CN117143741A - 一种免疫增强剂的制备及应用 - Google Patents
一种免疫增强剂的制备及应用 Download PDFInfo
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- CN117143741A CN117143741A CN202310931519.0A CN202310931519A CN117143741A CN 117143741 A CN117143741 A CN 117143741A CN 202310931519 A CN202310931519 A CN 202310931519A CN 117143741 A CN117143741 A CN 117143741A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种免疫增强剂的制备及应用。本发明属于食用菌发酵技术领域,提供了一种牛樟芝发酵提取物的制备方法:将菌种接种于发酵培养基发酵并流加油乳培养基,获得发酵液;灭菌后固液分离并干燥获得发酵物干粉;再以乙酸乙酯提取;然后经柱层析分离纯化即得。本发明的液体发酵过程牛樟芝菌丝不成团、提高了传质效率;通过加入油乳培养基使得高产泛醌类物质,克服了牛樟芝在液体深层发酵条件下无法中生产泛醌类物质的难题;使用的培养基中无价格昂贵原料,降低了牛樟芝发酵液的成本,适于工业化生产。本发明将牛樟芝发酵液以乙酸乙酯提取,最终分离纯化获得了泛醌类物质Antroquinonol;该提取物能增强家禽免疫系统和免疫反应,拓展了该物质的应用范围。
Description
技术领域
本发明属于食用菌发酵技术领域,具体涉及一种从牛樟芝发酵液中提取的免疫增强剂。
背景技术
过去的几十年中,以抗生素为代表的具有抗菌、促生长功效的添加剂被大量用于畜禽养殖,以达到缩短生长周期、降低发病率的目的。但在实际应用中,抗药细菌出现的速度逐渐超出了新型抗生素的研制速度,并且通过动物-食物链条影响了人类的健康,耐药性如今已成为全社会最为关注的公共卫生问题。随着越来越多的国家开始禁止在畜禽养殖中使用抗生素,疫病的防控又成为养殖业高质量发展的瓶颈。近年来,研究人员一直在寻找合适的替抗产品,以实现在不使用抗生素的条件下提高动物的生长性能和免疫性能。益生菌、有机酸、活性肽、蛋白酶、药用真菌等都是研究热点,其中药用真菌以其独特的功效成为新型饲料添加剂和免疫增强剂的潜在研究目标。相关研究表明,多种真菌活性成分或制剂能够有效促进动物的免疫性能,降低发病率、提高成活率。
牛樟芝(Antrodiacamphorata)是我国台湾省特有的珍稀药用真菌,隶属于担子菌门薄孔菌属。牛樟芝菌中含有多种生理活性物质,主要包括樟芝多糖、三萜类物质、泛醌类衍生物、马来酸与琥珀酸等,具有保肝、抗炎、抗氧化、降血压、抑制肿瘤、调节免疫等多种功效,如此优良的营养价值和保健作用,使得樟芝菌的开发前景十分广阔。当前对于牛樟芝活性物质的研究主要集中在多糖、萜类物质、马来酸与琥珀酸及其衍生物和泛醌类化合物。泛醌类化合物自2007年被发现以来,共鉴定出7种物质,分别为安卓奎诺尔(Antroquinonol)、Antroquinonol-B、4-acetyl-Antroquinonol-B、Antroquinonol-D、Antroquinonol LT1、Antroquinonol LT2和Antroquinonol LT3。
研究发现,Antroquinonol具有抗癌、抗炎、抗氧化应激的作用,但对于其是否具有免疫增强作用尚不清楚。相反,在其抗炎生理功能中,展现的是免疫抑制功能。此外,在牛樟芝所有的功能成分中,就免疫增强作用而言,多糖中的β-1,3-D-葡聚糖、萜类物质中的三萜类是主要成分,但应用也局限于体外细胞和小鼠。所以,以家禽为研究对象进行Antroquinonol免疫增强作用的研究至今尚属空白,且已有研究并不支持相关工作的开展。
发明内容
针对目前家禽养殖业缺乏安全、高效、绿色免疫增强剂和Antroquinonol功效研究的缺失及应用范围有限的问题,本发明提供一种免疫增强剂,以牛樟芝深层发酵的发酵液为原料,主要成分为安卓奎诺尔(Antroquinonol),对家禽的免疫系统和免疫反应均具有显著的增强作用。
为实现上述目的,本发明采用如下技术方案。
一种牛樟芝液体发酵方法,包括以下步骤:
(1)将菌种接种在固体种子培养基上培养;
(2)将步骤(1)获得的菌种在液体种子培养基中扩繁培养;
(3)将步骤(2)获得的菌种接种于发酵培养基在发酵罐内通气搅拌培养并流加油乳培养基,发酵至终点获得牛樟芝发酵液。
所述液体种子培养基或发酵培养基由以下成分组成:蛋白胨0.5-3 wt%,葡萄糖0.5-4 wt%,麦芽糖0.5-2 wt%,土豆淀粉1-3 wt%,KH2PO40.1-0.5 wt%,MgSO4·7H2O 0.05-0.2 wt%,维生素B1 50 mg/L-100 mg/L,大豆油0.1-0.5 wt%,pH 6-7。
所述油乳培养基由以下成分组成:大豆油30-90 L,枸橼酸30-50 g,丙三醇0.5-1L,吐温-80 50-100 mL,预胶化淀粉5-10 g,黄原胶10-20 g。
所述发酵培养基和油乳培养基的体积比为100:(6-10)。
优选地,油乳培养基在发酵第24小时起每隔12小时加入一次,分2-5次加入。
所述固体种子培养基由以下成分组成:200 g马铃薯煮出滤汁1 L,葡萄糖10-12g/L,麦芽糖8-10 g/L,KH2PO43 g/L,Mg2SO4·7H2O 1.5 g/L,维生素B1 50-100 mg/L,琼脂15-20 g/L;pH 6.5。
步骤(1)-(3)中,培养的温度为25℃-35℃。较高的温度虽然菌种生长速度较快,但是容易有杂菌污染或菌种劣变,优选的培养温度为25℃-30℃,更加优选为25℃-27℃。
为了降低牛樟芝菌种培养期间的杂菌污染、提高生长速度,步骤(1)中,将牛樟芝培养于并联的试剂瓶中通气培养;步骤(2)中,当固体种子培养基上牛樟芝菌落长至2/3以上器皿直径时,将液体种子培养基加入到长有牛樟芝的固体种子培养基中进行液体发酵培养。
为了适应发酵规模,获得足够的生产用种,步骤(2)扩繁可经过2-4次。
步骤(2)或(3)中,接种量为1%-15%,可以选择1%、3%、5%、7.5%、10%、12%、15%。为了更快更好的发酵获得目标产物,优选的接种量为5%-15%。
为了增加溶氧,还可以根据情况进行搅拌或/和通气,具体搅拌速度和通气量根据发酵的容量以菌丝生长正常为标准进行调节。优选地,通气量为0.1-0.5 vvm;如,对于1 L及以下的发酵可通气0.1-0.5 L/min静置培养或者100-200 r/min摇床培养,10 L左右的发酵可在1-5 L/min通气量下以100-300 r/min搅拌培养;100 L左右的发酵可在10-50 L/min通气量下以100-300 r/min搅拌培养。步骤(3)中,通气量为100-500 L/min;搅拌速度为100-300 r/min。
采用上述方法进行牛樟芝液体发酵,发酵液中的Antroquinonol含量可达100-500mg/L。在优选的条件下,发酵液中的Antroquinonol含量可达300 mg/L以上。
一种牛樟芝发酵提取物的制备方法,包括以下步骤:
(i)将菌种接种于发酵培养基在发酵罐内通气搅拌发酵并流加油乳培养基,发酵至终点获得发酵液;然后灭菌后固液分离获得发酵滤液,干燥后获得发酵物干粉;
(ii)将发酵物干粉以乙酸乙酯提取后去除溶剂获得粗提物;
(iii)将粗提物以乙酸乙酯溶解进行硅胶柱层析,梯度洗脱剂洗脱:正己烷:乙酸乙酯体积比为10:0、9:1、8:2、7.5:2.5,洗脱体积分别为2.5柱体积、2.5柱体积、5柱体积、2.5柱体积;
收集正己烷:乙酸乙酯体积比为7.5:2.5洗脱的流出液,旋转蒸发,用适量乙酸乙酯溶解后进行二次硅胶柱层析,梯度洗脱剂洗脱:石油醚(60-90馏程):乙酸乙酯体积比为10:0、9:1、8:2、7.5:2.5、7:3,洗脱体积分别为1.5柱体积、1.5柱体积、1.5柱体积、2.25柱体积、2.25柱体积;收集石油醚(60-90馏程):乙酸乙酯体积比为7:3洗脱的流出液,旋蒸蒸发干燥后,即得。
步骤(i)中,发酵培养基由以下成分组成:蛋白胨0.5-3 wt%,葡萄糖0.5-4 wt%,麦芽糖0.5-2 wt%,土豆淀粉1-3 wt%,KH2PO40.1-0.5 wt%,MgSO4·7H2O 0.05-0.2 wt%,维生素B1 50 mg/L-100 mg/L,大豆油0.1-0.5%,pH 6-7。所述油乳培养基由以下成分组成:大豆油30-90 L,枸橼酸30-50 g,丙三醇0.5-1 L,吐温-80 50-100 mL,预胶化淀粉5-10 g,黄原胶10-20 g。所述发酵培养基和油乳培养基的体积比为100:(6-10)。优选地,油乳培养基在发酵第24小时起每隔12小时加入一次,分2-5次加入。
步骤(i)中,发酵的温度为25℃-35℃,优选的发酵温度为25℃-30℃,更加优选为25℃-27℃。
步骤(i)中,接种量为1%-15%,如,1%、3%、5%、7.5%、10%、12%、15%;优选的接种量为5%-15%。
步骤(i)中,通气量为100-500 L/min;搅拌速度为100-300 r/min。
步骤(i)中固液分离的主要目的是分离牛樟芝的菌丝体,具体的方法可以根据产物规模选择,如滤纸过滤或抽滤,离心机离心,板框过滤等。
步骤(ii)中,发酵物干粉和乙酸乙酯的质量体积比为1:(8-30)g/mL,如,1:8、1:10、1:15、1:20、1:30;为了提高提取率可以提取2-3次。
一种上述方法制备的牛樟芝发酵提取物。该提取物中Antroquinonol的含量可达90 wt%以上。
一种上述牛樟芝提取物可作为免疫增强剂,应用于制备食品或药品。所述食品或药品的应用对象为家禽。
本发明具有以下优点:
本发明提供的牛樟芝液体发酵方法,通过培养基中加入淀粉使牛樟芝菌丝不成团、提高了传质效率;通过加入油乳培养基使得牛樟芝能够高产泛醌类物质,克服了牛樟芝在液体深层发酵条件下无法中生产泛醌类物质的难题;使用的培养基中无价格昂贵原料,降低了牛樟芝发酵液的成本,适于工业化生产。此外,本发明将牛樟芝发酵液以乙酸乙酯提取,最终分离纯化获得了高纯度的泛醌类物质Antroquinonol;并以家禽为研究对象,证实该提取物对其免疫系统和免疫反应均具有显著的增强作用,揭示了其新的生理功能;将其作为免疫增强剂在家禽中进行应用,拓展了的应用范围。
附图说明
图1为实施例1中初级扩繁菌种发酵系统示意图;
图2为提取物的1H NMR谱图;
图3为提取物的13C NMR谱图;
图4为HI抗体效价的动力学曲线;
图5为胸腺(a)、脾脏(b)和法氏囊(c)的器官指数;
图6为外周血CD4+(a)和CD8+(b)T细胞含量;
图7为外周血IL2(a)、IFN-γ(b)和IL4的表达量;
图8为外周血IFN-γ/IL4的比值。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 牛樟芝发酵菌种的制备
(1)按照以下方法配制改良PDA培养基:1 L水中加入200 g马铃薯块沸煮30min后8层纱布过滤获得滤汁,加入20 g琼脂加热融化补足至1 L,晾至65℃左右,加入10 g葡萄糖,10 g麦芽糖,3 g KH2PO4,1.5 g Mg2SO4·7H2O,50 mg维生素B1,pH值调整为6.5,分装到培养瓶中,使厚度为1 cm左右,121℃灭菌30 min备用;
按照图1所示组装无菌的初级扩繁菌种发酵系统:将5个750 mL的广口瓶作为培养瓶,瓶口塞硅胶塞,胶塞打三个直径5 mm圆孔,一孔用外径5 mm的通气管通入瓶底,另一端连接空气过滤器,用于通入压缩空气;第二个孔用外径5 mm的加液管插入瓶子中部、不接触培养基,上端连接阀门,用于注入液体培养基;第三孔用外径5 mm的排气管连接空气过滤器,用于排气;5个瓶子的通气管分别接到空压机上,加液管分别连接到液体培养基罐;
挑取斜面菌种接种在培养瓶的固体种子培养基上,在27℃下以3 L/min的量通入空气静置培养17天至牛樟芝的菌丝体扩展至培养瓶底直径的4/5左右。
(2)按照以下组成配制液体种子培养基:蛋白胨1 wt%,葡萄糖1 wt%,麦芽糖1wt%,土豆淀粉1 wt%,KH2PO40.1 wt%,MgSO4·7H2O 0.05 wt%,维生素B1 80 mg/L,大豆油0.3 wt%,调整pH 6.5,121℃灭菌20 min;
无菌条件下将上述培养基加入图1的液体培养基罐中,牛樟芝菌落在固体培养基上长至合适大小后向罐中泵入无菌空气,打开培养瓶的加液管阀门,使液体培养基流入培养瓶,至约500 mL,没过通气管开口,在27℃下以3 L/min的量通入空气静置培养7天完成初级扩繁;
将初级扩繁的菌种按照7.5%的接种量接种于10 L液体种子培养基中在25℃下按照通气量1 L/min以150 r/min搅拌发酵7天完成2级扩繁;
将2级扩繁的菌种按照10%的接种量接种于100 L液体种子培养基中在25℃下按照通气量25 L/min以150 r/min搅拌发酵7天完成3级扩繁,获得工作菌种。
相比传统的打取菌饼-摇瓶发酵的初级扩繁方式,上述方法能够快速完成扩繁,完成100 L工作菌种的发酵可比传统方法节省一次扩繁过程。此外,该系统降低了先试管培养后菌瓶扩大培养的工作量及转移中的污染风险,各个培养瓶均为独立且可视单元,即使在发酵过程中有某几个单元发生污染也可独立取出而不影响其他单元的菌种培养,从而实现了菌种的快速高效培养。
实施例2 牛樟芝液体发酵
(1)按照以下组成配制发酵培养基:蛋白胨1 wt%,葡萄糖1 wt%,麦芽糖1 wt%,土豆淀粉1 wt%,KH2PO40.1 wt%,MgSO4·7H2O 0.05 wt%,维生素B1 50 mg/L,大豆油0.5 wt%,调整pH 6.5,121℃灭菌20 min;
按照以下组成配制油乳培养基:大豆油50 L,枸橼酸45 g,丙三醇900 mL,吐温-8090 mL,预胶化淀粉9 g,黄原胶18 g,灭菌后备用;
(2)将100 L工作菌种接种到1000 L的发酵培养基中进行发酵,发酵条件如下:发酵温度(25±1)℃,通气量100 L/min,搅拌速度200 r/min;
将90 L油乳培养基在发酵第24 h起每隔12 h加入一次,分3次从发酵罐底部以细小液流的方式泵入;
(3)发酵10天后,将发酵罐温度升至91℃,保温4 h,降温后板框过滤,滤液喷雾干燥,获得发酵液粉末。
实施例3 牛樟芝发酵提取物的制备
(1)称取实施例2中牛樟芝发酵液粉末100 g,按照质量体积比1:10(g/mL)加入乙酸乙酯,超声波提取90 min,过滤,滤液旋转蒸发后用少量乙酸乙酯溶解,获得粗提物溶液;
(2)将粗提物溶液进行柱层析:层析柱规格30 mm×900 mm,硅胶300目,干料装柱,梯度洗脱:正己烷:乙酸乙酯体积比为10:0、9:1、8:2、7.5:2.5,洗脱体积分别为2.5柱体积、2.5柱体积、5柱体积、2.5柱体积;收集正己烷:乙酸乙酯=7.5:2.5的流出液,旋转蒸发,用适量乙酸乙酯溶解,获得精提物溶液;
(3)将精提物溶液进行柱层析:层析柱规格30 mm×300 mm,硅胶100目,干料装柱,梯度洗脱:石油醚(60-90):乙酸乙酯体积比为10:0、9:1、8:2、7.5:2.5、7:3,洗脱体积分别为1.5柱体积、1.5柱体积、1.5柱体积、2.25柱体积、2.25柱体积;收集石油醚(60-90):乙酸乙酯=7:3的流出液,旋转蒸发干燥后,即得到纯品85.6mg,纯度为91.03%。
将获得的纯品进行检测,其1H NMR和如图2-3所示,获得的纯品分子式为C24H38O4,结构为,与报道的牛樟芝提取的泛醌类物质中的Antroquinonol相同。
对比例1 Antroquinonol的制备
(1)按照以下组成配制发酵培养基:蛋白胨1 wt%,葡萄糖1 wt%,麦芽糖1 wt%,土豆淀粉1 wt%,KH2PO40.1 wt%,MgSO4·7H2O 0.05 wt%,维生素B1 50 mg/L,大豆油0.5 wt%,调整pH 6.5,121℃灭菌20 min;
(2)将实施例1中获得的工作菌种接种到1000 L的发酵培养基中进行发酵,发酵条件如下:发酵温度(25±1)℃,通气量100 L/min,搅拌速度200 r/min;
将90 L大豆油在发酵第24 h起每隔12 h加入一次,分3次从发酵罐底部以细小液流的方式泵入;
(3)发酵10天后,将发酵罐温度升至91℃,保温4 h,降温后板框过滤,滤液喷雾干燥,获得发酵液粉末。
(4)按照实施例3中的方法制备提取物,并未获取有效量的精提物。
这说明,实施例2中的油乳培养基能够诱导牛樟芝在液体培养条件下产生以Antroquinonol为代表的的泛醌类物质。
实施例4 牛樟芝发酵提取物作为家禽免疫增强剂
动物试验共分为3个组,分别为Antroquinonol组、免疫组和对照组,每组1日龄SPF鸡20只,分别饲养于隔离器中。免疫组和对照组饲喂普通饲料,Antroquinonol组在普通饲料中添加0.8%的Antroquinonol。饲养期49天,期间自由采食。饲喂7天后,Antroquinonol组、免疫组分别免疫H9亚型禽流感疫苗,操作按照说明书进行,皮下注射,每只0.3 mL,对照组不免疫。
免疫后每隔7天(day post inoculation, dpi)采集各组鸡的外周血,分离血清,经3000 r/min离心10 min分离血清,进行H9亚型禽流感特异性HI抗体的检测,记录HI抗体效价(图4)。分别于21和42 dpi进行免疫器官(胸腺、脾脏和法氏囊)指数的测定:鸡称重后进行宰杀,Antroquinonol组和免疫组各3只,分别采集胸腺、脾脏和法氏囊并称重,按照免疫器官指数(%)=免疫器官质量/活体质量×100%的计算公式得到免疫器官指数。以器官指数为纵坐标作两实验组胸腺、脾脏和法氏囊的柱状图,结果分别见图5。
结果表明,免疫后Antroquinonol组和免疫组鸡的抗体效价逐步提升,至28 dpi趋于稳定,直至42 dpi仍处于高位,而对照组抗体效价始终处于基准水平,表明所使用的商品化疫苗免疫效果良好;与免疫组相比,在饲料中添加Antroquinonol后,HI抗体在每个检测时间点均比免疫组高出1个左右的滴度,在免疫效果良好疫苗的基础上实现抗体效价的再提升,说明Antroquinonol有效激活了机体的免疫系统,实现了免疫反应的增强。为了证实这一点,进一步测定了两免疫组动物免疫器官指数,结果如图5所示。结果表明,无论是21dpi还是42 dpi,Antroquinonol均能够显著促进免疫器官的发育。胸腺和脾脏指数的变化趋势相似,21 dpi时,Antroquinonol组的器官指数显著高于免疫组,至42 dpi时升高数值有所降低,但仍然差异显著;法氏囊在两个时间的变化趋势与之相反,即42 dpiAntroquinonol对法氏囊的提升效果要好于21 dpi。结果表明,Antroquinonol通过促进免疫器官的发育,使得免疫反应更强。脾脏和胸腺分别是体液免疫和细胞免疫细胞产生的主要器官,脾脏功能的增强直接反应在抗体效价的提升方面,这与HI抗体的测定结果相一致。胸腺指数的升高是否也增强了细胞免疫反应的强度,为了验证这一点,随之进行了21 dpi和42 dpi外周血中CD4+和CD8+T细胞含量以及相关细胞因子IL-2、IFN-γ和IL4表达量的检测(图6-图8)。结果表明,无论是CD4+/ CD8+T细胞的含量还是IL-2、IFN-γ和IL4细胞因子的表达量,在Antroquinonol的作用下,含量均显著上调,说明无论是细胞免疫还是体液免疫的反应强度均得到提升。在免疫应答过程中,IL-2是细胞免疫的重要指标,它刺激Thp细胞向Th0细胞分化,Th0又分泌IL-2、IL-4和IFN-γ等细胞因子,故通常以IL-2衡量Th反应强度,IL-4衡量体液免疫水平,IFN-γ衡量细胞免疫水平,同时以IFN-γ/IL-4的比值反应Th1/Th2细胞的平衡状态。值得注意的是,虽然Antroquinonol组的IFN-γ和IL4含量在不同时间点均高于免疫组,但升高的比例不尽相同,所以IFN-γ/IL4的比值在21 dpi时免疫组高于Antroquinonol组,但到42 dpi时结果相反,表明Antroquinonol具有一定的免疫调节作用,作用前期偏向于提高体液免疫反应强度,而后期随着体液免疫到达稳定期,又偏向性的增强提升细胞免疫。
综上,Antroquinonol添加于基础日粮,能够有效提升家禽的免疫器官发育、增强免疫细胞的分化和免疫反应的强度,是效果良好的禽用免疫增强剂,这与之前的Antroquinonol发挥免疫抑制作用的研究结果相反,这可能与Antroquinonol的应用对象不同密切相关。
Claims (10)
1.一种牛樟芝液体发酵方法,其特征在于,包括以下步骤:
(1)将菌种接种在固体种子培养基上培养;
(2)将步骤(1)获得的菌种在液体种子培养基中扩繁培养;
(3)将步骤(2)获得的菌种接种于发酵培养基在发酵罐内通气搅拌培养并流加油乳培养基,发酵至终点获得牛樟芝发酵液;
所述发酵培养基由以下成分组成:蛋白胨0.5-3 wt%,葡萄糖0.5-4 wt%,麦芽糖0.5-2wt%,土豆淀粉1-3 wt%,KH2PO4 0.1-0.5 wt%,MgSO4·7H2O 0.05-0.2 wt%,维生素B1 50mg/L-100 mg/L,大豆油0.1-0.5 wt%,pH 6-7;
所述油乳培养基由以下成分组成:大豆油30-90 L,枸橼酸30-50 g,丙三醇0.5-1 L,吐温-80 50-100 mL,预胶化淀粉5-10 g,黄原胶10-20 g。
2.根据权利要求1所述的牛樟芝液体发酵方法,其特征在于,所述发酵培养基和油乳培养基的体积比为100:(6-10)。
3.根据权利要求1所述的牛樟芝液体发酵方法,其特征在于,油乳培养基在发酵第24小时起每隔12小时加入一次,分2-5次加入。
4.根据权利要求1所述的牛樟芝液体发酵方法,其特征在于,步骤(1)中,将牛樟芝培养于并联的试剂瓶中通气培养;且,
步骤(2)中,当固体种子培养基上牛樟芝菌落长至约2/3以上器皿直径时,将液体种子培养基加入到长有牛樟芝的固体种子培养基中进行液体发酵培养。
5.根据权利要求1所述的牛樟芝液体发酵方法,其特征在于,所述固体种子培养基由以下成分组成:200 g马铃薯煮出滤汁1 L,葡萄糖10-12 g/L,麦芽糖8-10 g/L,KH2PO4 3 g/L,Mg2SO4·7H2O 1.5 g/L,维生素B1 50-100 mg/L,琼脂15-20 g/L;pH 6.5;
所述液体种子培养基由以下成分组成:蛋白胨0.5-3 wt%,葡萄糖0.5-4 wt%,麦芽糖0.5-2 wt%,土豆淀粉1-3 wt%,KH2PO4 0.1-0.5 wt%,MgSO4·7H2O 0.05-0.2 wt%,维生素B150 mg/L-100 mg/L,大豆油0.1-0.5 wt%,pH 6-7;
步骤(1)-(3)中,培养的温度为25℃-35℃;优选的培养温度为25℃-30℃;更加优选为25℃-27℃;
步骤(2)中,扩繁次数为2-4次;
步骤(2)或(3)中,接种量为1%-15%;优选的接种量为5%-15%;
步骤(2)或(3)中,通气量为0.1-0.5 vvm,搅拌速度为100-300 r/min。
6.一种牛樟芝发酵提取物的制备方法,其特征在于,包括以下步骤:
(i)将菌种接种于发酵培养基在发酵罐内通气搅拌发酵并流加油乳培养基,发酵至终点获得发酵液;灭菌后固液分离获得发酵滤液,干燥后获得发酵物干粉;
(ii)将发酵物干粉以乙酸乙酯提取后去除溶剂获得粗提物;
(iii)将粗提物以乙酸乙酯溶解进行硅胶柱层析,梯度洗脱剂洗脱:正己烷:乙酸乙酯体积比为10:0、9:1、8:2、7.5:2.5,洗脱体积分别为2.5柱体积、2.5柱体积、5柱体积、2.5柱体积;
收集正己烷:乙酸乙酯体积比为7.5:2.5洗脱的流出液,旋转蒸发,用适量乙酸乙酯溶解后进行二次硅胶柱层析,梯度洗脱剂洗脱:石油醚(60-90馏程):乙酸乙酯体积比为10:0、9:1、8:2、7.5:2.5、7:3,洗脱体积分别为1.5柱体积、1.5柱体积、1.5柱体积、2.25柱体积、2.25柱体积;收集石油醚(60-90馏程):乙酸乙酯体积比为7:3洗脱的流出液,旋蒸蒸发干燥后,即得;
步骤(i)中,发酵培养基由以下成分组成:蛋白胨0.5-3 wt%,葡萄糖0.5-4 wt%,麦芽糖0.5-2 wt%,土豆淀粉-3 wt%,KH2PO4 0.1-0.5 wt%,MgSO4·7H2O 0.05-0.2 wt%,维生素B150 mg/L-100 mg/L,大豆油0.1-0.5%,pH 6-7;
所述油乳培养基由以下成分组成:大豆油30-90 L,枸橼酸30-50 g,丙三醇0.5-1 L,吐温-80 50-100 mL,预胶化淀粉5-10 g,黄原胶10-20 g。
7.根据权利要求6所述的制备方法,其特征在于,步骤(i)中,所述发酵培养基和油乳培养基的体积比为100:(6-10);油乳培养基在发酵第24小时起每隔12小时加入一次,分2-5次加入;
步骤(ii)中,发酵物干粉和乙酸乙酯的质量体积比为1:(8-30)g/mL;提取次数为2-3次。
8.根据权利要求6所述的制备方法,其特征在于,步骤(i)中,发酵的温度为25℃-35℃,优选的发酵温度为25℃-30℃,更加优选为25℃-27℃;
步骤(i)中,接种量为1%-15%;优选的接种量为5%-15%;通气量为100-500 L/min;搅拌速度为100-300 r/min;
步骤(i)中固液分离的方法选自滤纸过滤或抽滤,离心机离心或板框过滤。
9.一种如权利要求6-8任一所述的制备方法获得的牛樟芝提取物。
10.一种上述牛樟芝提取物作为免疫增强剂在制备食品或药品中的应用。
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