CN101437414B - 抗微生物制剂 - Google Patents
抗微生物制剂 Download PDFInfo
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- CN101437414B CN101437414B CN2007800112221A CN200780011222A CN101437414B CN 101437414 B CN101437414 B CN 101437414B CN 2007800112221 A CN2007800112221 A CN 2007800112221A CN 200780011222 A CN200780011222 A CN 200780011222A CN 101437414 B CN101437414 B CN 101437414B
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- alanine
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- amino acid
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Abstract
本发明涉及一种抑制革兰氏阴性细菌的食品保藏方法，该方法包括在食品中添加含有甘氨酸和丙氨酸的抗微生物制剂。本发明还涉及所述抗微生物制剂在控制人体和动物体肠道菌群中的应用。
Description
发明领域
本发明涉及一种抗微生物制剂以及一种保藏食品的方法。特别地，本发明涉及采用包括至少两种氨基酸的组合物保藏食品的方法。本发明还涉及该制剂在控制人体和动物体肠道菌群中的应用。
发明背景
与食用有传染性的和可产生毒素的微生物所污染的食品相关的发病率和死亡率不应该被低估。这正是食品制造商、消费者和政府机构经常关注食品中微生物的性质和安全的原因。此外，因微生物而起的腐败和腐烂会危及食物的营养价值并会导致重大的经济损失。食品微生物腐败是由不受控制的微生物增殖或活性而引起的。为防止这种现象在保藏中出现，确保食品质量和微生物安全的技术已经被研发出来。这些技术多种多样，包括(i)防止微生物接触食品的步骤；(ii)使微生物失活的步骤；以及(iii)防止或减缓微生物生长的步骤。消费者需求和食品法规的变化促使食品科技人员经常改进和完善现有的食品加工技术以及创新和发展新技术。食品制造的发展趋势并不仅在于外观吸引人、新鲜和更天然，而是与使用方便性相结合，例如较长贮藏期和制备简易性。此外，消费者也对风味、结构、外观和安全提出了高标准。实现全部上述要求对食品科技工作者而言是个巨大的挑战。为了在食品中减缓或防止微生物生长，新型和更天然的化学抗微生物物质的新应用已在稳步进行。然而，这些新型的化学防腐剂应该能满足若干要求：(i)它们应该对广范围的不同腐败菌和食源性致病菌具有有效的杀菌或抑菌活性；(ii)它们不应该影响食品的外观、口感、风味和结构；以及(iii)它们不应该对消费者有毒。众所周知，某些氨基酸具有杀菌或抑菌性能(例如shive，W.；C.G.Skinner(1963)氨基酸类似物.代谢性抑制剂。综合性专著(Hochster，R.M.；J.H.(Quastel eds).学术出版社.纽约，I卷，1-73页)。
此外，许多氨基酸在食品中天然存在，其逐渐被认为是安全的并被批准用于食品中。正因为如此，一些氨基酸尤其是甘氨酸作为防腐剂的商业应用已经被发现。
用丝氨酸保存食物的方法已经在US2711976和US6602532中公开。在US3615703中，食品或包含水果或果汁，蔬菜或蔬菜汁的饮料，或者啤酒的风味的保存是把食品或饮料密封在含有赖氨酸、鸟氨酸、组氨酸或它们的盐类的容器中。GB1510942公开了一种保存食品的方法，尤其是含有10-60％糖的食品，例如果冻、果酱和牛奶蛋糊，该方法通过在食品中掺入麦芽糖和甘氨酸以保护其不会腐败。甘氨酸的适合用量为0.3至2％。尽管甘氨酸目前广泛用作商业防腐剂，但该化合物也被认为不是强效的防腐剂并需要相对高的浓度以抑制细菌生长。然而，高浓度产生了一系列新问题。众所周知，甘氨酸、丙氨酸、丝氨酸和苏氨酸均有不同程度的甜味，赖氨酸和鸟氨酸都带有苦和甜的气味，同时精氨酸是强烈的苦味。氨基酸在味觉上的影响限制了这些化合物作为食品防腐剂的应用。
为了使甘氨酸作为食品防腐剂能得到更有效的应用，因此甘氨酸需要与其他物质结合使用以利于提高其抗微生物功效。Lee等人研究发现甘氨酸联合六偏磷酸盐、EDTA、胆酸和单癸酸丙三醇对一些革兰氏阴性菌和阳性菌群具有抑制作用(Lee，J.K.；K.Tatsuguchi；M.Tsutsumi；T.Watanabe，食品卫生期刊.日本26：279-284(1985))。WO01/56408公开了丙氨酸或甘氨酸与1，5-D-脱水果糖共同用作食品保持剂，该保持剂包括高度安全的抗微生物物质，并且因此改善食品的保藏质量，而不会对食品口感、风味产生不良影响。
尽管由单一氨基酸与一种或多种其他非氨基酸化合物组成的抗微生物混合物的方法(见上文)已经被公开，但是有关这些氨基酸混合物的抗微生物性能却了解甚少。
JP2000-224976公开了一种抑制微生物的方法，这些微生物可为在pH值≥6.5的食品中存在的乳酸肠球菌属，而使用乳酸钙、甘氨酸和其他有机酸盐类的混合物几乎不会破坏食品的质量。尽管在该申请中还公开了甘氨酸部分可以被丙氨酸替代，但是数据显示丙氨酸以及丙氨酸和甘氨酸的混合物对***乳酸肠球菌属的抑制效果差于单独使用甘氨酸。丙氨酸能够削弱甘氨酸的抑制效果不仅仅体现在与革兰氏阳性菌紧密相关的氏肠球菌、乳酸乳球菌上，还体现在革兰氏阴性菌大肠杆菌上(Snell，E.E.；B.M.Guirard(1943)美国国家 科学院院刊：29：66-73；Hishinuma，F.；K.Izaki；H.Takahashi(1969)甘氨酸和D-氨基酸对各类微生物生长的影响，农业生物化学.33：1577-1586)。众所周知，一种氨基酸可以消除另一种氨基酸引起的抑制作用，并且已经观察到这种现象存在于一些革兰氏阳性菌和革兰氏阴性菌种群中，例如在革兰氏阳性菌的炭疽芽孢杆菌和单核细胞增多性李斯特菌；以及革兰氏阴性菌的大肠杆菌中(Gladstone，G.P.(1939)Brit.J.Exp.Pathol.20：189-200；Friedman，M.E.；W.G.Roessler(1961)J.Bacteriol.82：528-533；de Felice等.(1979)Microbiol.Rev.43：42-58)。
在US2001/033884中，一种包含甘氨酸和丝氨酸的制剂被用于保存食品以抑制革兰氏阳性菌和阴性菌。
J-53050361的摘要也公开了包含甘氨酸和丝氨酸的组合物，该组合物能调节食品pH值。
US2001/039264和US2002/144946描述了包含了至少5种或更多种类氨基酸的其他组合物，该组合物分别用于别的用途例如与剧烈运动或减缓疲劳相关的氨基酸血浓度降低以及用于血液透析。
目前已经发现甘氨酸和丙氨酸的结合令人惊奇的产生了抑制革兰氏阴性菌的协同作用。
发明内容
因此，本发明涉及一种直接抑制革兰氏阴性菌的包含至少甘氨酸和丙氨酸的抗微生物制剂。
因此包含甘氨酸和丙氨酸混合物的所述抗微生物尤其适合于抑制大肠杆菌O157:H7和沙门氏菌群的生长。
要强调的是这两种氨基酸的组合还能与第三种氨基酸进行结合。已经发现这些结合物的抑菌效果大于基于从其他氨基酸中分离出来的一种氨基酸的预想效果，即显示了协同效果。
优选的丙氨酸是D-丙氨酸。最优选的，抗微生物包含DL-丙氨酸。如果还有其他氨基酸存在，最优选的是DL-丙氨酸。
另一方面，本发明涉及一种抑制细菌保存食品的方法，该方法包括在食品中添加上述提及的含有甘氨酸和丙氨酸的抗微生物。
根据本发明的方法尤其适用于保存食品，以使其对肠道沙门氏菌和大肠杆菌O157:H7的至少一种起抑制作用。
这些食品的例子是肉制品(腌制和/或未腌制，新鲜和/或煮制)，色拉和其他蔬菜制品，饮料和乳制品，半加工食品，方便食品如即食肉类和干制食品。这个方法对于那些没有经过任何热处理或经过不足以杀灭细菌的热处理的新鲜肉制品直接用于食用(例如美国鱼片(filet americain)、鞑靼牛排、寿司或生牛肉片)特别有意义。其他肉制品仅经过了局部的热处理后就被食用，有意的应用例如半熟牛排，或由于对食品的不当制备或不当处理而无意的应用。
本发明的抗微生物包括含有甘氨酸/丙氨酸的制剂，其还可被用于其他用途，例如通过抑制革兰氏阴性菌如沙门氏和大肠菌群为革兰氏阳性菌如乳酸菌提供选择有利性来控制肠道细菌；例如与益生菌共同施用。
以下培养物会在研究中使用：大肠杆菌血清型O157:H7(ATCC 700728)，肠道沙门氏菌(ATCC 13311)。所有培养物每天被转移到含有10ml脑心浸液肉汤(Oxoid，Basingstoke，英国)的螺旋盖管中(100×16mm)。培养物在没有搅拌、30℃的条件下进行培养。脑心浸液肉汤的使用提高了两种氨基酸的数量。在10 50mM阶段氨基酸浓度范围从0到450mM。这是在100个不同的培养基中得到的结果。培养基pH调整为6.1-6.2。培养基被制备成10ml的量并且过滤除菌(0.45μm孔径的Sartorius硝酸纤维素膜)。300μl的每种培养基被转移到100孔Bioscreen无菌蜂窝孔板上。在孔板中，用无菌Hamilton 5μl重复分液器(Hamilton，Bonaduz，瑞士)接种5μl在脑心浸液肉汤中生长过夜的培养物。生长速度用Bioscreen C(Labsystems，赫尔辛基，芬兰)进行测定，即用立式分光光度计测定浊度的动力学变化。孔板在37℃培养16-24小时，培养物的光密度采用宽带滤波器在420-580nm每30分钟测量一次。Bioscreen测定了固定时间间隔下的培养物光密度。根据这些数据，Bioscreen计算出了最大比生长速度。
数据进一步加工的目的是为了确定两种氨基酸是各自起独立作用或是在抑制作用中相互刺激(协同)或者削弱彼此的抑制效果(拮抗)。当某一确定化合物对生物没有影响时，该生物的比生长速度(μ)可以被表达为生长限制底物浓度(s)的函数(f)，例如以μ＝μmax·s/(KS+s)表示的莫诺方程式，其中μmax表示最大比生长速度，s代表培养基中的生长限制底物的恒定浓度以及Ks代表当μ＝0.5μmax时的底物浓度。然而，当抑制剂P的存在影响了细胞生长时，μ的函数f必定变化，即μ＝f(s，p)，其中p代表抑制剂P的浓度。细菌生长抑制动力学的大量研究表明，许多抑制剂表现为非竞争性抑制剂。这意味着只有最大比生长速度(μmax)的值受到影响，而亲和力(Ks)不受影响。因此，在抑制剂存在时，比生长速度可被写作：μ＝μi·s/(Ks+s)，其中的μi是抑制剂P存在时的最大比生长速度。μi和μmax以及抑制剂P浓度之间的关系用Logistic剂量反应方程表示：μi/μmax＝1/(1+(p/p0.5)b)(Jungbauer，A.(2001)。Logistic剂量反应函数：抗差拟合函数表示生命科学的转化观象。J.ClinicalLigand Assay 24：270-274)。在该方程式中，p代表抑制剂P的浓度，p0.5表示当μ1＝0.5μmax时P的浓度；μmax是最大比生长速度也就是在没有抑制剂P存在时的比生长速度，b是无量纲，其决定μi和p的关系。结合莫诺方程式，Logistic剂量反应方程可表示为：μ＝μmax(s/(Ks+s)/(1+(p/p0.5)b)。在分批培养中，其中s经常高于Ks许多倍，该方程式简化为μ＝μmax/(1+(p/p0.5)b)。
当比较在同样条件下生长的不同微生物，或者在不同条件下生长的同类微生物时，使用相对生长速度而不是绝对生长速度作为比较基准将会更有意义。相对生长速度(O)是生长速度(μ)和最大生长速度(μmax)的比值，即O＝μ/μmax。由此可以看出由于μ和μmax是(时间)-1的维数，它们的比值O为无量纲，即一个单纯的数值。类似地，我们可以定义相对抑制剂浓度ε为p/p0.5的比值。简化的莫诺和Logistic剂量反应方程现在可表示为：O＝1/(1+εb)。对于两种抑制剂X和Y，举例来说可定义用于O的以下两种表达方式：
Ox＝1/(1+εb1)和Oy＝1/(1+εb2)。
Ox和Oy可以通过测定X或Y对目标微生物生长速度的抑制效果进行实验估算。得知Ox和Oy的估算函数，理论上的独立效果定义为Ox·Oy。实验观测的X和Y组合物对相对生长速度的影响被定义为Oxy。如果X和Y各自独立地对某种微生物起作用，这种假设在数学上可翻译为Oxy/Ox·Oy＝1。否定这种假设意味着X和Y的结合效果不是累加效应而是协同或者是拮抗作用。假如抑制剂X和Y对目标微生物起协同作用，Oxy/Ox·Oy＜1(但＞0)。当抑制剂X和Y的结合效果是拮抗作用时，Oxy/Ox·Oy＞1。协同、独立效果和拮抗可以用Oxy与Ox·Oy的图表视视觉化。
在图1的示例中，其中给出的曲线是大肠杆菌O157:H7(ATCC 700728)的O甘氨酸·丙氨酸(实验观测的相对生长速度)对O甘氨酸·O丙氨酸(预测相对生长速度)曲线，结果表明甘氨酸和DL-丙氨酸之间为协同抑制。图表中的实线表示当实验观测相对生长速度(O甘氨酸·丙氨酸)等于预测相对生长速度(O甘氨酸·O丙氨酸)的线条，此时甘氨酸和丙氨酸作为独立抑制剂。
DL-丙氨酸和甘氨酸的组合物对肠道沙门氏菌和大肠杆菌O157:H7的抑制尤其有效(协同作用)。
大肠杆菌O157:H7(ATCC 700728)和植物乳杆菌(DSM 20174)(混合培养物A)之间的竞争，以及肠道沙门氏菌(ATCC 13311)和植物乳杆菌(DSM 20174)(混合培养物B)之间的竞争在肉汤培养基中进行研究。大肠杆菌，肠道沙门氏菌，以及植物乳杆菌每天被转移到含有10ml脑心浸液肉汤(Oxoid，Basingstoke，英国)的螺旋盖管中(100×16mm)。培养物在没有搅拌、30℃的条件下进行培养。500μl的大肠杆菌过夜培养液和5μl的植物乳杆菌培养物被转移到含有10ml新鲜制备的脑心浸液肉汤的螺旋盖管中，或被转移到含有200mM的DL-丙氨酸和200mM甘氨酸或400mM的DL-丙氨酸和400mM甘氨酸(混合培养物A，第一次转移)的10ml脑心浸液肉汤中。500μl的肠道沙门氏菌和5μl植物乳杆菌过夜培养液被转移到含有10ml新鲜制备的脑心浸液肉汤螺旋盖管中，或被转移到含有200mM的DL-丙氨酸和200mM甘氨酸，或400mM的DL-丙氨酸和400mM甘氨酸(混合培养物B，第一次转移)的10ml脑心浸液肉汤中。混合培养物都在30℃进行温育。24小时后培养物被转移到新鲜培养基(第二次转移)，以及铺板到紫红胆盐琼脂(Oxod，Basingstoke，CM0485)和MRS琼脂(Oxod，Basingstoke，CM0361)。第二次转移物在30℃培养24小时，随后铺板到紫红胆盐琼脂和MRS琼脂。表1总结的分析结果表明植物乳杆菌在含有DL-丙氨酸和甘氨酸的大肠杆菌和肠道沙门氏菌的混合培养基中具有竞争优势。
表1：第一次转移和第二次转移每ml肉汤的菌落形成单位(cfu)
本发明用于保存食品。以下例子是本发明的实施例。用于接种液体培养物的鼠伤寒沙门氏菌ATCC 13311和大肠杆菌O157:H7 ATCC 700728的培养物原液常规被保存在琼脂平板上，该平板上含有脑心浸液(OxoidCM225，Basingstoke，英国)，并用1.5％琼脂的进行强化固定。大肠杆菌和鼠伤寒沙门氏菌的培养物在含有10ml脑心浸液肉汤的螺旋盖管(100×16mm)中生长并在30℃培养过夜。在食品接种之前，培养物用含有0.1％蛋白胨和0.85％NaCl的溶液进行稀释。
牛乳
灭菌脱脂乳从当地超市购得。添加适量的氨基酸。
意大利面条(烤宽面条)
即食烤宽面条从当地超市购得并用台式混合机充分均质。把适量的氨基酸添加到500g均质的烤宽面条中。该混合物被真空密封，随后进行辐照(10kGray)。辐照的烤宽面条被保存在-30℃直到日后使用。
新鲜肉类
含有约20％脂肪的新鲜切片牛肉(胸肉)用装有1/8”研磨平板的PrimusMEW 613肉类研磨机(Maschinenfabrik Dorhan，Dorhan，德国)进行研磨。在整个过程中温度保持在10℃。把适量的氨基酸添加到500g研磨后的肉类中。肉和氨基酸混合物随后进行辐照(10kGray)。辐照的肉类被保存在-30℃直到日后使用。
烤宽面条和新鲜肉类的接种
深度冷冻的火鸡腿、烤宽面条或者新鲜肉类在-4(±1)℃解冻过夜。500g仍然冷冻的产品被快速切成2-4cm片状并转移到厨房用食品加工机的桶中(Tefal Kaleo食品加工型67604)。添加1.5ml适度稀释的细菌培养物并且整个混合物混合大约30-60秒。在这个阶段，混合物的温度仍然低于0℃。混合后，约25g双份混合物被快速转移到袋滤器(Interscience，St Nom，法国)。接种了大肠杆菌或鼠伤寒沙门氏菌的产品在12℃培养两周。
牛乳的接种
牛乳被冷却到10℃，随后接种适度稀释的鼠伤寒沙门氏菌培养物。接种后的牛乳样品在12℃进行培养。
微生物分析
食品样品的微生物分析按如下进行：打开一个密封袋并往其中添加2倍净重的无菌稀释液(8.5％(w/w)NaCl和0.1％(w/v)细菌蛋白胨)。样品在Stomacher400实验室混合机中进行1min的均化(Seward医学，伦敦，英国)，将50μl匀浆用Eddyjet型1.23加速细菌接种仪(IUL仪器，巴塞罗那，西班牙)铺板在合适的琼脂培养基上。大肠杆菌O157:H7和鼠伤寒沙门氏菌铺板在VRBG琼脂(Oxiod，CM0485，Basingstoke，美国)上。平板在30℃培养24小时后计数。
结果
甘氨酸，DL-丙氨酸单独添加物以及甘氨酸(0.67％)与DL-丙氨酸(0.8％)的混合物对在12℃牛乳中鼠伤寒沙门氏菌的生长的影响如表I所示：
上表显示，在肉汤培养基中，100mM浓度的甘氨酸和100mM浓度的DL-丙氨酸对鼠伤寒沙门氏菌的生长速度具有微小的影响或没有影响。另一方面，它们的组合对抑制该微生物在牛乳中的生长非常有效。
Claims (7)
1.一种抑制革兰氏阴性细菌的食品保藏方法，该方法包括在食品中添加一种含有甘氨酸和丙氨酸的抗微生物制剂，其中所述丙氨酸是D-丙氨酸或DL-丙氨酸。
2.根据权利要求1所述的方法，其中所述食品被保护以抑制沙门氏菌和大肠杆菌中的至少一种。
3.根据权利要求2所述的方法，其中所述食品被保护以抑制肠道沙门氏菌和大肠杆菌0157:H7中的至少一种。
4.抗微生物制剂，其包含两种或三种至少选自甘氨酸和丙氨酸的氨基酸，用于通过抑制革兰氏阴性细菌而控制人体或动物体肠道菌群，其中所述丙氨酸是D-丙氨酸或DL-丙氨酸。
5.权利要求4的抗微生物制剂，包含益生菌和两种或三种至少选自甘氨酸和丙氨酸的氨基酸。
6.一种通过施用至少包含甘氨酸和丙氨酸的抗微生物制剂，通过抑制革兰氏阴性细菌以控制人体或动物肠道菌群的方法。
7.权利要求6所述的方法，其中被施用的所述抗微生物制剂至少包含甘氨酸，丙氨酸以及益生菌。
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| EP06112286.7 | 2006-04-06 | ||
| EP06112286A EP1842437A1 (en) | 2006-04-06 | 2006-04-06 | Antimicrobial preparations |
| PCT/EP2007/053407 WO2007113333A2 (en) | 2006-04-06 | 2007-04-05 | Antimicrobial preparations |
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| WO2012043052A1 (ja) * | 2010-09-28 | 2012-04-05 | テルモ株式会社 | 術野洗浄液 |
| JP5675251B2 (ja) * | 2010-10-01 | 2015-02-25 | 博衛 小川 | 生食用生肉の風味改良変質防止剤及びその方法、それを含有する生肉 |
| CA3052047A1 (en) * | 2017-02-13 | 2018-08-16 | Wintermute Biomedical, Inc. | Anti-pathogenic therapeutic compositions |
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| JPH0873351A (ja) * | 1994-06-30 | 1996-03-19 | Ajinomoto Co Inc | 炎症性腸疾患用栄養組成物 |
| US6506552B2 (en) * | 1998-08-24 | 2003-01-14 | Meiji Dairies Corporation | Amino acid-trehalose composition |
| JP2000224976A (ja) * | 1999-02-03 | 2000-08-15 | Shinkoo Gijutsu Kaihatsu Center:Kk | 食品用保存剤およびそれを利用した食品の製造法 |
| EP1252827B1 (en) * | 2000-01-31 | 2007-05-23 | Nihon Starch Co., Ltd. | Process for producing foods having good keeping qualities and food keeping agents |
| US6602532B2 (en) * | 2000-03-03 | 2003-08-05 | F.G.A. Laboratories Flavourence Corporation | Process of preserving food and food preservative |
| US6881424B1 (en) * | 2000-09-05 | 2005-04-19 | Mionix Corporation | Highly acidic metalated organic acid |
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| JP4210037B2 (ja) * | 2001-02-16 | 2009-01-14 | 村樫石灰工業株式会社 | 抗菌性組成物 |
| FR2869543B1 (fr) * | 2004-04-30 | 2006-07-28 | Expanscience Laboratoires Sa | Medicament comprenant un extrait peptidique d'avocat destine au traitement et la prevention des maladies liees a une deficience du systeme immunitaire |
| PL368179A1 (en) * | 2004-05-24 | 2005-11-28 | Sgp & Sons Ab | Proteollytic method for processing proteins and protein hydrolysis products, enriched with exogenous, semi-exogenous and conditionally exogenous amino acids for making therapeutic preparation used in normal and medicinal diets |
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| EP2001313A2 (en) | 2008-12-17 |
| BRPI0709874A2 (pt) | 2011-07-26 |
| EP2001313B1 (en) | 2014-08-20 |
| JP2009532049A (ja) | 2009-09-10 |
| EP1842437A1 (en) | 2007-10-10 |
| JP5133336B2 (ja) | 2013-01-30 |
| WO2007113333A2 (en) | 2007-10-11 |
| CN101437414A (zh) | 2009-05-20 |
| ES2521265T3 (es) | 2014-11-12 |
| WO2007113333A3 (en) | 2007-12-21 |
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