CN112566512A - 作为食物和营养补充剂的蔗糖异构酶 - Google Patents
作为食物和营养补充剂的蔗糖异构酶 Download PDFInfo
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Abstract
蔗糖异构酶用作营养补充剂,或可与粉状食品/饮料制剂混合。当动物包括人食用蔗糖时,蔗糖异构酶将作用于食品中存在的蔗糖,并将蔗糖转化为其他糖。这导致所述食品的血糖指数降低而无需改变所述食品的制剂。
Description
技术领域
本发明涉及作为人和动物的营养补充剂的蔗糖异构酶的用途。蔗糖异构酶可以在食用之后酶促地减少食物中蔗糖的量,并且因此降低食品的血糖指数。蔗糖异构酶补充剂特别有益于降低血糖、降低食用的食品和/或饮料的血糖指数以及管理体重或减轻体重。
背景技术
高血糖在全球疾病负担中排名很高,并可能导致肥胖和2型糖尿病。含有快速吸收的碳水化合物(如糖或淀粉)的食品和饮料可导致血糖快速增加,接着胰岛素释放达到峰值,导致血糖快速降低。缺乏此类碳水化合物的食品和饮料导致血糖增加较慢且较低,且胰岛素释放不迅速达到峰值。血糖中的这种应答表述为食品的血糖指数(Glycemic Index,GI),其被表示为相对于对含有葡萄糖的参考物或白面包的摄入的应答(设定为100)。已经确定了许多食品的GI。已显示,以消化、吸收和代谢更慢的碳水化合物食品为基础的饮食(即,低GI饮食)与高GI饮食相比得到更好的胰岛素敏感性。与高GI饮食相比,低GI饮食降低2型糖尿病和心血管疾病的风险。已提出了与高GI饮食相比,低GI饮食在饱腹感、体重维持和饮食相关疾病的预防方面的作用。血糖负荷(GL)通过将食品中可用的碳水化合物的克数乘以食品的GI,然后除以100来计算。
饮食中的蔗糖含量丰富,并且占饮食中所有碳水化合物的约35-40%。对于希望降低血糖负荷的人来说,一种挑战是许多优选的放纵(indulgent)食品和饮料都含有大量蔗糖。例如,一些糖果条含有多达30克蔗糖(50重量%);一罐(330cc)可乐含有39克蔗糖;巧克力奶在450cc中含有58克蔗糖;并且冰淇淋通常含有28重量%蔗糖。但是,降低这些食品中的糖含量通常对甜味、口感和放纵特性产生负面影响。将此类产品中的蔗糖用GI较低的其他糖(例如,塔格糖、阿洛酮糖或帕拉金糖)、糖醇(例如,山梨糖醇、甘露糖醇或木糖醇)或高效甜味剂(例如,阿斯巴甜、三氯蔗糖或甜菊)代替将得到甜味较低的最终产品,或对食品或饮料的质地性质和/或口味产生负面效应,从而降低产品的放纵特性。因此,此类蔗糖代替物在食品产品中不经常使用,并且其使用主要集中在甜味饮料上。
异麦芽酮糖可以从Beneo以商品名PalatinoseTM获得并在食品工业中作为糖代替物用于食品中。PalatinoseTM在小肠中完全可用,但水解慢4-5倍,导致低血糖应答和较低的胰岛素水平。已经显示,用PalatinoseTM取代蔗糖导致胰岛素峰值较低并且运动时的脂肪燃烧增加。Beneo在市场上出售PalatinoseTM,其用于体重管理,具有不生龋齿的特性,使运动的耐力表现增强,预防妊娠糖尿病,使认知功能持续并且改善老年人的代谢概况(Beneo-institute,2017http://www.beneo.com/Expertise/BENEO-Institute/News_Papers/BENEO_paper_palatinose_US_201708v1_web_U SLetter_1.pdf)。
对于海藻酮糖(在专利文献中也称为“Vitalose”),预见到类似的益处,因为它也被肠蔗糖酶缓慢消化,但可用的临床数据较少。血糖水平和胰岛素应答与异麦芽酮糖的血糖水平和胰岛素应答相似或与其相比甚至略微更温和(如欧洲专利EP2418971B1中所报道)。
文献中提出了这些糖对小肠中的蔗糖酶/淀粉酶活性的抑制作用(Kashimura等人,2008 J.Agric.Food Chem.56:5892-5898),但是对此仍然没有确凿证据。这种抑制作用可以减慢蔗糖和淀粉的转化,从而进一步降低血糖负荷。因此,当与含有淀粉的食品组合时,异麦芽酮糖和/或海藻酮糖消耗可通过抑制肠中的蔗糖酶/淀粉酶活性来对降低血糖负荷而具有额外的益处。
在食品和饮料中使用异麦芽酮糖或海藻酮糖的一个问题是,在每克基础上,这些糖的甜度远低于蔗糖的甜度。因此,将蔗糖用这些蔗糖异构体代替将要求食品或饮料的组成的巨大改变。较低的甜度必须通过例如添加额外的人造甜味剂来补偿,这可能影响最终产品的口味。而且,异麦芽酮糖与蔗糖相比相对昂贵,因此可能出于经济原因不将其添加至食品或饮料产品中。
因此,社会上需要食用之后不引起高血糖应答的甜味放纵食品和饮料。有意识的消费者可能希望预防食用含有蔗糖的食品/饮料之后的高血糖指数和血糖增加。此外,在胃中将高血糖指数碳水化合物转化为低血糖指数碳水化合物对于患有1型和2型糖尿病的患者的血糖控制具有临床上显著的益处。降低血糖指数的营养疗法可以使2型糖尿病患者的糖化血红蛋白(A1C)降低1.0%至2.0%,并且当与糖尿病护理的其他组分一起使用时,可以进一步改善临床和代谢结果。
发明内容
我们惊讶地发现,将蔗糖异构酶用作营养补充剂或用作医学饮食或医学饮食补充剂的一部分,将降低与酶一起食用的甜味食品和/或饮料中的蔗糖含量。因此,用作营养补充剂或用作干燥食品或饮料(诸如预混物等)的一部分的蔗糖异构酶将降低此类食品在肠道中的血糖指数,而不影响食用之前食品或饮料的组成和性质。因此,作为营养补充剂的蔗糖异构酶可用于在不改变饮食习惯的情况下降低2型糖尿病和心血管疾病的风险。而且,通过降低血糖负荷,作为营养补充剂的蔗糖异构酶可适合体重维持的方案,并且可以预防与高糖饮食相关的疾病。作为营养补充剂的蔗糖异构酶还可以通过减慢糖的摄入来用于运动营养。蔗糖异构酶还可包括在建议或指示低碳水化合物饮食的糖尿病患者或前驱糖尿病患者的现用现混(ready-to-mix)代餐中,而不干扰代餐的碳水化合物含量。
因此,本发明的一个实施方案为一种降低食用包含蔗糖的食品或饮料的动物(包括人)的胰岛素水平的方法,所述方法包括在食用所述食品或饮料之前或过程中向所述动物或人施用有效量蔗糖异构酶营养保健品、膳食补充剂或药物。本发明的另一实施方案为一种降低动物(包括人)食用的包含蔗糖的食品或饮料的血糖指数的方法,其包括向所述动物或人施用呈营养保健品、膳食补充剂或药物的形式的蔗糖异构酶。本发明的另一实施方案为蔗糖异构酶制造降低食品或饮料的血糖指数的营养补充剂、干燥和/或粉状食品或饮料或药物的用途。本发明的另一实施方案为作为降低食品或饮料的血糖指数的营养补充剂的蔗糖异构酶。
新的研究表明,运动员的耐力持续的关键可能不在于所谓的“快碳水化合物”的消耗,而是在于“慢碳水化合物”的消耗,其平衡血糖而不是以血糖的形式提供一股快速的能量。出于本发明的目的,“耐力运动”意指增加呼吸和心率的运动,诸如走路、慢跑、游泳或骑自行车等。因此,本发明的另一实施方案为一种在一定时间段内使糖吸收减慢或持续以增强运动员进行耐力运动的能力的方法,其包括在耐力运动期间向进行耐力运动的、还食用含有蔗糖的食品或饮料的人施用有效量蔗糖异构酶。本发明的另一实施方案为一种增强运动员的耐力持续的方法,其包括向参与体育耐力活动的、还食用含有蔗糖的食品或饮料的人施用有效量蔗糖异构酶。本发明的另一实施方案为蔗糖异构酶增加人进行耐力运动的能力的用途。
本发明的另一实施方案为一种在含有蔗糖的餐后使糖吸收持续和/或减慢以使能量释放持续并使血糖升高和所谓的餐后“情绪消沉(dip)”最小化的方法,其包括向也食用含有蔗糖的食品的健康或(前驱)糖尿病人施用有效量蔗糖异构酶。这在人进餐(诸如午餐)并希望在食用之后数小时保持警觉并避免瞌睡期的情况下是特别有益的。
本发明的另一实施方案为一种帮助动物(包括人)减轻体重或维持体重减轻的方法,其包括向所述动物或人施用有效量蔗糖异构酶。
在一个实施方案中,蔗糖异构酶用于人营养。
在另一实施方案中,蔗糖异构酶用于有益于可食用蔗糖的动物,优选地伴侣动物(诸如猫、狗、马和通常用作宠物的驯养的猪)。伴侣动物经常易患肥胖症并且遭受其不良后果。本发明的蔗糖异构酶提供了在不需要依靠昂贵且不方便的胰岛素注射的情况下对抗伴侣动物的糖尿病、体重增加和相关联的代谢紊乱的方式。
优选的是,在食用含有蔗糖的食品或饮料之前摄取蔗糖异构酶至少一天一次。还优选的是,就在食用之前摄取(即,食用之前少于一小时,且更优选地,食用之前少于30分钟。特别优选的是,食用之前立即摄取或在食用期间摄取)。在另一实施方案中,在进餐2小时内摄取蔗糖异构酶。
附图说明
图1为如实施例1中详述的用于分离糖的HPLC的读出。
蔗糖异构酶用于从蔗糖工业生产异麦芽酮糖。据我们所知,尚没有将蔗糖异构酶用作补充剂的描述,其中,蔗糖异构酶可以在涉及制造片剂或其他合适的制剂的过程以及人消化过程中留存下来,以使得其在胃和/或消化道中仍保留活性。尽管蔗糖异构酶为本领域中已知的,但是它们的活性仅在实验室缓冲液的情况下才被观察到。
蔗糖异构酶将蔗糖(2-O-α-D-吡喃葡萄糖基-D-果糖)转化成低血糖的异麦芽酮糖(6-O-α-D-吡喃葡萄糖基-D-果糖)和/或海藻酮糖(1-O-α-D-吡喃葡萄糖基-D-果糖)(Mu等人(2014)Appl Microbiol Biotechnol 98:6569-6582)。
如实施例4-6中所示,发现尽管通过不同机理但也同样作用于蔗糖的另一种酶,即糖基转移酶,当进行模拟人消化系统的条件时是非活性的。因此,以蔗糖为底物的酶适于作为营养补充剂的用途是不可预测的。
本发明的蔗糖异构酶可来自任何来源,前提是它的稳健性足以留存于制剂并且还足以留存于消化过程,以使得可以保留有效量的酶作用于摄取的糖。有至少5类本领域公认的蔗糖异构酶(参见Goulter等人2012 Enz and Microb Technol 50:57-64):
第I组:包括普利茅斯沙雷菌(Serratia plymuthica)和红色精朊杆菌(Protaminobacter rubrum)
第II组:包括大黄欧文氏菌(Erwinia rhapontici)
第III组:包括肠杆菌属种(Enterobacter sp)、植生拉乌尔菌(Roaultellaplanticola)和新加坡克雷伯氏菌(Klebsiella singaporensis)
第IV组:包括分散泛菌(Pantoea dispersa);以及
第V组:包括中嗜酸性假单胞菌(Pseudomonas mesoacidophilia)和根瘤菌属种(Rhizobium sp.)。
优选的蔗糖异构酶的实例包括在以下生物中存在的那些:
红色精朊杆菌,包括被鉴定为Uniprot:D0VX20的酶,
分散泛菌,包括被鉴定为Uniprot:Q6XNK6的酶,
植生拉乌尔菌,包括被鉴定为Uniprot:Q6XKX6的酶,
中嗜酸性假单胞菌,包括被鉴定为Uniprot:Q2PS28的酶,
肠杆菌,包括被鉴定为Uniprot:B5ABD8的酶;以及
胡萝卜果胶杆菌(Pectobacterium carotovorum),包括被鉴定为Uniprot:S5YEW8的酶。
当存在时,它们的信号肽被甲硫氨酸(M)替代,并且这产生了分别在SEQ ID NO:1-6中描述的蛋白质序列。
优选的蔗糖异构酶包括在实施例中被鉴定为Sis4、Sis10、Sis12、Sis14和Sis15的那些。特别优选的蔗糖异构酶是Sis4。
如本文所述,本发明避免了先前的在食品和饮料制剂中使用异麦芽酮糖、海藻酮糖或任何其他低血糖糖替代品的问题。通过供应蔗糖异构酶作为营养成分,食品或饮料制剂不需要进行改变,并且异麦芽酮糖和/或海藻酮糖仅在胃或上消化道中消化期间形成。
与本发明有关的优选的含有蔗糖的食品/饮料包括诸如以下的放纵食品:
·甜点类–冰淇淋、布丁、乳蛋糕、酸乳酪
·糖食类–糖果、巧克力
·烘焙类–饼干、糕点、派、甜甜圈、早餐麦片
·饮料类–软饮料、能量饮料、果汁、调味奶
·水果和蔬菜类–玉米、热带水果、海枣、香蕉、甜菜根、南瓜
·涂抹酱类–果酱、甜果酱、巧克力酱、花生酱
·伴侣动物的食物:以犒赏食物或耐嚼小吃的形式
制剂
根据本发明的膳食和药物组合物可为适用于向身体施用的任何盖伦制剂形式(galenic form),尤其是方便口服施用的任何形式,例如:固体形式,诸如食品或饲料的添加剂/补充剂、食品或饲料预混物、强化食品或饲料、片剂、药丸、颗粒剂、糖衣丸(dragée)、胶囊和泡腾制剂(诸如粉剂和片剂);或液体形式,诸如溶液、乳液或混悬液,诸如以饮料、糊剂和混悬液的形式。可以将糊剂封装在硬壳或软壳胶囊中,由此胶囊具有例如鱼、诸、家禽或奶牛明胶基质、植物蛋白质或木质素磺酸盐的特征。膳食和药物组合物可以为受控释放制剂或延缓释放制剂的形式。本发明的组合物不是局部施用(诸如涂敷于鼻腔通道)的。
根据本发明的膳食组合物可还含有保护性水胶体(诸如树胶、蛋白质、改性的淀粉)、粘合剂、成膜剂、封装剂/材料、壁/壳材料、基质化合物、包衣、乳化剂、表面活性剂、增溶剂(油、脂肪、蜡、卵磷脂等)、吸附剂、载体、填充剂、共化合物、分散剂、润湿剂、加工助剂(溶剂)、流动剂、掩味剂、增重剂、胶冻剂、凝胶形成剂、抗氧化剂和抗微生物剂。
此外,根据本发明的组合物可还含有常规的药物添加剂和助剂、赋形剂或稀释剂,包括但不限于水、任何来源的明胶、植物胶、木质素磺酸盐、滑石、糖、淀粉、阿拉伯树胶、植物油、聚亚烷基二醇、调味剂、防腐剂、稳定剂、乳化剂、缓冲剂、润滑剂、着色剂、湿润剂、填充剂等。
剂量
酶作为营养补充剂的剂量为每100g摄取的蔗糖0.1-500mg纯蔗糖异构酶,优选地每100g摄取的蔗糖0.5-100mg、2-50mg、10-25mg蔗糖异构酶。当然,剂量根据每天、或每餐、或每种饮料所摄取的糖量而变化。例如,如果一个人每天食用75克添加的蔗糖,这在一些西方国家为常见量,那么每日酶的优选量将为7.5-20mg纯蔗糖异构酶蛋白。例如,如果一个人饮用300ml含有100g/l蔗糖的饮料,那么待与饮料一起摄入的酶的优选量将为3-7.5mg纯蔗糖异构酶蛋白。
具有蔗糖异构酶的典型组合物可含有二氧化硅、(微)纤维素(例如AvicelPH102)、硬脂酸镁、硬脂酸、聚乙烯吡咯烷酮(例如Crospovidone)和/或麦芽糖糊精。每片300mg这种组合物的片剂可含有例如60mg干燥的酶制剂(例如,含有7.5-20mg纯蔗糖异构酶加麦芽糖糊精直至60mg)、15mg交聚维酮、2.5mg硬脂酸镁和222.5mg Avicel PH102。
此外,组合物可为干燥食品、软饮粉末或代餐粉。通常,这种干燥组合物的水活度(Aw)<0.5。典型的等渗运动能量饮料粉末可含有每100g粉末多达90g碳水化合物,其中75g可为糖(主要是蔗糖和葡萄糖)。此外,每100g粉末还将含有2.15g矿物盐(主要是氯化钾、柠檬酸钾、柠檬酸钠、氯化钠和柠檬酸镁),此外还含有一些柠檬酸、调味料和色素。优选地,以每100g这种粉末7.5-20mg纯干燥酶添加蔗糖异构酶。
酶
同源性
出于本公开的目的,在此定义了,为了确定两个氨基酸序列或两个核酸序列的序列同源性或序列同一性的百分比,出于最佳比较目的,对序列进行比对。为了优化两个序列之间的比对,可在比较的两个序列中的任一个中引入空位。可在正比较的序列的全长上进行这种比对。替代地,可在较短长度内进行比对,例如在约20、约50、约100或更多个核酸/碱基或氨基酸上进行比对。序列同一性为报道的比对基因上两个序列之间相同匹配的百分比。
可以使用数学算法完成序列的比较和两个序列之间的序列同一性百分比的测定。技术人员应知道以下事实,若干不同的计算机程序可用于比对两个序列并测定两个序列之间的同一性(Kruskal,J.B.(1983)An overview of sequence comparison In D.Sankoffand J.B.Kruskal,(编),Time warps,string edits and macromolecules:the theoryand practice of sequence comparison,第1-44页Addison Wesley)。两个氨基酸序列之间或两个核苷酸序列之间的序列同一性百分比可使用比对两个序列的Needleman和Wunsch算法来测定(Needleman,S.B.和Wunsch,C.D.(1970)J.Mol.Biol.48,443-453)。两个氨基酸序列和核苷酸序列可通过所述算法来比对。Needleman-Wunsch算法已经在计算机程序NEEDLE中实现。出于本公开的目的,使用EMBOSS包的NEEDLE程序(2.8.0版或更高版本,EMBOSS:The European Molecular Biology Open Software Suite(2000)Rice,P.Longden,I.和Bleasby,A.Trends in Genetics 16,(6)第276—277页,http://emboss.bioinformatics.nl/)。对于蛋白质序列,将EBLOSUM62用于取代矩阵。对于核苷酸序列,使用EDNAFULL。使用的可选参数是空位开放罚分10和空位延伸罚分0.5。技术人员将理解,所有这些不同的参数将产生略有不同的结果,但是当使用不同的算法时,两个序列的总体同一性百分比不显著改变。
在如上所述通过程序NEEDLE进行比对之后,查询序列与本公开序列之间的序列同一性百分比计算如下:比对中显示两个序列的相同氨基酸或相同核苷酸的对应位置的数目除以减去比对的空位总数之后比对的总长度。如本文所定义的同一性可通过使用NOBRIEF选项从NEEDLE获得并且在程序的输出中标记为“最长同一性”。
本公开的核酸和蛋白质序列还可以用作“查询序列”以对公共数据库进行搜索,以例如鉴定其他家族成员或相关序列。这类搜索可以使用Altschul等人(1990)J.Mol.Biol.215:403—10的BLASTN和BLASTX程序(2.0版)进行。BLAST核苷酸搜索可用BLASTN程序来进行,得分=100,字长=12,以获得与本公开的核酸分子同源的核苷酸序列。BLAST蛋白质搜索可用BLASTX程序来进行,得分=50,字长=3,以获得与本公开的蛋白质分子同源的氨基酸序列。为了获得用于比较目的的空位比对,可以利用如Altschul等人,(1997)Nucleic Acids Res.25(17):3389-3402中所述的Gapped BLAST。当利用BLAST和Gapped BLAST程序时,可以使用对应程序(例如,BLASTX和BLASTN)的默认参数。美国国家生物技术信息中心(National Center for Biotechnology Information)的主页参见http://www.ncbi.nlm.nih.gov/。
如本文所用,术语“变体”、“衍生物”、“突变体”或“同源物”可以互换使用。它们可以指多肽或核酸。变体包括在相对于参考序列的一个或多个位置处的取代、插入、缺失、截短、颠换和/或倒位。可以通过例如位点饱和诱变、扫描诱变、插入诱变、随机诱变、定点诱变和定向进化以及各种其他重组方法来产生变体。变体多肽可能与参考多肽相差少量氨基酸残基,并且可以由它们与参考多肽的一级氨基酸序列同源性/同一性的水平来定义。优选地,变体多肽与参考多肽具有至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或甚至至少99%氨基酸序列同一性。用于测定同一性百分比的方法为本领域中已知的并且本文有所描述。通常,变体保留了参考多肽的特征实质,但是在一些特定方面具有改变的性质。例如,变体可具有改变的pH最佳值、改变的底物结合能力、改变的对酶促降解或其他降解的抗性、增加或降低的活性、改变的温度或氧化稳定性,但是保留其特征功能性。变体还包括具有化学修饰的多肽,所述化学修饰改变了参考多肽的特征。
关于核酸,所述术语是指编码变体多肽,与参照核酸具有指定程度的同源性/同一性或在严格条件下与参照核酸或其互补体杂交的核酸。优选地,变体核酸与参考核酸具有至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或甚至至少99%核酸序列同一性。用于测定同一性百分比的方法为本领域中已知的并且本文有所描述。
本发明通过以下实施例进一步进行说明。
实施例:
实施例1
蔗糖异构酶
从Uniprot数据库选择了六种注释为蔗糖异构酶的蛋白质。序列来源于红色精朊杆菌(Uniprot:D0VX20)、分散泛菌(Uniprot:Q6XNK6)、植生拉乌尔菌(Uniprot:Q6XKX6)、中嗜酸性假单胞菌(Uniprot:Q2PS28)、肠杆菌(Uniprot:B5ABD8)和胡萝卜果胶杆菌(Uniprot:S5YEW8)。通过SignalP 4.1预测软件预测革兰氏阴性菌的推定信号肽(Petersen,Nature Methods,8:785-786,2011)。当存在时,这些信号肽被甲硫氨酸(M)替代,并且这产生了在SEQ ID NO:1-6中描述的蛋白质序列。
如WO2017050652(A1)中所述,蛋白质序列(SEQ ID NO:1-6)在大肠杆菌中表达。根据DNA2.0(技术)的算法对编码推定蔗糖异构酶的合成DNA序列进行密码子优化以在大肠杆菌中表达。为了克隆目的,将含有NdeI位点的DNA序列引入在5'末端,并且将含有终止密码子和AscI位点的DNA序列引入在3'末端。经由5'NdeI和3'AscI限制位点将编码推定蔗糖异构酶的合成DNA克隆到阿拉伯糖诱导型大肠杆菌表达载体中,所述载体含有阿拉伯糖诱导型启动子PBAD和调节子araC(Guzman(1995)J.Bact.177:4121-4130)、卡那霉素抗性基因Km(R)和pBR322的复制起点ori327(Watson(1988)Gene.70:399-403)。使用化学感受态细胞转化带有额外的ampC和araB缺失的大肠杆菌宿主RV308(lacIq-,su-,ΔlacX74,gal IS II::OP308,strA,http://www.ebi.ac.uk/ena/data/view/ERP005879)。对来自SEQ ID NO:1-6的若干克隆进行序列验证,并在含有100μg/ml新霉素(O/N)的2xPY中培养。使用预培养(1/100vol)将发酵接种在MagicMediaTM大肠杆菌表达培养基(ThermoFisher Scientific Inc)和100μg/ml新霉素(24孔MTP,3ml体积,可透气密封件,550RPM80%RH)中,并且在30℃下生长4小时之后,用0.02%阿拉伯糖(最终浓度)诱导培养物并且在30℃下继续孵育48小时。通过高速离心来分离细胞沉淀物并冷冻直至进一步使用。通过重悬冷冻的细胞沉淀物并在37℃下与1.2ml裂解缓冲液(Tris-HCl 50mM、DNaseI 0.1mg/ml、溶菌酶2mg/ml、MgSO4 25μM)孵育1小时来制备无细胞提取物(CFE)。通过离心来除去细胞碎片,并将CFE在-20℃下储存直至进一步表征。
葡聚糖蔗糖酶
本研究中所使用的葡聚糖蔗糖酶获自商业供应商获得(Sigma Aldrich的肠膜明串珠菌(Leuconostoc mesenteroides)葡聚糖蔗糖酶;NZYTech的变形链球菌(Streptococcus mutans)葡聚糖蔗糖酶)。
本研究中所使用的所有酶在表1中陈述。
表1.本研究中使用的酶。
*红色精朊杆菌最可能重新命名为普利茅斯沙雷菌,并且中嗜酸性假单胞菌被指配为根瘤菌属种(Goulter等人(2012)Enzyme Microb.Technol.50,57-64)。
序列SEQ ID NO 1-6:
SEQ ID NO:1
MTIPKWWKEAVFYQVYPRSFKDTNGDGIGDINGIIEKLDYLKALGIDAIWINPHYDSPNTDNGYDIRDYRKIMKEYGTMEDFDRLISEMKKRNMRLMIDVVINHTSDQNEWFVKSKSSKDNPYRGYYFWKDAKEGQAPNNYPSFFGGSAWQKDEKTNQYYLHYFAKQQPDLNWDNPKVRQDLYAMLRFWLDKGVSGLRFDTVATYSKIPDFPNLTQQQLKNFAAEYTKGPNIHRYVNEMNKEVLSHYDIATAGEIFGVPLDQSIKFFDRRRDELNIAFTFDLIRLDRDSDQRWRRKDWKLSQFRQIIDNVDRTAGEYGWNAFFLDNHDNPRAVSHFGDDRPQWREPSAKALATLTLTQRATPFIYQGSELGMTNYPFKAIDEFDDIEVKGFWHDYVETGKVKADEFLQNVRLTSRDNSRTPFQWDGSKNAGFTSGKPWFKVNPNYQEINAVSQVTQPDSVFNYYRQLIKIRHDIPALTYGTYTDLDPANDSVYAYTRSLGAEKYLVVVNFKEQMMRYKLPDNLSIEKVIIDSNSKNVVKKNDSLLELKPWQSGVYKLNQ
SEQ ID NO:2
MASPLTKPSTPIAATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASGKVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLN
SEQ ID NO:3
MAPSVNQNIHVHKESEYPAWWKEAVFYQIYPRSFKDTNDDGIGDIRGIIEKLDYLKSLGIDAIWINPHYDSPNTDNGYDISNYRQIMKEYGTMEDFDNLVAEMKKRNMRLMIDVVINHTSDQHPWFIQSKSDKNNPYRDYYFWRDGKDNQPPNNYPSFFGGSAWQKDAKSGQYYLHYFARQQPDLNWDNPKVREDLYAMLRFWLDKGVSSMRFDTVATYSKIPGFPNLTPEQQKNFAEQYTMGPNIHRYIQEMNRKVLSRYDVATAGEIFGVPLDRSSQFFDPRRHELNMAFMFDLIRLDRDSNERWRHKSWSLSQFRQIISKMDVTVGKYGWNTFFLDNHDNPRAVSHFGDDRPQWREASAKALATITLTQRATPFIYQGSELGMTNYPFRQLNEFDDIEVKGFWQDYVQSGKVTATEFLDNVRLTSRDNSRTPFQWNDTLNAGFTRGKPWFHINPNYVEINAEREETREDSVLNYYKKMIQLRHHIPALVYGAYQDLNPQDNTVYAYTRTLGNERYLVVVNFKEYPVRYTLPANDAIEEVVIDTQQQATAPHSTSLSLSPWQAGVYKLR
SEQ ID NO:4
MEEAVKPGAPWWKSAVFYQVYPRSFKDTNGDGIGDFKGLTEKLDYLKGLGIDAIWINPHYASPNTDNGYDISDYREVMKEYGTMEDFDRLMAELKKRGMRLMVDVVINHSSDQHEWFKSSRASKDNPYRDYYFWRDGKDGHEPNNYPSFFGGSAWEKDPVTGQYYLHYFGRQQPDLNWDTPKLREELYAMLRFWLDKGVSGMRFDTVATYSKTPGFPDLTPEQMKNFAEAYTQGPNLHRYLQEMHEKVFDHYDAVTAGEIFGAPLNQVPLFIDSRRKELDMAFTFDLIRYDRALDRWHTIPRTLADFRQTIDKVDAIAGEYGWNTFFLGNHDNPRAVSHFGDDRPQWREASAKALATVTLTQRGTPFIFQGDELGMTNYPFKTLQDFDDIEVKGFFQDYVETGKATAEELLTNVALTSRDNARTPFQWDDSANAGFTTGKPWLKVNPNYTEINAAREIGDPKSVYSFYRNLISIRHETPALSTGSYRDIDPSNADVYAYTRSQDGETYLVVVNFKAEPRSFTLPDGMHIAETLIESSSPAAPAAGAASLELQPWQSGIYKVK
SEQ ID NO:5
MAYSAETSVTQSIQTQKESTLPAWWKEAVFYQIYPRSFKDTNGDGIGDIRGIIEKLDYLKSLGIDAIWINPHYDSPNTDNGYDIRDYEKIMQEYGTMEDFDTLVSEMKKRNMRLMIDVVINHTSDQHPWFIQSKSSKENPYREYYFWRDGKDNQPPNNYPSFFGGSAWQKDDKTGQYYLHYFARQQPDLNWDNPKVRGDLYAMLRFWLDKGVSGMRFDTVATYSKIPGFPDLTPEQQKNFAEQYTTGPNIHRYLQEMKQEVLSRYDVVTAGEIFGVPLERSSDFFDRRRNELDMSFMFDLIRLDRDSNERWRHKKWTLSQFRQIINKMDSNAGEYGWNTFFLDNHDNPRAVSHFGDDSPQWIEPSAKALATIILTQRATPFIFQGSELGMTNYPFKKLNEFDDIEVKGFWQDYVQTGKVSAEEFIDNVRLTSRDNSRTPFQWNDRKNAGFTSGKPWFRINPNYVEINADKELIRNDSVLNYYKEMIKLRHKTPALIYGTYKDISPEDDSVYAYTRTLGKERYLVVINFTEKTVRYPLPENNVIKSILIEANQNKTAEKQSTVLTLSPWQAGVYELQ
SEQ ID NO:6
MATNHNEQDTKTVIAVNDGVSAHPVWWKEAVFYQVYPRSFKDSNGDGIGDLKGLTEKLDYLKTLGINAIWINPHYDSPNTDNGYDIRDYRKIMKEYGTMDDFDNLIAEMKKRDMRLMIDVVVNHTSNEHKWFVESKKSKDNPYRDYYIWRDGKDGTPPNNYPSFFGGSAWQKDNVTQQYYLHYFGVQQPDLNWDNPKVREEVYDMLRFWIDKGVSGLRMDTVATFSKNPAFPDLTPEQLKNFAYTYTQGPNLHRYIQEMHQKVLAKYDVVSAGEIFGVPLEEAAPFIDQRRKELDMAFSFDLIRLDRAVEERWRRNDWTLSQFRQINNRLVDMAGQYGWNTFFLSNHDNPRAVSHFGDDRPEWRIRSAKALATLALTQRATPFIYQGDELGMTNYPFTSLSEFDDIEVKGFWQDFVETGKVKPDVFLENVKQTSRDNSRTPFQWSNAEQAGFTTGTPWFRINPNYKNINAEDQTQNPDSIFHFYRQLIALRHATPAFTYGAYQDLDPNNNEVLAYTRELNQQRYLVVVNFKEKPVHYALPKTLSIKQTLLESGQKDKVAPNATSLELQPWQSGIYQLN
酶量化
使用SDS-PAGE,接着使用考马斯染色来使样品中存在的酶可视化。为了量化各个酶,着眼于正确分子质量的带。带的染色强度通过ImageQuant软件来量化。将所选带的蛋白质染色强度与在相同凝胶上进行的牛血清白蛋白(BSA)的已知量进行比较,并计算回原始的酶储备溶液
糖鉴定和量化
使用配备有CarboPac PA20柱的Dionex HPLC(HPAEC BioLC系统,Dionex 5000)分析含有蔗糖的溶液与酶一起孵育之后的糖组成概况。孵育之后,将样品稀释,过滤并使用表2中所述的程序分离糖组分。
表2:用于在Dionex上分离糖的HPLC方法。
步骤 | 时间(min) | NaOH(mM) | 注释 |
1 | -15 | 25 | 平衡 |
2 | 0 | 25 | 进样 |
3 | 15 | 25 | 等梯度运行 |
4 | 30 | 30 | 轻微的梯度 |
5 | 40 | 126 | 洗涤柱(还用乙酸) |
6 | 50 | 25 | 准备下一次运行 |
通过示踪获自Merck Millipore的蔗糖、葡萄糖、果糖、异麦芽酮糖、明串珠菌二糖、海藻酮糖和异麦芽糖(范围为2至75mg/ml)的纯溶液,对HPLC上的峰进行指配和量化。图1显示了使用这种技术分离不同糖的实例。对检测糖浓度的峰面积进行积分并绘制浓度对峰面积的线性曲线拟合来计算每种糖的响应因数。响应因数用于计算每种样品中存在的糖的绝对量。通过将样品中测量的每种糖的绝对量除以样品中检测到的所有糖的总量并乘以100来计算以百分比计的相对糖浓度。
血糖指数的计算
计算这些实验中的血糖指数(GI),假定不同糖的GI;蔗糖:65;果糖:15;葡萄糖:100;异麦芽酮糖:32;海藻酮糖:32。Wolever(European Journal of Clinical Nutrition(2013)67,1229–1233)指出,根据加拿大法规,GI>70被视为高,并且GI<55为低。将产品中每种糖的百分比含量除以100,并且乘以其GI。将所有数字相加,以计算不同处理的产品的GI。
实施例2
不同pH下蔗糖异构酶的活性
为了测试不同pH下不同蔗糖异构酶对蔗糖的活性,我们将20%蔗糖/250mM磷酸钠缓冲液与10%稀释(0.07-0.21mg蛋白质/ml)的不同酶一起孵育。溶液的pH设定为4.5和6.0,并且在37℃下孵育6小时,之后通过在99℃下加热5分钟来停止反应。使用Dionex HPLC方法量化蔗糖向不同糖的转化率。结果示出于表3中,表示为获自相应酶的2-4种不同制备剂的实验相对于孵育之后样品中检测到的所有糖的总量的平均百分比。
表3:使用蔗糖异构酶在各种pH下蔗糖向各种糖的转化率
从表3清楚可见,所有测试的酶均能够在pH 6.0和pH 4.5下几乎完全转化蔗糖。产物形成一定程度地取决于酶,但是在所有蔗糖异构酶的情况下,最突出的产物是异麦芽酮糖和海藻酮糖,在大多数情况下占总糖的60-100%。
实施例3
不同pH下葡聚糖蔗糖酶的活性
为了测试不同pH下不同葡聚糖蔗糖酶对蔗糖的活性,我们将20%蔗糖/250mM磷酸钠缓冲液与10%稀释的不同酶一起孵育。溶液的pH设定为4.5和6.0,并且在37℃下孵育6小时,之后通过在99℃下加热5分钟来停止反应。使用Dionex HPLC方法量化糖组成。结果示出于以下表4中,表示为获自相应酶的实验相对于转化之后的总糖百分比。因为葡聚糖可以以不同形式存在,所以难以使用这些实验中使用的HPLC方法进行量化。因此,在校正不添加酶的空白样的果糖和葡萄糖含量之后,由果糖和葡萄糖的增加的差计算总葡聚糖形成。因此,所示数字仅为形成的葡聚糖总量的粗略估计值。
表4:使用葡聚糖蔗糖酶在各种pH下蔗糖向各种糖的转化率
从表4清楚可见,一些葡聚糖蔗糖酶(70B和70C)能够在pH6.0和pH 4.5下转化几乎所有蔗糖,而其他葡聚糖蔗糖酶显示活性非常小。产物形成一定程度地取决于酶,并且在这些条件下,葡聚糖产率最大为约30-40%。由这些酶形成的其他糖主要是明串珠菌二糖和一些异麦芽酮糖。
实施例4
可乐中蔗糖异构酶的活性
对蔗糖异构酶和葡聚糖蔗糖酶在可乐(当地超市)中的活性进行了测试。对于这项实验,再次将酶以10%稀释添加到此基质中,并在37℃下孵育130分钟,之后通过在90℃下加热5分钟来停止反应。可乐中测量出约100g/L总糖。因为可乐酸性很强(pH2.6),所以一部分蔗糖转化成葡萄糖和果糖,特别是在加热步骤期间,并且测量的总蔗糖量可能较低,果糖和葡萄糖量较高,然后存在于可乐中。再次,使用Dionex HPLC方法量化蔗糖向不同糖的转化率。结果示出如下,表示为孵育之后样品中检测到的所有糖的总量的百分比。如以下表5所示,使用Sis14和Sis15蔗糖异构酶,可使总糖的40-50%转化成低血糖的糖异麦芽酮糖和海藻酮糖,导致低的血糖指数。如实施例2的缓冲液实验中已经看到,Sis14似乎优先形成异麦芽酮糖,而Sis15优先形成海藻酮糖。Sis2在可乐中未显示任何活性,而Sis10、Sis12和Sis4显示8-15%转化率。
表5:可乐中的糖转化率
葡聚糖蔗糖酶在可乐中均不显示显著活性。
实施例5:
在模拟的胃条件下蔗糖异构酶在巧克力奶中的活性
对蔗糖异构酶和葡聚糖蔗糖酶在巧克力奶中的活性进行了测试。撇去浮沫的巧克力奶(Friesland Campina)具有中性pH(pH6.4)并且含有约100g/L蔗糖。在这项实验中,将100ml巧克力奶在设定为37℃的水浴中在20rpm的搅拌下孵育,并且通过添加HCl逐步降低pH。在实验开始时,添加>250u/mg固体的猪胃粘膜粉末的胃蛋白酶(Sigma;P7000),最终浓度为0.02mg/ml,并且添加0.1%(v/v)蔗糖异构酶。孵育0.5小时之后,将pH设定为4.0,1小时之后,设定为pH 3.0,并且1.5小时之后通过添加HCl将设定为pH 2.0。在5-10分钟(t=0)、1小时(t=1)和2小时(t=2)孵育之后,抽出1ml样品,并且通过加热(99℃,5分钟)使酶活性立即失活。
如上文所述使用HPLC方法分析样品,并且对不同糖进行量化并表述为样品中检测到的所有糖的总量的百分比,并显示在以下表6中。再次,同样如可乐实验中所观察的,样品显示通过在低pH下加热蔗糖向葡萄糖和果糖的(化学)转化(尤其在t=2样品中普遍)。
表6:巧克力奶中的糖转化率
从表6清楚可见,在模拟的胃条件下,尤其是Sis4能够将巧克力奶中总蔗糖的75%转化成低血糖的糖,如异麦芽酮糖和海藻酮糖。Sis10可以特别地将约40%蔗糖转化成异麦芽酮糖,而Sis15产生总计约30%主要海藻酮糖,并且Sis12产生总计约20%两种蔗糖异构体。Sis2和Sis14在这项实验中均未显示出效果。所以,即使在模拟胃消化的条件下酶剂量低得多,这些酶的大多数也导致常规食品产品如巧克力奶的血糖指数降低。
在这项实验中,葡聚糖蔗糖酶在巧克力奶中均不显示显著活性。
实施例6:
在模拟的胃条件下蔗糖异构酶在冰淇淋中的活性
对蔗糖异构酶和葡聚糖蔗糖酶在冰淇淋中的活性进行了测试。冰淇淋(AlbertHeijn Roomijs香草味)具有中性pH(pH6.5)并且含有约230g/kg蔗糖。完全如实施例4中所述进行实验,包括酶剂量、胃蛋白酶添加、pH设置、取样时间和取样量以及在HPLC上的糖分析。
再次,对不同糖进行量化并表述为样品中检测到的总糖量的百分比。糖分析的结果出于下表7中。
表7:冰淇淋中糖的转化率
从表7清楚可见,在模拟的胃条件下,Sis4能够将冰淇淋中总蔗糖的25-35%转化成低血糖的糖,如异麦芽酮糖和海藻酮糖。Sis10可以特别地将约15%蔗糖转化成异麦芽酮糖,而且Sis12和Sis15产生一些蔗糖异构体。同样,Sis2和Sis14在这些条件下没有活性。所以,即使在模拟胃消化的条件下酶剂量低得多,这些酶的大多数也导致常规食品产品如冰淇淋的血糖指数降低。
在这项实验中,葡聚糖蔗糖酶在冰淇淋中均不显示显著活性。
序列表
<110> 帝斯曼知识产权资产管理有限公司(DSM IP Assets B.V.)
<120> 作为食物和营养补充剂的蔗糖异构酶
<130> 33197-EP-EPA
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 559
<212> PRT
<213> 红色精朊杆菌
<400> 1
Met Thr Ile Pro Lys Trp Trp Lys Glu Ala Val Phe Tyr Gln Val Tyr
1 5 10 15
Pro Arg Ser Phe Lys Asp Thr Asn Gly Asp Gly Ile Gly Asp Ile Asn
20 25 30
Gly Ile Ile Glu Lys Leu Asp Tyr Leu Lys Ala Leu Gly Ile Asp Ala
35 40 45
Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly Tyr
50 55 60
Asp Ile Arg Asp Tyr Arg Lys Ile Met Lys Glu Tyr Gly Thr Met Glu
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Asp Phe Asp Arg Leu Ile Ser Glu Met Lys Lys Arg Asn Met Arg Leu
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Met Ile Asp Val Val Ile Asn His Thr Ser Asp Gln Asn Glu Trp Phe
100 105 110
Val Lys Ser Lys Ser Ser Lys Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe
115 120 125
Trp Lys Asp Ala Lys Glu Gly Gln Ala Pro Asn Asn Tyr Pro Ser Phe
130 135 140
Phe Gly Gly Ser Ala Trp Gln Lys Asp Glu Lys Thr Asn Gln Tyr Tyr
145 150 155 160
Leu His Tyr Phe Ala Lys Gln Gln Pro Asp Leu Asn Trp Asp Asn Pro
165 170 175
Lys Val Arg Gln Asp Leu Tyr Ala Met Leu Arg Phe Trp Leu Asp Lys
180 185 190
Gly Val Ser Gly Leu Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys Ile
195 200 205
Pro Asp Phe Pro Asn Leu Thr Gln Gln Gln Leu Lys Asn Phe Ala Ala
210 215 220
Glu Tyr Thr Lys Gly Pro Asn Ile His Arg Tyr Val Asn Glu Met Asn
225 230 235 240
Lys Glu Val Leu Ser His Tyr Asp Ile Ala Thr Ala Gly Glu Ile Phe
245 250 255
Gly Val Pro Leu Asp Gln Ser Ile Lys Phe Phe Asp Arg Arg Arg Asp
260 265 270
Glu Leu Asn Ile Ala Phe Thr Phe Asp Leu Ile Arg Leu Asp Arg Asp
275 280 285
Ser Asp Gln Arg Trp Arg Arg Lys Asp Trp Lys Leu Ser Gln Phe Arg
290 295 300
Gln Ile Ile Asp Asn Val Asp Arg Thr Ala Gly Glu Tyr Gly Trp Asn
305 310 315 320
Ala Phe Phe Leu Asp Asn His Asp Asn Pro Arg Ala Val Ser His Phe
325 330 335
Gly Asp Asp Arg Pro Gln Trp Arg Glu Pro Ser Ala Lys Ala Leu Ala
340 345 350
Thr Leu Thr Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly Ser
355 360 365
Glu Leu Gly Met Thr Asn Tyr Pro Phe Lys Ala Ile Asp Glu Phe Asp
370 375 380
Asp Ile Glu Val Lys Gly Phe Trp His Asp Tyr Val Glu Thr Gly Lys
385 390 395 400
Val Lys Ala Asp Glu Phe Leu Gln Asn Val Arg Leu Thr Ser Arg Asp
405 410 415
Asn Ser Arg Thr Pro Phe Gln Trp Asp Gly Ser Lys Asn Ala Gly Phe
420 425 430
Thr Ser Gly Lys Pro Trp Phe Lys Val Asn Pro Asn Tyr Gln Glu Ile
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Asn Ala Val Ser Gln Val Thr Gln Pro Asp Ser Val Phe Asn Tyr Tyr
450 455 460
Arg Gln Leu Ile Lys Ile Arg His Asp Ile Pro Ala Leu Thr Tyr Gly
465 470 475 480
Thr Tyr Thr Asp Leu Asp Pro Ala Asn Asp Ser Val Tyr Ala Tyr Thr
485 490 495
Arg Ser Leu Gly Ala Glu Lys Tyr Leu Val Val Val Asn Phe Lys Glu
500 505 510
Gln Met Met Arg Tyr Lys Leu Pro Asp Asn Leu Ser Ile Glu Lys Val
515 520 525
Ile Ile Asp Ser Asn Ser Lys Asn Val Val Lys Lys Asn Asp Ser Leu
530 535 540
Leu Glu Leu Lys Pro Trp Gln Ser Gly Val Tyr Lys Leu Asn Gln
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<210> 2
<211> 578
<212> PRT
<213> 分散泛菌
<400> 2
Met Ala Ser Pro Leu Thr Lys Pro Ser Thr Pro Ile Ala Ala Thr Asn
1 5 10 15
Ile Gln Lys Ser Ala Asp Phe Pro Ile Trp Trp Lys Gln Ala Val Phe
20 25 30
Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Ser Asn Gly Asp Gly Ile
35 40 45
Gly Asp Ile Pro Gly Ile Ile Glu Lys Leu Asp Tyr Leu Lys Met Leu
50 55 60
Gly Val Asp Ala Ile Trp Ile Asn Pro His Tyr Glu Ser Pro Asn Thr
65 70 75 80
Asp Asn Gly Tyr Asp Ile Ser Asp Tyr Arg Lys Ile Met Lys Glu Tyr
85 90 95
Gly Ser Met Ala Asp Phe Asp Arg Leu Val Ala Glu Met Asn Lys Arg
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Gly Met Arg Leu Met Ile Asp Ile Val Ile Asn His Thr Ser Asp Arg
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His Arg Trp Phe Val Gln Ser Arg Ser Gly Lys Asp Asn Pro Tyr Arg
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Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Gln Gly Gln Ala Pro Asn Asn
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Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln Leu Asp Lys Gln Thr
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Asp Gln Tyr Tyr Leu His Tyr Phe Ala Pro Gln Gln Pro Asp Leu Asn
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Trp Asp Asn Pro Lys Val Arg Ala Glu Leu Tyr Asp Ile Leu Arg Phe
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Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe Asp Thr Val Ala Thr
210 215 220
Phe Ser Lys Ile Pro Gly Phe Pro Asp Leu Ser Lys Ala Gln Leu Lys
225 230 235 240
Asn Phe Ala Glu Ala Tyr Thr Glu Gly Pro Asn Ile His Lys Tyr Ile
245 250 255
His Glu Met Asn Arg Gln Val Leu Ser Lys Tyr Asn Val Ala Thr Ala
260 265 270
Gly Glu Ile Phe Gly Val Pro Val Ser Ala Met Pro Asp Tyr Phe Asp
275 280 285
Arg Arg Arg Glu Glu Leu Asn Ile Ala Phe Thr Phe Asp Leu Ile Arg
290 295 300
Leu Asp Arg Tyr Pro Asp Gln Arg Trp Arg Arg Lys Pro Trp Thr Leu
305 310 315 320
Ser Gln Phe Arg Gln Val Ile Ser Gln Thr Asp Arg Ala Ala Gly Glu
325 330 335
Phe Gly Trp Asn Ala Phe Phe Leu Asp Asn His Asp Asn Pro Arg Gln
340 345 350
Val Ser His Phe Gly Asp Asp Ser Pro Gln Trp Arg Glu Arg Ser Ala
355 360 365
Lys Ala Leu Ala Thr Leu Leu Leu Thr Gln Arg Ala Thr Pro Phe Ile
370 375 380
Phe Gln Gly Ala Glu Leu Gly Met Thr Asn Tyr Pro Phe Lys Asn Ile
385 390 395 400
Glu Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp Asn Asp Tyr Val
405 410 415
Ala Ser Gly Lys Val Asn Ala Ala Glu Phe Leu Gln Glu Val Arg Met
420 425 430
Thr Ser Arg Asp Asn Ser Arg Thr Pro Met Gln Trp Asn Asp Ser Val
435 440 445
Asn Ala Gly Phe Thr Gln Gly Lys Pro Trp Phe His Leu Asn Pro Asn
450 455 460
Tyr Lys Gln Ile Asn Ala Ala Arg Glu Val Asn Lys Pro Asp Ser Val
465 470 475 480
Phe Ser Tyr Tyr Arg Gln Leu Ile Asn Leu Arg His Gln Ile Pro Ala
485 490 495
Leu Thr Ser Gly Glu Tyr Arg Asp Leu Asp Pro Gln Asn Asn Gln Val
500 505 510
Tyr Ala Tyr Thr Arg Ile Leu Asp Asn Glu Lys Tyr Leu Val Val Val
515 520 525
Asn Phe Lys Pro Glu Gln Leu His Tyr Ala Leu Pro Asp Asn Leu Thr
530 535 540
Ile Ala Ser Ser Leu Leu Glu Asn Val His Gln Pro Ser Leu Gln Glu
545 550 555 560
Asn Ala Ser Thr Leu Thr Leu Ala Pro Trp Gln Ala Gly Ile Tyr Lys
565 570 575
Leu Asn
<210> 3
<211> 571
<212> PRT
<213> 植生拉乌尔菌
<400> 3
Met Ala Pro Ser Val Asn Gln Asn Ile His Val His Lys Glu Ser Glu
1 5 10 15
Tyr Pro Ala Trp Trp Lys Glu Ala Val Phe Tyr Gln Ile Tyr Pro Arg
20 25 30
Ser Phe Lys Asp Thr Asn Asp Asp Gly Ile Gly Asp Ile Arg Gly Ile
35 40 45
Ile Glu Lys Leu Asp Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp
50 55 60
Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile
65 70 75 80
Ser Asn Tyr Arg Gln Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe
85 90 95
Asp Asn Leu Val Ala Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile
100 105 110
Asp Val Val Ile Asn His Thr Ser Asp Gln His Pro Trp Phe Ile Gln
115 120 125
Ser Lys Ser Asp Lys Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg
130 135 140
Asp Gly Lys Asp Asn Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly
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Gly Ser Ala Trp Gln Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His
165 170 175
Tyr Phe Ala Arg Gln Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val
180 185 190
Arg Glu Asp Leu Tyr Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val
195 200 205
Ser Ser Met Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly
210 215 220
Phe Pro Asn Leu Thr Pro Glu Gln Gln Lys Asn Phe Ala Glu Gln Tyr
225 230 235 240
Thr Met Gly Pro Asn Ile His Arg Tyr Ile Gln Glu Met Asn Arg Lys
245 250 255
Val Leu Ser Arg Tyr Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val
260 265 270
Pro Leu Asp Arg Ser Ser Gln Phe Phe Asp Pro Arg Arg His Glu Leu
275 280 285
Asn Met Ala Phe Met Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn
290 295 300
Glu Arg Trp Arg His Lys Ser Trp Ser Leu Ser Gln Phe Arg Gln Ile
305 310 315 320
Ile Ser Lys Met Asp Val Thr Val Gly Lys Tyr Gly Trp Asn Thr Phe
325 330 335
Phe Leu Asp Asn His Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp
340 345 350
Asp Arg Pro Gln Trp Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Ile
355 360 365
Thr Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu
370 375 380
Gly Met Thr Asn Tyr Pro Phe Arg Gln Leu Asn Glu Phe Asp Asp Ile
385 390 395 400
Glu Val Lys Gly Phe Trp Gln Asp Tyr Val Gln Ser Gly Lys Val Thr
405 410 415
Ala Thr Glu Phe Leu Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser
420 425 430
Arg Thr Pro Phe Gln Trp Asn Asp Thr Leu Asn Ala Gly Phe Thr Arg
435 440 445
Gly Lys Pro Trp Phe His Ile Asn Pro Asn Tyr Val Glu Ile Asn Ala
450 455 460
Glu Arg Glu Glu Thr Arg Glu Asp Ser Val Leu Asn Tyr Tyr Lys Lys
465 470 475 480
Met Ile Gln Leu Arg His His Ile Pro Ala Leu Val Tyr Gly Ala Tyr
485 490 495
Gln Asp Leu Asn Pro Gln Asp Asn Thr Val Tyr Ala Tyr Thr Arg Thr
500 505 510
Leu Gly Asn Glu Arg Tyr Leu Val Val Val Asn Phe Lys Glu Tyr Pro
515 520 525
Val Arg Tyr Thr Leu Pro Ala Asn Asp Ala Ile Glu Glu Val Val Ile
530 535 540
Asp Thr Gln Gln Gln Ala Thr Ala Pro His Ser Thr Ser Leu Ser Leu
545 550 555 560
Ser Pro Trp Gln Ala Gly Val Tyr Lys Leu Arg
565 570
<210> 4
<211> 562
<212> PRT
<213> 中嗜酸性假单胞菌
<400> 4
Met Glu Glu Ala Val Lys Pro Gly Ala Pro Trp Trp Lys Ser Ala Val
1 5 10 15
Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr Asn Gly Asp Gly
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Ile Gly Asp Phe Lys Gly Leu Thr Glu Lys Leu Asp Tyr Leu Lys Gly
35 40 45
Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr Ala Ser Pro Asn
50 55 60
Thr Asp Asn Gly Tyr Asp Ile Ser Asp Tyr Arg Glu Val Met Lys Glu
65 70 75 80
Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Met Ala Glu Leu Lys Lys
85 90 95
Arg Gly Met Arg Leu Met Val Asp Val Val Ile Asn His Ser Ser Asp
100 105 110
Gln His Glu Trp Phe Lys Ser Ser Arg Ala Ser Lys Asp Asn Pro Tyr
115 120 125
Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Gly His Glu Pro Asn
130 135 140
Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glu Lys Asp Pro Val
145 150 155 160
Thr Gly Gln Tyr Tyr Leu His Tyr Phe Gly Arg Gln Gln Pro Asp Leu
165 170 175
Asn Trp Asp Thr Pro Lys Leu Arg Glu Glu Leu Tyr Ala Met Leu Arg
180 185 190
Phe Trp Leu Asp Lys Gly Val Ser Gly Met Arg Phe Asp Thr Val Ala
195 200 205
Thr Tyr Ser Lys Thr Pro Gly Phe Pro Asp Leu Thr Pro Glu Gln Met
210 215 220
Lys Asn Phe Ala Glu Ala Tyr Thr Gln Gly Pro Asn Leu His Arg Tyr
225 230 235 240
Leu Gln Glu Met His Glu Lys Val Phe Asp His Tyr Asp Ala Val Thr
245 250 255
Ala Gly Glu Ile Phe Gly Ala Pro Leu Asn Gln Val Pro Leu Phe Ile
260 265 270
Asp Ser Arg Arg Lys Glu Leu Asp Met Ala Phe Thr Phe Asp Leu Ile
275 280 285
Arg Tyr Asp Arg Ala Leu Asp Arg Trp His Thr Ile Pro Arg Thr Leu
290 295 300
Ala Asp Phe Arg Gln Thr Ile Asp Lys Val Asp Ala Ile Ala Gly Glu
305 310 315 320
Tyr Gly Trp Asn Thr Phe Phe Leu Gly Asn His Asp Asn Pro Arg Ala
325 330 335
Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp Arg Glu Ala Ser Ala
340 345 350
Lys Ala Leu Ala Thr Val Thr Leu Thr Gln Arg Gly Thr Pro Phe Ile
355 360 365
Phe Gln Gly Asp Glu Leu Gly Met Thr Asn Tyr Pro Phe Lys Thr Leu
370 375 380
Gln Asp Phe Asp Asp Ile Glu Val Lys Gly Phe Phe Gln Asp Tyr Val
385 390 395 400
Glu Thr Gly Lys Ala Thr Ala Glu Glu Leu Leu Thr Asn Val Ala Leu
405 410 415
Thr Ser Arg Asp Asn Ala Arg Thr Pro Phe Gln Trp Asp Asp Ser Ala
420 425 430
Asn Ala Gly Phe Thr Thr Gly Lys Pro Trp Leu Lys Val Asn Pro Asn
435 440 445
Tyr Thr Glu Ile Asn Ala Ala Arg Glu Ile Gly Asp Pro Lys Ser Val
450 455 460
Tyr Ser Phe Tyr Arg Asn Leu Ile Ser Ile Arg His Glu Thr Pro Ala
465 470 475 480
Leu Ser Thr Gly Ser Tyr Arg Asp Ile Asp Pro Ser Asn Ala Asp Val
485 490 495
Tyr Ala Tyr Thr Arg Ser Gln Asp Gly Glu Thr Tyr Leu Val Val Val
500 505 510
Asn Phe Lys Ala Glu Pro Arg Ser Phe Thr Leu Pro Asp Gly Met His
515 520 525
Ile Ala Glu Thr Leu Ile Glu Ser Ser Ser Pro Ala Ala Pro Ala Ala
530 535 540
Gly Ala Ala Ser Leu Glu Leu Gln Pro Trp Gln Ser Gly Ile Tyr Lys
545 550 555 560
Val Lys
<210> 5
<211> 576
<212> PRT
<213> 肠杆菌属种
<400> 5
Met Ala Tyr Ser Ala Glu Thr Ser Val Thr Gln Ser Ile Gln Thr Gln
1 5 10 15
Lys Glu Ser Thr Leu Pro Ala Trp Trp Lys Glu Ala Val Phe Tyr Gln
20 25 30
Ile Tyr Pro Arg Ser Phe Lys Asp Thr Asn Gly Asp Gly Ile Gly Asp
35 40 45
Ile Arg Gly Ile Ile Glu Lys Leu Asp Tyr Leu Lys Ser Leu Gly Ile
50 55 60
Asp Ala Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn
65 70 75 80
Gly Tyr Asp Ile Arg Asp Tyr Glu Lys Ile Met Gln Glu Tyr Gly Thr
85 90 95
Met Glu Asp Phe Asp Thr Leu Val Ser Glu Met Lys Lys Arg Asn Met
100 105 110
Arg Leu Met Ile Asp Val Val Ile Asn His Thr Ser Asp Gln His Pro
115 120 125
Trp Phe Ile Gln Ser Lys Ser Ser Lys Glu Asn Pro Tyr Arg Glu Tyr
130 135 140
Tyr Phe Trp Arg Asp Gly Lys Asp Asn Gln Pro Pro Asn Asn Tyr Pro
145 150 155 160
Ser Phe Phe Gly Gly Ser Ala Trp Gln Lys Asp Asp Lys Thr Gly Gln
165 170 175
Tyr Tyr Leu His Tyr Phe Ala Arg Gln Gln Pro Asp Leu Asn Trp Asp
180 185 190
Asn Pro Lys Val Arg Gly Asp Leu Tyr Ala Met Leu Arg Phe Trp Leu
195 200 205
Asp Lys Gly Val Ser Gly Met Arg Phe Asp Thr Val Ala Thr Tyr Ser
210 215 220
Lys Ile Pro Gly Phe Pro Asp Leu Thr Pro Glu Gln Gln Lys Asn Phe
225 230 235 240
Ala Glu Gln Tyr Thr Thr Gly Pro Asn Ile His Arg Tyr Leu Gln Glu
245 250 255
Met Lys Gln Glu Val Leu Ser Arg Tyr Asp Val Val Thr Ala Gly Glu
260 265 270
Ile Phe Gly Val Pro Leu Glu Arg Ser Ser Asp Phe Phe Asp Arg Arg
275 280 285
Arg Asn Glu Leu Asp Met Ser Phe Met Phe Asp Leu Ile Arg Leu Asp
290 295 300
Arg Asp Ser Asn Glu Arg Trp Arg His Lys Lys Trp Thr Leu Ser Gln
305 310 315 320
Phe Arg Gln Ile Ile Asn Lys Met Asp Ser Asn Ala Gly Glu Tyr Gly
325 330 335
Trp Asn Thr Phe Phe Leu Asp Asn His Asp Asn Pro Arg Ala Val Ser
340 345 350
His Phe Gly Asp Asp Ser Pro Gln Trp Ile Glu Pro Ser Ala Lys Ala
355 360 365
Leu Ala Thr Ile Ile Leu Thr Gln Arg Ala Thr Pro Phe Ile Phe Gln
370 375 380
Gly Ser Glu Leu Gly Met Thr Asn Tyr Pro Phe Lys Lys Leu Asn Glu
385 390 395 400
Phe Asp Asp Ile Glu Val Lys Gly Phe Trp Gln Asp Tyr Val Gln Thr
405 410 415
Gly Lys Val Ser Ala Glu Glu Phe Ile Asp Asn Val Arg Leu Thr Ser
420 425 430
Arg Asp Asn Ser Arg Thr Pro Phe Gln Trp Asn Asp Arg Lys Asn Ala
435 440 445
Gly Phe Thr Ser Gly Lys Pro Trp Phe Arg Ile Asn Pro Asn Tyr Val
450 455 460
Glu Ile Asn Ala Asp Lys Glu Leu Ile Arg Asn Asp Ser Val Leu Asn
465 470 475 480
Tyr Tyr Lys Glu Met Ile Lys Leu Arg His Lys Thr Pro Ala Leu Ile
485 490 495
Tyr Gly Thr Tyr Lys Asp Ile Ser Pro Glu Asp Asp Ser Val Tyr Ala
500 505 510
Tyr Thr Arg Thr Leu Gly Lys Glu Arg Tyr Leu Val Val Ile Asn Phe
515 520 525
Thr Glu Lys Thr Val Arg Tyr Pro Leu Pro Glu Asn Asn Val Ile Lys
530 535 540
Ser Ile Leu Ile Glu Ala Asn Gln Asn Lys Thr Ala Glu Lys Gln Ser
545 550 555 560
Thr Val Leu Thr Leu Ser Pro Trp Gln Ala Gly Val Tyr Glu Leu Gln
565 570 575
<210> 6
<211> 578
<212> PRT
<213> 胡萝卜果胶杆菌
<400> 6
Met Ala Thr Asn His Asn Glu Gln Asp Thr Lys Thr Val Ile Ala Val
1 5 10 15
Asn Asp Gly Val Ser Ala His Pro Val Trp Trp Lys Glu Ala Val Phe
20 25 30
Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Ser Asn Gly Asp Gly Ile
35 40 45
Gly Asp Leu Lys Gly Leu Thr Glu Lys Leu Asp Tyr Leu Lys Thr Leu
50 55 60
Gly Ile Asn Ala Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr
65 70 75 80
Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys Ile Met Lys Glu Tyr
85 90 95
Gly Thr Met Asp Asp Phe Asp Asn Leu Ile Ala Glu Met Lys Lys Arg
100 105 110
Asp Met Arg Leu Met Ile Asp Val Val Val Asn His Thr Ser Asn Glu
115 120 125
His Lys Trp Phe Val Glu Ser Lys Lys Ser Lys Asp Asn Pro Tyr Arg
130 135 140
Asp Tyr Tyr Ile Trp Arg Asp Gly Lys Asp Gly Thr Pro Pro Asn Asn
145 150 155 160
Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln Lys Asp Asn Val Thr
165 170 175
Gln Gln Tyr Tyr Leu His Tyr Phe Gly Val Gln Gln Pro Asp Leu Asn
180 185 190
Trp Asp Asn Pro Lys Val Arg Glu Glu Val Tyr Asp Met Leu Arg Phe
195 200 205
Trp Ile Asp Lys Gly Val Ser Gly Leu Arg Met Asp Thr Val Ala Thr
210 215 220
Phe Ser Lys Asn Pro Ala Phe Pro Asp Leu Thr Pro Glu Gln Leu Lys
225 230 235 240
Asn Phe Ala Tyr Thr Tyr Thr Gln Gly Pro Asn Leu His Arg Tyr Ile
245 250 255
Gln Glu Met His Gln Lys Val Leu Ala Lys Tyr Asp Val Val Ser Ala
260 265 270
Gly Glu Ile Phe Gly Val Pro Leu Glu Glu Ala Ala Pro Phe Ile Asp
275 280 285
Gln Arg Arg Lys Glu Leu Asp Met Ala Phe Ser Phe Asp Leu Ile Arg
290 295 300
Leu Asp Arg Ala Val Glu Glu Arg Trp Arg Arg Asn Asp Trp Thr Leu
305 310 315 320
Ser Gln Phe Arg Gln Ile Asn Asn Arg Leu Val Asp Met Ala Gly Gln
325 330 335
Tyr Gly Trp Asn Thr Phe Phe Leu Ser Asn His Asp Asn Pro Arg Ala
340 345 350
Val Ser His Phe Gly Asp Asp Arg Pro Glu Trp Arg Ile Arg Ser Ala
355 360 365
Lys Ala Leu Ala Thr Leu Ala Leu Thr Gln Arg Ala Thr Pro Phe Ile
370 375 380
Tyr Gln Gly Asp Glu Leu Gly Met Thr Asn Tyr Pro Phe Thr Ser Leu
385 390 395 400
Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp Gln Asp Phe Val
405 410 415
Glu Thr Gly Lys Val Lys Pro Asp Val Phe Leu Glu Asn Val Lys Gln
420 425 430
Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln Trp Ser Asn Ala Glu
435 440 445
Gln Ala Gly Phe Thr Thr Gly Thr Pro Trp Phe Arg Ile Asn Pro Asn
450 455 460
Tyr Lys Asn Ile Asn Ala Glu Asp Gln Thr Gln Asn Pro Asp Ser Ile
465 470 475 480
Phe His Phe Tyr Arg Gln Leu Ile Ala Leu Arg His Ala Thr Pro Ala
485 490 495
Phe Thr Tyr Gly Ala Tyr Gln Asp Leu Asp Pro Asn Asn Asn Glu Val
500 505 510
Leu Ala Tyr Thr Arg Glu Leu Asn Gln Gln Arg Tyr Leu Val Val Val
515 520 525
Asn Phe Lys Glu Lys Pro Val His Tyr Ala Leu Pro Lys Thr Leu Ser
530 535 540
Ile Lys Gln Thr Leu Leu Glu Ser Gly Gln Lys Asp Lys Val Ala Pro
545 550 555 560
Asn Ala Thr Ser Leu Glu Leu Gln Pro Trp Gln Ser Gly Ile Tyr Gln
565 570 575
Leu Asn
Claims (10)
1.一种营养保健、药物或营养补充剂组合物,其包含蔗糖异构酶。
2.根据权利要求1所述的组合物,其适用于人。
3.根据权利要求2所述的组合物,其适用于伴侣动物。
4.根据权利要求1所述的组合物,其包含与序列SEQ ID NO:1-6中任一个具有至少60%同一性,优选地90%、95%、98%、99%、100%同一性的蔗糖异构酶。
5.根据权利要求4所述的组合物,其包含与序列SEQ ID NO:4具有至少60%同一性,优选地90%、95%、98%、99%、100%同一性的蔗糖异构酶。
6.一种降低摄取蔗糖的动物包括人的血糖水平的增加的方法,其包括向有需要的所述动物包括人施用如权利要求1所述的营养保健品、药物或营养补充剂。
7.一种降低人或伴侣动物食用的食物或饲料的血糖指数的方法,其包括向所述人或伴侣动物施用如权利要求1所述的包含蔗糖异构酶的营养保健品、药物或营养补充剂。
8.一种减轻人或伴侣动物的体重或维持其体重减轻的方法,其包括向所述人或伴侣动物施用如权利要求1所述的包含蔗糖异构酶的营养保健品、药物或营养补充剂。
9.包含蔗糖异构酶的营养保健品、药物或营养补充剂用于实现动物包括人的以下状况的用途,所述状况选自由以下组成的组:
a)降低血糖水平的增加;
b)进行耐力运动的动物的耐力增加;
c)体重减轻或维持体重减轻
d)降低摄取的食物或饲料的血糖指数;以及
e)在含有蔗糖的餐后持续能量释放和/或防止或最小化快速的血糖升高和所谓的餐后“情绪消沉”。
10.一种干燥食品或饮料混合物,其包含蔗糖异构酶。
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PCT/EP2019/072310 WO2020038966A1 (en) | 2018-08-22 | 2019-08-21 | Sucrose isomerases as food and nutritional supplements |
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BR112021002996A2 (pt) | 2021-05-11 |
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