CN101341255A - 生产寡糖的方法 - Google Patents
生产寡糖的方法 Download PDFInfo
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- CN101341255A CN101341255A CNA2006800416294A CN200680041629A CN101341255A CN 101341255 A CN101341255 A CN 101341255A CN A2006800416294 A CNA2006800416294 A CN A2006800416294A CN 200680041629 A CN200680041629 A CN 200680041629A CN 101341255 A CN101341255 A CN 101341255A
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Abstract
用产半乳糖苷酶的细菌由乳糖生产半乳寡聚糖的益生素混合物的方法,其中所述细菌细胞可在合成反应中重复用而不损失产量。
Description
本发明涉及生产半乳寡聚糖的益生素混合物的方法。
益生素被定义为不可消化的食物成分,其通过选择性刺激结肠中一种或有限数目的细菌的生长和/或活性来有益地影响哺乳动物宿主,由此造成宿主健康的改善。
半乳寡聚糖是不可消化的碳水化合物,其抗哺乳动物胃肠消化酶但可被特定结肠细菌发酵。它们在近端结肠和横结肠显示出有很好的益生素活性。
GB 2 412 380描述了新的双歧双歧杆菌(Bifidobacterium bifidum)菌株,其能产生半乳糖苷酶酶活性,将乳糖转化成新的含Gal(α1-6)-Gal、Gal(β1-6)-Gal(β1-4)-Glc、Gal(β1-3)-Gal(β1-4)-Glc、Gal(β1-6)-Gal(β1-6)-Gal(β1-4)-Glc和Gal(β1-6)-Gal(β1-6)-Gal(β1-6)-Gal(β1-4)-Glc的半乳寡聚糖混合物。该菌株在2003年3月31日保藏于英国阿伯丁的国家工业和海洋细菌保藏所,保藏号NCIMB 41171。
该保藏的双歧双歧杆菌菌株、或其生物功能等同物可用于在本发明的方法中生产如上定义的半乳寡聚糖混合物。短语“生物功能等同物”解释为是指双歧双歧杆菌菌株,其产生半乳糖苷酶酶活性,将乳糖转化成如上定义的半乳寡聚糖混合物。
为了生产如上定义的半乳寡聚糖混合物,将乳糖或含乳糖的底物用如上定义的双歧双歧杆菌菌株处理。
合适的含乳糖的底物可选自商业途径获得的乳糖、全脂奶、半脱脂奶、脱脂奶、乳清和换脂奶。该乳产品可得自奶牛、水牛、绵羊或山羊。换脂奶被定义为脱去乳脂的全脂奶,然后乳脂通过添加植物脂肪或油来替换。
已发现,所述保藏的双歧双歧杆菌菌株产生的多数半乳糖苷酶是细胞结合的,使之可能利用整个细胞来合成半乳寡聚糖混合物。已出人意料地发现了,所述细菌细胞(生物质)可通过离心回收并在连续合成反应中重复使用多至8次,而不显著损失生物量或改变反应时间并产生相同量的寡糖产物。
根据本发明,提供有合成含二糖Gal(α1-6)-Gal、至少一种选自Gal(β1-6)-Gal(β1-4)-Glc、Gal(β1-3)-Gal(β1-4)-Glc的三糖、四糖Gal(β1-6)-Gal(β1-6)-Gal(β1-4)-Glc和五糖Gal(β1-6)-Gal(β1-6)-Gal(β1-6)-Gal(β1-4)-Glc的半乳寡聚糖混合物的方法,其中Gal表示半乳糖残基而Glc表示葡萄糖残基,其中向乳糖或含乳糖的底物加入双歧双歧杆菌细胞培养物,而且所述细菌细胞在至多8次的连续合成反应中重复使用而不损失所述半乳寡聚糖混合物的产量。
8次合成反应后产生的寡糖稍稍减少,12次重复使用后,产生的寡糖是最初反应中形成的总产物的10%。
本发明将进一步通过参考以下实施例的方式来描述。
实施例
材料和方法
所有该研究中所用的化学和培养基制品都来自Sigma(Dorset,英国)、WR(Dorset,英国)、Oxoid(Basingstoke,英国)。
微生物生长和酶的产生
双歧双歧杆菌NCIMB 41171分离自人粪便样品。工作培养物在肉汤中繁殖,所述肉汤含胰蛋白胨15g/l、Lab Lemco(常规的肉类提取物)2.5g/l、酵母提取物7.5g/l、K2HPO4 4.5g/l、半胱氨酸-HCl 0.05g/l,乳糖2.5g/l、葡萄糖7.5g/l和吐温801ml/l。生长培养基的pH调节到6.7,然后高压灭菌并在厌氧条件下(10∶10∶80;H2∶CO2∶N2)于37℃进行孵育。
双歧双歧杆菌酶的生产发酵在7和150L发酵罐中进行,采取所有必要措施以保证无菌操作。最大酶产生所用的培养基含胰蛋白胨7.5g/l、LabLemco(常规的肉类提取物)7.5g/l、酵母提取物7.5g/l、K2HPO4 2g/l、半胱氨酸-HCl 0.5g/l、乳糖4g/l、葡萄糖6g/l和吐温800.5ml/l。发酵桶中的无氧条件通过在灭菌后冷却期间向培养基注入无氧氮气并且还通过在生长期间在培养物上覆盖氮气来获得。接种水平是5%(v v-1),温度保持在37°C,以100rpm搅拌,pH用氢氧化钠溶液(2M)调节在6.7。
观察到双歧双歧杆菌NCIMB 41171所产生的大于三分之二的半乳糖苷酶活性结合在微生物的细胞壁上,而剩余的分泌到培养上清液中。因此并为了易于通过离心(以7,000xg)收集生物质,收集结合到微生物细胞的半乳糖苷酶并用作GOS合成的酶制剂。为了帮助收集生物质,使培养物的pH(在指数生长期的大多时候调节在6.7)在生长稳定期时落到介于5和5.5的值,其能诱导细胞絮凝。
收集的细胞团重悬于0.1M磷酸盐缓冲液(pH 6.8)中,洗两次,然后用甲苯处理。根据Onishi、Yamashiro和Yokozeki,Appl.& Env.Microbiol(1995),61(11),4002-4025,用甲苯处理双歧双歧杆菌生物质能增加细胞渗透性并由此增加观察到的半乳糖苷酶活性。该处理通过将从11培养物中收集的细胞重悬于80ml 0.1M磷酸盐缓冲液(pH 6.8)中并向该悬浮液加入0.16ml甲苯来进行。该制品置于20℃摇动水浴中1h。然后用缓冲液洗细胞3次,冷冻并冻干。该冻干的生物质制剂用于GOS合成。
用去离子水洗并于105℃干燥4h后称重留在0.2μm滤膜上的细胞,由此进行发酵时的生物量监测。细菌数通过于Wilkins-Chalgreen厌氧生物琼脂上铺板来监测。
测定α-和β-半乳糖苷酶活性、最佳pH和温度确定
用4-硝基苯基β-D-半乳吡喃糖苷作为底物,在0.1M磷酸盐缓冲溶液(pH6.8)中于40℃来进行测定双歧双歧杆菌生物质中所含的β-半乳糖苷酶活性。用四硼酸二钠(0.2M)终止酶反应并显色。作为通过420nm吸光度而确定的释放出的O-硝基酚的函数来测定酶活性。考虑底物和生物质干扰的校正。一单位β-半乳糖苷酶被定义为在上述指定条件下每分钟释放出1μmol O-硝基酚的酶量。
双歧双歧杆菌细胞中β-半乳糖苷酶活性的最佳pH通过进行标准生物质制剂在不同pH值(介于4和8)时的酶活性测定(如上所述)来确定。10mM 2-硝基苯基-β-D-半乳吡喃糖苷溶液用0.1M调节在所需pH的磷酸盐和柠檬酸盐-磷酸盐缓冲液来制备。
双歧双歧杆菌细胞中所含的β-gal活性的最佳温度通过进行标准生物质制剂在介于30至55℃的不同温度时的酶活性测定(如上所述)来确定。
用与β相同的方式,但使用4-硝基苯基-α-D-半乳吡喃糖苷作为底物来测定并定义α-半乳糖苷酶活性。
GOS合成和副产物抑制
用纯的乳糖和超滤奶酪乳清透过物溶液来进行GOS合成。
当纯的乳糖用作底物(450,500mg/ml)时,在0.1M磷酸盐(pH 6.8)和0.1M柠檬酸/柠檬酸钠(6.2)缓冲溶液中、于40±0.5℃、以100rpm搅拌,由此进行合成。乳糖溶解并使温度平衡于40℃后,每100ml合成混合物中加入2.5g冻干的酶(344Ug-1)。追踪反应24h。将样品煮沸10分钟以使酶失活,然后分析它们的碳水化合物含量。由于在温度降到40℃时观察到乳糖结晶,因此更高的乳糖合成浓度不适用。
在上述GOS合成条件(以450mg/ml底物浓度)下,在6h的时间段上观察到最佳的寡糖浓度。为了测试重复使用相同生物质进行重复合成反应的可能性,进行实验,其中用以7,000rpm离心收集的相同生物质重复进行450mg/ml合成反应。在6天的时间段中用中间时间间隔时储存于2-4℃的生物质进行一系列12次连续6h合成反应。离心以避免降低生物质浓度,然后收集样品进行碳水化合物分析。
浓缩的乳清超滤透过物(粉末形式)由Volac国际有限公司(Liverpool,英国)馈赠。所提供的制品含0-0.5%(w/w)脂肪、4.5-7.5%蛋白质、8-10%灰分、82%乳糖和稀释在水中时介于5-5.5的pH值。合成前,所有乳清透过物制剂加热到95℃以溶解结晶的乳糖,并以7,000rpm离心10分钟去除因存在的肽热变性而观察到的沉淀。在去除所用的条件下,该沉淀占总溶液重的2.6%(w/w)。去除该蛋白质沉淀被认为是必要的,由此能通过离心收集双歧双歧杆菌生物质并在接下来的合成反应中重复使用它。合成条件和酶浓度如对纯乳糖合成反应所述的。
为了测试葡萄糖和半乳糖对GOS产生的影响,进行一系列实验,其中与使用乳糖(400mg/ml)作为底物同时,最初将不同浓度的葡萄糖和半乳糖(100或150mg/ml)加入到反应混合物中。在pH 6.8(0.1M磷酸盐缓冲液)、于40±0.5℃、以100rpm搅拌的条件,来进行这些实验,每100ml合成混合物中加入2.5g冻干的生物质(344U/g))。
一式两份进行所有以上GOS合成反应。
从GOS混合物中选择性去除单糖
尝试通过酵母发酵从混合物中产生的单糖中选择性地纯化以上产生的寡糖。使用酿酒酵母(Saccharomyces cerevisiae)菌株,因为选择性发酵的特征在于它显示针对不同的糖。葡萄糖和半乳糖是GOS合成期间的单糖副产物,由乳糖水解和半乳糖转移到用作为转半乳糖基接受体的水分子而形成。
纯化本研究中产生的寡糖和商品化寡糖混合物(Vivinal GOS,其得自Borculo Domo Ingredients,Zwolle,荷兰;57%(ww-1)GOS,23%乳糖,22%葡萄糖和0.8%半乳糖)。在0.1M磷酸盐缓冲液(pH 6.8)中制备450mg/ml糖浓度的碳水化合物混合物溶液,由此保持适于酵母代谢的pH,并过滤除菌。发酵于30℃在摇瓶中进行,每100ml溶液加入1g冻干的酵母(29×109cfug-1)。追踪发酵32h并分析样品的碳水化合物、乙醇和蛋白质含量。在CM129胰蛋白胨大豆琼脂板上进行酵母细胞计数。一式两份进行所有GOS纯化发酵。
分析样品的碳水化合物和乙醇含量。
通过高效液相层析(HPLC),利用Bio-Rad Laboratories有限公司(Hertfordshire,英国)提供的基于Aminex HPX-87C Ca+2树脂的柱(300×7.7mm)和与折光率检测器连用的HPLC分析仪,分析合成和酵母发酵的样品。柱保持在85℃并将HPLC级水用作流动相,其流速为0.6ml分钟-1。在这些条件下,寡糖洗脱为两个不能很好分辨的峰,然后是二糖(一个峰)和单糖,其中葡萄糖和半乳糖作为分开的峰出现。用该柱通过标准校准曲线可测定乙醇,因为它单独洗脱。
分别通过麦芽三糖、乳糖、葡萄糖和半乳糖标准校准曲线定量测定寡糖(聚合度(DP)≥3)、二糖、和单糖。
为了定量如HPLC分析所确定的二糖合并峰中所含的转半乳糖化的二糖量,合成样品还通过与脉冲电流检测连用的高效阴离子交换层析(HPAEC-PAD)来分析。使用来自Dionex Chromatography(Surrey,英国)的基于薄膜阴离子交换树脂的柱CarboPac PA-I。用梯度流动相浓度的氢氧化钠和乙酸钠溶液在20±0.5℃以1ml/分钟的流速洗下碳水化合物。在这种情况下,作为单独的峰洗下的乳糖允许利用标准校准曲线进行定量测定,所述标准校准曲线结合HPLC数据能定量测定转半乳糖基化的二糖。
用在吡啶中的氯化羟胺衍生成糖肟并用六甲基二硅氮烷和三氟乙酸全硅烷化(persilyation)后,所选的样品进一步通过气相层析质谱分析。分析中所用的柱是J&W Scientific(美国)的DB-17MS(长30m,I.D.0.25mm,薄膜0.25μm)。
结果和讨论
发酵生产双歧双歧杆菌NCIMB 41171半乳糖苷酶
在发酵产生双歧双歧杆菌NCIMB 41171期间,观察到7-8h的指数生长期,细菌数量从13×106上升至43×108cfu ml-1。测得稳定期一开始有2.68g L-1冻干的生物质含量。观察到最大半乳糖苷酶活性时培养物仍在稳定期,显示出培养物(上清液加细胞)有1U ml-1β-半乳糖苷酶活性。这最终会给出205.5Ug-1冻干的生物质的活性。该制剂的α-半乳糖苷酶活性测定为3.05U g-1。7L和试验工场(150L)发酵之间的重复性非常好,而且该生物质用甲苯处理、冷冻、冻干并接着用于所有的合成反应。对双歧双歧杆菌NCIMB 41171生物质的冷冻和冻干不影响半乳糖苷酶活性,但它影响细菌的存活性(某与预期用途无关)。用甲苯处理双歧双歧杆菌细胞,然后冻干,能增加细胞渗透性,导致增加α-和β-半乳糖苷酶活性,观察到分别有5.04和344Ug-1。
合成GOS
用双歧双歧杆菌NCIMB 41171细胞结合的酶进行GOS合成。超过一种半乳糖苷酶存在于双歧双歧杆菌菌株中,本研究中产生的寡糖被认为是它们组合活性的产物。图1显示通过HPLC分析得到的由样品产生GOS期间的典型时间进程。寡糖浓度最初增加到最大值,然后在转半乳糖基化活性变得不如水解活性显著时下降。由乳糖水解形成主更量的葡萄糖和半乳糖。
寡糖浓度随着乳糖浓度增加而增加,因为随着底物浓度增加,合成溶液的水活度下降,使半乳糖转移至水分子的反应发生可能性下降。在表1中,显示了在pH 6.8、6.2和用乳清透过物粉作为乳糖源时以最大可能底物浓度的合成反应的碳水化合物组成。由此可见,存在于混合物中的转半乳糖基化的二糖(除了乳糖之外的二糖)量非常接近于产生的更高聚合度(DP≥3)寡糖的浓度。合成的pH从6.8降至6.2和5.4,乳清透过物粉用作底物时,观察到水解产物的量增加。固定乳清透过物底物的反应pH于较高的值,被证明是不合需要的,因为存在肽和氨基酸,在升高的pH将产生广泛的Maillard褐化。
用HPAEC-PAD测得的实际乳糖浓度确定最大寡糖浓度时的乳糖转化(表1),在约80至85%乳糖转化时观察到最高寡糖浓度。随着用于合成的乳糖浓度增加,观察到最大寡糖浓度时的底物转化值也增加了。用纯乳糖作为底物时寡糖产量在39和43%之间变化,乳清透过物作为乳糖源时在36和38%之间。不同起始底物浓度间的产量值中没有观察到显著差异。
在图2中,显示产生的寡糖混合物的有代表性的HPAEC-PAD层析图。碳水化合物的分子量增加时,多种不同GOS产量下降。重要的发现是在与α(1-6)半乳二糖标准品相同保留时间洗下的二糖。为了确证该结果,衍生成其糖肟后,样品通过气相层析质谱分析。在所指定的分析条件下存在两个很好分辨的峰,保留时间为27.7和29.0分钟,这再次确证存在α-连接的二糖。比较每个峰的主要光谱比率显示在标准品和合成样品之间非常小的差异,这再次确证了存在该碳水化合物。在测试重复使用双歧双歧杆菌生物质进行连续合成反应的可能性的实验中,相同量的生物质成功地在8次随后的450mg/ml(乳糖)合成反应中重复使用,在近似的反应时间段产生了相同量的寡糖产物(如表1所示)。
从这点开始观察到产生的寡糖有轻微减少,12次重复使用后,产生的寡糖为最初反应中形成的总产物的10%。
Claims (4)
1.合成含二糖Gal(α1-6)-Gal、至少一种选自Gal(β1-6)-Gal(β1-4)-Glc、Gal(β1-3)-Gal(β1-4)-Glc的三糖、四糖Gal(β1-6)-Gal(β1-6)-Gal(β1-4)-Glc和五糖Gal(β1-6)-Gal(β1-6)-Gal(β1-6)-Gal(β1-4)-Glc的半乳寡聚糖混合物的方法,其中Gal表示半乳糖残基而Glc表示葡萄糖残基,其中向乳糖或含乳糖的底物加入双歧双歧杆菌细胞培养物,其特征在于所述细胞在至多8次的连续合成反应中重复使用而不损失所述半乳寡聚糖混合物的产量。
2.权利要求1所述的方法,其中所述双歧双歧杆菌培养物是在2003年3月31日保藏于英国阿伯丁的国家工业和海洋细菌保藏所的菌株NCIMB 41171或本文所定义的生物功能等同物的培养物。
3.权利要求1或权利要求2所述的方法,其中含乳糖的底物选自由全脂奶、半脱脂奶、脱脂奶、乳清和换脂奶组成的组。
4.权利要求3所述的方法,其中奶得自牛、水牛、绵羊或山羊。
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2008
- 2008-05-06 NO NO20082094A patent/NO20082094L/no not_active Application Discontinuation
- 2008-05-07 ZA ZA200803921A patent/ZA200803921B/xx unknown
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103080329A (zh) * | 2010-07-19 | 2013-05-01 | 阿尔拉食品公司 | 含有半乳寡聚糖的组合物及其生产方法 |
CN103080329B (zh) * | 2010-07-19 | 2016-08-03 | 阿尔拉食品公司 | 含有半乳寡聚糖的组合物及其生产方法 |
Also Published As
Publication number | Publication date |
---|---|
WO2007054459A2 (en) | 2007-05-18 |
GB0522740D0 (en) | 2005-12-14 |
NO20082094L (no) | 2008-05-29 |
CA2628671A1 (en) | 2007-05-18 |
AU2006311107A1 (en) | 2007-05-18 |
GB2445137A (en) | 2008-06-25 |
AU2006311107A2 (en) | 2008-06-19 |
BRPI0618300A2 (pt) | 2011-08-23 |
JP2009514543A (ja) | 2009-04-09 |
GB0807808D0 (en) | 2008-06-04 |
WO2007054459A3 (en) | 2007-07-26 |
RU2008122918A (ru) | 2009-12-20 |
US20090155860A1 (en) | 2009-06-18 |
EP1945787A2 (en) | 2008-07-23 |
ZA200803921B (en) | 2009-04-29 |
KR20080086979A (ko) | 2008-09-29 |
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