CN113647591B - 蜂王浆酶解物及其制备方法与应用 - Google Patents
蜂王浆酶解物及其制备方法与应用 Download PDFInfo
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- CN113647591B CN113647591B CN202110962958.9A CN202110962958A CN113647591B CN 113647591 B CN113647591 B CN 113647591B CN 202110962958 A CN202110962958 A CN 202110962958A CN 113647591 B CN113647591 B CN 113647591B
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- royal jelly
- enzymolysis
- protease
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- zymolyte
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Abstract
本发明公开了一种蜂王浆酶解物,是蜂王浆经蛋白酶M‑P001酶解得到的酶解物;所述蛋白酶M‑P001来源于结核分枝杆菌(Mycobacterium tuberculosis),氨基酸序列如SEQ IDNO.1所示。所述酶解条件为:用蛋白酶M‑P001酶解蜂王浆水溶液,蜂王浆水溶液中蜂王浆浓度为5~20%;以1500~2500U/g(以蜂王浆原料计)的添加量添加蛋白酶M‑P001,pH6.0~8.0,33~36℃下酶解20~25小时。本发明蜂王浆酶解物,能够显著提高小鼠单核—巨噬细胞廓清能力,提高小鼠NK细胞活性,具有提高免疫力活性的功效,可以以其为原料用于制备具有提高免疫力活性功效的保健品、药品或食品,可显著提升蜂王浆的产品价值。
Description
技术领域
本发明涉及一种蜂王浆酶解物及其制备方法与应用,属于食品加工技术领域。
背景技术
蜂王浆是工蜂分泌的一种浆状物质,是蜜蜂饲养幼虫的重要营养物质,决定着蜜蜂幼虫的分化方向。蜂王浆化学成分复杂,含有丰富的蛋白质、脂肪酸、糖类等活性成分,是一种高端的营养食品。但蜂王浆在食用过程中存在口感酸涩、蛋白吸收缓慢、功能性成分难以发挥效果等问题,若能进一步加工利用,可以大大提高蜂王浆的产品价值。
蛋白酶水解技术是食品加工常用的技术,不同的蛋白酶酶解由于其水解模式的不同,往往会带来不同的产物功能活性。例如,中国发明专利申请CN 107136458 A公开了酶解蜂王浆的制备方法及其产品和应用,其采用蛋白酶K在pH7.5下对蜂王浆进行酶解,得到的酶解物具有降糖功效。中国发明专利CN 108251487 A公开了抗氧化活性新疆黑蜂蜂王浆谷蛋白酶解产物的制备方法,其采用胃蛋白酶、胰蛋白酶、胰凝蛋白酶酶解蜂王浆和谷蛋白,得到的酶解产物具有抗氧化活性。
因此,利用新型蛋白酶对蜂王浆进行酶解加工,有可能得到功能独特的蜂王浆酶解产品,对于提升蜂王浆的营养价值和产品价值具有显著的推动作用。
发明内容
针对上述现有技术,本发明提供了一种蜂王浆酶解物,及其制备方法与应用。
本发明是通过以下技术方案实现的:
一种蜂王浆酶解物,是蜂王浆经蛋白酶M-P001酶解得到的酶解物;所述蛋白酶M-P001的氨基酸序列如下所示,如SEQ ID NO.1所示。
蛋白酶M-P001的氨基酸序列:
MQTAHRRFAAAFAAVLLAVVCLPANTAAADDKLPLGGGAGIVVNGDTMCTLTTIGHDKNGDLIGFTSAHCGGPGAQIAAEGAENAGPVGIMVAGNDGLDYAVIKFDPAKVTPVAVFNGFAINGIGPDPSFGQIACKQGRTTGNSCGVTWGPGESPGTLVMQVCGGPGDSGAPVTVDNLLVGMIHGAFSDNLPSCITKYIPLHTPAVVMSINADLADINAKNRPGAGFVPVPA。
所述蛋白酶M-P001,来源于结核分枝杆菌(Mycobacterium tuberculosis)。
进一步地,所述酶解条件为:用蛋白酶M-P001酶解蜂王浆水溶液,蜂王浆水溶液中蜂王浆浓度为5~20%(质量体积比,单位g:mL),优选10%;条件为:以1500~2500U/g(以蜂王浆原料计)的添加量添加蛋白酶M-P001,pH 6.0~8.0,33~36℃下酶解20~25h。
所述蜂王浆酶解物的制备方法,包括以下步骤:
(1)将蜂王浆原料溶解于水中,料液比为1:5~20(质量体积比,单位g:mL),优选1:10,得蜂王浆水溶液;
(2)将蛋白酶M-P001加入蜂王浆水溶液中,添加量为1500~2500U/g(以蜂王浆原料计),调节pH为6.0~8.0,在温度33~36℃下酶解20~25h,酶解后,100℃保温5min灭酶,8000r/min离心取上清,即得蜂王浆酶解液,浓缩、干燥,即得蜂王浆酶解物;
所述蛋白酶M-P001,氨基酸序列如SEQ ID NO.1所示,来源于结核分枝杆菌(Mycobacterium tuberculosis)。
进一步地,所述步骤(1)中,调节蜂王浆水溶液的pH至7.0,用碳酸氢钠溶液或盐酸调节。
进一步地,所述步骤(2)中,蛋白酶M-P001的添加量为2000U/g(以蜂王浆原料计)。
进一步地,所述步骤(2)中,酶解温度为35℃,酶解时间24h。
上述蜂王浆酶解物,经实验研究表明,能够显著提高小鼠单核-巨噬细胞廓清能力,提高小鼠NK细胞活性,具有提高免疫力活性的功效,可以以其为原料用于制备具有提高免疫力活性功效的保健品、药品或食品。
本发明的蜂王浆酶解物,是以蛋白酶M-P001酶解蜂王浆得到的,具有提高免疫力活性的功效(这说明经蛋白酶M-P001酶解后,由于水解模式、酶切位点等的不同,赋予了酶解物不同于一般蜂王浆酶解物的功能活性),显著提升了蜂王浆的产品价值。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1:本发明的蛋白酶M-P001纯化后的纯酶SDS-PAGE电泳图,其中,M为标准蛋白Marker;1为纯化后的酶蛋白。
图2:不同pH条件下蜂王浆酶解液多肽含量示意图。
图3:不同加酶量的蜂王浆酶解液多肽含量示意图。
图4:不同酶解温度的蜂王浆酶解液多肽含量示意图。
图5:不同酶解时间的蜂王浆酶解液多肽含量示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1获得蛋白酶M-P001
本发明的发明人从结核分枝杆菌(Mycobacterium tuberculosis)基因组序列中发掘到一段蛋白酶编码序列(结核分枝杆菌因其高侵染性和繁殖力,其蛋白酶可能存在与现有蛋白酶不同的特性,因此选择该蛋白酶进行研究),序列经密码子优化后如SEQ IDNO.2所示,其编码的蛋白酶命名为M-P001(氨基酸序列如SEQ ID NO.1所示)(本发明为该蛋白酶的首次表达、纯化,并对其应用进行了研究)。该基因片段存在于结核分枝杆菌(Mycobacterium tuberculosis)中,可以以结核分枝杆菌(Mycobacterium tuberculosis)基因组为模板通过PCR扩增得到,也可以根据序列通过人工基因合成序列得到。
密码子优化后的基因序列(如SEQ ID NO.2所示):5’-
1 ATGCAAACTG CACACCGTCG TTTTGCAGCA GCATTCGCAG CGGTTCTGCT GGCAGTTGTTTGTCTGCCGG
71 CTAACACGGC AGCCGCAGAC GATAAACTGC CGCTGGGTGG CGGTGCTGGT ATTGTTGTAAACGGTGACAC
141 TATGTGTACC CTGACTACCA TCGGCCATGA TAAAAACGGT GATCTGATCG GTTTCACCTCTGCACACTGC
211 GGCGGTCCTG GTGCTCAGAT CGCAGCAGAA GGTGCTGAAA ACGCTGGTCC AGTTGGCATCATGGTTGCTG
281 GTAATGACGG CCTGGACTAT GCTGTAATCA AGTTCGATCC GGCTAAAGTT ACCCCTGTTGCCGTGTTCAA
351 CGGCTTCGCG ATCAACGGTA TCGGTCCGGA TCCGTCTTTC GGCCAAATCG CGTGTAAACAGGGCCGCACC
421 ACTGGCAATT CTTGCGGTGT TACCTGGGGT CCGGGTGAAT CTCCGGGTAC CCTGGTAATGCAGGTATGTG
491 GTGGTCCGGG TGATTCTGGC GCTCCGGTTA CGGTGGACAA CCTGCTGGTG GGTATGATCCATGGTGCTTT
561 CTCTGACAAC CTGCCGTCTT GCATCACTAA ATATATCCCG CTGCACACGC CGGCGGTTGTGATGTCTATT
631 AACGCTGACC TGGCTGACAT CAACGCTAAA AACCGTCCGG GCGCGGGTTT CGTTCCAGTTCCTGCT-3’。
将该基因片段重组于载体pET-21a(+)上,以大肠杆菌BL21为宿主进行异源表达。重组工程菌发酵培养,培养温度20℃,摇床转速200r/min,培养基为ZYP-5052,诱导培养48h,发酵结束后离心取菌体沉淀。使用pH 7.0的磷酸缓冲液进行重悬与超声破碎,破碎液于10000r/min,4℃下离心10min后取上清。上清液使用镍柱进行纯化,使用50mM咪唑洗脱,使用截留分子量5KD超滤膜浓缩洗脱液并脱盐,将浓缩液冷冻干燥,制得蛋白酶M-P001酶粉,蛋白酶M-P001蛋白电泳结果见图1,由图1可以看到,分子量约为23kDa,确定为蛋白酶M-P001。
实施例2蜂王浆酶解液酶解条件研究
最适酶解体系pH选择:
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用盐酸、碳酸钠溶液分别调节体系pH至4.0、5.0、6.0、7.0、8.0,以2000U/g(以蜂王浆原料计)量添加实施例1制得的蛋白酶至蜂王浆溶液中,控制酶解温度30℃,酶解18h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,测定酶解液多肽含量。
结果如图2所示。由图2结果可知,在不同pH条件下,酶解液多肽含量有较大区别,其中在pH 7.0条件下酶解液多肽含量最高,因此选择pH 7.0为最适酶解pH进行酶解加工。
体系最适加酶量选择:
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节体系pH至7.0,分别以1000U/g、2000U/g、3000U/g、4000U/g、5000U/g(以蜂王浆原料计)量添加实施例1制得的蛋白酶至蜂王浆溶液中,控制酶解温度30℃,酶解18h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,测定酶解液多肽含量。
结果如图3所示。由图3结果可知,不同的加酶量对酶解液中的多肽含量有较大影响,加酶量少时,蛋白不能充分酶解,所得多肽含量偏低;加酶量过高时,酶解程度偏大,部分多肽被进一步酶解成游离氨基酸,影响多肽含量。根据实验结果,选择2000U/g为最适酶添加量。
体系最适酶解温度选择:
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节体系pH至7.0,以2000U/g(以蜂王浆原料计)量添加实施例1制得的蛋白酶至蜂王浆溶液中,控制酶解温度分别为25℃、30℃、35℃、40℃、45℃,酶解18h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,测定酶解液多肽含量。
结果如4所示。由图4结果可知,在不同温度下酶解所得多肽含量不同,温度偏低,蛋白酶活性不高,温度偏高,蛋白酶容易失活。根据实验结果,选择35℃为最适酶解温度。
体系最佳酶解时间选择:
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节体系pH至7.0,以2000U/g(以蜂王浆原料计)量添加实施例1制得的蛋白酶至蜂王浆溶液中,控制酶解温度为35℃,分别酶解6h、12h、18h、24h、30h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,测定酶解液多肽含量。
结果如图5所示。由图5结果可知,随着酶解时间的增长,体系多肽含量呈现先升高后降低的趋势,在24h内,多肽含量随着时间的增加浓度不断升高,超过24h后,部分多肽被水解成游离氨基酸,导致多肽含量降低,综上结果,选择24h为最适酶解时间。
实施例3制备蜂王浆酶解液
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节pH至7.0,得到蜂王浆水溶液;以2000U/g(以蜂王浆原料计)量添加实施例1制得的蛋白酶至蜂王浆溶液中,控制酶解温度35℃,酶解24h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,得蜂王浆酶解液Ⅰ。
实施例4制备蜂王浆酶解液
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节pH至7.0(最适pH),得到蜂王浆水溶液;以2000U/g(以蜂王浆原料计)量添加菠萝蛋白酶(庞博生物,100万U/g)至蜂王浆溶液中,控制酶解温度55℃(最适温度),酶解24h,酶解后于100℃保温5min灭酶,后于8000r/min离心取上清,得蜂王浆酶解液Ⅱ。
实施例5制备蜂王浆酶解液
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节pH至7.0(最适pH),得到蜂王浆水溶液;以2000U/g(以蜂王浆原料计)量添加木瓜蛋白酶(庞博生物,100万U/g)至蜂王浆溶液中,控制酶解温度50℃(最适温度),酶解24h,酶解后于100℃保温5min灭酶,后于8000r/min离心取上清,得蜂王浆酶解液Ⅲ。
实施例6制备蜂王浆酶解液
取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节pH至7.0,得到蜂王浆水溶液;以2000U/g(以蜂王浆原料计)量添加中性蛋白酶(庞博生物,20万U/g)至蜂王浆溶液中,控制酶解温度50℃(最适温度),酶解24h,酶解后于100℃保温5min灭酶,后于8000r/min离心取上清,得蜂王浆酶解液Ⅳ。
小鼠免疫效果实验
使用昆明种小鼠进行免疫效果实验,小鼠选择雌性,SPF级,30日龄,体重20.51±3.32g,在饲养环境下适应性饲养5天,每10只小鼠分成一组实验组,共分为6组:阴性对照组、蜂王浆水溶液饲喂组、蜂王浆酶解液Ⅰ饲喂组、蜂王浆酶解液Ⅱ饲喂组、蜂王浆酶解液Ⅲ饲喂组和蜂王浆酶解液Ⅳ饲喂组。各实验组使用灌胃方式给予蜂王浆水溶液和蜂王浆酶解液,灌胃剂量为10.0mL/kg·BW,阴性对照组给予同剂量去离子水,每日灌胃1次,连续灌胃30天。实验周期结束后,处死小鼠,取出胸腺与脾脏,计算胸腺体重比值及脾脏体重比值;取免疫细胞,进行小鼠碳廓清实验和小鼠NK细胞活性测定实验,相关实验按照《保健食品检验与评价技术规范》(2003版)规定的方法实施。
胸腺体重比值及脾脏体重比值的结果如表1所示。
表1不同实验组的小鼠胸腺、脾脏与体重比
注:不同字母表示实验组结果差异显著(P<0.05)
由表1可以看出,经过灌胃实验30天后,各给药实验组的小鼠胸腺体重比与脾脏体重比与阴性对照组相比均无显著性差异,结果表明各实验样品对小鼠的免疫器官与体重的比值无显著影响。
小鼠碳廓清实验结果如表2所示。
表2不同实验组的小鼠单核-巨噬细胞廓清能力
注:不同字母表示实验组结果差异显著(P<0.05)
由表2可以看出,与阴性对照组相比,各实验组的小鼠单核-巨噬细胞碳廓清能力都有显著上升。其中蜂王浆酶解液Ⅰ饲喂组小鼠单核-巨噬细胞廓清能力提升最为显著,与其他实验组相比,其小鼠单核-巨噬细胞廓清能力最高,表明酶解处理对于饲喂小鼠单核-巨噬细胞廓清能力具有促进作用。
蜂王浆酶解液Ⅰ为使用蛋白酶M-P001酶解制备,蜂王浆酶解液Ⅱ、蜂王浆酶解液Ⅲ和蜂王浆酶解液Ⅳ分别为使用市场上的商品化菠萝蛋白酶、木瓜蛋白酶和中性蛋白酶制备,可以看出使用蛋白酶M-P001酶解制备的蜂王浆酶解液其对饲喂小鼠单核-巨噬细胞廓清能力的促进作用最明显。由于不同的蛋白酶其酶切作用的位点不同,导致所得酶解产物的多肽含量和序列各有不同,从而使得酶解产物的生理活性也有差别。使用蛋白酶M-P001制得的蜂王浆酶解产物其提高免疫力效果较其他蛋白酶有较明显的提升作用。
小鼠NK细胞活性测定实验结果如表3所示。
表3不同实验组的小鼠NK细胞活性
注:不同字母表示实验组结果差异显著(P<0.05)
由表3可以看出,与阴性对照组相比,各实验组的小鼠NK细胞活性都有显著上升。其中蜂王浆酶解液Ⅰ饲喂组小鼠单核-巨噬细胞廓清能力提升最为显著,与其他实验组相比,其小鼠单核-巨噬细胞廓清能力最高,表明酶解处理对于饲喂小鼠单核-巨噬细胞廓清能力具有促进作用,且酶的来源影响显著。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
序列表
<110> 山东丰采健康产业有限公司
<120> 蜂王浆酶解物及其制备方法与应用
<141> 2021-08-20
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 232
<212> PRT
<213> Mycobacterium tuberculosis
<400> 1
Met Gln Thr Ala His Arg Arg Phe Ala Ala Ala Phe Ala Ala Val Leu
1 5 10 15
Leu Ala Val Val Cys Leu Pro Ala Asn Thr Ala Ala Ala Asp Asp Lys
20 25 30
Leu Pro Leu Gly Gly Gly Ala Gly Ile Val Val Asn Gly Asp Thr Met
35 40 45
Cys Thr Leu Thr Thr Ile Gly His Asp Lys Asn Gly Asp Leu Ile Gly
50 55 60
Phe Thr Ser Ala His Cys Gly Gly Pro Gly Ala Gln Ile Ala Ala Glu
65 70 75 80
Gly Ala Glu Asn Ala Gly Pro Val Gly Ile Met Val Ala Gly Asn Asp
85 90 95
Gly Leu Asp Tyr Ala Val Ile Lys Phe Asp Pro Ala Lys Val Thr Pro
100 105 110
Val Ala Val Phe Asn Gly Phe Ala Ile Asn Gly Ile Gly Pro Asp Pro
115 120 125
Ser Phe Gly Gln Ile Ala Cys Lys Gln Gly Arg Thr Thr Gly Asn Ser
130 135 140
Cys Gly Val Thr Trp Gly Pro Gly Glu Ser Pro Gly Thr Leu Val Met
145 150 155 160
Gln Val Cys Gly Gly Pro Gly Asp Ser Gly Ala Pro Val Thr Val Asp
165 170 175
Asn Leu Leu Val Gly Met Ile His Gly Ala Phe Ser Asp Asn Leu Pro
180 185 190
Ser Cys Ile Thr Lys Tyr Ile Pro Leu His Thr Pro Ala Val Val Met
195 200 205
Ser Ile Asn Ala Asp Leu Ala Asp Ile Asn Ala Lys Asn Arg Pro Gly
210 215 220
Ala Gly Phe Val Pro Val Pro Ala
225 230
<210> 2
<211> 696
<212> DNA
<213> Artificial Sequence
<400> 2
atgcaaactg cacaccgtcg ttttgcagca gcattcgcag cggttctgct ggcagttgtt 60
tgtctgccgg ctaacacggc agccgcagac gataaactgc cgctgggtgg cggtgctggt 120
attgttgtaa acggtgacac tatgtgtacc ctgactacca tcggccatga taaaaacggt 180
gatctgatcg gtttcacctc tgcacactgc ggcggtcctg gtgctcagat cgcagcagaa 240
ggtgctgaaa acgctggtcc agttggcatc atggttgctg gtaatgacgg cctggactat 300
gctgtaatca agttcgatcc ggctaaagtt acccctgttg ccgtgttcaa cggcttcgcg 360
atcaacggta tcggtccgga tccgtctttc ggccaaatcg cgtgtaaaca gggccgcacc 420
actggcaatt cttgcggtgt tacctggggt ccgggtgaat ctccgggtac cctggtaatg 480
caggtatgtg gtggtccggg tgattctggc gctccggtta cggtggacaa cctgctggtg 540
ggtatgatcc atggtgcttt ctctgacaac ctgccgtctt gcatcactaa atatatcccg 600
ctgcacacgc cggcggttgt gatgtctatt aacgctgacc tggctgacat caacgctaaa 660
aaccgtccgg gcgcgggttt cgttccagtt cctgct 696
Claims (4)
1.一种蜂王浆酶解物,其特征在于:是蜂王浆经蛋白酶M-P001酶解得到的酶解物;所述蛋白酶M-P001的氨基酸序列如下所示,如SEQ ID NO.1所示;
蛋白酶M-P001的氨基酸序列:
MQTAHRRFAAAFAAVLLAVVCLPANTAAADDKLPLGGGAGIVVNGDTMCTLTTIGHDKNGDLIGFTSAHCGGPGAQIAAEGAENAGPVGIMVAGNDGLDYAVIKFDPAKVTPVAVFNGFAINGIGPDPSFGQIACKQGRTTGNSCGVTWGPGESPGTLVMQVCGGPGDSGAPVTVDNLLVGMIHGAFSDNLPSCITKYIPLHTPAVVMSINADLADINAKNRPGAGFVPVPA;
是通过以下方法制备得到的:取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节pH至7.0,得到蜂王浆水溶液;以2000U/g量添加蛋白酶M-P001至蜂王浆溶液中,控制酶解温度35℃,酶解24h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,得蜂王浆酶解液。
2.根据权利要求1所述的蜂王浆酶解物,其特征在于,所述蛋白酶M-P001是通过以下方法制备得到的:将核苷酸序列如SEQ ID NO.2所示的基因片段重组于载体上,以细菌为宿主进行异源表达;重组工程菌发酵培养,发酵结束后离心取菌体沉淀;重悬,超声破碎,离心取上清,纯化,干燥,制得蛋白酶M-P001酶粉。
3.权利要求1或2所述的蜂王浆酶解物的制备方法,其特征在于:取蜂王浆原料100g,以料液比1:10剪切溶解于去离子水中,使用碳酸钠溶液调节pH至7.0,得到蜂王浆水溶液;以2000U/g量添加蛋白酶M-P001至蜂王浆溶液中,控制酶解温度35℃,酶解24h,酶解后于100℃保温5min灭酶,于8000r/min离心取上清,得蜂王浆酶解液。
4.权利要求1或2所述的蜂王浆酶解物在制备具有提高免疫力活性功效的保健品、药品或食品中的应用。
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