CN116042727A - 一种鹿茸素及其制备方法与应用 - Google Patents
一种鹿茸素及其制备方法与应用 Download PDFInfo
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- CN116042727A CN116042727A CN202211724768.4A CN202211724768A CN116042727A CN 116042727 A CN116042727 A CN 116042727A CN 202211724768 A CN202211724768 A CN 202211724768A CN 116042727 A CN116042727 A CN 116042727A
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- fermentation
- antler
- freezing
- lactobacillus
- pilose antler
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Abstract
本发明公开了一种鹿茸素及其制备方法与应用,涉及鹿茸素制备技术领域。本发明提供了一种鹿茸素的制备方法,包括以下步骤:(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;(2)将所述鹿茸切片继续进行冷冻干燥后粉碎,得到鹿茸微粉,将鹿茸微粉溶于水后冷冻,后恢复至15‑25℃,得到混合液;(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后破碎处理,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;(4)将所述鹿茸料浆冷冻干燥后,得到所述鹿茸素。
Description
技术领域
本发明涉及鹿茸素制备技术领域,尤其是一种鹿茸素及其制备方法与应用。
背景技术
鹿茸为鹿科动物梅花鹿Cervus nippon Temminck或马鹿Cervus elaphusLinnaeus的雄鹿未骨化密生茸毛的幼角,内含有胶质等前成骨组织,富含血管和神经。古代医家认为,鹿之精气全在于角,而茸为角之嫩芽,气体全而未发泄,故补阳益血之力最盛。明代李时珍在《本草纲目》上称鹿茸“善于补肾壮阳,生精益血,补髓健骨”。现代科学研究证明,鹿茸含有20多种氨基酸、多种激素、超氧歧化酶(SOD)和多胺类物质,还含有磷脂类、多糖类、多肽、维生素A、鹿茸胰岛素样生长因子(IGF-1)、鹿茸生长激素(HGH)、鹿茸促生长释放因子(GHRF)、鹿茸神经生长因子(NGF)、鹿茸表皮生长因子(EGF)、鹿茸成纤维生长因子(FGF)等活性肽成份以及磷酸钙、硫酸软骨素等许多生物活性因子。鹿茸是一味性甘咸温、入肝肾经的药物,有补肾壮元阳、益精髓、强筋骨、补气血、通督脉之功效;可促进人体生长发育和新陈代谢、提高机体的抗氧化能力、延缓衰老;缓解青春期性机能障碍及中老年前列腺萎缩,改善性机能;对长期不愈合或新生不良的溃疡伤口,均能增强其再生过程,并可促进骨折的愈合;还能起到抗应激、抗炎和抗肿瘤等多种作用。研究还证实,鹿茸可以提高机体的细胞免疫和体液免疫功能,促进淋巴细胞的转化,具有免疫促进剂的作用,是一种优良的中药和保健品原料。
鹿茸加工的主要目的是脱水、干燥、防腐、消毒、保形、保色,提高质量,利于保存。长期以来,我国鹿茸加工一直采用沸水煮炸和高温烘烤技术,但是长时间的高温煮炸,一方面会使很多水溶性成分损失掉,另一方面蛋白质、多肽等热敏活性成分不同程度的流失、破坏或损失殆尽,使产品质量下降;也常常出现破皮、空头、酸败、焦化、腐败变质等缺陷,影响药效,造成经济损失。
《中华人民共和国药典》2010年版一部所载鹿茸饮片的炮制方法是将鹿茸用热白酒润透或灌白酒稍蒸即切片,其缺点是切制难度受鹿茸骨化程度的增大而增大,饮片孔隙度不均匀,且饮片厚、质脆,煎药时鹿茸片的水溶性蛋白、总磷脂及总多糖不易溶出,口服时鹿茸片渣滓粗糙,口感差。
鹿茸口含片作为保健用品被消费者所青睐,但是因鹿茸中含有丰富的蛋白质、多肽、氨基酸,而这些物质也极易在微生物分解酶的作用下发生复杂的反应,形成多种具有不良气味物质,从而使鹿茸提取物散发出腥臭气味。有文献或专利采用对鹿茸粉进行水提或醇提,申请公布号CN110063967A的发明专利“鹿茸提取物的制备方法、鹿茸提取物及其应用”提及了采用溶剂回流方法对鹿茸中的有效成分进行提取,提取的温度80-85℃;申请公布号CN112715935A的发明专利“一种鹿茸口含片及其制备方法”提及采用超声水提+醇提的提取方式对鹿茸进行提取,再与微晶纤维素、山梨醇、乳糖、交联羧甲基纤维素钠、硬脂酸镁等进行复合;申请公布号CN108969538A的发明专利“一种鹿茸含片及其衍生鹿茸多肽的生产工艺”采用两次水浴提取鹿茸活性成分,提取温度不低于80℃。授权公告号CN106551391B的发明专利“一种鹿茸深加工方法”采用蛋白酶对鹿茸进行酶解,获得鹿茸骨粉,再制备去骨化鹿茸脂质体。上述这些方法解决了鹿茸腥臭气味的问题,但是都存在工艺复杂,最终所获得的活性成分丢失较多的缺陷,主要是提取的高温、有机溶剂、酶等会破坏鹿茸的有效成分。
发明内容
基于此,本发明的目的在于克服上述现有技术的不足之处而提供一种鹿茸素及制备方法与应用。
为实现上述目的,本发明所采取的技术方案为:一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
(2)将所述鹿茸切片继续进行冷冻干燥后粉碎,得到鹿茸微粉,将鹿茸微粉溶于水后冷冻,后恢复至15-25℃,得到混合液;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后破碎处理,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;
(4)将所述鹿茸料浆冷冻干燥后,得到所述鹿茸素。
优选地,所述步骤(1)中,新鲜鹿茸选择健康3-6龄梅花鹿公鹿,采茸时间为每年端午节前后两星期。
优选地,所述步骤(1)中,封口的具体方式为淀粉封口、壳聚糖粉封口、火焰烘烤封口、电烙铁封口中的一种;进一步优选地,所述步骤(1)中,封口的具体方式为食品级壳聚糖粉封口,以壳聚糖粉均匀撒至切面,至无可见鲜血。
本发明封口的目的是保留茸血,茸血中含有丰富的氨基酸、磷脂、矿物质、胶原蛋白、蛋白聚糖、硫酸软骨素、硫酸葡萄糖胺、葡萄胺聚糖、透明质酸、核苷酸、神经节苷脂、生长激素和生长素等,应不使其流失。本发明优选食品级壳聚糖粉封口,原因是食品级壳聚糖来源于甲壳素,是自然界中唯一带有正电荷的多糖,具有一定的抑菌防腐功能。
优选地,所述步骤(1)中,清洗采用超声波清洗机,超声波清洗机的频率为20-40KHz,功率为100-200W,清洗的时间为5-15min,清洗的温度为15-30℃;干燥的时间为1-3h,干燥的温度为20-25℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为30-60min,浸入无水乙醇的时间为15-30min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为1-3h。
本发明清洗的目的是清除鹿茸表面的污垢、杂质及寄生微生物等。浸入无水乙醇的目的是让鹿茸片的结构松散。
进一步优选地,切片后鹿茸厚度为2-5mm,液氮冷冻为浸渍冷冻。
优选地,所述步骤(2)中,冷冻干燥至水分含量为≤15%,粉碎至粒径大于400目的组分>70%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:(5-10),所述水优选为电阻率达到18MΩ*cm(25℃)的超纯水;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为48-72h,冷冻的温度为-10~-30℃。
本发明鹿茸微粉溶于水后进行冷冻的目的,是低温下鹿茸细胞中含有的水份体积增大,使细胞结构松散,便于后续发酵及细胞破碎。
优选地,所述步骤(2)中,冷冻干燥的设备为工作压力0.4-1.0Mpa的冷冻干燥机,冷冻干燥的时间为3-8h。
优选地,所述步骤(3)中,所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为3×105-10×107CFU/ml,所述的乳酸杆菌的接种量为1.5×105-5.0×107CFU/ml;所述酵母菌为产朊假丝酵母、巴斯德毕赤酵母、克鲁维氏酵母、扣囊复膜孢酵母中的至少一种,所述的酵母菌的接种量为1.0×105-1.0×107CFU/ml;
进一步优选地,所述的双歧杆菌为长双歧杆菌(Bifidobacterium longum)、长双歧杆菌亚种(Bifidobacterium longum subsp.longum Reuter)、婴儿双歧杆菌(Bifidobacterium infantis)、短双歧杆菌(Bifidobacterium breve)、青春双歧杆菌(Bifidobacterium adolensentis)、两歧双歧杆菌(Bifidobacterium bifidum)中的至少一种;所述乳酸杆菌为嗜酸乳杆菌(Lactobacillus acidophilus)、副干酪乳杆菌(Lactobacillus paracasei)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、短乳杆菌(Lactobacillus brevis)、保加利亚乳杆菌(Lactobacillus bulgaricus)、罗伊氏乳杆菌(Lactobacillus reuteri)、瑞士乳杆菌(lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)中的至少一种。
优选地,所述步骤(3)中,所述发酵过程中,本领域技术人员可以根据需要,向鹿茸微粉溶液中添加碳源、氮源等发酵菌所需的培养基物质。所述碳源为葡萄糖、乳糖、半乳糖、海藻糖、糊精、麦芽糖、麦芽糖醇、甘露糖中的至少一种;所述氮源为乳蛋白、丝素蛋白、小麦蛋白、燕麦蛋白、玉米蛋白中的至少一种;
优选地,所述步骤(3)中,向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.1-0.2%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.15-0.25%。
本领域技术人员可以根据所用发酵菌的种类和接种量,调整发酵的温度、时间、搅拌速度、氧气的供应与否等具体条件。
优选地,所述步骤(3)中,乳酸菌厌氧发酵时,发酵温度为20-45℃,发酵时间为3-8h,搅拌速度为50-100rpm;酵母菌好氧发酵时,发酵温度为30-42℃,发酵时间为2-6h,搅拌速度为10-50rpm,发酵液氧气含量为0.5-1.0mg/L。
进一步优选地,乳酸菌厌氧发酵时,发酵温度为30-40℃,发酵时间为4-6h,搅拌速度为60-80rpm;酵母菌好氧发酵时,发酵温度为35-40℃,发酵时间为3-5h,搅拌速度为20-40rpm。
本发明复合发酵有利于大分子量的鹿茸蛋白质、聚多糖等物质的水解,生成小分子肽、多糖等降解产物,极大提高最终鹿茸素中分子量≤1000道尔顿的多肽的含量,同时获得氨基酸、生物酶、核苷酸类等活性物质。
优选地,所述步骤(3)中,破碎处理采用低温超高压连续流细胞破碎机,利用超高压能量使样品通过狭缝瞬间释放,在剪切效应、空穴效应、碰撞效应的作用下,使其细胞破碎、物质均质、分散。工作时料体温度为4-6℃,压力为207MPa;所述的低温超高压连续流细胞破碎机处理次数为2-5遍。
优选地,所述步骤(4)中,冷冻干燥的时间为6-8h,所述鹿茸素水分含量为≤1%,粉粒径小于100μm的组分>99%。
此外,本发明提供了采用上述的鹿茸素的制备方法制备得到的鹿茸素。
进一步地,本发明提供了所述的鹿茸素在制备食品、药品、保健品中的应用。
优选地,所述的鹿茸素在应用时可以添加辅料,如糊精、淀粉、微晶纤维素、甘露聚糖等,本发明所述鹿茸素的剂型为片剂、粉剂、颗粒、胶囊中的一种。
相对于现有技术,本发明的有益效果为:
(1)本发明提供的鹿茸素,含有鹿茸全部的营养成分,包括蛋白质、多肽、氨基酸、多糖、脂肪酸、甾类、磷脂、核苷、生物胺以及微量元素。具体地,本发明提供的鹿茸素,含有鹿茸全部的营养成分,包括64.72-68.55%蛋白质、2.2-3.4%水溶性氨基酸总量、2600-3000mg/Kg水溶性总多糖、1.1-1.8%脂肪、4.0-6.5g/Kg核苷类总量、18-32%灰分、水分≤1%。(2)本发明整个加工环节,在室温或低于室温下进行,温度不超过30℃,避免了鹿茸中热敏活性成分的破坏。(3)加工工艺不涉及水提取、溶剂提取、烘烤等方法,保留鹿茸中全部的活性成分不流失。(4)本发明在常规超微粉碎的基础上,结合了复合菌种共生发酵和低温超高压连续流细胞破碎技术,鹿茸素全粉活性成分含量高。(5)本发明所获得的鹿茸素全粉蛋白质水解度高,产物口感及吸收度好,应用便捷,可作为保健品直接应用或与其他辅料复合后应用。
附图说明
图1为组织形态图;其中(a)为空白组组织形态图,(b)为实施例1组织形态图,(c)为对比例8组织形态图;
图2为FLG免疫荧光图;其中(d)为空白组FLG免疫荧光图,(e)为实施例1FLG免疫荧光图,(f)为对比例8FLG免疫荧光图;
图3为LOR免疫荧光图;其中(g)为空白组LOR免疫荧光图,(h)为实施例1LOR免疫荧光图,(i)为对比例8LOR免疫荧光图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。
实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明乳酸菌和酵母菌,皆为标准菌株,购自广东省微生物研究所。
实施例1
一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切成2-5mm片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
其中,新鲜鹿茸选择健康4龄梅花鹿公鹿,封口的具体方式为壳聚糖粉封口;清洗采用超声波清洗机,声波清洗机的频率为40KHz,功率为100W,清洗的时间为10min,清洗的温度为20℃;干燥的时间为2h,干燥的温度为20℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为45min,浸入无水乙醇的时间为20min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为2h;
(2)将所述鹿茸切片继续进行冷冻干燥后在气流式超微粉碎机中粉碎,得到鹿茸微粉,将鹿茸微粉溶于超纯水后冷冻,后恢复至20℃,得到混合液;其中,冷冻干燥至水分含量为8%,冷冻干燥的设备为工作压力0.6Mpa的冷冻干燥机,冷冻干燥的时间为4h,粉碎至粒径大于400目的组分至80%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:8;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为60h,冷冻的温度为-20℃;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后采用低温超高压连续流细胞破碎机破碎处理3遍,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为8×106CFU/ml,所述的乳酸杆菌的接种量为4×106CFU/ml;所述酵母菌为扣囊复膜孢酵母,接种量为1.0×106CFU/ml;向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.15%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.2%;乳酸菌厌氧发酵时,发酵温度为35℃,发酵时间为3h,搅拌速度为70rpm;酵母菌好氧发酵时,发酵温度为40℃,发酵时间为4h,搅拌速度为15rpm,发酵液氧气含量为0.8mg/L。
(4)将所述鹿茸料浆在0.6Mpa的工业级冷冻干燥设备中7h后,得到所述鹿茸素水分含量为0.6%,粉粒径小于100μm的组分为99.5%,得到所述鹿茸素。
实施例2
一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切成2mm片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
其中,新鲜鹿茸选择健康3龄梅花鹿公鹿,封口的具体方式为淀粉封口;清洗采用超声波清洗机,声波清洗机的频率为30KHz,功率为200W,清洗的时间为5min,清洗的温度为22℃;干燥的时间为1h,干燥的温度为22℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为30min,浸入无水乙醇的时间为15min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为1h;
(2)将所述鹿茸切片继续进行冷冻干燥后在气流式超微粉碎机中粉碎,得到鹿茸微粉,将鹿茸微粉溶于超纯水后冷冻,后恢复至15℃,得到混合液;其中,冷冻干燥至水分含量为10%,冷冻干燥的设备为工作压力0.4Mpa的冷冻干燥机,冷冻干燥的时间为3h,粉碎至粒径大于400目的组分85%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:5;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为48h,冷冻的温度为-10℃;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后采用低温超高压连续流细胞破碎机破碎处理2遍,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为3×105CFU/ml,所述的乳酸杆菌的接种量为1.5×105CFU/ml;所述酵母菌为产朊假丝酵母,接种量为1.0×105CFU/ml;向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.1%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.15%;乳酸菌厌氧发酵时,发酵温度为20℃,发酵时间为4h,搅拌速度为50rpm;酵母菌好氧发酵时,发酵温度为30℃,发酵时间为2h,搅拌速度为10rpm,发酵液氧气含量为0.5mg/L。
(4)将所述鹿茸料浆在0.4Mpa的工业级冷冻干燥设备中6h后,得到所述鹿茸素水分含量为0.7%,粉粒径小于100μm的组分99.3%,得到所述鹿茸素。
实施例3
一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切成5mm片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
其中,新鲜鹿茸选择健康5龄梅花鹿公鹿,封口的具体方式为淀粉封口;清洗采用超声波清洗机,声波清洗机的频率为20KHz,功率为150W,清洗的时间为15min,清洗的温度为25℃;干燥的时间为3h,干燥的温度为25℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为60min,浸入无水乙醇的时间为18min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为3h;
(2)将所述鹿茸切片继续进行冷冻干燥后在气流式超微粉碎机中粉碎,得到鹿茸微粉,将鹿茸微粉溶于超纯水后冷冻,后恢复至18℃,得到混合液;其中,冷冻干燥至水分含量为9%,冷冻干燥的设备为工作压力1.0Mpa的冷冻干燥机,冷冻干燥的时间为8h,粉碎至粒径大于400目的组分75%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:10;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为50h,冷冻的温度为-15℃;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后采用低温超高压连续流细胞破碎机破碎处理2-5遍,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为5×106CFU/ml,所述的乳酸杆菌的接种量为2.5×106CFU/ml;所述酵母菌包括产朊假丝酵母和扣囊复膜孢酵母,所述的产朊假丝酵母的接种量为0.5×107CFU/ml,扣囊复膜孢酵母的接种量为0.5×107CFU/ml;向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.12%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.18%;乳酸菌厌氧发酵时,发酵温度为32℃,发酵时间为4.5h,搅拌速度为65rpm;酵母菌好氧发酵时,发酵温度为40℃,发酵时间为5h,搅拌速度为30rpm,发酵液氧气含量为0.6mg/L。
(4)将所述鹿茸料浆在1Mpa的工业级冷冻干燥设备中8h后,得到所述鹿茸素水分含量为0.8%,粉粒径小于100μm的组分99.8%,得到所述鹿茸素。
实施例4
一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切成4mm片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
其中,新鲜鹿茸选择健康6龄梅花鹿公鹿,封口的具体方式为淀粉封口;清洗采用超声波清洗机,声波清洗机的频率为40KHz,功率为100W,清洗的时间为8min,清洗的温度为23℃;干燥的时间为1.5h,干燥的温度为23℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为40min,浸入无水乙醇的时间为25min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为1.5h;
(2)将所述鹿茸切片继续进行冷冻干燥后在气流式超微粉碎机中粉碎,得到鹿茸微粉,将鹿茸微粉溶于超纯水后冷冻,后恢复至20℃,得到混合液;其中,冷冻干燥至水分含量为7%,冷冻干燥的设备为工作压力0.5Mpa的冷冻干燥机,冷冻干燥的时间为5h,粉碎至粒径大于400目的组分80%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:6;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为55h,冷冻的温度为-25℃;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后采用低温超高压连续流细胞破碎机破碎处理4遍,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为10×107CFU/ml,所述的乳酸杆菌的接种量为5.0×107CFU/ml;所述酵母菌为扣囊复膜孢酵母,接种量为1.0×107CFU/ml;向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.2%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.25%;乳酸菌厌氧发酵时,发酵温度为45℃,发酵时间为8h,搅拌速度为100rpm;酵母菌好氧发酵时,发酵温度为42℃,发酵时间为6h,搅拌速度为20rpm,发酵液氧气含量为1.0mg/L。
(4)将所述鹿茸料浆在0.5Mpa的工业级冷冻干燥设备中6.5h后,得到所述鹿茸素水分含量为0.5%,粉粒径小于100μm的组分为99.6%,得到所述鹿茸素。
实施例5
一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切成2.5mm片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
其中,新鲜鹿茸选择健康3龄梅花鹿公鹿,封口的具体方式为淀粉封口;清洗采用超声波清洗机,声波清洗机的频率为30KHz,功率为200W,清洗的时间为12min,清洗的温度为24℃;干燥的时间为2.5h,干燥的温度为24℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为50min,浸入无水乙醇的时间为30min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为2.5h;
(2)将所述鹿茸切片继续进行冷冻干燥后在气流式超微粉碎机中粉碎,得到鹿茸微粉,将鹿茸微粉溶于超纯水后冷冻,后恢复至25℃,得到混合液;其中,冷冻干燥至水分含量为8%,冷冻干燥的设备为工作压力0.8Mpa的冷冻干燥机,冷冻干燥的时间为6h,粉碎至粒径大于400目的组分85%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:7;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为72h,冷冻的温度为-30℃;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后采用低温超高压连续流细胞破碎机破碎处理3遍,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为2×107CFU/ml,所述的乳酸杆菌的接种量为1×107CFU/ml;所述酵母菌为扣囊复膜孢酵母,接种量为0.5×107CFU/ml;向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.14%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.2%;乳酸菌厌氧发酵时,发酵温度为38℃,发酵时间为5.5h,搅拌速度为70rpm;酵母菌好氧发酵时,发酵温度为30℃,发酵时间为4h,搅拌速度为50rpm,发酵液氧气含量为0.8mg/L。
(4)将所述鹿茸料浆在0.8Mpa的工业级冷冻干燥设备中7.5h后,得到所述鹿茸素水分含量为0.6%,粉粒径小于100μm的组分99.5%,得到所述鹿茸素。
实施例6
一种鹿茸素的制备方法,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切成3.5mm片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
其中,新鲜鹿茸选择健康4龄梅花鹿公鹿,封口的具体方式为淀粉封口、壳聚糖粉封口、火焰烘烤封口、电烙铁封口中的一种;清洗采用超声波清洗机,声波清洗机的频率为20KHz,功率为150W,清洗的时间为10min,清洗的温度为21℃;干燥的时间为2h,干燥的温度为21℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为35min,浸入无水乙醇的时间为22min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为2h;
(2)将所述鹿茸切片继续进行冷冻干燥后在气流式超微粉碎机中粉碎,得到鹿茸微粉,将鹿茸微粉溶于超纯水后冷冻,后恢复至22℃,得到混合液;其中,冷冻干燥至水分含量为6%,冷冻干燥的设备为工作压力1.0Mpa的冷冻干燥机,冷冻干燥的时间为7h,粉碎至粒径大于400目的组分75%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:9;所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为68h,冷冻的温度为-17℃;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后采用低温超高压连续流细胞破碎机破碎处理2遍,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为10×106CFU/ml,所述的乳酸杆菌的接种量为5.0×106CFU/ml;所述酵母菌包括产朊假丝酵母和扣囊复膜孢酵母,所述的产朊假丝酵母的接种量为0.5×105CFU/ml,扣囊复膜孢酵母的接种量为0.5×105CFU/ml;向混合液中添加乳糖作为碳源,乳糖的添加量为混合液质量的0.18%。向鹿茸微粉溶液中添加乳蛋白作为氮源,乳蛋白的添加量为鹿茸微粉溶液质量的0.22%;乳酸菌厌氧发酵时,发酵温度为35℃,发酵时间为6h,搅拌速度为75rpm;酵母菌好氧发酵时,发酵温度为38℃,发酵时间为3h,搅拌速度为25rpm,发酵液氧气含量为0.5mg/L。
(4)将所述鹿茸料浆在1Mpa的工业级冷冻干燥设备中8h后,得到所述鹿茸素水分含量为0.8%,粉粒径小于100μm的组分99.3%,得到所述鹿茸素。
对比例1
对比例1与实施例1一种鹿茸素的制备方法相比,仅步骤(1)不同,不进行第二次冷冻,其余制备方法完全相同。
对比例2
对比例2与实施例1一种鹿茸素的制备方法相比,仅步骤(1)不同,切片后不浸入无水乙醇中,其余制备方法完全相同。
对比例3
对比例3与实施例1一种鹿茸素的制备方法相比,仅步骤(2)不同,所述鹿茸切片继续进行冷冻干燥后不进行粉碎处理,其余制备方法完全相同。
对比例4
对比例4与实施例1一种鹿茸素的制备方法相比,仅步骤(2)不同,将鹿茸微粉溶于超纯水后不进行冷冻处理,其余制备方法完全相同。
对比例5
对比例5与实施例1一种鹿茸素的制备方法相比,仅步骤(3)不同,只采用乳酸菌进行厌氧发酵,不添加酵母菌进行好氧发酵,其余制备方法完全相同。
对比例6
对比例6与实施例1一种鹿茸素的制备方法相比,仅步骤(3)不同,只采用酵母菌进行好氧发酵,不添加乳酸菌进行厌氧发酵,其余制备方法完全相同。
对比例7
对比例7与实施例1一种鹿茸素的制备方法相比,仅步骤(3)不同,发酵结束后采用不低温超高压连续流细胞破碎机破碎处理,采用气流式超微粉碎处理,其余制备方法完全相同。
对比例8
对比例8与实施例1一种鹿茸素的制备方法相比,以传统工艺制备鹿茸素,工艺为:水煮、烘烤、风干、切片、超微粉碎,具体可参考[李和平,王春生.生态养鹿[M].北京:中国农业出版社,2011]。
性能测试实施例及对比例的鹿茸素指标检测
测试标准:
蛋白质总量采用全自动凯氏定氮仪测定,方法参考《GB 5009.5-2016食品安全国家标准食品中蛋白质的测定》;
游离氨基酸总量,以质量比1:10加入纯水,搅拌15min,高速离心,上清液过0.45μm,采用甲醛滴定法测定;
水溶性总多糖以水提-醇沉法制备,采用硫酸-咔唑法测定;
脂肪总量采用索式抽提法测定,方法参考《GB 5009.6-2016食品安全国家标准食品中脂肪的测定》;
核苷类总量以超高效液相色谱(UPLC)测定,统计胞嘧啶、尿嘧啶、腺嘌呤、鸟嘌呤、次黄嘌呤、黄嘌呤、尿苷、胸腺嘧啶、肌苷、鸟苷、腺苷、2′-脱氧鸟苷、β-胸苷总量;
水分含量采用水分仪测定,方法参考《GB 5009.3-2016食品安全国家标准食品中水分的测定》;
灰分含量采用马弗炉灼烧测定,方法参考《GB 5009.4-2016食品安全国家标准食品中灰分的测定》。
测试结果如表1-2所示。
表1
表2
由表1-2可知,本发明实施例制备的鹿茸素在蛋白质总量、水溶性氨基酸总量、水溶性多糖总量、脂肪总量、核苷类总量上含量均很高,尤其高于用传统方法得到的鹿茸素。
应用测试1急性经口毒性测试
试验方法:
KM小鼠在本实验室屏障环境动物房中预饲养3天,以适应环境。试验前,KM小鼠禁食过夜,不限制饮水。采用一次限量法,灌胃剂量为5000mg/kg·bw,水溶液给药体积为2mL/100g,染毒前称量动物体重并记录,染毒后继续禁食3h。染毒后对每只动物单独全面的记录,染毒第1天定时观察KM小鼠的中毒表现和死亡情况,其后每天进行一次仔细检查。观察期限为14天,观察期内存活KM小鼠每周称重,观察期结束存活KM小鼠称重,处死后进行尸检。对KM小鼠进行大体解剖学检查,并记录全部大体病理改变。对死亡和存活24h和24h以上动物并存在大体病理改变的器官进行病理组织学检查。
试验结果:
KM小鼠在染毒14天内未见任何中毒症状和死亡;雌雄动物的平均体重未见异常。试验观察期结束,对受试动物进行大体解剖检查也未见异常变化。因此,该受试物对KM小鼠的急性经口LD50>5000mg/kg·bw。
表3
应用测试2超氧化物歧化酶(SOD)活性测试
超氧化物歧化酶(SOD)活性测试采用邻苯三酚自氧化法,25℃时抑制邻苯三酚自氧化速率50%时所需的SOD量为一个活力单位。
原理:在碱性条件下,邻苯三酚会发生自氧化,可根据SOD抑制邻苯三酚自氧化能力确定SOD活力。
试剂:0.1mol/L Tris-HCl-EDTA缓冲液(pH8.2):1.2114gTris和37.2mg EDTA溶于62.4ml/L盐酸溶液中,用蒸馏水定容至100ml。45mmol/L邻苯三酚溶液:称取邻苯三酚56.7mg溶于10mmol/L盐酸溶液,并定容至100ml。10mmol/L盐酸溶液,0.200mg/ml超氧化歧化酶(SOD),蒸馏水。
仪器:紫外-可见分光光度计,精密酸度计(0.01pH),10ml比色管。
试样的制备:在试管加入缓冲液和双蒸水,于25℃恒温20min后先加入一定量的0.200mg/ml超氧化歧化酶,再加入25℃预热过的邻苯三酚(对照管用10mmol/L盐酸代替),迅速摇匀,立即倾入比色杯中,在波长325nm处每30s测定一次吸光值。
测试结果如表4所示。
表4
从上表可知,实施例所制备的鹿茸素中超氧化物歧化酶(SOD)的含量大大高于对比例所制备的鹿茸素。
应用测试3抗氧化活性
(1)DPPH自由基抑制考察
DPPH是一种很稳定的氮自由基,其甲醇或乙醇溶液呈深紫红色,向DPPH自由基溶液中加入自由基清除剂时,深紫色的DPPH自由基被还原成黄色DPPH-H非自由基形式,其褪色程度与所接受的电子数量成定量关系,因而可以通过吸光度的变化进行定量分析。
精确称量实施例及对比例,加入双蒸水99g,37℃恒温震荡1h,滤液以0.45μm微滤膜过滤,待用。取100μL样品溶液,加入100μL 0.05%DPPH乙醇溶液,混合摇匀在暗箱反应1h后,于517nm测定吸光值,重复三次取平均值。对照管以100μL蒸馏水做对照;空白管以100μL无水乙醇加100μL对应样品做空白,计算DPPH自由基清除率。
清除率(%)=[1-(A样品-A空白)/A对照]×100%
测试结果如表5所示。
表5
从实验结果可以看出,本发明实施例制备的鹿茸素DPPH自由基清除率高于80%以上;对比例清除率略低,而市售炮制鹿茸片清除率最低,只有20.17%。因此,本发明采用的加工方法防止了鹿茸在加工过程中营养和功能活性流失,生物活性高,清除DPPH自由基的能力强。
(2)羟基自由基抑制考察
利用Fe3+-EDTA、抗坏血酸及H2O2发生Fenton反应生成羟自由基(·OH),其能够使脱氧核糖降解。在酸性环境中,降解产物可与TBA(硫代巴比妥酸)反应,形成粉红色物质,可用比色法测知脱氧核糖的降解情况。通过分析自由基(·OH)脱氧核糖分子的氧化情况,确定样品是否具有抑制羟自由基(·OH)的作用。
精确称量实施例和对比实施例产品,加入双蒸水99g,37℃恒温震荡1h,滤液以0.45μm微滤膜过滤,待用。
在试管中依次加入0.40ml 50mmol/L pH7.5的磷酸缓冲液,1.04mmol/L的EDTA溶液,1mmol/L FeCl3,一定浓度的样品0.10mL(对照管以蒸馏水替代)和60mmol/L脱氧核糖(空白不加)及10mmol/L H2O2各0.10m L混匀。37℃恒温水浴反应1小时后取出,立即加入1.0mL 20%TCA(三氯乙酸)终止反应,加入1.0mL 0.8%TBA(硫代巴比妥酸)显色剂,在沸水浴中反应15min,立即用冰水冷却,在532nm测定吸光度,计算清除率。
清除率(%)=[1-(A样品-A空白)/(A对照-A空白)]×100%
测试结果如表6所示。
表6
从实验结果可以看出,本发明实施例制备的鹿茸素对羟自由基(·OH)清除率较好,对比实施例清除率略低,而市售炮制鹿茸片清除率最低。
应用测试3抗疲劳动物测试
疲劳是机体复杂的生理生化变化过程,是指脑力或体力到达一定阶段时必然出现的一种正常的生理现象。它既标志着机体原有工作能力的暂时下降,又可能是机体发展到疾病状态的先兆。1982年第五届国际运动生化会议统一了疲劳的概念:机体生理过程不能持续其机能在一特定水平或各器官不能维持预定的运动强度。从中枢神经系统到骨骼肌细胞再到细胞内的物质代谢过程,中间任何一个环节或者过程综合变化,都可造成疲劳。据WHO调查,全球有35%以上的人处于疲劳状态,中年男性人群疲劳状态达60%。疲劳的出现,可引起运动能力降低、工作效率降低、差错事故增多、战斗力减退。疲劳发生后如果得不到及时恢复,逐渐积累,还会导致“过劳”,出现“慢性疲劳综合征(chronic fatiguesyndrome,CFS)”、“过度训练综合症”等,使机体发生内分泌紊乱、免疫力下降,甚至出现器质性疾病,威胁人类健康。以动物测试评测本发明鹿茸素的抗疲劳功能。
以小鼠灌胃给药进行测试,实验方法参考:王文龙,提高免疫力抗疲劳鹿茸片的研究[D],吉林大学,2018。分别取0.5、1.0、3.0g实施例1所制备的鹿茸素样品加蒸馏水配置为100mL试剂作为低、中、高剂量组,同时使用蒸馏水为空白对照组。每组小鼠数量≥30只,各组小鼠灌胃量均为0.2mL/10g BW d,连续灌胃30d。第30日进行负重游泳试验,将实验小鼠置于游泳箱中进行游泳,设计游泳箱中水深约30厘米,室温下进行,在实验小鼠尾根部固定5%体重的重物以加速实验进程,记录实验小鼠自游泳开始至下沉的时间(min)。
结果如表7所示。
表7
剂量 | 小鼠只数 | 游泳时间 |
空白对照 | 33 | 12.5±1.5 |
高剂量组 | 33 | 18.8±2.1 |
中剂量组 | 33 | 15.4±1.3 |
低剂量组 | 33 | 13.7±1.8 |
从实验结果可以看出,通过灌胃给予实验鼠不同剂量实施例1所制备的鹿茸素样品30天,中、高剂量组游泳时间长于空白对照组,且差异均有显著性(P<0.05),说明样品延长实验小鼠负重游泳时间。
以中等剂量给药为标准,考察不同鹿茸素样品对实验鼠的影响,每组小鼠数量≥10只。
结果如表8所示。
表8
序号 | 小鼠只数 | 游泳时间 |
实施例2 | 10 | 14.5±0.5 |
实施例3 | 10 | 16.3±0.7 |
实施例4 | 10 | 15.2±1.1 |
实施例5 | 10 | 15.8±1.6 |
实施例6 | 10 | 16.0±0.8 |
对比例1 | 10 | 12.2±1.4 |
对比例2 | 10 | 13.6±2.3 |
对比例3 | 10 | 11.0±1.5 |
对比例4 | 10 | 12.2±0.9 |
对比例5 | 10 | 11.7±2.2 |
对比例6 | 10 | 12.8±0.7 |
对比例7 | 10 | 12.6±1.3 |
对比例8 | 10 | 13.1±0.4 |
实验结果表明:按照中等剂量饲味实验小鼠,实施例所制备的鹿茸素对实验小鼠提高免疫力、抗疲劳方面大大高于对比例所制备的鹿茸素。
应用测试4体外皮肤屏障保护、抗炎测试
皮肤接触刺激性较强的一些待测物或紫外线等,会导致皮肤屏障发生临床性的急性损伤,导致皮肤干燥,出现红斑现象。阴离子表面活性剂SLS具有双亲性(亲水性和亲脂性)特征,在较大浓度下接触皮肤后能够损伤皮肤屏障,尤其是屏障中的脂质成分以及细胞膜。组织形态是经过H&E染色后的组织微观生理学结构分析,通过模型组织学形态,可以看出不同处理条件下皮肤屏障的变化情况,例如SLS损伤后皮肤屏障变得疏松,活细胞层厚度降低,而活性物处理后能够改善这种损伤。角质胞封(CE)是皮肤渗透屏障的结构基础。兜甲蛋白(LOR)是CE组装过程的关键成分,含量占80%,起着关键的加固作用,LOR蛋白含量的降低是皮肤屏障功能弱化的主要因素。丝聚蛋白(FLG)是CE组装过程中关键的成分,FLG除了是皮肤屏障的结构性组成外,还能够被Caspase-14水解形成天然保湿因子。因此,可以通过给药后观测模型组织形态、兜甲蛋白(LOR)、丝聚蛋白(FLG)含量的变化,评估样品对皮肤的保护能力。
皮肤屏障损伤最典型的临床症状是皮肤泛红,表明发生炎症反应。炎症因子和炎性介质是表征炎症反应的相关指标,SLS刺激后炎症因子大量分泌,而活性物作用后炎症因子有了显著性下降,从而可以达到修复皮肤屏障损伤的效果。IL-1α为一种促炎因子,为一种无疏水性的多肽,只有细胞膜损伤后才能释放到细胞外,待测物作用后炎症因子含量降低说明具有舒缓的功效。前列腺素PGE2为花生四烯酸代谢产物,主要是由环氧合酶介导合成的,PGE2能够扩张血管,导致红斑产生,同时还能诱发炎性疼痛的临床症状。待测物作用后PGE2含量降低,说明具有舒缓的功效。因此,可以通过给药后观测促炎因子IL-1α以及炎性介质PGE2含量的变化来评价样品抗炎能力。
测试结果如表9所示。
表9
从上表及图1-3实验结果可以看出,本发明制备的鹿茸素对皮肤屏障具有显著的修复作用,能够抑制炎症因子的释放。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种鹿茸素的制备方法,其特征在于,包括以下步骤:
(1)将未骨化的新鲜鹿茸切面封口后清洗,干燥后进行第一次冷冻,切片后浸入无水乙醇中,干燥后进行第二次冷冻,得到鹿茸切片;
(2)将所述鹿茸切片继续进行冷冻干燥后粉碎,得到鹿茸微粉,将鹿茸微粉溶于水后冷冻,后恢复至15-25℃,得到混合液;
(3)将乳酸菌和酵母菌加入到所述混合液中发酵,发酵结束后破碎处理,得到鹿茸料浆;其中,发酵为2段式发酵,先由乳酸菌进行厌氧发酵,再由酵母菌进行好氧发酵;
(4)将所述鹿茸料浆冷冻干燥后,得到所述鹿茸素。
2.如权利要求1所述的鹿茸素的制备方法,其特征在于,所述步骤(1)中,清洗采用超声波清洗机,清洗的时间为5-15min,清洗的温度为15-30℃;干燥的时间为1-3h,干燥的温度为20-25℃;第一次冷冻采用液氮冷冻,第一次冷冻的时间为30-60min,浸入无水乙醇的时间为15-30min,第二次冷冻采用液氮冷冻,第二次冷冻的时间为1-3h。
3.如权利要求1所述的鹿茸素的制备方法,其特征在于,所述步骤(2)中,冷冻干燥至水分含量为≤15%,粉碎至粒径大于400目的组分>70%,所述鹿茸微粉和水的质量比为鹿茸微粉:水=1:(5-10);所述步骤(2)中,鹿茸微粉溶于水后冷冻的时间为48-72h,冷冻的温度为-10~-30℃。
4.如权利要求1所述的鹿茸素的制备方法,其特征在于,所述步骤(3)中,所述乳酸菌包括双歧杆菌和乳酸杆菌,所述双歧杆菌的接种量为3×105-10×107CFU/ml,所述的乳酸杆菌的接种量为1.5×105-5.0×107CFU/ml;所述酵母菌为产朊假丝酵母、巴斯德毕赤酵母、克鲁维氏酵母、扣囊复膜孢酵母中的至少一种,所述的酵母菌的接种量为1.0×105-1.0×107CFU/ml。
5.如权利要求4所述的鹿茸素的制备方法,其特征在于,所述双歧杆菌为长双歧杆菌、长双歧杆菌亚种、婴儿双歧杆菌、短双歧杆菌、青春双歧杆菌、两歧双歧杆菌中的至少一种;所述乳酸杆菌为嗜酸乳杆菌、副干酪乳杆菌、鼠李糖乳杆菌、短乳杆菌、保加利亚乳杆菌、罗伊氏乳杆菌、瑞士乳杆菌、植物乳杆菌中的至少一种。
6.如权利要求1所述的鹿茸素的制备方法,其特征在于,所述步骤(3)中,乳酸菌厌氧发酵时,发酵温度为20-45℃,发酵时间为3-8h,搅拌速度为50-100rpm;酵母菌好氧发酵时,发酵温度为30-42℃,发酵时间为2-6h,搅拌速度为10-50rpm,发酵液氧气含量为0.5-1.0mg/L。
7.如权利要求1所述的鹿茸素的制备方法,其特征在于,所述步骤(4)中,冷冻干燥的时间为6-8h,所述鹿茸素水分含量为≤1%,粉粒径小于100μm的组分>99%。
8.一种如权利要求1-7任一项所述的鹿茸素的制备方法制备得到的鹿茸素。
9.一种如权利要求8所述的鹿茸素在制备食品、药品、保健品中的应用。
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