CN111448322B - 使用苹果乳杆菌制造转果糖基化甜菊醇配糖物的方法 - Google Patents
使用苹果乳杆菌制造转果糖基化甜菊醇配糖物的方法 Download PDFInfo
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- CN111448322B CN111448322B CN201880079763.6A CN201880079763A CN111448322B CN 111448322 B CN111448322 B CN 111448322B CN 201880079763 A CN201880079763 A CN 201880079763A CN 111448322 B CN111448322 B CN 111448322B
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- steviol glycoside
- transglucosylated
- lactobacillus
- rebaudioside
- malus
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Abstract
本申请涉及使用苹果乳杆菌菌株的粗酶液制备转葡糖基化甜菊醇配糖物的方法。
Description
技术领域
本公开涉及使用苹果乳杆菌(Lactobacillus mali)菌株的粗酶液以制备转葡糖基化甜菊醇配糖物(steviol glycoside)的方法。
背景技术
随着世界卫生组织(WHO)出于对糖消耗引起疾病(肥胖)的担忧,建议降低每日糖摄入量,发达国家政府正在积极讨论旨在减少糖摄入量的各种政策。因此,随着市场上开发各种替代增甜剂的需求不断增加,替代增甜剂正在不断被开发和商业化。作为替代增甜剂,这些是以下列的形式持续变化:合成高强度增甜剂(例如,糖精、阿斯巴甜、三氯蔗糖(Sucralose)等)、合成糖醇(例如,麦芽糖醇和木糖醇)、和高强度增甜剂(例如,新蛇菊苷(Rebaudioside)A和甘草)。然而,由于对合成增甜剂的安全性的担忧,消费者对天然增甜剂的需求一直在稳定增加;然而,因为天然增甜剂特有的风味特性(即,异味(off-oder)和败味(off-flavor))的限制,所以天然增甜剂不能完全取代现有的以合成增甜剂为基础的低卡路里和零卡路里产品。
近年来备受关注的天然高强度增甜剂是从甜叶菊(Stevia rebaudiana Bertoni)的叶中提取的甜菊糖苷(stevia)。甜菊糖苷是天然材料,其甜度是糖的甜度的200至300倍。进一步,甜菊糖苷由蛇菊苷(Stevioside)、新蛇菊苷A、B、C、D、E和M等组成。此外,因为据报道,甜菊糖苷不产生卡路里,其对血糖和胰岛素水平具有正面作用,而且它对人体没有副作用,所以甜菊糖苷具有作为替代增甜剂的潜在用途;然而,甜菊糖苷有苦味,其在使用上有一定的局限性。
迄今为止,已经有三种方法来改善甜菊糖苷的甜味:(1)与糖类增甜剂、氨基酸、或氨基酸盐混合的方法,(2)包括诸如环糊精的物质的物理方法;以及(3)使用酶转移葡萄糖的方法。作为使用酶转移葡萄糖的方法,使用环糊精葡萄糖基转移酶(CGTase)将1至12个葡萄糖分子转移到甜菊醇配糖物的方法在本领域广泛使用(韩国专利申请号10-1991-0020769)。然而,这种方法具有以下有缺点:所有转移到甜菊醇配糖物的葡萄糖都被肠道微生物降解,从而增加了卡路里。
已知一般乳酸杆菌在与β-葡萄糖苷酶反应时产生甜茶苷(rubusoside)(韩国专利申请号10-17676060000)。此外,特定的基因组路氏乳杆菌(Lactobacillus reuteri)180是已知的唯一能识别甜菊醇配糖物以产生转葡糖基化甜菊醇配糖物的乳酸杆菌。
发明内容
[技术问题]
在这种情况下,本发明人通过发现苹果乳杆菌通过α-(1,6)-键将葡萄糖转葡糖基化到甜菊醇配糖物以产生难以消化的转葡糖基化甜菊醇配糖物来完成了本公开。
[技术手段]
本公开的目的是提供使用苹果乳杆菌微生物或其培养物制备转葡糖基化甜菊醇配糖物的方法。
本公开的另一目的是提供经由上述制备方法制备的转葡糖基化甜菊醇配糖物。
本公开的又一目的是提供用于生产转葡糖基化甜菊醇配糖物的组合物,其包含苹果乳杆菌微生物或其培养物。
本公开的又一目的是提供增甜剂,其包含使用苹果乳杆菌微生物或其培养物通过上述制备方法制备的转葡糖基化甜菊醇配糖物。
[有益效果]
本公开的用于制备转葡糖基化甜菊醇配糖物的方法,可具体地利用苹果乳杆菌微生物或其培养物生产转葡糖基化甜菊醇配糖物。此外,苹果乳杆菌微生物或其培养物具有从甜菊醇配糖物转化成转葡糖基化甜菊醇配糖物的高转化率,且因此可有效地生产转葡糖基化甜菊醇配糖物。根据本公开的转葡糖基化甜菊醇配糖物是具有改善苦味的高强度增甜剂的材料,并且其卡路里含量与已知的转葡糖基化甜菊醇配糖物相比并不高,且从而可被用于各种领域。
附图说明
图1至9显示了使用苹果乳杆菌的粗酶液制备的转葡糖基化甜菊醇配糖物的HPLC结果。
图10至16显示了使用苹果乳杆菌菌株的粗酶液制备的转葡糖基化甜菊醇配糖物的HPLC/MS结果。
图17是显示转葡糖基化甜菊醇配糖物(蛇菊苷和新蛇菊苷A)根据温度的转化率的图表。
图18是显示转葡糖基化甜菊醇配糖物(蛇菊苷和新蛇菊苷A)根据pH的转化率的图表。
图19是显示转葡糖基化甜菊醇配糖物根据甜菊醇配糖物(蛇菊苷和新蛇菊苷A)的浓度的转化率的图表。
[技术方案]
在下文,将详细描述本公开。同时,本文公开的每个解释和示例性实施方式可应用于其它解释和示例性实施方式。即,本文公开的各种要素的所有组合都属于本公开的范围。此外,本公开的范围不应受下文提供的具体公开的限制。
为了实现本公开的目的,本公开的一方面提供了使用苹果乳杆菌微生物或其培养物制备转葡糖基化甜菊醇配糖物的方法。
如本文所用,术语“甜菊醇配糖物”是指具有连接到化学式1的13-OH或19-OH的葡萄糖、鼠李糖、木糖等的天然增甜剂:
[化学式1]
通常,在化学式1中,在R1处,可结合氢(H),或可经由β-键结合1至3个葡萄糖分子;以及在R2处,可经由β-键结合葡萄糖、木糖和鼠李糖中的任一个,并且可以经由β-键与其结合0至2个葡萄糖分子,但这些不限于此。
甜菊醇配糖物的优点在于,与糖相比,其具有更少的卡路里,且其甜度为糖的甜度的约200至300倍;但缺点在于其伴随着独特的涩味或苦味。因此,一直努力改善甜菊醇配糖物的甜度。
α-/β-糖苷键通过异头位置(anomeric position)和离单糖的1-碳最远的立体中心(stereocenter)的相对立体化学(R型或S型)来区分。一般来说,当两个碳具有相同的立体化学时,形成α-糖苷键,而当两个碳具有不同的立体化学时,出现β-糖苷键。
本发明人首次发现了,苹果乳杆菌微生物及其培养物利用糖和甜菊醇配糖物作为底物将糖分解成葡萄糖,并且通过α-键将1至4个葡萄糖分子选择性地连接到甜菊醇配糖物。此外,本发明人首次发现了,源自本公开的苹果乳杆菌的酶的优点在于,它们在转化成转葡糖基化甜菊醇配糖物的方面具有优良转化率,并且与现有甜菊醇配糖物相比,其气味减少且其甜度显著增加。
如本文所用,术语“转葡糖基化甜菊醇配糖物”可指具有以下形式的甜菊醇配糖物,在该形式中通过使用糖和甜菊醇配糖物作为底物,将1至4个葡萄糖分子通过苹果乳杆菌经由α-键直接添加到甜菊醇配糖物的19-OH位点。更具体地,转葡糖基化甜菊醇配糖物可以处于以下形式,在该形式中1至4个葡萄糖分子通过α-(1,6)键被添加到与甜菊醇配糖物的19-OH位点连接的葡萄糖,但不限于此。
将详细描述制备转葡糖基化甜菊醇配糖物的方法的每个步骤。首先,在该方法中,可以制备苹果乳杆菌微生物或其培养物。
在制备转葡糖基化甜菊醇配糖物的方法的下一步骤中,糖可以在苹果乳杆菌微生物或其培养物的存在下与甜菊醇配糖物反应。
为了本公开的目的,培养物可以是指含有细胞的培养基或不含细胞的粗酶液。具有糖水解活性的酶可发挥选择性地将1至4个葡萄糖分子与甜菊醇配糖物的19-OH位点连接的葡萄糖α-结合的作用,但不限于此。
本文,甜菊醇配糖物可以是选自蛇菊苷、甜茶苷、卫矛醇苷A(Dulcoside A)、新蛇菊苷A、新蛇菊苷C、新蛇菊苷D、新蛇菊苷E、新蛇菊苷F、和新蛇菊苷M中的一种或多种,但不限于此。
进一步,使糖与甜菊醇配糖物反应的步骤可以在1至10的pH下、更具体地在2至9的pH下、或在3至8的pH下进行,但pH不限于此。
此外,使糖与甜菊醇配糖物反应的步骤可以在1℃至80℃的温度下进行、更具体地在5℃至70℃、10℃至60℃、或25℃至50℃的温度下进行,但温度不限于此。
此外,具有转葡糖基化甜菊醇配糖物特征的苹果乳杆菌的特征在于,与含有已知酶的其它微生物相比,具有从甜菊醇配糖物到转葡糖基化甜菊醇配糖物的更高转化率。
转化成本公开的转葡糖基化甜菊醇配糖物的转化率可以为40%至90%,但不限于此。更具体地,转化率可以是40%至90%、50%至80%、50%至85%、60%至85%、60%至80%、70%至85%、或70%至80%,但不限于此。
更具体地,转化率在以下条件下被测量:其中将在30℃下培养24小时至48小时的其中具有转糖基化甜菊醇配糖物特征的苹果乳杆菌的培养基在4000rpm至8000rpm下离心1分钟至20分钟,以分离细胞以及使粗酶液与含有甜菊醇配糖物和糖的底物溶液反应。
为了实现本公开的目的,本公开的另一方面提供了通过上述制备方法制备的转葡糖基化甜菊醇配糖物。转葡糖基化甜菊醇配糖物可以处于以下形式,在该形式中将1至4个葡萄糖分子经由α-键直接添加到甜菊醇配糖物的19-OH位点;以及更具体地,可以处于以下形式,在该形式中将1至4个葡萄糖分子经由α-(1,6)键直接添加到甜菊醇配糖物的19-OH位点,但不限于此。
更具体地,根据上述方法制备的转葡糖基化甜菊醇配糖物可以是选自转葡糖基化蛇菊苷、转葡糖基化甜茶苷、转葡糖基化卫矛醇苷A、转葡糖基化新蛇菊苷A、转葡糖基化新蛇菊苷C、转葡糖基化新蛇菊苷D、转葡糖基化新蛇菊苷E、转葡糖基化新蛇菊苷F、和转葡糖基化新蛇菊苷M中的一种或多种,但不限于此。
本公开的又一方面提供了用于生产转葡糖基化甜菊醇配糖物的组合物,其包含苹果乳杆菌微生物或其培养物。
本公开的又一方面提供了增甜剂,其包含通过该方法制备的转葡糖基化甜菊醇配糖物。为了本公开的目的,增甜剂的特征在于具有减少的异味和增强的甜度。
本公开的又一方面提供了增强增甜剂甜度的方法,其包括使用苹果乳杆菌微生物或其培养物生产转葡糖基化甜菊醇配糖物。
具体实施方式
在下文,将结合随附的示例性实施方式详细描述本公开。然而,本文公开的示例性实施方式仅用于说明性目的,并且不应被解释为限制本公开的范围。
实施例1:具有糖水解活性的新型源自苹果乳杆菌的酶的制备方法
在含有酵母提取物和玉米浆作为氮源、糖(纯度为99%或更高的Beksul白糖)作为碳源、氨基酸等的营养培养基中,在30℃下单独培养了苹果乳杆菌微生物(DSM20444、ATCC27054、ATCC 27304)24小时。将培养基(培养物)在8000rpm下离心10分钟,以分离细胞和上清液,以及然后仅收集了上清液。使粗酶液与糖反应,这导致糖水解活性,并且这确认了具有糖水解活性的新型酶存在于粗酶液中。
实施例2:从甜菊醇配糖物转化成转葡糖基化甜菊醇配糖物的评价
将甜菊醇配糖物和糖溶解在0.05M乙酸盐缓冲溶液中,并且向其中添加了实施例1中已经制备的苹果乳杆菌菌株的粗酶液,然后在40℃下反应24小时。反应后,将反应溶液在100℃下灭活,以及然后通过HPLC确认了转葡糖基化甜菊醇配糖物的产生。使用的甜菊醇配糖物是蛇菊苷、甜茶苷、卫矛醇苷A、和新蛇菊苷A/C/D/E/F/M,并且通过HPLC确定了是否它们产生转葡糖基化蛇菊苷、转葡糖基化甜茶苷、转葡糖基化卫矛醇苷A、和转葡糖基化新蛇菊苷A/C/D/E/F/M。具体地,基于图1至9为了鉴定通过苹果乳杆菌菌株的粗酶液与甜菊醇配糖物之间的反应而新产生的物质,通过进行HPLC/MS分析确定了该物质是转葡糖基化甜菊醇配糖物。进行HPLC/MS分析的甜菊醇配糖物是蛇菊苷、新蛇菊苷A/C/D/E/F/M、甜茶苷和卫矛醇苷A。
图10至16显示了使用苹果乳杆菌菌株的粗酶液制备的转葡糖基化甜菊醇配糖物的HPLC/MS分析的结果。通过图10至16确认了在使用苹果乳杆菌菌株的粗酶液制备的转葡糖基化甜菊醇配糖物中,1至4个葡萄糖分子被转移到甜菊醇配糖物(蛇菊苷、新蛇菊苷A/C/E/F、甜茶苷、和卫矛醇苷A)。
实施例3:温度对转葡糖基化甜菊醇配糖物合成的影响
在通过源自苹果乳杆菌菌株的粗酶液的转葡糖基化甜菊醇配糖物的生产中,评价了温度的影响。将甜菊醇配糖物(蛇菊苷和新蛇菊苷A)和糖溶解于0.05M乙酸缓冲溶液(pH5.0)中,并且向其中添加了粗酶液,然后在10℃至80℃下反应24小时。反应后,通过HPLC分析了反应溶液中转葡糖基化甜菊醇配糖物的量。
图17是显示转葡糖基化甜菊醇配糖物(蛇菊苷和新蛇菊苷A)根据温度的转化率的图表。基于图17,通过苹果乳杆菌菌株的粗酶液向转葡糖基化甜菊醇配糖物的转化率在10℃至50℃下高达10%至70%。
实施例4:pH对转葡糖基化甜菊醇配糖物合成的影响
在通过苹果乳杆菌菌株的粗酶液的转葡糖基化甜菊醇配糖物的生产中,评价了pH的影响。将甜菊醇配糖物和糖溶解在0.05M乙酸缓冲溶液(pH 2.0至pH 5.0)、磷酸盐缓冲溶液(pH 6.0)、Tris缓冲溶液(pH 7.0至pH 8.0)、和碳酸氢钠缓冲溶液(pH 9.0)中。此后,粗酶液被添加至其中,在pH 2至pH 9下进行反应24小时。反应后,通过HPLC分析了转糖基化甜菊醇配糖物的量。
图18是显示转葡糖基化甜菊醇配糖物根据pH的合成转化率的图表。基于图18,通过苹果乳杆菌菌株的粗酶液向转葡糖基化甜菊醇配糖物的转化率在pH 4.0至pH 7.0下高,且特别地,在pH 5.0下显示了最高转化率90%。
实施例5:根据甜菊醇配糖物浓度的转葡糖基化甜菊醇配糖物的分析
评价了通过苹果乳杆菌的粗酶液,根据甜菊醇配糖物的浓度的转葡糖基化甜菊醇配糖物的生产。将糖和甜菊醇配糖物(蛇菊苷和新蛇菊苷A)溶解于乙酸缓冲溶液(pH 5.0)中,以及在40℃下进行反应24小时。反应后,通过HPLC对转葡基化甜菊醇配糖物的生产进行了分析。
图18是显示转葡糖基化甜菊醇配糖物根据甜菊醇配糖物的浓度的转化率的图表。结果,确认了通过苹果乳杆菌的粗酶液向转葡糖基化甜菊醇配糖物的转化率在0%(w/w)到4%(w/w)是高的,且特别地,在6%(w/w)是最高的。
图18显示了转葡糖基化新蛇菊苷A根据新蛇菊苷A的浓度的合成转化率。结果,确认了通过苹果乳杆菌的粗酶液向转葡糖基化新蛇菊苷A的转化率在0%(w/w)至32%(w/w)是高的,且特别地,在6%(w/w)是最高的。
实施例6:转葡糖基化甜菊醇配糖物的核磁共振(NMR)分析
将糖和甜菊醇配糖物溶解在乙酸缓冲溶液(pH 5.0)中,以及然后向其中添加了粗酶液,然后在40℃下反应24小时。将反应溶液在100℃下灭活,以及然后使用0.45μm过滤器移除杂质。利用HP20树脂,对其中转移了一个葡萄糖的甜菊醇配糖物(蛇菊苷和新蛇菊苷A)中的每个,进行了纯分离。通过1H/13C NMR、同核相关谱(homonuclear correlationspectroscopy,COSY)、全相关谱(total correlation spectroscopy,TOCSY)、异核单量子相干谱(heteronuclear single-quantum coherence,HSQC)、和异核多键相关谱(heteronuclear multiple-bond correlation,HMBC)对分离的转葡糖基化蛇菊苷和转葡糖基化新蛇菊苷A的键结构进行了分析。另外,其结果(1H/13C NMR、COSY和HMBC)示于表1和表2中。
另外,作为鉴定转葡糖基化蛇菊苷和转葡糖基化新蛇菊苷A的结构的结果,确认了这些是新型化合物,如转葡糖基化蛇菊苷是13-[(2-O-β-D-吡喃葡糖(苷基)-β-D-吡喃葡糖(苷基))氧基]对映-贝壳杉-16-烯-19-酸6-O-α-D-吡喃葡糖(苷基)-β-D-吡喃葡糖(苷基)酯(13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oicacid6-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester)和转葡糖基化新蛇菊苷A是13-[(2-O-β-D-吡喃葡糖(苷基)-3-O-β-D-吡喃葡糖(苷基)-β-D-吡喃葡糖(苷基))氧基]对映-贝壳杉-16-烯-19-酸6-O-α-D-吡喃葡糖-β-D-吡喃葡糖(苷基)酯(13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 6-O-α-D-glucopyranose-β-D-glucopyranosyl ester)。
<转葡糖基化蛇菊苷的图>
<转葡糖基化新蛇菊苷A的图>
[表1]
[表2]
虽然已经参考具体的说明性实施方式描述了本公开,但是本公开所属领域的技术人员将理解,在不脱离本公开的技术精神或本质特征的情况下,可以以其他具体形式来体现本公开。因此,上面描述的实施方式在所有方面都被认为是说明性的而不是限制性的。此外,本公开的范围由所附权利要求而不是详细描述来限定,并且应当理解,从本公开的含义和范围及其等同物得出的所有修改或变化都被包括在所附权利要求的范围内。
Claims (6)
1.制备转葡糖基化甜菊醇配糖物的方法,
其中所述方法包括在苹果乳杆菌(Lactobacillus mali)微生物或其培养物的存在下使甜菊醇配糖物与糖反应,
其中所述苹果乳杆菌微生物具有由甜菊醇配糖物转化成转葡糖基化甜菊醇配糖物的特征,
其中所述苹果乳杆菌微生物选自DSM20444、ATCC 27054和ATCC 27304。
2.根据权利要求1所述的方法,其中所述甜菊醇配糖物是选自蛇菊苷、甜茶苷、卫矛醇苷A、新蛇菊苷A、新蛇菊苷C、新蛇菊苷D、新蛇菊苷E、新蛇菊苷F和新蛇菊苷M中的一种或多种。
3.根据权利要求1所述的方法,其中使所述甜菊醇配糖物与所述糖反应是在10℃至60℃的温度下在pH 3至pH 8下进行的。
4.根据权利要求1所述的方法,其中所述转葡糖基化甜菊醇配糖物是甜菊醇配糖物,其中通过凭借与连接到所述甜菊醇配糖物的19-OH位点的葡萄糖经由α-(1,6)键连接来添加葡萄糖。
5.根据权利要求4所述的方法,其中所述转葡糖基化甜菊醇配糖物包含1至4个葡萄糖分子。
6.组合物用于产生所述转葡糖基化甜菊醇配糖物的用途,所述组合物包含苹果乳杆菌微生物或其培养物,
其中所述苹果乳杆菌微生物具有由甜菊醇配糖物转化成转葡糖基化甜菊醇配糖物的特征,
其中所述苹果乳杆菌微生物选自DSM20444、ATCC 27054和ATCC 27304。
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