CN114717219B - 一种具有抗氧化和降低胆固醇功能的罗伊氏乳杆菌制剂 - Google Patents
一种具有抗氧化和降低胆固醇功能的罗伊氏乳杆菌制剂 Download PDFInfo
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Abstract
本发明提供一种罗伊氏乳杆菌制剂,所述的菌制剂包含有芯材和壁材,其中芯材是保藏号为CCTCC NO:M2019779的罗伊氏乳杆菌VHProbi M07株。本发明提供的罗伊氏乳杆菌制剂可以作为益生菌添加到食品中,长期服用不会有副作用及过量的风险。所述罗伊氏乳杆菌VHProbi M07株抗氧化能力较强,该菌株还能有效降解胆固醇。本发明制备得到的罗伊氏乳杆菌制剂中单位活菌量高,且经冷冻干燥后菌的存活率高达97.0%。所述罗伊氏乳杆菌制剂抗逆性强,经过人工胃液和人工肠液消化后仍有较高的活菌量,活菌存活率高达84.2%。
Description
技术领域
本发明属于益生菌应用技术领域,具体涉及一种具有抗氧化和降低胆固醇功能的罗伊氏乳杆菌制剂。
背景技术
益生菌是一类活的微生物,当施以足够数量时能够对宿主健康产生益处。近二十年来,科学界对肠道菌群的研究呈现出爆发性增长。研究发现益生菌可以帮助消化吸收,促进肠道菌群平衡,维护人体健康。越来越多的证据表明,益生菌对于人体的健康影响不仅局限于肠道,还有更广泛的作用范围,如内分泌平衡调节、免疫平衡调节、神经系统调节、呼吸系统调剂等。
心血管疾病已经成为现代人死亡的主要原因之一,降低血液胆固醇、提高机体抗氧化能力已经成为预防心血管疾病的重要途经之一。益生菌可以水解胆汁盐、吸收胆固醇、调节胆固醇转运,降低氧化应激水平。因此,通过服用益生菌来预防心血管疾病得到了越来越多的关注。。
益生菌活菌量是功能性益生菌菌株发挥益生作用的前提,只有施以一定量的功能性菌株才会对宿主产生健康益处。发酵乳标准中规定所标注活菌数不得低于106CFU/g。通过将益生菌制备成菌粉制剂,不仅能够提高其单位活菌量和抗逆性能,还能保证益生菌货架期内的稳定性,有力降低生产成本,为益生菌株开发成更多的功能性食品奠定了基础。
发明内容
本发明的目的是提供一种罗伊氏乳杆菌制剂及其制备方法。所提供的菌制剂具有抗氧化和降解胆固醇功能。
本发明提供一种益生菌制剂,所述的菌制剂包含有芯材和壁材,其中芯材为罗伊氏乳杆菌(Lactobacillus reuteri)VHProbi M07株;所述的罗伊氏乳杆菌(Lactobacillusreuteri)VHProbi M07株,已于2019年10月08日保藏于位于中国武汉的武汉大学的中国典型培养物保藏中心,其保藏号为CCTCC NO:M2019779。
所述的壁材,其组分包含有大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙;
优选的,大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙的质量份数比为1:4:1:1:0.1:8;
所提供的菌制剂,其一种制备方法,是将大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙加水搅拌混匀,并与菌悬液充分混匀制成冷冻混合液;将混合液进行冷冻干燥获得益生菌制剂。
本发明提供的罗伊氏乳杆菌VHProbi M07可以作为益生菌添加到食品中,长期服用不会有副作用及过量的风险。所述罗伊氏乳杆菌VHProbi M07株抗氧化能力较强,菌体抗脂质过氧化抑制率为73.1%,DPPH清除率达到50.26%,HRS清除率达到64.79%。该菌株还能有效降解胆固醇,降解率达到27.86%。本发明制备得到的罗伊氏乳杆菌制剂中单位活菌量高,且经冷冻干燥后菌的存活率高达97.0%。所述罗伊氏乳杆菌制剂抗逆性强,经过人工胃液和人工肠液消化后仍有较高的活菌量,活菌存活率高达84.2%。
本发明提供的罗伊氏乳杆菌制剂的制备工艺过程简单,包被材料便宜,产业化成本低,能有效提高了罗伊氏乳杆菌VHProbi M07在存储加工运输过程中的稳定性,有力提高了罗伊氏乳杆菌VHProbi M07的实际使用效果,可广泛应用于食品、保健品等领域。
附图说明
图1为M07菌株Riboprinter指纹图谱;
图2为M07菌株的RAPD指纹图谱;
图3为M07菌株的rep-PCR指纹图谱;
图4为各组别小鼠白细胞分类计数结果图;
图5为各组别小鼠细肺泡灌洗液细胞因子测定结果图;
图6为各组别小鼠肺部组织病理染色结果图。
具体实施方式
申请人在之前研究中发现筛选获得的罗伊氏乳杆菌(Lactobacillus reuteri)VHProbi M07株具有缓解小鼠过敏性哮喘的功效;进一步研究发现M07株还具有抗氧化和降解胆固醇的功效,并制备相关的菌制剂。
下面对本发明所使用的罗伊氏乳杆菌(Lactobacillus reuteri)VHProbi M07株的筛选,以及缓解过敏性哮喘的功效进行描述。
实施例1:罗伊氏乳杆菌VHProbi M07分离筛选、理化性质及缓解过敏性哮喘的功效
一、罗伊氏乳杆菌VHProbi M07的分离筛选
1、初筛
依据2019版《人类遗传资源库伦理规范》,与样本提供者签订项目承诺书和知情同意书后,按照生物样本库标准操作规范,取1mL半年内未食用过益生菌制剂的哺乳期产妇的新鲜母乳,经无菌生理盐水稀释后放入无菌样品袋中,用匀浆仪拍打混匀;取100μL混匀液梯度稀释,涂布于MRS琼脂培养基后于37℃厌氧培养48h,待平板长出单菌落镜检。
根据镜检结果,申请人共筛选出7株潜在乳酸杆菌,分别命名为M01,M02,M03,M04,M05,M06和M07。
2、复筛
将筛选得到的7株乳酸杆菌按6%接种量分别接种于耐酸性培养基中,37℃条件下厌氧静置培养48h,取发酵液进行菌量计数。
结果显示,所述7株乳酸杆菌发酵液中活菌量的对数值分别为7.23、6.94、6.76、7.56、6.33、5.39、8.78Log CFU/mL,其中M07菌株经耐酸性培养基复筛后活菌量最多,对数值高达8.78Log CFU/mL;从而说明M07号菌株耐酸能力最高。
将M07菌株接种于MRS琼脂培养基上,37℃厌氧培养24h后,可见M07单菌落呈乳白色,菌落直径在1.5-3mm左右,表面湿润,显微镜下末端呈圆形的弯曲杆菌。
盐度耐受性试验结果表明M07菌株最大耐受盐浓度为1%。
过氧化氢酶实验结果表明M07菌株不产生气泡,是阴性反应。
对M07株进行碳源代谢试验,具体结果见表1。
表1:M07菌株碳源代谢结果表
注:“+”阳性反应;“-”阴性反应。
通过测序获得M07菌株的16s rDNA序列SEQ ID NO:1,并将该序列在NCBI数据库中进行比对,初步确定M07菌株为罗伊氏乳杆菌。二、罗伊氏乳杆菌M07的分子生物学特征
M07菌株的Riboprinter指纹图谱结果如图1所示,RAPD指纹图谱如图2所示,rep-PCR指纹图谱如图3所示。
将M07菌株的菌落形态以及生理生化特性结果上传至网站http://www.tgw1916.net/bacteria_logare_desktop.htmL,同时结合文献De Clerck E,etal.Systematic and applied microbiology,2004,27(1)50公布的结果,进行比对。综合分子生物学的鉴定结果,可以得出结论,M07菌株为一株新型的罗伊氏乳杆菌,将其命名为罗伊氏乳杆菌VHProbi M07。三、罗伊氏乳杆菌VHProbi M07株对人工胃液和人工肠液的耐受性试验
按照国标《GB4789.35-2016-食品微生物检验乳酸菌检验》进行检测,确定菌株经过人工肠液消化后的活菌量(Log CFU/mL)
表2:人工胃肠液消化后的活菌量表
四、罗伊氏乳杆菌VHProbi M07的溶血性实验
罗伊氏乳杆菌VHProbi M07不能生长,血细胞平板没有变化,说明罗伊氏乳杆菌VHProbi M07不产生溶血素,不能够溶解血细胞。
五、抗生素耐受性实验
从表3的结果可以看出,罗伊氏乳杆菌VHProbi M07对红霉素和氨苄西林等常见抗生素敏感,生物安全性良好。
表3:罗伊氏乳杆菌VHProbi M07的抗生素MIC值(μg/mL)
六、疏水性细胞表面测试
结果显示本发明提供的罗伊氏乳杆菌VHProbi M07细胞表面疏水性为64.48%,标准差为2.16%。
七、罗伊氏乳杆菌VHProbi M07在缓解小鼠过敏性哮喘中的应用1.1实验材料
BALB/c小鼠SPF级,24只,雌性,8-9周,体重19~25g。实验动物饲养管理的环境条件:室温20~26℃,日温差≤4℃,相对湿度40~70%,明暗交替时间为12/12h。动物饲养于标准小鼠笼具中,每笼6只。
1.2实验方法
1.2.1菌液制备
将单菌落划线MRS平板上,37℃厌氧培养24~48h,挑取单菌落于MRS肉汤培养基扩大培养16h后,收集菌液调整其浓度为109CFU/mL菌悬液。
1.2.2分组
小鼠适应性饲养7天后随机分为空白对照组、OVA过敏模型组、益生菌预处理组、益生菌后处理组,每组6只小鼠,益生菌预处理组和后处理组按照0.2mL/10g灌胃给予益生菌菌液。
1.2.3造模及益生菌干预方案
益生菌预处理组小鼠造模前开始提前灌胃给予益生菌,连续给予10天,其他组不处理。除空白对照组外,OVA过敏模型组,益生菌预处理组,益生菌后处理组小鼠在第0,6天腹腔注射过敏原溶液200μl,每只小鼠注射50μg OVA,800μg氢氧化铝,空白对照组注射PBS;第12,14,17,20,23,27天对OVA过敏模型组,益生菌预处理组,益生菌后处理组小鼠以2%OVA雾化激发,空白对照组以生理盐水代替,最后一次雾化激发24h后处死小鼠,进行肺泡灌洗,收集肺泡灌洗液,离心,涂片,瑞氏染色,进行白细胞分类计数,上清液用于细胞因子浓度检测;取肺组织,固定,HE染色,观察病理改变。
1.3指标观察
1.3.1小鼠肺泡灌洗液白细胞分类计数及IL-5、IL-10、IL-13、MCP-1、TNF-α、INF-γ、eotaxin细胞因子浓度测定
每只小鼠给予2%的戊巴比妥钠溶液(0.045ml/g)腹腔注射麻醉,切开小鼠颈部皮肤,钝性分离颈部肌肉及结缔组织,暴露气管。用自制穿刺针气管插管并连接1ml注射器,用4℃预冷PBS灌洗全肺肺泡,每次0.8ml,充分回收至少0.6ml,反复3次。将收集的BALF于4℃,1200r/min,离心10min。收集上清,-20℃保存。沉淀用于制备细胞涂片,瑞氏染色,进行白细胞分类计数;收集的上清,通过Elisa方法检测IL-5、IL-10、IL-13、MCP-1、TNF-α、INF-γ、eotaxin细胞因子浓度。
1.3.2组织病理检查
雾化激发24h后,取肺组织,4%多聚甲醛固定,取材,脱水,石蜡包埋,切片,HE染色观察肺组织病理学改变。
1.3.3数据统计处理方法
所有实验数据以均数±标准差表示,用Microsoft EXCEL进行数据统计和作图,两组数据间比较采用t检验,以P<0.05判定为有显著性差异。
1.4实验结果
1.4.1小鼠肺泡灌洗液白细胞分类计数及IL-5、IL-10、IL-13、MCP-1、TNF-α、INF-γ、eotaxin细胞因子浓度测定
末次激发后与空白对照组比较,OVA过敏模型组中嗜酸性粒细胞、中性粒细胞显著增加(P<0.05),证明模型构建成功。与OVA模型组比较,益生菌预处理组嗜酸性粒细胞和中性粒细胞减少,具有显著性差异(P<0.05);益生菌后处理组中性粒细胞减少,具有显著性差异(P<0.05),嗜酸性粒细胞减少。各组小鼠肺泡灌洗液白细胞分类计数如下表4,比较结果如图4。
表4:小鼠肺泡灌洗液中白细胞分类计数结果表
PBS:空白对照组,OVA:OVA过敏模型组,Pre:益生菌预处理组,Pos:益生菌后处理组。与空白对照组比较*,P<0.05;与OVA过敏模型组比较#,P<0.05
末次激发后与空白对照组比较,OVA过敏模型组小鼠肺泡灌洗液中IL-5、IL-13、MCP-1、TNF-α、eotaxin细胞因子浓度升高且具有显著性差异(P<0.05),IL-10、INF-γ浓度降低且具有显著性差异(P<0.05),说明OVA致小鼠过敏性哮喘模型构建成功。与OVA过敏模型组比较,益生菌后处理组小鼠肺泡灌洗液中IL-5、IL-13、MCP-1、TNF-α、eotaxin浓度均降低且有显著性差异(P<0.05),IL-10、INF-γ浓度升高且有显著性差异(P<0.05);益生菌预处理组肺泡灌洗液中IL-5、IL-13、MCP-1、TNF-α、eotaxin浓度均降低且有显著性差异(P<0.05),IL-10、INF-γ浓度升高且有显著性差异(P<0.05)。各组小鼠肺泡灌洗液中IL-5、IL-10、IL-13、MCP-1、TNF-α、INF-γ、eotaxin细胞因子浓度数据如下表5,比较如图5所示。
表5:各组小鼠肺泡灌洗液中细胞因子浓度检测结果比较表
PBS:空白对照组;OVA:OVA过敏模型组;Pre:益生菌预处理组;Pos:益生菌后处理组。与空白对照组比较:*P<0.05,**P<0.01;与OVA模型组比较:#P<0.05,##P<0.01
1.5.2组织病理学检查
光学显微镜下可见,空白对照组肺内支气管各级分支被覆正常的呼吸道上皮,肺泡由I型和II型肺泡细胞围成,并见各级支气管和肺泡之间的少量间质内及其血管周围,无炎性细胞浸润;OVA模型组肺脏表现为终末细支气管周围套袖样的炎性增生,终末细支气管向较大支气管周围蔓延,肺泡巨噬细胞增多明显;益生菌预处理组肺脏的主要病理变化表现为除肺泡巨噬细胞增多外,未见其它明显炎性反应;益生菌后处理组肺脏的主要病理变化表现为肺血管扩张,炎性细胞浸润、肺泡巨噬细胞增多等炎性反应;典型病理病变见图6。
由上述结果可知,与OVA过敏模型组相比,益生菌预处理组小鼠气道炎症减轻,嗜酸性粒细胞和中性粒细胞分别降低了77.0%和37.1%。Th1/Th2型免疫反应趋于平衡,IL-5降低了26.8%,IL-13降低了29.1%、MCP-1降低了14.7%、TNF-α降低了43.0%、eotaxin浓度降低了27.5%,IL-10升高了17.2%、INF-γ浓度升高了10.8%;与OVA过敏模型组相比,益生菌后处理组小鼠气道炎症减轻,嗜酸性粒细胞和中性粒细胞分别降低了72.3%和36.2%。Th1/Th2型免疫反应趋于平衡,IL-5降低了20.7%,IL-13降低了38.0%、MCP-1降低了13.2%、TNF-α降低了59.4%、eotaxin浓度降低了31.9%,IL-10升高了34.5%、INF-γ浓度升高了11.7%;病理切片结果显示,益生菌预处理组小鼠的未见明显炎症反应,而益生菌后处理组小鼠炎性细胞浸润现象减轻。
上述结果表明本发明提供的罗伊氏乳杆菌VHProbi M07对人工肠胃液具有很强的耐受性,在人工肠胃液中的存活率达到86.2%;该菌株对红霉素和氨苄西林等常见的抗生素敏感,不产生溶血素,不能够溶解血细胞。具有良好的生物安全性;最大耐受盐浓度为1%,过氧化氢酶反应呈阴性。
实施例2:罗伊氏乳杆菌VHProbi M07抗氧化功能测定
一、菌株清除DPPH(1,1-二苯基-2-三硝基苯肼)和羟基自由基(HRS)能力测定
1、PBS菌悬液制备
将生长状态优良的单菌落接种于3mL的MRS液体培养基中,37℃条件下培养24h,以此培养液为接种液,按照2%的接种量接种于50mL的MRS液体培养基中,静置培养24h,获得菌株的培养液。吸取1mL菌液收集菌体后用1mLPBS缓冲液洗涤菌体2遍后再加入2mLPBS溶液重悬菌体备用。
2、菌株清除DPPH自由基能力的测定
取1mL待测菌株的PBS菌悬液,加入1mL 0.4mM的现配的DPPH自由基溶液,混合均匀后然后置于室温温度下遮光反应30min,然后测定样品在波长517nm处的吸光度A样本,测3次平行。对照组样品以等体积PBS溶液和DPPH·乙醇混合液,并以等体积PBS菌悬液和乙醇混合液空白调零。清除率按下列公式计算:清除率%=[1-(A样品-A空白)/A对照]×100%。结果见表6。
表6:DPPH自由基清除率表
3、菌株清除HRS能力的测定
将100μL 5mM的水杨酸钠-乙醇溶液,100μL 5mM的硫酸亚铁,500μL去离子水和200μL乳酸菌PBS菌悬液混匀后加入100μL过氧化氢溶液(3mM),37℃水浴15min后在510nm波长处测量样品吸光度。羟自由基清除率按照下列公式进行计算。
清除率%=(A样品-A控制)/(A空白-A控制)×100%,其中A控制为去离子水替代样品,A空白为去离子水替代样品和H2O2,结果见表7。
表7:HRS自由基清除率表
二、菌株抗脂质过氧化实验鉴定
1、乳酸菌的培养及发酵上清液的制备:
乳酸菌在MRS液体培养基中37℃培养24h,传3代后,6000rpm/min,4℃离心10min,收集上清液即为发酵上清液。收集的菌体用PBS缓冲液(pH 7.4)于6000r/min离心10min,洗涤3次。将菌体重悬于PBS缓冲液,调整菌体浓度为1.0×109cells/mL,获得菌悬液。
2、亚油酸乳化液制备:0.1mL亚油酸,0.2mL Tween 20,19.7mL去离子水。
3、0.5mL的PBS溶液(pH 7.4)中加入1mL亚油酸的乳化液,1mLFeSO4(1%),再加入0.5mL样品,37℃水浴1.5h,混合液加入0.2mL TCA(4%),2mL TBA(0.8%),100℃水浴30min,迅速冷却,4000rpm/min离心15min,收集上清液在532nm下测吸光度即为A;对照组以0.5mL蒸馏水代替样品即为A0。抑制率/%=(A0-A)/A0×100%
注:A为样品组吸光度;A0为对照组吸光度,结果见表8。
表8:抗脂质过氧化抑制率表
实施例3:罗伊氏乳杆菌VHProbi M07体外胆固醇降解实验
1、胆固醇胶束溶液的配制:准确称取1g胆固醇,溶于无水乙醇中,并定容至100mL,在无菌条件下用0.22μm微孔滤膜过滤除菌。
2、称取蛋白胨10.0g,牛肉膏10.0g,酵母膏5.0g,柠檬酸氢二铵2.0g,葡萄糖20.0g,吐温80 1.0mL,乙酸钠5.0,硫酸镁0.1,硫酸锰0.05,磷酸氢二钾2.0g,胆盐1g,蒸馏水1000mL调节pH值7.3,115℃灭菌30min,然后加入胆固醇溶液使胆固醇终浓度为0.1%。
按照0.1%的接种量接种新鲜菌液,37℃静止培养48h,然后取0.2mL菌液,加入1.8mL无水乙醇,混匀,静止10分钟,3000转离心5分钟,取上清液用于测定胆固醇含量。胆固醇测定方法按照GB/T5009.128-2003<食品中胆固醇的测定>。
结果显示:本发明提供的罗伊氏乳杆菌VHProbi M07对胆固醇的降解率达到27.86%(此为不含胆盐的数据)。
因此,罗伊氏乳杆菌VHProbi M07株抗氧化能力较强,菌体抗脂质过氧化抑制率为73.1%,DPPH清除率达到50.26%,HRS清除率达到64.79%。该菌株还能有效降解胆固醇,降解率达到27.86%。
实施例4:一种罗伊氏乳杆菌VHProbi M07的制剂及其制备方法
1、制备罗伊氏乳杆菌发酵液
在无菌条件下,将罗伊氏乳杆菌VHProbi M07种子液以3%的体积比接种于发酵培养基中,所述发酵培养基中各组分及其含量分别为:玉米淀粉10g/L、葡萄糖0.5/L、豆粕5/L、碳酸钙0.1/L、氯化铵0.1/L、磷酸氢二钾0.1g/L、硫酸镁0.01g/L和硫酸锰0.01g/L;37℃培养24h,转速为180rpm,通风量为1:1.2,罐压为0.05MPa。发酵完成后,即得到罗伊氏乳杆菌VHProbi M07发酵液,其中罗伊氏乳杆菌的活菌量为1010-1011CFU/mL。
2、制备冷冻干燥混合液
将罗伊氏乳杆菌VHProbi M07发酵液在4℃,5000r/min条件下离心10min,收集菌体后用无菌生理盐水洗涤2次,重悬菌体制成菌悬液。
按1:4:1:1:0.1:8的重量比分别称取大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙六种包被材料,加水搅拌混匀,并与上述菌悬液充分混匀,得冷冻混合液;所述冷冻混合液中各种包被材料的重量体积比(g/mL)分别为大豆蛋白2.5%、脱脂奶粉10%、玉米淀粉2.5%、蔗糖2.5%、阿拉伯胶0.25%、乳酸钙20%;混合液中罗伊氏乳杆菌VHProbiM07的终浓度为1.5×109CFU/mL。
3、冷冻干燥处理
将混合液置于冷冻干燥机中进行冷冻干燥,冷冻干燥程序为:预冷温度-40℃,预冷时间3h,以1℃/min的速率升温至-30℃以进行一次干燥,时间持续800min,然后以1℃/min的速率升温至25℃进行二次干燥,时间持续2h,冷阱温度-80℃左右,真空度20Pa,获得冻干菌粉。
实施例5:一种罗伊氏乳杆菌制剂及其制备方法
1、制备罗伊氏乳杆菌发酵液
在无菌条件下,将罗伊氏乳杆菌VHProbi M07种子液以5%的体积比接种于发酵培养基中,所述发酵培养基中各组分及其含量分别为:玉米淀粉20g/L、葡萄糖1.5g/L、豆粕20g/L、碳酸钙1.0g/L、氯化铵0.5g/L、磷酸氢二钾0.5g/L、硫酸镁0.5g/L和硫酸锰0.5g/L;37℃培养30h,转速为180~200rpm,通风量为1:1.3,罐压为0.06MPa。发酵完成后即得到罗伊氏乳杆菌VHProbi M07发酵液,其中罗伊氏乳杆菌的活菌量为1010-1011CFU/mL。
2、制备冷冻混合液
将罗伊氏乳杆菌VHProbi M07发酵液在4℃,5000r/min条件下离心10min,收集菌体后用无菌生理盐水洗涤2次,重悬菌体制成菌悬液。
按5:6:2:3:0.4:10的重量比分别称取大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙六种包被材料,加水搅拌混匀,并与上述菌悬液充分混匀,得冷冻混合液;所述冷冻混合液中各种包被材料的重量体积比(g/mL)分别为:大豆蛋白12.5%、脱脂奶粉15%、玉米淀粉5%、蔗糖7.5%、阿拉伯胶1%、乳酸钙25%;所述冷冻混合液中罗伊氏乳杆菌VHProbi M07的终浓度为1.1×1010CFU/mL;
3、冷冻干燥处理
将混合液置于冷冻干燥机中进行冷冻干燥,冷冻干燥程序为:预冷温度-50℃,预冷时间2.5h,以1℃/min的速率升温至-20℃以进行一次干燥,时间持续1000min,然后以1.5℃/min的速率升温至30℃进行二次干燥,时间持续1.2h,冷阱温度-80℃左右,真空度20Pa,获得冻干菌粉。
实施例6:一种罗伊氏乳杆菌制剂及其制备方法
1、制备罗伊氏乳杆菌发酵液
在无菌条件下,将罗伊氏乳杆菌VHProbi M07种子液以5%的体积比接种于发酵培养基中,所述发酵培养基中各组分及其含量分别为:玉米淀粉40g/L、葡萄糖3g/L、豆粕25g/L、碳酸钙1.5g/L、氯化铵1.0g/L、磷酸氢二钾1.0g/L、硫酸镁0.7g/L和硫酸锰0.7g/L;37℃培养36h,转速为200rpm,通风量为1:1.5,罐压为0.05~0.08MPa。发酵完成后即得到罗伊氏乳杆菌VHProbi M07发酵液,其中罗伊氏乳杆菌的活菌量为1010-1011CFU/mL。
2、制备冷冻混合液
将罗伊氏乳杆菌VHProbi M07发酵液在4℃,5000r/min条件下离心10min,收集菌体后用无菌生理盐水洗涤2次,重悬菌体制成菌悬液。
按2:5:4:2:1:5的重量比分别称取大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙六种包被材料,加水搅拌混匀,并与上述菌悬液充分混匀,得冷冻混合液;所述冷冻混合液中各种包被材料的重量体积比(g/mL)分别为:大豆蛋白5%、脱脂奶粉12.5%、玉米淀粉10%、蔗糖5%、阿拉伯胶2.5%、乳酸钙12.5%;所述冷冻混合液中罗伊氏乳杆菌VHProbi M07的终浓度为3.5×109CFU/mL;
3、冷冻干燥处理
将混合液置于冷冻干燥机中进行冷冻干燥,冷冻干燥程序为:预冷温度-60℃,预冷时间1h,以1℃/min的速率升温至-40℃以进行一次干燥,时间持续500min,然后以1℃/min的速率升温至20℃进行二次干燥,时间持续2.5h,冷阱温度-80℃左右,真空度20Pa,获得冻干菌粉。
实施例:7罗伊氏乳杆菌制剂性能检测
1、菌株存活率检测
取实施例4-6制备得到的罗伊氏乳杆菌制剂各1g,经生理盐水梯度稀释后,采用倾注法计数所述制剂中的活菌数,计算制剂中单位活菌量及罗伊氏乳杆菌的存活率,结果见表9。
表9:罗伊氏乳杆菌制剂中单位活菌量及菌株的存活率比较
罗伊氏乳杆菌制剂 | 单位活菌量 | 罗伊氏乳杆菌的存活率 |
实施例4 | 4.29×1010CFU/g | 97.0% |
实施例5 | 2.18×1010CFU/g | 90.1% |
实施例6 | 3.05×1010CFU/g | 94.9% |
从表9的结果可以看出,实施例6提供的罗伊氏乳杆菌制剂单位活菌量高,且罗伊氏乳杆菌VHProbi M07经冷冻干燥后存活率可以达到97.0%,从而说明本发明选用的包被材料和冷冻干燥制备工艺对罗伊氏乳杆菌VHProbi M07的伤害非常小,效果显著。
2、抗逆性检测
2.1人工胃液的配制
分别称取蛋白胨5g、酵母提取物2.5g、葡萄糖1g和NaCl 2g,加入1000mL蒸馏水,用稀盐酸调pH3.0,然后115℃灭菌20min。然后使用前加入3.2g猪粘膜胃蛋白酶,摇匀溶解,置37℃水浴摇床中温水浴1h,以模拟人体温度。
2.2人工肠液的配制
分别称取蛋白胨5g、酵母提取物2.5g、葡萄糖1g、KH2PO4 6.8g和牛胆盐3.0g,加入77mL的0.2mol/L的NaOH溶液,定容至1000mL,用稀盐酸或者NaOH溶液调pH6.8±0.1,115℃灭菌20min。然后使用前加入1g胰酶,摇匀溶解,置37℃水浴摇床中温水浴1h,以模拟人体温度。
2.3试验方法
取2g实施例6制备得到的罗伊氏乳杆菌制剂用2mL生理盐水重悬,作为接种液。取1mL接种液,加入到9mL提前温水浴1h的人工胃液中,置于37℃水浴摇床以200rpm/min转速振荡2h,分别于0h和2h时取样1mL,检测活菌量。然后取1mL消化2h后的人工胃液,加入到24mL人工肠液中,置于37℃水浴摇床(200rpm/min)3h,取样1mL,检测活菌量。活菌计数方法按照国标《GB4789.35-2016-食品微生物检验乳酸菌检验》测定菌量,该菌制剂经过人工胃液和人工肠液消化后的活菌量(Log CFU/mL)见表10。
表10:罗伊氏乳杆菌制剂对人工胃液和人工肠液的耐受效果表
从表10的结果可以看出,实施例6制备的罗伊氏乳杆菌制剂经过人工胃液和人工肠液消化后仍有较高的活菌量,活菌存活率为84.2%,说明本发明制得的罗伊氏乳杆菌制剂具有很强的耐胃酸耐胆碱能力。
本发明提供的罗伊氏乳杆菌制剂的制备工艺过程简单,包被材料便宜,产业化成本低,能有效提高了罗伊氏乳杆菌VHProbi M07在存储加工运输过程中的稳定性,有力提高了罗伊氏乳杆菌VHProbi M07的实际使用效果,可广泛应用于食品、保健品等领域。
序列表
<110> 青岛蔚蓝生物股份有限公司
青岛蔚蓝生物集团有限公司
<120> 一种具有抗氧化和降低胆固醇功能的罗伊氏乳杆菌制剂
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1315
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgactttggg cgttacaaac tcccatggtg tgacgggcgg tgtgtacaag gcccgggaac 60
gtattcaccg cggcatgctg atccgcgatt actagcgatt ccgacttcgt gtaggcgagt 120
tgcagcctac agtccgaact gagaacggct ttaagagatt agcttactct cgcgagtttg 180
cgactcgttg taccgtccat tgtagcacgt gtgtagccca ggtcataagg ggcatgatga 240
tctgacgtcg tccccacctt cctccggttt gtcaccggca gtctcactag agtgcccaac 300
tcaatgctgg caactagtaa caagggttgc gctcgttgcg ggacttaacc caacatctca 360
cgacacgagc tgacgacgac catgcaccac ctgtcattgc gtccccgaag ggaacgcctt 420
atctctaagg ttagcgcaag atgtcaagac ctggtaaggt tcttcgcgta gcttcgaatt 480
aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt tcaaccttgc 540
ggtcgtactc cccaggcgga gtgcttaatg cgttagctcc ggcactgaag ggcggaaacc 600
ctccaacacc tagcactcat cgtttacggc atggactacc agggtatcta atcctgttcg 660
ctacccatgc tttcgagcct cagcgtcagt tgcagaccag acagccgcct tcgccactgg 720
tgttcttcca tatatctacg cattccaccg ctacacatgg agttccactg tcctcttctg 780
cactcaagtc gcccggtttc cgatgcactt cttcggttaa gccgaaggct ttcacatcag 840
acctaagcaa ccgcctgcgc tcgctttacg cccaataaat ccggataacg cttgccacct 900
acgtattacc gcggctgctg gcacgtagtt agccgtgact ttctggttgg ataccgtcac 960
tgcgtgaaca gttactctca cgcacgttct tctccaacaa cagagcttta cgagccgaaa 1020
cccttcttca ctcacgcggt gttgctccat caggcttgcg cccattgtgg aagattccct 1080
actgctgcct cccgtaggag tatggaccgt gtctcagttc cattgtggcc gatcagtctc 1140
tcaactcggc tatgcatcat cgccttggta agccgttacc ttaccaacta gctaatgcac 1200
cgcaggtcca tcccagagtg atagccaaag ccatctttca aacaaaagcc atgcggcttt 1260
tgttgttatg cggtattagc atctgtttcc aaatgttatc ccccgctccg gggca 1315
Claims (6)
1.一种益生菌制剂,其特征在于,所述的益生菌制剂包含有芯材和壁材,其中芯材是保藏号为CCTCC NO:M2019779的罗伊氏乳杆菌。
2.如权利要求1所述的益生菌制剂,其特征在于,所述的壁材的组分包含有大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙。
3.如权利要求2所述益生菌制剂,其特征在于,所述的大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙的质量份数比为1:4:1:1:0.1:8。
4.如权利要求2-3任一项所述的益生菌制剂,其特征在于,所述的菌制剂是将大豆蛋白、脱脂奶粉、玉米淀粉、蔗糖、阿拉伯胶和乳酸钙加水搅拌混匀,并与菌悬液充分混匀制成冷冻混合液;将混合液进行冷冻干燥获得益生菌制剂。
5.权利要求1-3任一项所述的益生菌制剂在制备抗氧化制品中的应用。
6.权利要求1-3任一项所述益生菌制剂在制备降解胆固醇的制品中的应用。
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