CN108486000B - 一种双歧杆菌单菌发酵乳制备方法及其应用 - Google Patents
一种双歧杆菌单菌发酵乳制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种双歧杆菌单菌发酵乳制备方法及其应用,属于乳制品加工领域。本发明中使用的产黏性胞外多糖的双歧杆菌菌株为长双歧杆菌长亚种YS108R菌株。所述双歧杆菌发酵乳含有≥108CFU/g的双歧杆菌活菌,胞外多糖含量为130mg/L。与商品发酵剂发酵制作的产品相比,本发明的双歧杆菌发酵乳具有更低的乳清析出率和更高的持水力,且口感粘滑、细腻。本发明的双歧杆菌发酵乳还具有缓解结肠炎的作用。
Description
技术领域
本发明涉及一种双歧杆菌单菌发酵乳制备方法及其应用,属于乳品加工领域。
背景技术
乳酸菌在发酵乳制品工业中具有重要的应用,菌株的特性对发酵乳产品的理化性质有显著的影响。通常用于发酵乳生产的乳酸菌为嗜热链球菌和保加利亚乳杆菌。然而,随着有关双歧杆菌保健作用的研究增加以及潜在的商业利益的发掘,双歧杆菌发酵乳的研究和相关产品的开发也越来越多。研究表明,部分双歧杆菌发酵乳具有改善血脂水平、缓解结肠炎、预防腹泻和便秘以及免疫调节等保健功能。
炎症性肠病(IBD)是一类肠道慢性炎症,具有反复发作的特点,主要包括溃疡性结肠炎(UC)和克罗恩病(CD)两种表型。在二十年前,我国居民中IBD患者还很少见,患病率远低于欧美发达国家,然而随着我国经济水平的提高,伴随而来的饮食、环境、生活压力的变化,我国的IBD发病率不断升高。IBD患者经常出现腹痛、腹泻、呕吐、发烧、便血等症状,给患者带来极大的痛苦,严重影响生活质量,损害患者身体健康,一些患者具有较高的罹患结直肠癌的风险。
目前,IBD的发病机制还尚未明确,但越来越多的研究表明,IBD的发病与易感基因、环境因素、免疫应答紊乱、肠道菌群失调、肠道屏障损伤等因素具有相关性。随着高通测序技术的普及和生物信息学分析手段的发展,越来越来多的研究结果表明,炎症性肠病患者普遍存在肠道微生态系统的失调以及肠道菌群多样性的降低。肠道菌群是人体重要组成部分,肠道微生物和肠道粘膜免疫之间的相互作用影响着免疫反应的启动和调节。肠道菌群的紊乱可能导致免疫反应过度或失调,从而造成肠道粘膜的损伤。
有研究表明,UC患者的结肠活检样本和粪便样本中双歧杆菌和乳杆菌的比例有下降的趋势。而双歧杆菌和乳杆菌通常被认为益生菌而被用于某些疾病的干预。发酵乳是人们普遍喜爱的一种发酵食品,双歧杆菌和乳杆菌作为乳酸菌的重要成员,可以应用于发酵乳的制备。目前用于治疗炎症性肠病的药物主要包括抑炎类药物、免疫抑制剂以及抗生素等,这些药物通常只能缓解疾病症状,并不能达到治疗的目的,且长期使用可能导致副作用,例如过敏和肝脏疾病。而双歧杆菌作为健康人肠道中的共生菌,对人体没有毒副作用。
微生物胞外多糖(EPS)在食品工业中有着广泛的应用,例如它可以作为增稠剂、乳化剂、胶凝剂以及稳定剂用于食品加工中。在发酵乳制品中,乳酸菌产生的胞外多糖可以减少乳清晰出、改善产品的黏度和流变特性以及提高发酵乳的口感。通常,工业生产中,为了改善发酵乳的品质,生产商会增加乳固形物水平或添加稳定剂,因而会在一定程度上增加生产成本,另外,一些稳定剂可能会对人体健康造成不利影响。乳酸菌在酸乳发酵过程中产生的胞外多糖可以作为天然增稠剂或稳定剂,从而减少甚至替代传统稳定剂的使用。因此,发酵剂对发酵乳产品的影响在一定程度上与所产生的胞外多糖的性质有关。胞外多糖(EPS)作为微生物合成并分泌到胞外的一类大分子聚合物,它是微生物影响宿主的重要机制之一。胞外多糖可以提高菌体对胃肠道环境的耐受能力,保护菌体免受宿主免疫系统的影响,同时,某些双歧杆菌产生的胞外多糖可以调节宿主的免疫反应,降低宿主的炎症水平,对结肠炎起到防治作用。
通常,双歧杆菌在牛乳中的生长和产酸速率较慢,且不利于凝乳,可能造成过量的乳清晰出。因此,筛选获得具有缓解结肠炎作用、同时又能够适用于发酵乳制备的双歧杆菌,对于改善发酵乳产品品质,以及提高产品的营养和保健作用,有重要意义和广阔前景。
发明内容
本发明的第一个目的是提供一种长双歧杆菌,所述长双歧杆菌(Bifidobacteriumlongum subsp.longum)为长双歧杆菌(Bifidobacterium longum subsp.longum)YS108R,于2018年1 月3日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCC No.60310。
在本发明的一种实施方式中,所述长双歧杆菌YS108R在制备有缓解结肠炎的作用的食品和药物上的应用。
本发明的第二个目的是提供一种产黏性胞外多糖的双歧杆菌单菌发酵乳的制备方法,主要步骤如下:
(1)发酵乳原料的准备:将乳粉、蔗糖、酵母粉与水混合,然后65~75℃杀菌处理20-30min
(2)将YS108R菌株在的MRS培养基中活化后,8000-1200g离心20~30min,收集菌体,用生理盐水清洗2~3次,以1×107~108CFU/mL的接种量添加到上述发酵乳原料中, 37~39℃厌氧培养5~8h。
在本发明的一种实施方式中,所述乳粉、蔗糖、酵母粉的添加量分别为100~150g/L、50~80 g/L、1~2g/L。
在本发明的一种实施方式中,所述MRS培养基是加了半胱氨酸盐酸盐的MRS培养基。
在本发明的一种实施方式中,所述胱氨酸盐酸盐的添加量为按质量分数0.02-0.08%。
在本发明的一种实施方式中,所述MRS培养基的组分为胰蛋白胨8-10g、牛肉膏8-10g、酵母粉4-6g、葡萄糖18-22g、无水乙酸钠1.5-2.5g、七水硫酸镁0.4-0.6g、一水硫酸锰0.25-0.30g、柠檬酸氢二铵1.5-2.5g、三水磷酸氢二钾2.4-2.8g、Tween80 1-2mL、半胱氨酸盐酸盐0.4-0.6g,加蒸馏水至1L,pH为7.0~7.2。
本发明的有益效果:
(1)本发明所述双歧杆菌发酵乳的发酵菌株分离自健康长寿老人的肠道菌群,是人体的正常共生菌,无病原性。
(2)该菌株用于发酵乳的制作,歧杆菌发酵乳含有≥108CFU/g的双歧杆菌活菌,胞外多糖含量为130mg/L,同时能够减少产品的乳清析出率、提高产品的黏度和持水力,是产品更加粘滑、细腻。
(3)该菌株制备的发酵乳具有改善结肠炎的功能,动物实验结果表明,本发明的双歧杆菌发酵乳能够显著降低DSS诱导期间小鼠的疾病活动指数,维持结肠组织结构的完整,并可降低炎症因子的表达,提高紧密连接蛋白及粘蛋白等与肠道屏障相关的基因表达水平,并且相对传统药物治疗具有一定的优势,对人体无副作用。
生物材料保藏
一种长双歧杆菌(Bifidobacterium longum subsp.longum)YS108R,于2018年1月3日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCC No.60310。
附图说明
图1为本发明菌株的菌落拉丝效果(A),光学显微镜观察结果(B),透射电镜观察结果 (C)。
图2为本发明的双歧杆菌发酵乳的外观照片。YS108R单菌发酵乳(A),BB12单菌发酵乳(B),保加利亚乳杆菌+嗜热链球菌发酵乳(C)。
图3为各组小鼠DSS造模期间的DAI指数变化。
图4为各组小鼠结肠组织的病理切片。
图5为各组小鼠结肠组织中炎症因子基因的表达水平。
图6为各组小鼠结肠组织中紧密连接蛋白ZO-1和Claudin-1基因的表达水平。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步的说明,但并不局限如此,凡是对本发明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精神和范围,均应涵盖在本发明的保护范围中。下列实施例中未注明具体条件的实验方法,均按照本领域常规方法和条件。
MRS培养基组分:胰蛋白胨10g、牛肉膏10g、酵母粉5g、葡萄糖20g、无水乙酸钠2g、七水硫酸镁0.5g、一水硫酸锰0.25g、柠檬酸氢二铵2g、三水磷酸氢二钾2.6g、Tween80 1mL、半胱氨酸盐酸盐0.5g,加蒸馏水至1L,pH为7.0~7.2。
实施例1:菌株的分离方法
双歧杆菌的分离方法,主要步骤如下:
将采集的长寿老人的粪便为样品,将样品梯度稀释后,选取适当的3个梯度稀释液,在添加了0.05%半胱氨酸盐酸盐和100mg/L莫匹罗星的MRS平板上进行涂布,将平板倒置于 37℃厌氧培养箱中培养48h;挑取具有良好拉丝效果的菌落进行划线纯化,进而接种到含 0.05%半胱氨酸盐酸盐的MRS液体培养基中厌氧培养24~48h,收集发酵液用于测定胞外多糖,同时保留少量发酵液,离心收集菌体后,用于提取基因组、PCR和16S rDNA测序。胞外多糖的测定:取10mL发酵液在4℃下10000g离心15min,收集上清液,经三氯乙酸沉淀法除去蛋白成分后,与三倍体积的冰乙醇混合均匀,4℃静止过夜,然后在4℃下12000 g离心10min,弃去上清液,沉淀用截留分子量为8000~14 000Da的透析袋透析48h后,冻干得到粗多糖。采用苯酚-硫酸法测定胞外多糖含量,从而筛选出一株高产黏性胞外多糖的菌株。
经生理生化鉴定和16S rDNA测序结果分析,该菌株鉴定为长双歧杆菌长亚种(Bifidobacterium longum subsp.longum),菌株号为YS108R。生理生化试验结果如表1所示, 16S rDNA测序结果如SEQ ID NO.1所示。
表1YS108R生理生化试验结果
试验项目 | 实验结果 | 试验项目 | 实验结果 |
葡萄糖 | + | L-阿拉伯糖 | + |
D-葡萄糖胺 | - | 松三糖 | - |
D-木糖 | + | 葡糖酸钠 | - |
阿拉伯树胶 | - | 棉子糖 | + |
F-果糖 | + | 水杨苷 | - |
纤维二塘 | - | D-甘露糖 | - |
蔗糖 | + | 麦芽糖 | + |
接触酶 | - | 革兰氏染色 | 阳性 |
实施例2:双歧杆菌YS108R的性质测定
双歧杆菌生物学特征的测定:
(1)菌落特征的观察方法:将菌株在MRS固体培养基上划线,培养至长出单菌落后,观察其菌落形态。
菌落特征:在MRS固体培养基上形成圆形、凸起的菌落,乳白色,不透明,表面光滑,菌落直径为1~3mm,有拉丝效果,如附图1(A)所示。
(2)菌体特征观察方法:挑取少量菌落涂在载玻片上,进行革兰氏染色,并于光学显微镜下观察。
菌体特征:革兰氏染色呈阳性,在pH 3.0~8.0环境下生长良好,不形成孢子,菌体约0.5~1.5 μm×2~8μm,呈杆状、棒状或分叉的Y形,光学显微镜和透射电镜观察结果显示菌体表面有明显的胞外多糖,如附图1(B)和1(C)所示。
(3)液体培养特征:在MRS液体培养基培养过程中,菌体均匀悬浮在培养中,不在底部沉积。
(4)模拟胃液中的存活率的测定方法:人工模拟胃肠液需新鲜配制。将胃蛋白酶溶于 pH 3.0的PBS中使其终浓度为3g/L,经0.22μm滤膜过滤后制备成模拟胃液。将胰蛋白酶溶于pH 8.0的PBS中使其终浓度为1g/L,经0.22μm滤膜过滤后制备成模拟肠液。将培养好的双歧杆菌在4℃下以6000rpm离心10min,收集菌泥,用0.85%生理盐水重悬后,在模拟胃液(pH 3.0)中将其菌液密度调节为1×109CFU/mL。混匀后将其放置于37℃培养2h后计活菌数。取1mL模拟胃液处理过的菌液加入至9mL模拟肠液(pH 8.0)中,混匀后于37℃培养,4h后检测活菌数。处理后的活菌数与初始活菌数比值的百分比为存活率。
在模拟胃液中的存活率为86.33%,在模拟肠液中的存活率为79.38%。
(5)耐盐能力的测定方法:将菌株分别接种于含有0%、2%、4%、8%NaCl的MRS培养基中,培养24h后测定OD值,含有0%、2%、4%、8%NaCl的MRS培养基中的OD值分别为2.13、0.58、0、0。
说明该菌株具有一定的耐盐能力,在2%NaCl浓度下生长良好。
(6)胞外多糖的测定方法:取10mL发酵液在4℃下10000g离心15min,收集上清液,经三氯乙酸沉淀法除去蛋白成分后,与三倍体积的冰乙醇混合均匀,4℃静止过夜,然后在4℃下12000g离心10min,弃去上清液,沉淀用截留分子量为8000~14 000Da的透析袋透析48h后,冻干得到粗多糖。采用苯酚-硫酸法测定胞外多糖含量。
在MRS液体培养基中培养时,胞外多糖产量为150mg/L。
实施例3:双歧杆菌发酵乳对DSS诱导结肠炎小鼠症状的缓解作用
准备雄性C57小鼠40只,随机分为5组:正常对照组(Control)、模型组(Model)、YS108R 发酵乳干预组(YS108R-FM)、BB12发酵乳干预组(BB12-FM组)和保加利亚乳杆菌+嗜热链球菌发酵乳干预组(SL-FM),每组8只。实验方案和各组小鼠的处理方式如表5所示,每只小鼠的灌胃剂量为0.2mL。第14天时,葡聚糖硫酸钠(DSS)用生理盐水配成浓度为2.5%的溶液,代替饮用水让小鼠饮用,用于造模,每两天配制并更换一次。DSS造模期间(15~21天),每天记录小鼠的体重、粪便状态、粪便隐血,用于计算疾病活动指数(DAI),评分标准如表3所示。
表2实验方案
表3DAI评分标准
DSS造模过程中各组小鼠的疾病活动指数如附图3所示。从造模第二天开始,除正常组小鼠外,其余三组小鼠的DAI指数开始上升,模型组DAI指数上升最快。造模结束时,YS108R 发酵乳干预组的DAI指数显著低于模型组,BB12发酵乳干预组的DAI指数略低于模型组,但差异无统计学意义,而商品发酵剂发酵乳干预组的DAI指数与模型组无差异,说明本发明的双歧杆菌发酵乳对结肠炎的症状有缓解作用。
实施例4:双歧杆菌发酵乳对结肠炎小鼠结肠组织的保护作用
小鼠经DSS诱导结肠炎后,结肠组织会出现明显的损伤,包括炎症细胞浸润、隐窝和腺体结构的破坏等。各组小鼠结肠组织的病理切片如图4所示。YS108R发酵乳干预组的结肠组织与正常组相似,隐窝结构完整,而模型组的结肠粘膜层有明显的炎症浸润,隐窝基本消失。
实施例5:双歧杆菌发酵乳对结肠炎小鼠结肠组织中炎症因子表达水平的影响
各组小鼠结肠组织中细胞因子TNF-α、IL-1β、IL-6和IL-10基因的表达水平如附图5所示。YS108R发酵乳干预组的TNF-α、IL-1β、IL-6三个促炎因子的基因表达水平都明显低于模型组,且YS108R发酵乳干预组的抑炎因子IL-10的表达水平显著高于其他组,说明本发明的双歧杆菌YS108R发酵乳可以有效缓解DSS诱导结肠炎模型的炎症水平。
实施例6:双歧杆菌发酵乳对结肠炎小鼠结肠组织中紧密连接蛋白表达水平的影响
紧密连接蛋白是组成肠道屏障的重要部分,对于维持肠上皮屏障功能的完整性具有重要作用。各组小鼠结肠组织中紧密连接蛋白ZO-1和Claudin-1基因的表达水平如附图6所示。 YS108R发酵乳干预组的ZO-1和Claudin-1基因的表达水平都明显高于模型组,商品发酵剂发酵乳干预组的ZO-1和Claudin-1基因的表达水平与模型组无明显差异,而BB12发酵乳干预组的ZO-1和Claudin-1基因的表达水平虽有所提升,但与YS108R发酵乳干预组相比,仍相对较低。说明本发明的双歧杆菌发酵乳对于维持结肠屏障结构具有较好的效果。
实施例2~6中的结果表明,本发明的双歧杆菌发酵乳可以通过抑制炎症因子表达和维持结肠屏障结构从而改善DSS诱导的结肠炎。
实施例7:高产黏性胞外多糖双歧杆菌YS108R单菌发酵乳的制作
(1)多糖双歧杆菌YS108R单菌发酵酸奶
发酵乳原料的准备:将乳粉、蔗糖、酵母粉分别按120g/L、50g/L、1g/L的比例与水混匀,然后70℃杀菌处理30min。
将YS108R菌株在添加了0.05%半胱氨酸盐酸盐的MRS培养基中活化3代后,8000g离心20min,收集菌体,用生理盐水清洗2次,以1×107CFU/mL的接种量添加到上述发酵乳原料中,37℃厌氧培养6h,制得发酵乳制品。
(2)乳双歧杆菌BB12单菌发酵乳的制作方法
制作方法同YS108R单菌发酵酸奶,将BB12菌株以1×107CFU/mL的接种量添加到上述发酵乳原料中,37℃厌氧培养6h,制得发酵乳制品。
(3)商品发酵剂发酵乳
将商品发酵剂(含保加利亚乳杆菌和嗜热链球菌)以0.01%的接种量添加到上述发酵乳原料中,42℃厌氧培养5h,制得发酵乳制品。
与商业化菌株乳双歧杆菌BB12制作的发酵乳以及市售酸奶发酵剂制作的发酵乳相比, YS108R菌株发酵乳具有较低的乳清析出率以及较高的持水力和表观粘度,具体性状指标如表4和附图2所示。
表4发酵乳的理化指标
YS108R菌株单菌发酵乳的胞外多糖含量为130mg/L,表观粘度为3210cP,乳清析出率为2.13%,持水力为21.44%,pH为4.65,酸度为70.22°T,含有≥108CFU/g的双歧杆菌活菌。上述发酵乳的感官品评结果如表5所示。产粘性胞外多糖双歧杆菌YS108R发酵乳的感官品评结果优于乳双歧杆菌BB12发酵乳,与市售保加利亚乳杆菌和嗜热链球菌发酵剂制作的发酵乳在风味上无明显差异,但口感更细腻。本发明的双歧杆菌发酵乳还具有缓解结肠炎的作用。
表5发酵乳的感官品评结果
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 一种双歧杆菌单菌发酵乳制备方法及其应用
<120> 江南大学
<170> PatentIn version 3.3
<210> 1
<211> 1514
<212> DNA
<213> 双歧杆菌(Bifidobacterium)
<400> 1
aaggaggtga tccagccgca ccttccggta cggctacctt gttacgactt agtcccaatc 60
acgagcctca ccttagacgg ctccatccca caaggggtta ggccaccggc ttcgggtgct 120
gcccactttc atgacttgac gggcggtgtg tacaaggccc gggaacgcat tcaccgcgac 180
gttgctgatt cgcgattact agcgactccg ccttcacgca gtcgagttgc agactgcgat 240
ccgaactgag accggttttc agggatccgc tccgcgtcgc cgcgtcgcat cccgttgtac 300
cggccattgt agcatgcgtg aagccctgga cgtaaggggc atgatgatct gacgtcatcc 360
ccaccttcct ccgagttaac cccggcggtc ccccgtgagt tcccggcata atccgctggc 420
aacacggggc gagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 480
gacgacgacc atgcaccacc tgtgaacccg ccccgaaggg aagccgtatc tctacgaccg 540
tcgggaacat gtcaagccca ggtaaggttc ttcgcgttgc atcgaattaa tccgcatgct 600
ccgccgcttg tgcgggcccc cgtcaatttc tttgagtttt agccttgcgg ccgtactccc 660
caggcgggat gcttaacgcg ttagctccga cacggaaccc gtggaacggg ccccacatcc 720
agcatccacc gtttacggcg tggactacca gggtatctaa tcctgttcgc tccccacgct 780
ttcgctcctc agcgtcagta acggcccaga gacctgcctt cgccattggt gttcttcccg 840
atatctacac attccaccgt tacaccggga attccagtct cccctaccgc actcaagccc 900
gcccgtaccc ggcgcggatc caccgttaag cgatggactt tcacaccgga cgcgacgaac 960
cgcctacgag ccctttacgc ccaataattc cggataacgc ttgcacccta cgtattaccg 1020
cggctgctgg cacgtagtta gccggtgctt attcaacggg taaactcact ctcgcttgct 1080
ccccgataaa agaggtttac aacccgaagg cctccatccc tcacgcggcg tcgctgcatc 1140
aggcttgcgc ccattgtgca atattcccca ctgctgcctc ccgtaggagt ctgggccgta 1200
tctcagtccc aatgtggccg gtcgccctct caggccggct acccgtcgaa gccacggtgg 1260
gccgttaccc cgccgtcaag ctgataggac gcgaccccat cccataccgc gaaagctttc 1320
ccagaagacc atgcgatcaa ctggagcatc cggcattacc acccgtttcc aggagctatt 1380
ccggtgtatg gggcaggtcg gtcacgcatt actcacccgt tcgccactct caccaccaag 1440
caaagcctga tggatcccgt tcgacttgca tgtgttaagc acgccgccag cgttcatcct 1500
gagccagaat cgaa 1514
Claims (6)
1.长双歧杆菌YS108R在制备发酵乳方面的应用,其特征在于,所述长双歧杆菌为长双歧杆菌(Bifidobacterium longum subsp.longum)YS108R,于2018年1月3日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCC No.60310。
2.长双歧杆菌YS108R在制备有缓解结肠炎的作用的食品和药物方面的应用,其特征在于,所述长双歧杆菌为长双歧杆菌(Bifidobacterium longum subsp.longum)YS108R,于2018年1月3日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCC No.60310。
3.一种长双歧杆菌单菌发酵制备发酵乳的方法,其特征在于,所述方法的主要步骤如下:
(1)发酵乳原料的准备:将乳粉、蔗糖、酵母粉与水混合,然后65~75℃杀菌处理20-30min;
(2)将权利要求1所述的YS108R菌株活化后,8000-1200g离心20~30min,收集菌体,用生理盐水清洗2~3次,以1×107~108CFU/mL的接种量添加到上述发酵乳原料中,37~39℃厌氧培养5~8h;
所述长双歧杆菌为长双歧杆菌(Bifidobacterium longum subsp.longum)YS108R,于2018年1月3日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所,保藏编号为GDMCC No.60310。
4.根据权利要求3所述的方法,其特征在于,所述乳粉、蔗糖、酵母粉的添加量分别为100~150g/L、50~80g/L、1~2g/L。
5.根据权利要求3或4所述的方法,其特征在于,所述YS108R菌株的活化是在MRS培养基中进行活化。
6.根据权利要求3或4所述方法制备得到的发酵乳。
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