CN110964780A - 一种靶向筛选抗菌肽的方法 - Google Patents
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Abstract
本发明公开了一种靶向筛选抗菌肽的方法,该方法用一种活体细菌与抗菌活性组分进行亲和吸附,利用反相液相色谱(RP‑HPLC)检测吸附前后的组分,分析图谱找出差异峰,靶向分离抗菌肽,再采用质谱技术鉴定抗菌肽。本发明基于肽——膜作用机制,直接利用活菌菌体与抗菌活性组分亲和吸附,再联合RP‑HPLC图谱靶向分离抗菌肽。同传统的抗菌肽分离方法相比,该法具有靶向高效、易分离抗菌肽的优点,同时克服了模拟细胞膜和脂质细胞膜色谱在分离抗菌肽中易失活、柱效低、膜提取难等缺点。本发明的方法的建立为靶向快捷筛选新型高纯度抗菌肽这类含量少、难分离的生物活性物质,提供一种高效靶向的分离途径。
Description
技术领域
本发明属于抗菌肽筛选技术领域,特别涉及一种靶向筛选抗菌肽的方法。
背景技术
由于传统抗生素的不当使用,微生物对其产生了越来越强的抗性,这也成为了目前全球公共卫生的难题。因此,寻找传统抗生素替代品已迫在眉睫。近些年,抗菌肽作为一种新型的抗菌剂引起了国内外相关研究者的广泛关注。研究已证实抗菌肽具有抗菌谱广、安全性高、不会产生抗药性等优点。如将其应用在食品保鲜上,利用抗菌肽替代部分抗生素来杀菌,可大大改善抗生素滥用、残留等安全问题,保证食品安全和提高人们健康水平。
抗菌肽分离纯化的难题一直约束着其发展和应用,传统的凝胶色谱、离子交换色谱和高效液相色谱技术无法靶向判定组分复杂样品中的目标组分,而且分析时间较长,样品流动相的耗费大。因此急需寻找一种快捷、易操作的方法来解决此问题。
细胞膜色谱技术是一种能从复杂组分中直接筛选识别目标组分的新技术。但传统模拟细胞膜、脂质膜存在制备步骤繁琐、膜提取不确定性大(如:没有准确的标准衡量细胞膜提取的是否完整、膜组分是否被破坏、胞内物是否除尽)、柱效短、膜易脱离等问题。
发明内容
本发明的目的在于提供一种靶向筛选抗菌肽的方法,以解决上述问题。
为实现上述目的,本发明采用以下技术方案:
一种靶向筛选抗菌肽的方法,包括以下步骤:
步骤1,制备能够识别复杂样品中微量目标组分的活体细菌菌体;
步骤2,制备具有抗菌活性的组分,选取出抗菌活性最强的组分;
步骤3,筛选抗菌活性最强的组分与活体细菌菌体亲和吸附,然后离心收集上清液,得到吸附后组分;
步骤4,利用反相液相色谱技术,简称:RP-HPLC,分析吸附前后差异峰,差异峰即为目标组分,纯化制备目标组分后利用质谱技术鉴定抗菌肽。
进一步的,步骤1中具体为:将从冻存的菌种接种至液体培养基中连续活化3代,将第3代细菌再次接种至液体培养基中于36℃~38℃条件下摇床培养18h~24h,取5mL~20mL菌悬液离心,弃上清液得沉淀菌体,无菌水清洗菌体4次~6次以除去残留培养基及代谢物。
进一步的,摇床培养的转速为110rpm。
进一步的,步骤2中,具有抗菌活性的组分的制备方法为:
1)用贯筋藤凝乳酶酶解酪蛋白制备酶解液;用pH 8.5,0.05mol/L Tris-HCl缓冲液配制酪蛋白浓度为20mg/mL,按酶底比为1:45~1:125(w/w)向酪蛋白溶液中加入贯筋藤凝乳酶,40℃~70℃下酶解1h~6h,酶解结束后,沸水灭酶10min,将其快速冷却至室温,冷冻离心,离心条件为:时间15min,转速10000rpm,离心后取上清液,0.45μm微滤,100Da透析12h~16h,获得酶解液;
2)将步骤1)中所得蛋白酶解液超滤,超滤条件为0.2MPa~0.5MPa,30min~60min,超滤步骤为:先用10kDa的膜超滤,滤出液在经1kDa的膜超滤;将超滤后获得的不同组分:>10kDa、1kDa~10kDa、<1kDa,真空冷冻干燥成粉,真空冷冻干燥步骤为:①预冻:将各个组分冻存于-80℃超低温冰箱中4h;②真空冷冻干燥:将各个冻存的组分放于真空冷冻干燥机中,设置参数为-50℃,压力0.03mBar~0.2mBar,时间为72h;将冻干成粉的组分保存于-20℃备用;用无菌超纯水溶解后不同超滤组分测定抑菌活性,筛选出活性最强的超滤组分。
进一步的,抗菌活性最强的组分浓度为5mg/mL~30mg/mL,用超纯水配制,0.22μm微滤备用。
进一步的,步骤3中抗菌活性最强的组分与活体细菌菌体亲和吸附步骤为:向步骤1中得到的菌体中分别加入体积10mL~20mL抗菌活性最强的组分,于37℃条件下亲和吸附0h~12h,离心取上清液,沉淀用超纯水洗涤3次~5次,保留洗涤液,将上清液和洗涤液合并,得到吸附后组分。
进一步的,步骤4具体为:利用RP-HPLC技术,检测吸附后组分和等体积稀释后的具有抗菌活性组分;根据RP-HPLC图谱比较两者的差异峰,若有抗菌肽在活体细菌上保留,吸附后的组分较未吸附组分的图谱则会有峰消失或明显的峰面积减少的情况,则认为消失或明显减小的峰为“目标物”的峰,纯化制备消失或峰面积明显减小的差异峰,并对其抗菌活力进验证和鉴定,制备得差异峰中的肽即为抗菌肽。
与现有技术相比,本发明有以下技术效果:
现有分离抗菌肽的细胞膜色谱技术,存在繁琐的细胞膜提取步骤、细胞膜提取是否完整没有标准、细胞膜的使用时效短等问题。本发明提供的活体细菌直接与活性组分亲和吸附联合RP-HPLC的靶向分离方法:(1)利用活体细菌直接吸附活性组分,克服了模拟细胞膜、脂质细胞膜在操作中膜提取繁琐、柱效短、易失活的缺点;(2)利用活体细菌直接吸附抗菌肽,能直观地反应抗菌肽对细菌的吸附情况,能大大提高抗菌肽筛选的靶向性,降低抗菌肽制备成本;(3)本发明通过比对经活体细菌吸附前后酶解液的差异峰,快速锁定具有抗菌活性的目标组分,大大简化了传统分离方法从成分复杂的酶解液中多步纯化抗菌肽的步骤。本发明对抗菌肽的快速分离纯化提供了新的技术手段,同时也为新型抗菌肽的发现具有重要意义。
本发明利用活体细菌对活性组分进行亲和吸附后利用RP-HPLC进行靶向分离,再利用质谱技术鉴定出抗菌肽。将活体细菌洗净后直接与活性组分进行亲和吸附,营造了真实的抗菌肽与细菌细胞膜作用的环境,从而靶向地分离出抗菌肽。
目前致病性大肠杆菌是导致人类健康问题的主要病原菌之一。所以本发明利用致病性大肠杆菌作为活体菌体膜吸附抗菌肽,能靶向地获得具有抗致病性大肠杆菌的抗菌肽。对于分离出的抗菌肽在抑制致病性大肠杆菌的应用上具有一定的指导意义。
附图说明
附图1为菌体与活性组分亲和吸附流程图。
附图2本发明的流程图。
附图3为亲和吸附前后液相图。
附图4为差异峰F1、F2、F3抑菌活性图。
附图5为抗菌肽BCp12的液相图及抑菌圈图。
附图6为抗菌肽BCp12的质谱鉴定图。
具体实施方式
以下结合附图对本发明进一步说明:
请参阅图1至图6,一种靶向筛选抗菌肽的方法,包括以下步骤:
步骤1,制备能够识别复杂样品中微量目标组分的活体细菌菌体;
步骤2,制备具有抗菌活性的组分,选取出抗菌活性最强的组分;
步骤3,筛选抗菌活性最强的组分与活体细菌菌体亲和吸附最佳条件,然后在最佳条件下离心收集上清液,得到吸附后组分;最佳条件就是筛选一下最佳的亲和吸附条件,而不是直接拿组分与菌体吸附。还要考察:他俩的比例,组分的浓度,吸附时间。
步骤4,利用反相液相色谱技术,简称:RP-HPLC,分析吸附前后差异峰,差异峰即为目标组分,纯化制备目标组分后利用质谱技术鉴定抗菌肽。
步骤1中具体为:将从冻存的菌种接种至液体培养基中连续活化3代,将第3代细菌再次接种至液体培养基中于36℃~38℃条件下摇床培养18h~24h,取5mL~20mL菌悬液离心,弃上清液得沉淀菌体,无菌水清洗菌体4次~6次以除去残留培养基及代谢物。
摇床培养的转速为110rpm。
步骤2中,具有抗菌活性的组分的制备方法为:
1)用贯筋藤凝乳酶酶解酪蛋白制备酶解液;用pH 8.5,0.05mol/L Tris-HCl缓冲液配制酪蛋白浓度为20mg/mL,按酶底比为1:45~1:125(w/w)向酪蛋白溶液中加入贯筋藤凝乳酶,40℃~70℃下酶解1h~6h,酶解结束后,沸水灭酶10min,将其快速冷却至室温,冷冻离心,离心条件为:时间15min,转速10000rpm,离心后取上清液,0.45μm微滤,100Da透析12h~16h,获得酶解液;
2)将步骤1)中所得蛋白酶解液超滤,超滤条件为0.2MPa~0.5MPa,30min~60min,超滤步骤为:先用10kDa的膜超滤,滤出液在经1kDa的膜超滤;将超滤后获得的不同组分:>10kDa、1kDa~10kDa、<1kDa,真空冷冻干燥成粉,真空冷冻干燥步骤为:①预冻:将各个组分冻存于-80℃超低温冰箱中4h;②真空冷冻干燥:将各个冻存的组分放于真空冷冻干燥机中,设置参数为-50℃,压力0.03mBar~0.2mBar,时间为72h;将冻干成粉的组分保存于-20℃备用;用无菌超纯水溶解后不同超滤组分测定抑菌活性,筛选出活性最强的超滤组分。
抗菌活性最强的组分浓度为5mg/mL~30mg/mL,用超纯水配制,0.22μm微滤备用。
步骤3中抗菌活性最强的组分与活体细菌菌体亲和吸附步骤为:向步骤1中得到的菌体中分别加入体积10mL~20mL抗菌活性最强的组分,于37℃条件下亲和吸附0h~12h,离心取上清液,沉淀用超纯水洗涤3次~5次,保留洗涤液,将上清液和洗涤液合并,得到吸附后组分。
步骤4具体为:利用RP-HPLC技术,检测吸附后组分和等体积稀释后的具有抗菌活性组分;根据RP-HPLC图谱比较两者的差异峰,若有抗菌肽在活体细菌上保留,吸附后的组分较未吸附组分的图谱则会有峰消失或明显的峰面积减少的情况,则认为消失或明显减小的峰为“目标物”的峰,纯化制备消失或峰面积明显减小的差异峰,并对其抗菌活力进验证和鉴定,制备得差异峰中的肽即为抗菌肽。
实施例:
本发明中活体细菌亲和吸附联合RP-HPLC靶向筛选抗菌肽的方法包含以下步骤:
(1)大肠杆菌菌体制备
将购买于中国工业微生物菌种保藏管理中心的大肠杆菌粉接种至LB肉汤培养基中于37℃条件下摇床(110rpm)培养18h,连续活化3代。将第3代大肠杆菌接种至LB肉汤培养基中于37℃条件下摇床(110rpm)扩培24h,再利用PCA培养基对扩培后的菌悬液进行计数。取15mL培养至对数期(1×108CFU/mL)的大肠杆菌悬液,离心(4℃,10000rpm,5min),弃上清液(主要是培养基和代谢物)得沉淀菌体。菌体用无菌水清洗5次,以除去残留物,得洁净的菌体。
(2)活体大肠杆菌亲和萃取联合RP-HPLC及质谱技术靶向筛选鉴定抗菌肽
取10mL抗菌活力最佳的样品组分与洗净的菌体按菌体:样品=1.5:1(v/v)混合均匀,于30℃条件下摇床(500rpm)吸附4h,离心(4℃,10000rpm,5min)收集上清液。沉淀用超纯水离心清洗3次,保留洗涤液(上清液)。将合上清液与3次洗涤液合并,即为吸附后的样液。向吸附前的酶解液中加入等体积洗涤超纯水,使吸附前后酶解液的浓度一致。利用反向高效液相色谱和质谱技术,检测吸附前后液相图上的差异峰,纯化制备差异峰,并对其抗菌活力进行验证和肽序分析,差异峰中的肽即为抗菌肽。
下面结合具体的实施例对本文作进一步的详细说明,所述是对本发明的解释而非限定。
实施例
细菌耐药性是威胁人类健康的重大公共卫生问题。大肠杆菌耐药性问题也不断威胁着人类、蓄群动物的健康。因此,本发明选用致泻性大肠杆菌吸附具有膜亲和作用的抗菌活性物质,能有效靶向地从复杂的组分中分离出抗菌肽。对抗菌肽的靶向分离具有重要意义。活体大肠杆菌亲和吸附联合RP-HPLC图谱法靶向筛选槟榔江水牛奶酪蛋白源抗菌肽的方法,包含以下操作(附图2):
(1)活体大肠杆菌
将培养至对数期(1×108CFU/mL)的大肠杆菌液体培养物15mL,离心(4℃,10000rpm,5min),弃上清液,得沉淀菌体,再用无菌水清洗菌体5次,以除去残留物,得洗净的菌体沉淀。
槟榔江水牛奶酪蛋白制备
新鲜的槟榔江水牛奶,冷藏4h,低温离心脱脂(4℃,4000rpm,20min),向脱脂奶中搅拌加入0.2mol/L柠檬酸溶液,使其pH下降至4.6,于磁力搅拌器上搅拌(600rpm)10min,低温离心(4℃,4000rpm,20min),弃上清液,将沉淀捣碎利用蒸馏水离心清洗沉淀3次。再次捣碎,利用1.0mol/L氢氧化钠溶液将其pH调节至7.0,沸水处理15min使牛奶中自身含有的酶失活。冷却至室温后,放入-80℃预冻6h,冷冻干燥成粉,-20℃保存备用。
贯筋藤凝乳酶水解酪蛋白制备抗菌肽
利用自制的贯筋藤凝乳酶酶解槟榔江水牛乳酪蛋白。配制20mg/mL酪蛋白溶液(溶于pH 8.5,0.05mol/L Tris-HCl缓冲液),酶底比1:45(w/w),54℃下酶解4.5h,酶解结束后,沸水灭酶10min,将其快速冷却至室温。离心(4℃,10000rpm,15min),收集上清液,微滤(0.45μm)除杂,再将其超滤(10kDa、1kDa超滤膜),超滤条件为0.25MPa,35min。收集活性最强的超滤组分(<1kDa)置于-80℃超低温冰箱中预冻,预冻时间为4h,随后进行真空冷冻干燥(-50℃,0.03~0.2mBar,72h),冻干粉于-20℃保存留用。
表1超滤组分抑菌活性
BC:水牛乳酪蛋白;BCH:水牛乳酪蛋白酶解液。
活体大肠杆菌亲和吸附具有抗菌活性的酶解液组分
大肠杆菌与样品吸附的比率、样品浓度、吸附时间都是本发明的关键。因此,以96孔板法测定亲和吸附后的样品对大肠杆菌及金黄色葡萄球菌的OD630为指标(根据OD630的大小来判断菌体吸附的强弱),利用不同菌样比、不同样品浓度、不同吸附时间三个因素来考察最佳的亲和吸附条件,最佳的菌肽比为1.5:1,最佳样品浓度为20mg/mL,最佳吸附时间为4h。
取10mL抗菌活力最好的超滤组分(<1kDa,用超纯水配制成20mg/mL)与洗净的大肠杆菌菌体混合,37℃条件下摇床(300rpm)吸附4h,离心(4℃,10000rpm,5min)收集上清液。沉淀用超纯水离心清洗3次,保留洗涤液(上清液)。将合上清液与3次洗涤液合并,即为吸附后的酶解液。
RP-HPLC图谱及质谱技术靶向筛选鉴定抗菌肽
向吸附前的酶解液中加入等体积洗涤超纯水,使吸附前后酶解液的浓度一致。利用反向高效液相色谱和质谱技术,检测吸附前后液相图上的差异峰,纯化制备差异峰,并对其抗菌活力进行验证和肽序分析,差异峰中的肽即为抗菌肽。
色谱条件:
液相:Agilent 1200
检测波长:215nm
表2液相条件
色谱柱:Aglient Eclipse XDB-C18,250mm×4.6mm,5μm
样品:吸附前后槟榔江水牛奶酪蛋白酶解液组分(<1kDa)
柱温:30℃
检测结果如图3所示:图3-A为样品溶剂组,图3-B-a为吸附前样品,图3-B-b为吸附后样品,对比a和b可明显地看出1,2,3这3个差异峰,吸附前后峰面积变化情况见图4。暂且锁定这三个峰为目标峰,将其制备后测定抑菌活性,结果见图5,峰3对大肠杆菌和金黄色葡萄球菌的抑菌效果均比峰1和峰2强。所以,选择峰3进行质谱鉴定,鉴定结果见图,抗菌肽BCp12的肽序列为YLGYLEQLLRLK(Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg-Leu-Lys),对大肠杆菌的最小抑菌浓度为1.60mg/mL,对金黄色葡萄球菌的最小抑菌浓度为0.80mg/mL。
上述结果表明,本发明所利用的活体细菌亲和吸附联合RP-HPLC图谱及质谱技术能够高效靶向地从复杂酶解液组分中分离出抗菌肽,提高分离效率。
Claims (7)
1.一种靶向筛选抗菌肽的方法,其特征在于,包括以下步骤:
步骤1,制备能够识别复杂样品中微量目标组分的活体细菌菌体;
步骤2,制备具有抗菌活性的组分,选取出抗菌活性最强的组分;
步骤3,筛选抗菌活性最强的组分与活体细菌菌体亲和吸附,然后离心收集上清液,得到吸附后组分;
步骤4,利用反相液相色谱技术,简称:RP-HPLC,分析吸附前后差异峰,差异峰即为目标组分,纯化制备目标组分后利用质谱技术鉴定抗菌肽。
2.根据权利要求1所述的一种靶向筛选抗菌肽的方法,其特征在于,步骤1中具体为:将从冻存的菌种接种至液体培养基中连续活化3代,将第3代细菌再次接种至液体培养基中于36℃~38℃条件下摇床培养18h~24h,取5mL~20mL菌悬液离心,弃上清液得沉淀菌体,无菌水清洗菌体4次~6次以除去残留培养基及代谢物。
3.根据权利要求2所述的一种靶向筛选抗菌肽的方法,其特征在于,摇床培养的转速为110rpm。
4.根据权利要求1所述的一种靶向筛选抗菌肽的方法,其特征在于,步骤2中,具有抗菌活性的组分的制备方法为:
1)用贯筋藤凝乳酶酶解酪蛋白制备酶解液;用pH 8.5,0.05mol/L Tris-HCl缓冲液配制酪蛋白浓度为20mg/mL,按酶底比为1:45~1:125(w/w)向酪蛋白溶液中加入贯筋藤凝乳酶,40℃~70℃下酶解1h~6h,酶解结束后,沸水灭酶10min,将其快速冷却至室温,冷冻离心,离心条件为:时间15min,转速10000rpm,离心后取上清液,0.45μm微滤,100Da透析12h~16h,获得酶解液;
2)将步骤1)中所得蛋白酶解液超滤,超滤条件为0.2MPa~0.5MPa,30min~60min,超滤步骤为:先用10kDa的膜超滤,滤出液在经1kDa的膜超滤;将超滤后获得的不同组分:>10kDa、1kDa~10kDa、<1kDa,真空冷冻干燥成粉,真空冷冻干燥步骤为:①预冻:将各个组分冻存于-80℃超低温冰箱中4h;②真空冷冻干燥:将各个冻存的组分放于真空冷冻干燥机中,设置参数为-50℃,压力0.03mBar~0.2mBar,时间为72h;将冻干成粉的组分保存于-20℃备用;用无菌超纯水溶解后不同超滤组分测定抑菌活性,筛选出活性最强的超滤组分。
5.根据权利要求5所述的一种靶向筛选抗菌肽的方法,其特征在于,抗菌活性最强的组分浓度为5mg/mL~30mg/mL,用超纯水配制,0.22μm微滤备用。
6.根据权利要求1所述的一种靶向筛选抗菌肽的方法,其特征在于,步骤3中抗菌活性最强的组分与活体细菌菌体亲和吸附步骤为:向步骤1中得到的菌体中分别加入体积10mL~20mL抗菌活性最强的组分,于37℃条件下亲和吸附0h~12h,离心取上清液,沉淀用超纯水洗涤3次~5次,保留洗涤液,将上清液和洗涤液合并,得到吸附后组分。
7.根据权利要求1所述的一种靶向筛选抗菌肽的方法,其特征在于,步骤4具体为:利用RP-HPLC技术,检测吸附后组分和等体积稀释后的具有抗菌活性组分;根据RP-HPLC图谱比较两者的差异峰,若有抗菌肽在活体细菌上保留,吸附后的组分较未吸附组分的图谱则会有峰消失或明显的峰面积减少的情况,则认为消失或明显减小的峰为“目标物”的峰,纯化制备消失或峰面积明显减小的差异峰,并对其抗菌活力进验证和鉴定,制备得差异峰中的肽即为抗菌肽。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06239888A (ja) * | 1993-02-18 | 1994-08-30 | Kanebo Ltd | 新規ペプチド及び鎮静剤並びに精神安定用食品 |
WO2008131008A2 (en) * | 2007-04-16 | 2008-10-30 | Solae, Llc | Protein hydrolysate compositions having improved sensory characteristics and physical properties |
CN102533912A (zh) * | 2011-12-09 | 2012-07-04 | 江南大学 | 一种快速准确发现、鉴定及制备蛋白质水解物源抗菌肽的方法 |
CN103642896A (zh) * | 2013-12-06 | 2014-03-19 | 江南大学 | 一种利用细菌膜脂构建模拟细胞膜快速筛选抗菌肽的方法 |
CN105132506A (zh) * | 2015-09-28 | 2015-12-09 | 昆明理工大学 | 一种辣椒籽抗菌肽的制备方法 |
CN107019046A (zh) * | 2017-06-14 | 2017-08-08 | 云南农业大学 | 一种抑菌抗氧化即食水牛奶奶羹花及其制备方法 |
-
2019
- 2019-12-24 CN CN201911347197.5A patent/CN110964780A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06239888A (ja) * | 1993-02-18 | 1994-08-30 | Kanebo Ltd | 新規ペプチド及び鎮静剤並びに精神安定用食品 |
WO2008131008A2 (en) * | 2007-04-16 | 2008-10-30 | Solae, Llc | Protein hydrolysate compositions having improved sensory characteristics and physical properties |
CN102533912A (zh) * | 2011-12-09 | 2012-07-04 | 江南大学 | 一种快速准确发现、鉴定及制备蛋白质水解物源抗菌肽的方法 |
CN103642896A (zh) * | 2013-12-06 | 2014-03-19 | 江南大学 | 一种利用细菌膜脂构建模拟细胞膜快速筛选抗菌肽的方法 |
CN105132506A (zh) * | 2015-09-28 | 2015-12-09 | 昆明理工大学 | 一种辣椒籽抗菌肽的制备方法 |
CN107019046A (zh) * | 2017-06-14 | 2017-08-08 | 云南农业大学 | 一种抑菌抗氧化即食水牛奶奶羹花及其制备方法 |
Non-Patent Citations (3)
Title |
---|
QIONG ZHAO 等: "Characterization of a novel antimicrobial peptide from buffalo casein hydrolysate based on live bacteria adsorption", 《J DAIRY SCI》 * |
王钰潭等: "水牛奶干酪功能活性肽的研究", 《中国奶牛》 * |
赵存朝等: "贯筋藤酶解山羊乳酪蛋白糖巨肽的工艺研究", 《中国乳品工业》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111870683A (zh) * | 2020-09-17 | 2020-11-03 | 北京欣颂生物科技有限公司 | 一种抗菌肽在制备药物或化妆品中的用途 |
CN111925422A (zh) * | 2020-09-17 | 2020-11-13 | 北京欣颂生物科技有限公司 | 抗菌肽及其在制备药物或化妆品中的应用 |
CN111925422B (zh) * | 2020-09-17 | 2021-05-18 | 张雪山 | 抗菌肽及其在制备药物或化妆品中的应用 |
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