积混合。
优选地,步骤(7)中所述的PEG-ACS/M-siRNA纳米复合物的粒径为150~250nm。
优选地,步骤(7)中所述的冷冻时间为4h,冷冻干燥时间为24h。
本发明还提供一种上述的PEG-ACS/M-siRNA纳米复合物在鱼粉贮藏中的应用。
进一步,每千克鱼粉中加入0.12~0.20mmol PEG-ACS/M-siRNA纳米复合物。
本发明还提供一种降低鱼粉贮藏中组胺含量的方法,在鱼粉中加入上述的PEG-ACS/M-siRNA纳米复合物,添加比例为每千克鱼粉中加入3~6g PEG-ACS/M-siRNA纳米复合物。
与现有技术相比,本发明具有如下优点:
本发明制备了一种用于降低鱼粉贮藏过程中组胺合成的PEG-ACS/M-siRNA纳米复合物,通过特异靶向摩氏摩根菌组氨酸脱羧酶基因,与其mRNA分子特异性结合,从而阻碍了组氨酸脱羧酶基因的表达,从根本上抑制了组胺的合成。本发明选用改性之后的壳聚糖作为传递siRNA的载体,构建的PEG-ACS/M-siRNA纳米复合物通过与鱼粉中主要的组胺产生菌摩氏摩根菌组氨酸脱羧酶基因的mRNA结合,能够有效抑制组氨酸脱羧酶的表达,进而抑制了鱼粉中组胺的合成,有效地延缓了鱼粉贮藏过程中的腐败进程,具有广泛的应用前景。
经实验证明,PEG-ACS/M-siRNA纳米复合物具有效率高、靶向性强的特点。使用本发明的方法对鱼粉中组胺的含量有显著的降低作用。与对照组相比,组胺含量显著下降了49~53%,可有效延长鱼粉的保质期。
附图说明
图1为实施例1中不同处理下鱼粉中组胺的含量。
图2为实施例2不同处理下鱼粉中组胺的含量。
图3为实施例1不同处理下鱼粉中每月组胺的增长量。
图4为实施例2不同处理下鱼粉中每月组胺的增长量。
图5为对比例1和实施例1相比,不同siRNA载体下组氨酸脱羧酶基因表达量随时间的变化情况。
具体实施方式
下面将结合具体实施实例,对本发明的技术方案进行完整的描述。本发明实施例添加不同含量的PEG-ACS/M-siRNA纳米复合物加入鱼粉中,产品组胺含量显著降低49~53%。
实施例1:
(1)设计M-siRNA序列。
正义链:5'-GGAUCGUAUUUCUGUUGAAAU-3',
反义链:5'-UUCAACAGAAAUACGAUCCAC-3'。
(2)M-siRNA的制备。
(3)ACS的制备:称取0.5g壳聚糖,溶于pH 5.0的TEMED/HCl缓冲溶液中,然后向该溶液中加入567mg EDC和340mg NHS偶联剂,搅拌均匀后,再加入与壳聚糖氨基80%摩尔量的精氨酸,在磁力搅拌下室温反应8h,然后经脱盐水透析和冷冻干燥处理得到精氨酸修饰的壳聚糖。
(4)PEG-ACS的制备:取2.5mL,10mg/mL精氨酸修饰的壳聚糖,加入25mg PEG-SPA,室温下反应4h。使用截留分子量为14000的透析袋透析,去掉未反应的PEG。即得PEG化精氨酸修饰的壳聚糖,冷冻干燥备用。
(5)siRNA溶液的制备:用DEPC水溶解siRNA,制备浓度为20μM的siRNA溶液,-20℃保存备用。
(6)PEG-ACS/M-siRNA的制备:PEG-ACS溶解于NaAc/HAc缓冲溶液(pH 5.5)中,至PEG-ACS的质量分数为0.02%。将PEG-ACS溶液和siRNA溶液分别置于50℃恒温水浴加热10min,然后将其等体积混合。迅速在涡旋混合器上混合30s,得到PEG-ACS/M-siRNA纳米复合物。
(7)通过动态光散射法测得纳米复合物的粒径平均为217nm。将制备的复合物于-20℃冷冻4h后冷冻干燥24h得到冻干粉。
(8)按每千克鱼粉5.5g PEG-ACS/M-siRNA纳米复合物的比例添加。
(9)用高效液相色谱(HPLC)检测鱼粉中组胺的含量,同时做空白对照,结果如表1和图1所示。
表1不同处理下鱼粉中组胺的含量(mg/100g)
由表1可得,在贮藏过程中,鱼粉中组胺的含量不断增加,在8月份时达到了饲料变质的标准(二级鱼粉组胺含量≤1000mg/kg)。而添加了PEG-ACS/M-siRNA纳米复合物的鱼粉即使在12月份组胺含量仍未达到变质标准,实验结果表明,可有效的延长鱼粉的保质期。结合结果,进一步比较分析可得,PEG-ACS/M-siRNA纳米复合物对鱼粉中组胺的抑制达到了51~53%。
由图1可得,在贮藏过程中鱼粉中组胺的含量不断增加,PEG-ACS/M-siRNA纳米复合物显著降低了2、3、5月份时鱼粉中组胺的含量,极显著降低了其他月份时鱼粉中组胺的含量。PEG-ACS/M-siRNA纳米复合物对鱼粉贮藏过程中组胺的合成有明显的抑制作用。
由图3可得,在贮藏过程中,鱼粉中组胺含量不断增加,其中7、8月份时组胺增长量相对较多。PEG-ACS/M-siRNA纳米复合物对鱼粉贮藏过程中组胺的合成有明显的抑制作用。
实施例2:
(1)设计M-siRNA序列。
正义链:5'-GGAUCGUAUUUCUGUUGAAAU-3',
反义链:5'-UUCAACAGAAAUACGAUCCAC-3'。
(2)M-siRNA的制备。
(3)ACS的制备:称取0.8g壳聚糖,溶于pH 4.5的TEMED/HCl缓冲溶液中,然后向该溶液中加入900mg EDC和550mg NHS偶联剂,搅拌均匀后,再加入与壳聚糖氨基等摩尔量的精氨酸,在磁力搅拌下室温反应7h,然后经脱盐水透析和冷冻干燥处理得到精氨酸修饰的壳聚糖。
(4)PEG-ACS的制备:取3mL,8mg/mL精氨酸修饰的壳聚糖,加入24mg PEG-SPA,室温下反应4h。使用截留分子量为14000的透析袋透析,去掉未反应的PEG。即得PEG化精氨酸修饰的壳聚糖,冷冻干燥备用。
(5)siRNA溶液的制备:用DEPC水溶解siRNA,制备浓度为18μM的siRNA溶液,-20℃保存备用。
(6)PEG-ACS/M-siRNA的制备:PEG-ACS溶解于NaAc/HAc缓冲溶液(pH 5.5)中,至PEG-ACS的质量分数为0.02%。将PEG-ACS溶液和siRNA溶液分别置于55℃恒温水浴加热15min,然后将其等体积混合。迅速在涡旋混合器上混合40s,即得PEG-ACS/M-siRNA纳米复合物。
(7)通过动态光散射法测得纳米复合物的粒径平均为215nm。将制备的复合物于-20℃冷冻5h后冷冻干燥20h得到冻干粉。
(8)按每千克鱼粉4g PEG-ACS/M-siRNA纳米复合物的比例添加。
(9)用HPLC检测鱼粉中组胺的含量,同时做空白对照,结果如表2和图2所示,可得PEG-ACS/M-siRNA纳米复合物对鱼粉贮藏过程中组胺的抑制达到49~51%左右。
表2不同处理下鱼粉中组胺的含量(mg/100g)
由表2可得,在贮藏过程中,鱼粉中组胺的含量不断增加,在8月份时达到了饲料变质的标准(二级鱼粉组胺含量≤1000mg/kg)。而添加了PEG-ACS/M-siRNA纳米复合物的鱼粉即使在12月份组胺含量仍未达到变质标准,实验结果表明,可有效的延长鱼粉的保质期。结合结果,进一步比较分析可得,PEG-ACS/M-siRNA纳米复合物对鱼粉中组胺的抑制达到了49~51%左右。
由图2可得,在贮藏过程中鱼粉中组胺的含量不断增加,PEG-ACS/M-siRNA纳米复合物显著降低了1、2、3月份时鱼粉中组胺的含量,极显著降低了其他月份时鱼粉中组胺的含量。PEG-ACS/M-siRNA纳米复合物对鱼粉贮藏过程中组胺的合成有明显的抑制作用。
由图4可得,在贮藏过程中,鱼粉中组胺含量不断增加,其中7、8月份时组胺增长量相对较多。PEG-ACS/M-siRNA纳米复合物对鱼粉贮藏过程中组胺的合成有明显的抑制作用。
对比例1
以壳聚糖作为载体的实验步骤:
(1)设计M-siRNA序列。
(2)M-siRNA的制备。
(3)CS溶液的制备:CS溶解于NaAc/HAc缓冲溶液(pH=4.5),得到1mg/mL的CS溶液,再用NaOH溶液将其pH调整至5.5。
(5)siRNA溶液的制备:siRNA溶液的制备:用DEPC水溶解siRNA,制备浓度为18~25μM的siRNA溶液,-20℃保存备用。
(4)CS/M-siRNA纳米复合物的制备:将CS溶液和siRNA溶液分别置于50~55℃恒温水浴加热10~15min,然后将其等体积混合。迅速在涡旋混合器上混合30~40s,即得CS/M-siRNA纳米粒。
(5)通过动态光散射法测得纳米复合物的粒径在170~240nm之间。将制备的复合物于-20℃冷冻3~6h后冷冻干燥20~26h得到冻干粉备用。
(6)CS/M-siRNA冻干粉按每千克鱼粉加入3.5~5g纳米复合物的比例加入鱼粉中。
由图5可得,用壳聚糖作为传递siRNA的载体时,虽能在一定程度上降低组氨酸脱羧酶的表达,但是具有效率低下、靶向性不强等缺点。而用PEG化精氨酸修饰的壳聚糖作为传递siRNA的载体时,组氨酸脱羧酶的表达量显著降低。说明PEG-ACS/M-siRNA纳米复合物效率高、靶向性强。
SEQUENCE LISTING
<110> 浙江工商大学
<120> PEG-ACS/M-siRNA纳米复合物及其应用和降低鱼粉贮藏中组胺含量的方法
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1137
<212> DNA
<213> 摩氏摩根菌(Morganella morganii subsp. morganii)
<400> 1
atgactctgt ctatcaatga tcaaaacaaa cttgatgcat tctgggctta ttgcgtaaaa 60
aaccagtatt tcaacatcgg ctatcctgaa tcagcggatt tcgattacac caacctggaa 120
cgtttcttac gtttctccat caacaactgc ggtgactggg gcgaatattg caactacctg 180
ctgaactctt tcgatttcga gaaagaagtg atggagtatt tcgcagacct gttcaaaatt 240
ccgtttgaac aaagctgggg ttatgtgacc aacggcggta ctgaaggtaa catgttcggt 300
tgctacctgg gccgtgaaat cttccctgac ggtaccctgt actattcaaa agatactcac 360
tattccgttg cgaaaatcgt taaattactg cgtatcaaat ctcaggttgt tgaatctctg 420
ccaaacggcg aaatcgacta tgacgatctg atgaaaaaaa tcgctgacga taaagaagcg 480
catccgatca ttttcgctaa catcggtacc actgtccgcg gtgctattga tgatatcgct 540
gaaatccaga aacgcctgaa agcagccggt atcaaacgtg aagattacta cctgcacgct 600
gatgcggcac tgagcggcat gatcctgcca ttcgttgatg atgcacagcc attcacgttt 660
gctgacggta tcgactcaat cggtgtttcc ggccataaaa tgattggttc gccaatccct 720
tgcggtatcg ttgttgcgaa gaaagaaaac gtggatcgta tttctgttga aatcgactac 780
atctccgcac acgacaaaac catcaccggt tcacgtaacg gtcacacacc actgatgctg 840
tgggaagcta tccgttcaca ttcaactgag gaatggaaac gccgcatcac ccgcagtctg 900
gatatggctc agtacgctgt tgaccgtatg cagaaagccg gtatcaatgc atggcgcaac 960
aaaaactcca tcactgttgt gttcccttgc ccgtcagaac gcgtctggag agaacattgc 1020
ctggcaacgt ccggcgacgt ggctcacctg atcaccaccg cgcaccatct ggataccgcg 1080
cagatcgaca aactgatcga cgacgttatc gcggatttta acttacacgc ggcataa 1137
Claims (7)
1.一种PEG-ACS/M-siRNA纳米复合物,其特征在于,根据鱼粉中主要的组胺产生菌摩氏摩根菌Morganella morganii subsp. morganii KT组氨酸脱羧酶基因MU9_RS17900序列设计M-siRNA,其中MU9_RS17900序列如SEQ ID NO:1所示,以PEG化精氨酸修饰的壳聚糖为载体,制备PEG-ACS/M-siRNA纳米复合物;
M-siRNA为双链,其序列为:
正义链:5'-GGAUCGUAUUUCUGUUGAAAU-3',
反义链:5'-UUCAACAGAAAUACGAUCCAC-3';
所述PEG化精氨酸修饰的壳聚糖采用以下步骤制备:
(1)精氨酸修饰壳聚糖的制备:壳聚糖溶于TEMED/HCl缓冲溶液中,然后向该溶液中加入1-乙基-3(3-二甲胺丙基)碳二亚胺、N-羟基-丁二酰亚胺偶联剂,搅拌均匀后,再加入与壳聚糖氨基摩尔量50~100%的精氨酸,在磁力搅拌下连续室温反应6~10h,然后经脱盐水透析和冷冻干燥处理得到精氨酸修饰的壳聚糖;
(2)PEG-ACS的制备:取精氨酸修饰的壳聚糖,加入PEG-SPA,室温下反应,使用截留分子量为14000的透析袋透析,去掉未反应的PEG,得到PEG化的精氨酸修饰的壳聚糖。
2.根据权利要求1所述的PEG-ACS/M-siRNA纳米复合物,其特征在于,步骤(1)中 1-乙基-3(3-二甲胺丙基)碳二亚胺与壳聚糖氨基等摩尔量,N-羟基-丁二酰亚胺偶联剂与壳聚糖氨基等摩尔量。
3.根据权利要求1所述的PEG-ACS/M-siRNA纳米复合物,其特征在于,PEG-ACS/M-siRNA复合物的制备包括以下步骤:
(a)siRNA溶液的制备:用 DEPC水溶解 siRNA,制备浓度为18~25 µM的siRNA溶液;
(b)将PEG-ACS溶解于NaAc/HAc缓冲溶液中,与siRNA溶液分别置于50~55 ℃恒温水浴加热10~15 min,然后将两种溶液等体积混合,迅速在涡旋混合器上混合30~40 s,得PEG-ACS/M-siRNA纳米复合物。
4.根据权利要求1所述的PEG-ACS/M-siRNA纳米复合物,其特征在于,所述的PEG-ACS/M-siRNA纳米复合物的粒径为150~250 nm。
5.如权利要求1-4任意一项所述的PEG-ACS/M-siRNA纳米复合物在鱼粉贮藏中的应用。
6.根据权利要求5所述的PEG-ACS/M-siRNA纳米复合物在鱼粉贮藏中的应用,其特征在于,每千克鱼粉中加入3~6 g PEG-ACS/M-siRNA纳米复合物。
7.一种降低鱼粉贮藏中组胺含量的方法,其特征在于,在鱼粉中加入如权利要求1-4任意一项所述的PEG-ACS/M-siRNA纳米复合物,添加比例为每千克鱼粉中加入3~6 g PEG-ACS/M-siRNA纳米复合物。
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