续——最近的一次作业（blogcn: 2006-10-18 22:46）

Gastrointestinal models
The Nordic network experiments with the TNO gastrointestinal tract model
focused on survival studies after gastric and ileal delivery. The SHIME-reactor
was chosen to illustrate the population dynamics in the small and large intestine.
Parameters such as pH, redox state, NH4, SCFA, gas composition, enzyme
activities and major bacterial groups were determined. As indicated already in
the discussion of adhesion, changes occurred in samples taken from different
parts of the TNO model system. Some of the non-viable bacteria recovered from
the TNO model also showed some immunological activity. Probiotic treatment
was shown to increase temporarily the numbers of lactic acid bacteria in
different parts of the SHIME ecosystem. Enterobacteriaceae decreased markedly
during treatment. The results indicated that further studies are necessary in order
to evaluate the repeatability of the SHIME system in the assessment of fatty acid
and enzymatic profile changes (Table 12.5).33

Anti-mutagenicity properties
Lactic acid bacteria or cultured dairy products have been reported to reduce the
mutagenicity of known chemical mutagens in in vitro tests.34 In in vivo trials
probiotic strains have occasionally been associated with the reduction of faecal
enzymatic activities involved in mutagen or carcinogen activation.35 Results
have been somewhat contradictory in trials studying the effects of orally
ingested probiotic strains on actual faecal mutagenicity. Lidbeck and co-workers
detected a decrease in faecal and urinary mutagenicity as a result of
Lactobacillus acidophilus NCFB 1748 consumption.36 No such effect was seen
in similar tests with another probiotic.37

12.2.2 The Probdemo strains
One of the first tasks of the Probdemo project was to establish selection criteria
for probiotic strains, and then apply these specific criteria for selecting the
project strains. The preliminary selection criteria included stability in in vitro
models simulating conditions in the upper gastrointestinal tract, where probiotic
bacteria are first exposed to an acidic and protease-rich environment in the
stomach before encountering bile acids in the small intestine. Other selection
criteria employed included the ability to adhere to intestinal mucosae, the ability
to inhibit intestinal pathogens and safety to the consumers.5, 11, 12 Application of
these selection criteria resulted in the selection of the following probiotic strains:
L. rhamnosus GG, Lactobacillus johnsonii LJ-1, Lactobacillus salivarius UCC
118, Lactobacillus crispatus M247, L. paracasei F19, and B. lactis Bb-12.
Bifidobacterium longum UCC 35624 was later included in the study due to its
promising positive influence on inflammatory bowel diseases.

Further characterisation of these strains, both at the phenotypic and genotypic
level, continued throughout the project. This led to the discovery of new
mechanisms for colonisation in the human intestinal tract such as expression of
co-aggregation proteins.38, 39 In safety studies it was demonstrated that the genes
encoding vancomycin resistance in L. rhamnosus GG were distinct from the
transferable genes in Enterococcus, indicating that they do not pose a safety
concern in this strain.40 The probiotic properties of the strains are now known to
be chromosomally encoded rather than being coded on potentially unstable
plasmids. Further research on the probiotic mechanisms of the project strains
(including molecular studies to determine how and where they adhere to
intestinal mucosae, inhibit pathogens and induce immunomodulation in the host)
has been performed, yielding further insights into how probiotic bacteria can be
selected and can benefit human health.

12.3 Pilot testing in clinical human trials
Probiotic strains should be safe and clinically tested prior to commercial human
use. Although this is an important aspect, no firm guidelines exist for safety
criteria. Examples of clinical and safety criteria for probiotic foods are listed in
Tables 12.6 and 12.7.16, 41
Guidelines for probiotic clinical trials (Probdemo approach) stem from the
trial design using volunteers (healthy or diseased in case of demonstration). In a
trial design volunteers should be randomly selected from a panel of a specific
population group targeted for the trial. A number of exclusion criteria were
employed when choosing the original healthy volunteer panel. These exclusion
criteria were the following:
• antibiotic treatment during the last month
• strong chronic intestinal disorders
• chronic inflammatory diseases
• chronic viral illness
• current drug therapy

上面的是原文，下面的是我的翻译。从来没做过这么多的翻译，7000+的字，贴在这里，全当日后的纪念。文中的表格和图都没有贴上来，经常出现在句末的奇怪的数字是文中的注解。

第十二章
益生菌类功能食品
T.Mattila-Sandholm and M.Saarela, VTT Biotechnology, Espoo

12．1 引言：益生菌类食品对于健康的益处
在欧洲，有益健康的食品这一领域被视为优先考虑的研究领域，这是基于对通过食物促进健康有着巨大潜力的认识。此外，饮食是公共健康策略的一个主要焦点，它针 对于终生维持最佳的健康状况，阻止诸如胃肠道疾病、心血管疾病、癌症和骨质疏松症等慢性疾病的早期发作，以及促进更加健康的老龄化。虽然人们对食物和健康 之间高度复杂的关系仍然知之甚少，但是近期在不同学科中的研究进展给人们提供了新的有希望的方法去增加对它的了解。对健康食品的越来越大的需求正在刺激国 际间的食品工业的革新以及新产品的发展，而的确，通过健康食品的供应和促销，食品工业在促进改良的饮食行为中占有中心地位。

益生菌是活的微生物食物的补充，它通过维持或改善肠内微生物的平衡而有益于消费者的健康。1 由于它们可感知的健康益处，在过去的二十年里，益生菌已经越来越多地含于酸奶和发酵牛奶中。通常情况下，它们是像嗜酸乳酸杆菌 （Lactobacillus acidophilus）这样的乳酸杆菌，以及像双叉乳杆菌这样的双歧杆菌（见表12.1）。2 功能食品的一个主要新生物就是包含有益生菌和可以增强肠道中有益健康的微生物菌落的益生助生质的食品。越来越多的科学证据支持维持健康的内脏微生物菌落可 能预防包括胃肠道感染、肠炎，甚至癌症在内的胃肠道疾病这一观念。益生菌培养的运用可以刺激一些微生物的优先生长，驱逐有潜在危害的细菌，并且加强人体的 自然防御机制。

益生菌要有益于人类健康，必须满足以下几个标准：它必须拥有良好的工艺学特性，这样才能够被 加工、混合进入食品产品中，并且不丧失生活力和功能性，不产生令人不悦的气味或质地；它必须能够在通过上胃肠道以及到达其作用地点的时候都能存活；它必须 能够在内脏环境中发挥功能。为了研究胃肠道中的益生菌株，我们必须建立从所摄入的构成胃肠生态系统的潜在的成千上万种菌株中鉴别出益生菌株的分子技术，还 要建立能够证实益生菌株对肠道内其他小型微生物群落的作用以及对宿主的重要作用的技术。这不仅要有积极的健康方面的利益，还要证明益生菌株没有任何的毒害 作用。有了这些知识的辅助，益生菌才能进入临床的初步研究中，以评估其对消费者临床健康益处的大小（表12.2）。

12．1．1 益生菌食品营养功能性的证明（FAIR CT96-1028）
欧洲拥有传统上益生菌市场的领先地位。但是，消费者对于益生菌有着相当多的混淆和怀疑，而与其有关的消费者组织和某些科学团体就把这与益菌性的产品联系起 来。这极大地妨碍了含有益生菌的功能食品的开发，也削弱了欧洲生产商面对竞争时的市场地位。要消除这些障碍，加快对益生菌食品科技的适应，以及增加新的益 生菌食品的吸引力，关键在于通过介绍益生菌、益生菌食品的健康和营养益处来证明目前市场要求这一基础，特别要将重点放在肠道的整体性和免疫调节性，发掘选 择新的益生菌和益生菌食品的有效方法，并且向由企业、当局和消费者组成的广大受众普及已有知识等方面。Probdmo工程的启动就是为了向欧洲的消费者证 明益生菌产品的价值，它的目标分为四个相互作用的任务
（表12.3）：
1． 建立一个普遍适用的为功能食品选择益生菌株的科学的基础的方法。代表乳酸杆菌（Lactobacillus）和双歧杆菌（Bifidobacterium）的六种益生菌株被选作证明之用。
2． 证明益菌性食品在包括儿童和成人在内的人类初步试验中的有益价值。初步的测试表明益生菌株对健康的儿童和成人没有任何毒害作用。此外，益生菌株在治疗婴儿的食物过敏以及幼童的由轮状病毒引起的腹泻方面显示出效果，它也被证明对成人的肠炎有疗效。
3． 证明并满足功能的和工艺的要求。这对于将益生菌作为功能食品进行工业生产是必需的。通过对益生菌株体外特性的研究并将结果反映在临床表现上，研究人员已经 实现了上述的要求。现在主要的焦点集中在通过人类活组织检查证明体外与体内试验间的联系，证明益生菌株产品的工艺标准，以及益生菌株试生产等方面上。
4． 向包括企业主、当局和消费者组织在内的广大受众普及有关益生菌的知识和试验结果。这一点已经通过年度的研讨会实现（关于益生菌安全的第一届研讨会于 1996年召开，关于益生菌研究工具的第二届研讨会于1997年召开，关于功能食品研究的第三届研讨会于1998年召开，关于功能食品的第四届研讨会于 2000年召开）。5-8

基于先前的EU项目在乳酸菌和益生菌方面的成果，工程的参与者和研究所在这个研究 领域内都有着丰富的经验，工业合作伙伴有着长期参与功能食品市场的惯例对益生菌产品来说也有着特殊的意义。芬兰的VTT生物科技（VTT Biotechnology）扮演着协调和普及相关活动和证明任务的角色，这些活动和任务是关于益生菌株特性、工艺特性和成人临床测试的；荷兰的瓦格宁根 大学（The University of Wageningen）在通过使用包括PCR、原位杂交、变性梯度凝胶电泳法和温度梯度凝胶电泳在内的分子方法来证明益生菌株在人类临床试验上的活性和生 存力方面扮演着关键的角色；意大利的皮亚琴察天主教大学（The Catholic University of Piacenza）扮演着展示将被证明的菌株的粘附和聚合特性的角色；爱尔兰的国立科克大学（The University of College Cork）在人类胃肠病学、成人临床检测和益生菌的免疫调节活性等方面有着造诣精深的专业技术；芬兰的图尔库大学（University of Turku）在儿童临床检测以及临床儿科和功能食品之间的联系方面有着长期的科学传统。Valio有限公司（芬兰）、Arla（瑞典）、Nestlé（瑞 士）和Christian Hansen实验室（丹麦）等工业合作伙伴有着益生菌产品工业生产的坚实基础、功能食品市场的丰富经验和此领域内进行的研究，它们在选择待证明的菌株、准 备待开发的产品以及验证它们的有益效果等方面是极为重要的。

12．2 选择益生菌株
表12.4和图12.1中所示的益生菌微生物的选择理论基础包括：
·安全
· 功能行为 【存活率，粘连，成菌落性，抗菌产品，免疫刺激，抗遗传毒性的活力，以及对诸如幽门螺旋杆菌（Helicobacter pylori）、沙门氏菌（Salmonella）、李斯特菌属（Listeria）和梭状芽胞杆菌（Clostridium）等病原体的预防】
·工艺方面（在牛奶中的生长，感官特性，稳定性，噬菌体抗性，在处理中的成活力）

总的来说，用于探索性试验的菌株应该基于已有的体外试验的科学性的数据来进行选择。自然地，益生菌株的安全成为了最重要的问题，而且新的指导方针也已经制定 出来。9-13 成功的益生菌目前的安全标准和功能特性在近期的综述中已经有了详细的说明，14-16 它们包括以下的技术条件：
·用于人类的菌株最好来自于人类
·它们是从健康人的胃肠道中分离散出来的
·它们即使在免疫妥协的宿主体内也有不致病的历史
·它们没有与诸如传染性心内膜炎和胃肠道疾病有联系的历史

研究人员最近在讨论人类来源的重要性，但是大多数（如果不是全部的话）目前成功的菌株都是人类来源的。同样的，研究人员也在争论菌落生长的能力在人类胃肠道 中的重要性。然而，大多数目前所报道的菌株能够在人体中耐受，至少暂时可以在摄入后在人的排泄物中检测到。酸和胆汁的稳定性对于任何可能会在肠道中有显著 效果的菌株来说都是应该具有的基本特性。粘附和耐受的能力也是与潜在的免疫效力关系极为密切的，很有可能需要某些粘附以及（或者）结合于肠道细胞的机制。 如此一来，像Caco-2细胞系这样的体外模型系统的对照比较研究就很重要了。17，18 由于有粘附性的益生菌株可能在肠道内停留更长的时间，进而比非粘附性的菌株有着更多的机会发挥代谢效果，它一出现便受到人们的青睐，并且至少有一种商业益 生菌株已经被证明可以粘附在体内的粘膜上。19

由于对结肠菌群有影响，对于益生菌株来说，通过抗菌产物或者竞争性排斥表现 出对病原菌的对抗状态是很重要的。虽然大量的研究成果集中在细菌素的研究上，但是，益生菌在内脏中的细菌素功能和有效性模式是未知的。虽然益生菌株可能在 体外生产细菌素，但是细菌素在病原菌抑制方面的作用在体内只是有限的，因为传统的细菌素只对与像其他的乳酸杆菌（Lactobacillus）密切相关的 种类有抑制作用，或者对诸如芽胞杆菌（Bacillus）或梭状芽胞杆菌（Clostridium）的孢子生成菌（sporeformer）有抑制作用。 然而，小分子量的代谢产物（以及次级代谢产物）可能是很重要的，因为它们表现出对诸如沙门氏菌（Salmonella）、大肠杆菌 （Escherichia coli）、梭状芽胞杆菌（Clostridium）和缠绕杆菌（Helicobacter）等有害微生物的广谱抑制作用。20-22

12．2．1 针对功能性益生菌食品培育的发展
包括培育的奶产品在内的益生菌奶制品及其培育在北欧食品工业产品中具有悠久的历史和巨大的消费量，并且其益生菌特性也已经被研究了数十年。北欧项目 （Nordic programme，1994年至1997年末）是为了确认工业益生菌株的体外功能性，菌株由工程的参与者Arla（瑞典）、Christian Hansen（丹麦）、Norwegian Dairies（挪威）和Valio Ltd（芬兰）提供。所研究的菌株有：类干酪乳杆菌类干酪亚种（Lactobacillus paracasei subsp. paracasei）菌株E-94522和E-94510，鼠李糖乳杆菌（Lactobacillus rhamnosus）菌株E-94509和E-94522，嗜酸性乳酸杆菌（Lactobacillus acidophilus）E-94507，植物乳杆菌（Lactobacillus plantarum）E-79098，乳酸乳球菌乳酸亚种（Lactococcus lactis subsp. lactis）E-90414，乳酸乳球菌乳脂亚种（L. lactis subsp. cremoris）E-94523，乳制品动物双歧杆菌（Bifidobacterium animalis lactis）E-94508和长双歧杆菌（Bifidobacterium longum）E-94505。研究包括体外的细胞因子释放效果、粘附特性、抗诱变性以及在胃肠道模型（荷兰的TNO胃肠道模型和比利时Ghent的 SHIME的生态系统）中的行为。此外，研究人员也在评估其工艺和产品特性。

粘附特性的评估
先粘附 在人类肠细胞上，随后在人类胃肠道中生长为菌落被认为是益生菌发挥其功能的重要的先决条件，粘附检验菌株寄居于肠道并在肠环境中生长的潜力，也提供了促进 与内脏接触、调节局部和系统免疫效力的淋巴组织的粘膜表面相互作用。因而，只有具粘附能力的益生菌可以诱导免疫效力并稳定肠粘膜屏障。23 北欧项目工程的体外粘附检测结果对于不同的检测和菌种间的不同及变化有一个清晰的指征，很明显，除了Caco-2细胞系试验之外，其余的检测系统也需要具 有粘附潜力及不同粘附机制的特征。粘附系统也用于研究益生菌株的抗侵入潜力。不同的益生菌株在侵入抑制方面表现出相对不同的行为，而且需要有新的方法从与 临床表现相关的角度去评估这些特性。粘附试验表明不同益生菌株生长为菌落的潜力是明显不同的，并且与临床数据联系起来之后，可能为以后益生菌株的选择和方 法的发展提供一个有益的基础。17，18 近来，粘附检测也被应用于人类回肠造口糖蛋白（用于小肠粘液的模型），并再次表现出不同的益生菌性特征。26

使用结肠活组织检查的体内粘附性研究
排泄物样品被用于益生菌的大多数菌落研究中。27，28 但是，这些反映的仅仅是排泄物质的细菌学表现，并没有给出关于这种表现的胃肠道不同部分或者内脏粘膜层的精确描绘。从结肠镜检查的病人上获取活组织检查物质就具有这样的优点：不仅可以从直肠S形区域，而且可以从大肠（升结肠、横结肠和降结肠）的其他部位获得组织样品。结果，通过使用活组织检查物质检测出一 种商业益生菌株【乳酸杆菌GG（Lactobacillus GG）】优先粘连于大结肠的下降部。在停止益生菌制剂的服用之后，甚至在菌株已经不能在排泄样品中检测出之后，这种益生菌株仍表现出在内脏上皮组织中存活的能力。19 Johansson和他的同事也证明了从服用发酵燕麦汤的志愿者体内获得的不同的乳酸杆菌（Lactobacillus）菌种对直肠粘膜活组织检查样品有 粘附作用。29

免疫学评估
与内脏相关的淋巴组织可能与粘附的益生菌制剂有联系，因此，粘附是引起免 疫效力的一种方法。北欧研究网络研究了益生菌株与用细胞因子产品【人类α-肿瘤坏死因子（human TNF-α），白细胞介素-6（interleukin-6），白细胞介素-10（interleukin-10），肿瘤生长因子-β（TGF-β）和 α-干扰素（interferon-α）】进行的奶制品培育【保加利亚乳酸杆菌（Lactobacillus bulgaricus），嗜热链球菌（Streptococcus thermophilus）】之间的相互影响。研究人员也检测了已经通过体外TNO胃肠道模型的益生菌株的诱导细胞因子产品【α-肿瘤坏死因子（TNF- α），白细胞介素-6（interleukin-6）】的能力，主要的目的是研究益生菌株在体内是否通过细胞因子刺激免疫系统。白细胞介素-6（IL- 6）产品在用活细菌进行的试验中表现出相当大的变化，测试菌株没有被观察到能够诱导白细胞介素-10（IL-10）。31 研究人员努力开发一种通过Northern杂交探测mRNA的新方法去检测早期的细胞因子应答。这种方法被证实是比ELISA敏感性更高的方法，并且益生 菌株确实生产出白细胞介素-10（IL-10）和白细胞介素-1b（IL-1b）。进一步的研究集中在对通过益生菌的细胞因子诱导通路的分析，对血清蛋白 效力的预测，以及益生菌和人类细胞表面分子之间的相互作用之上。32

胃肠道模型
北欧研究网络对 TNO胃肠道模型的试验集中于益生菌在胃和回肠的运送之后的生存力研究上。SHIME-反应物（SHIME-reactor）被选作阐明小肠和大肠群体动 力学的对象。研究人员确定了pH、氧化还原状态、NH4、SCFA（short-chain fatty acid，短链脂肪酸）、气体组成、酶活性和主要细胞群等参数。如同已经在粘附部分中讨论的一样，来自TNO模型系统不同部分的样品会有不同，一些无法生 存的细菌从TNO模型中复原后也表现出一定的免疫学活性。益生菌治疗表现出可以暂时性地增加SHIME生态系统中不同部分的乳酸菌数量，肠杆菌科细菌在治 疗过程中显著减少。这样的结果表明，为了评价SHIME系统在评估脂肪酸和酶轮廓变化中的重复性，进一步的研究是必须的（表12.5）。33

抗诱变特性
有报道说乳酸菌或培育的奶制品在体外检测中可以减少已知化学诱变剂的诱变性。34 在体内试验中，益生菌株偶而会与排泄物中与诱变或致癌物活化有关的酶活力相关。35 这样的结果在某种程度上与试验中研究的口服益生菌株后实际的排泄物诱变性的效果相矛盾。Lidbeck和他的同事检测到排泄物和尿的诱变性减小，这是由嗜 酸性乳酸杆菌（Lactobacillus acidophilus）NCFB 1748所引起的。36 而在使用其他益生菌的相似的检测中并没有得到这样的结果。

12．2．2 Probdemo菌株
Probdemo 工程的首要工作之一是建立益生菌株的选择标准，之后应用这些特殊的标准去选择功能菌株。初步的选择标准包括模拟胃肠道上部（在小肠中遇到胆汁前，这里是益 生菌最先暴露于酸的和富含蛋白酶的环境）条件的体内模型的稳定性。其他应用的选择标准还包括粘附在肠粘膜上的能力，抑制肠内病原菌的能力以及对消费者的安 全性。5，11，12 在这些选择标准下，以下的益生菌株将被选择：
鼠李糖乳杆菌GG（L. rhamnosus GG）, 约氏乳杆菌（Lactobacillus johnsonii） LJ-1，唾液乳杆菌（Lactobacillus salivarius）UCC118，卷曲乳杆菌（Lactobacillus crispatus）M247，类干酪乳杆菌（L. paracasei）F19, 和乳制品双歧杆菌（B. lactis）Bb-12。长双歧杆菌（Bifidobacterium longum）UCC 35624 由于其对于胃肠炎的可能存在的有利影响，后来也被加入到研究行列中。

在表现型和基因型 水平上对这些菌株的描述通过这项工程继续着，这导致了人体肠道内新的菌落机制的发现，如共聚集蛋白的表达。38，39 在安全性的研究中，已经证明编码的万古霉素（vancomycin）在鼠李糖乳杆菌GG（L. rhamnosus GG）中的抗性基因与肠道球菌（Enterococcus）的可转移基因是截然不同的，这表明它们在这个菌株中并未引起安全性的关注。40 目前所知，菌株的益生菌特性更可能是染色体编码的，而非潜在的不稳定的质粒所编码。进一步针对工程菌株的益生菌机制的研究（包括决定它们是如何以及在何处 粘附于肠粘膜上、抑制病原体并诱导宿主体内的免疫调节的分子研究）已经开展了，并着眼于如何选择益生菌和益生菌如何能够有益于人体健康。

12．3 临床人体试验中的探索性试验
益生菌株应该是安全的，并且经过预先的临床试验证明是可以进行与人相关的商业使用的。虽然这是一个重要的方面，但是仍然没有一个严格的针对安全标准的指导方针存在。益生菌食品的临床和安全标准的例子列于表12.6和表12.7中。16，41

益生菌临床试验的指导方针（Probdemo方法）来源于使用志愿者（健康的或者未知带病的）的试验设计。在试验设计中，志愿者应该从针对试验的特殊人群中随机选择。在选择原始的健康志愿者时，研究人员应用了许多排除标准。这些排除标准包括：
·在过去的一个月中的抗生素治疗
·严重的慢性肠道疾病
·慢性的炎症
·慢性的病毒性疾病
·目前的药物疗法

最近的一次作业——翻译（blogcn: 2006-10-18 22:12）

12
Probiotic functional foods
T. Mattila-Sandholm and M. Saarela, VTT Biotechnology, Espoo

12.1 Introduction: the health benefits of probiotic foods
The area of food for health has been identified as a priority area for research in
Europe. This is based on the recognition that there is enormous potential for
improving health through food. Furthermore, diet is a major focus of public
health strategy aimed at maintaining optimum health throughout life, preventing
early onset of chronic diseases such as gastrointestinal disorders, cardiovascular
disease, cancer and osteoporosis, as well as promoting healthier ageing.
Although the highly complex relationship between food and health is still poorly
understood, recent research advances in different disciplines provide promising
new approaches to improve our understanding. The growing demand for
‘healthy’ foods is stimulating innovation and new product development in the
food industry internationally. Indeed, the food industry has a central role in
facilitating improved eating practices through the provision and promotion of
healthy foods.

Probiotics are live microbial food supplements which benefit the health of
consumers by maintaining or improving their intestinal microbial balance.1 Due
to their perceived health benefits probiotic bacteria have been increasingly
included in yoghurts and fermented milks during the past two decades. Most
commonly they have been lactobacilli such as Lactobacillus acidophilus, and
bifidobacteria often referred to as ‘bifidus’ (see Table 12.1).2 A major
development in functional foods pertains to foods containing probiotics and
prebiotics which enhance health-promoting microbial flora in the intestine.
There is growing scientific evidence to support the concept that the maintenance
of healthy gut microflora may provide protection against gastrointestinal
disorders including gastrointestinal infections, inflammatory bowel diseases and
even cancer. The use of probiotic bacterial cultures stimulates the growth of
preferred micro-organisms, crowds out potentially harmful bacteria and
reinforces the body’s natural defence mechanisms.

Before a probiotic can benefit human health it must fulfil several criteria: it
must have good technological properties so that it can be manufactured and
incorporated into food products without losing viability and functionality or
creating unpleasant flavours or textures; it must survive passage through the
upper gastrointestinal tract and arrive alive at its site of action; and it must be
able to function in the gut environment. To study the probiotic strain in the
gastrointestinal (GI) tract, molecular techniques must be established for
distinguishing the ingested probiotic strain from the potentially thousands of
other bacterial strains that make up the gastrointestinal ecosystem. Techniques
are also required to establish the effect of the probiotic strain on other members
of the intestinal microbiota and importantly on the host. This includes not only
positive health benefits, but also demonstration that probiotic strains do not have
any deleterious effects. Armed with this knowledge, the probiotics can then
enter human clinical pilot studies that attempt to assess their clinical health
benefits to consumers (Table 12.2).3, 4

12.1.1 Demonstration of Nutritional Functionality of Probiotic Foods
(FAIR CT96-1028)
Europe has traditionally had a leading position on the probiotic market.
Considerable confusion and scepticism, however, exists on the side of consumers,
consumer organisations and certain quarters of the scientific community about the
claims associated with probiotic products. This greatly hampers further
exploitation of functional foods containing probiotic bacteria and weakens the
market position of European producers in the face of competition. To eliminate
these hurdles, to speed up adaptation of the probiotic food technology and to
enhance the attractiveness of new probiotic foods, it is essential to demonstrate
the up-to-date basis for marketable claims by presenting the health and nutritional
benefits of probiotic bacteria and foods. Special emphasis should be put on
intestinal integrity and immune modulation, exploitation of validated methods for
the selection of novel probiotic bacteria and foods, and dissemination of the
obtained knowledge to the extended audiences consisting of industries,
authorities and consumers. The Probdemo project was initiated to demonstrate
the value of probiotic products to European consumers. The project objectives
were divided into four interactive tasks (Table 12.3):

1. To establish a scientifically based selection of probiotic bacterial strains
currently available for functional foods. Six probiotic strains representing
Lactobacillus and Bifidobacterium species were chosen for demonstration
purposes.
2. To demonstrate the beneficial value of probiotic products in human pilot
trials both in children and adults. Initial tests showed that probiotic strains
did not have any deleterious effects in healthy children or adults.
Furthermore, probiotic strains were shown to be effective in the treatment
of infants with food allergy and small children with rotavirus diarrhoea. The
effect of probiotics was also demonstrated in adults with inflammatory
bowel disease (IBD).
3. To demonstrate and meet the functional and technological requirements
essential for the industrial production of probiotics as functional foods. This
has been established by studying probiotic strain properties in vitro and
reflecting these results to the clinical situations. The main focus has been on
demonstrating adhesion in vitro and in vivo using human biopsies, on
demonstrating the technological criteria for probiotic products, and on pilot
production of probiotic strains.
4. To disseminate the knowledge and results to extended audiences consisting
of industrial users, authorities and consumer organisations. This has been
established by annual workshops (Workshop 1 was held on Safety of
Probiotics in 1996, Workshop 2 on Probiotic Research Tools in 1997,
Workshop 3 on Functional Food Research in 1998, Workshop 4 on
Functional Foods in 2000).5–8

The project participants and institutes collectively have wide experience in
this research area, building on the results of former EU programmes on lactic
acid bacteria and probiotics. The industrial partners have long traditions in the
markets of functional foods with special reference on probiotic products. VTT
Biotechnology, Finland, has the role of coordination and dissemination of
activities and demonstration tasks on probiotic strain properties, technological
properties and clinical testing on adults. The University of Wageningen,
Netherlands, has the key role of demonstrating the activity and viability of
probiotic strains in human clinical trials by using molecular methods including
PCR, in situ hybridisation and DGGE/TGGE. The Catholic University of
Piacenza, Italy, has the role of showing the adhesive and aggregation properties
of the strains to be demonstrated. The University of College Cork, Ireland, has
profound expertise on human gastroenterology, clinical testing with adults and
immune modulation activities of probiotics. University of Turku, Finland, has a
long scientific tradition of clinical testing with children and the links between
clinical pediatrics and functional foods. The industrial partners Valio Ltd.
(Finland), Arla (Sweden), Nestle´ (Switzerland) and Christian Hansen Laboratories
(Denmark) have sound basis of industrial production of probiotic products
and long experience on functional foods market as well as research in this area.
This industrial role is of utmost importance in selecting the strains to be
demonstrated, preparing the products to be developed and in verifying their
beneficial effects.

12.2 Selecting probiotic strains
The theoretical basis for selection of probiotic micro-organisms illustrated in
Table 12.4 and Fig. 12.1 includes:
• safety
• functional behaviour (survival, adherence, colonisation, anti-microbial
production, immune stimulation, anti-genotoxic activity and prevention of
pathogens such as Helicobacter pylori, Salmonella, Listeria and Clostridium)
• technological aspects (growth in milk, sensory properties, stability, phage
resistance, viability in processes).

In general, strains for pilot testing should be selected based on established in
vitro scientific data. Naturally, the safety of probiotic strains has been of prime
importance and new guidelines have been developed.9–13 Current safety criteria
and functional properties for successful probiotics have been defined in recent
reviews.14–16 These include the following specifications:
• Strains for human use are preferably of human origin.
• They are isolated from healthy human GI tract.
• They have a history of being non-pathogenic even in immunocompromised
hosts.
• They have no history of association with diseases such as infective
endocarditis or GI disorders.

The significance of human origin has been debated recently, but most if not
all current successful strains are indicated to be of human origin. Similarly, the
importance of the ability to colonise the human gastrointestinal tract has been
questioned. However, most current strains are reported to persist in humans at
least temporarily as measured by faecal counts following ingestion. Acid and
bile stability are self-evident properties for any strain expected to have effects in
the intestinal tract. Ability to adhere and persist are also closely related to
potential immune effects. It is likely that some mechanisms of adhering and/or
binding to the intestinal cells are required. Thus controlled comparable studies
on in vitro model systems, such as the Caco-2 cell line, are of importance.17, 18
Adherent strains of probiotic bacteria are favoured since they are likely to persist
longer in the intestinal tract and thus have better possibilities of showing
metabolic effects than non-adhering strains. At least one of the commercial
probiotic strains has been demonstrated to adhere to the colonic mucosae in
vivo.19

To have an impact on colon flora it is important for probiotic strains to show
antagonism against pathogenic bacteria via anti-microbial substance production
or competitive exclusion. Enormous research efforts have focused on bacteriocin
research. However, the mode of action and efficacy of bacteriocins in the gut is
not known for probiotic bacteria. Although probiotic strains may produce
can only be limited, since traditional bacteriocins have an inhibitory effect only
against closely related species such as other Lactobacillus or on sporeformers
such as Bacillus or Clostridium. However, low molecular weight metabolites
(and secondary metabolites) may be more important since they show wide
inhibitory spectrum against many harmful organisms like Salmonella,
Escherichia coli, Clostridium and Helicobacter.20–22

12.2.1 Development of cultures aimed for functional probiotic foods
Probiotic dairy foods and cultures have a long history and large consumption in
the Nordic diet. Industrial products, including cultured dairy products, and their
probiotic properties have been studied for many decades. The objective of the
Nordic programme (from 1994 to end of 1997) was to validate industrial
probiotic strains with regard to in vitro functionality. Strains were provided by
project participants Arla (Sweden), Christian Hansen (Denmark), Norwegian
Dairies (Norway) and Valio Ltd (Finland). Strains studied included the
following: Lactobacillus paracasei subsp. paracasei strains E-94506 and E-
94510, Lactobacillus rhamnosus strains E-94509 and E-94522, Lactobacillus
acidophilus E-94507, Lactobacillus plantarum E-79098, Lactococcus lactis
subsp. lactis E-90414, L. lactis subsp. cremoris E-94523, Bifidobacterium
animalis (lactis) E-94508 and Bifidobacterium longum E-94505. The studies
included research on the in vitro cytokine release effects, adhesive properties,
anti-mutagenicity and behaviour in the gastrointestinal tract models (TNO
gastrointestinal tract model in the Netherlands, and the SHIME ecosystem at
Ghent, Belgium). Also technological and production properties were assessed.

Assessment of adhesion properties
Adhesion of probiotic strains to human intestinal cells and the following
colonisation of the human gastrointestinal tract has been suggested as an
important prerequisite for probiotic action. Adhesion verifies the potential of the
strain to inhabit the intestinal tract and to grow in intestinal conditions. Adhesion
also provides an interaction with the mucosal surface facilitating contact with
gut-associated lymphoid tissue mediating local and systemic immune effects.
Thus, only adherent probiotics have been thought to induce immune effects and
to stabilise intestinal mucosal barrier.23 The Nordic programme project results of
in vitro adhesion assays gave a clear indication of differences and variation
between assays and different strains.24, 25 It was evident that in addition to Caco-
2 cell line experiments, other test systems were also needed to characterise the
adhesion potential and different adhesion mechanisms. The adhesion system was
also used to study the anti-invasion potential of probiotic stains. Different
probiotic strains show relatively different behaviour in invasion inhibition and
novel methodologies are needed to assess these properties in a way that relates
them to clinical situations. Adhesion experiments indicate clear differences in
the colonisation potential of different probiotic strains and, when later connected
with clinical data, may provide a useful basis for selection and method
development for future probiotic strains.17, 18 Lately adhesion assays have also
been applicated to human ileostomy glycoproteins (modelling for small
intestinal mucus), showing once again different characteristics of the probiotic
features.26

In vivo adhesion studies using colonic biopsies
Faecal samples have been used in most colonisation studies on probiotic
bacteria.27, 28 These, however, reflect only the bacteriological situation in faecal
material and do not give an accurate picture about the situation in different parts
of the gastrointestinal tract or in the mucosal layer of the gut. There are
advantages in taking biopsy material from colonoscopy patients: in this way
tissue samples have been obtained, not only from the rectal-sigmoidal region,
but also from other parts of large intestine (ascending, transverse and descending
colon). As a result the preferential adhesion of a commercial probiotic strain
(Lactobacillus GG) to the descending part of large colon was detected by using
biopsy material. This probiotic strain was shown to survive in the gut epithelium
for several days after consumption of the probiotic preparation was stopped and
even after the strain could no longer be detected in faecal samples.19 Johansson
and co-workers have also demonstrated the adhesion of different Lactobacillus
strains to rectal mucosal biopsy samples obtained from volunteers who had
consumed fermented oatmeal soup.29

Immunological assessment
Gut-associated lymphoid tissue may have contact with adhesive probiotic
preparations and therefore adhesion is one way of provoking immune effects.
The Nordic network studied the interactions of probiotic strains and dairy
cultures (Lactobacillus bulgaricus, Streptococcus thermophilus) with cytokine
production (human TNF-, interleukin-6, interleukin-10, interleukin-12, TGF-,
and interferon-). Probiotic strains which had passed through the in vitro TNO
gastrointestinal tract model were also assayed for their ability to induce cytokine
production (TNF-, interleukin-6).30 The main goal was to investigate whether
probiotic strains stimulate the immune system in vitro through cytokines. IL-6
production showed considerable variation between experiments performed with
live bacteria. Test strains were not observed to induce IL-10.31 Efforts were
made to develop new methods to measure early cytokine responses by detection
of mRNA by Northern hybridisation. This method proved more sensitive than
the ELISA and has demonstrated that probiotic strains indeed produce IL-10 and
IL-1b. Further investigations focused on analysis of the pathway of cytokine
induction by probiotics, estimation of the effect of serum proteins, interaction
between probiotics and human cell surface molecules.32

原来我遮住了自己生活中的阳光（blogcn: 2006-10-18 18:47）

食品工程的课结束了，没有考试，最后的考核是两个班的人一共翻译两本书，然后按小组用PPT讲一下自己的主要内容。于是，我害怕了。我从来不喜欢这种分组的形式，因为我的组员都不是 我选的。事实按照我为自己设想的最坏的思路发展下去，七个人的小组，我做了内容最多、最难翻译的一部；今天下午的所谓的答辩，根本没有人关心，有人连来也 不来。本来各自讲各自的内容，成了我跟一个要好的朋友的活儿。恶心的结果是可想而知的。我没有什么权力去要求别人；事实上，我根本不忍去要求别人，别人要 考研，别人根本不在乎自己的成绩，那对他们完全没有用，90分和60分的成绩没有区别。我请自己不要难过，好吗？

辅修成绩出来了，我进入了能够参加最后双位学选拔考试的大名单。但是成绩依旧有问题。我的一门课的成绩本来是95分，结果也不知道为什么录成了80分，去找 教务处的老师，她让我去找任课老师；可是，任课老师在辅修的第一学期成绩出来的时候已经明确地告诉我，是95分，80分确实是有问题的。然而，问题还没有 解决，辅修的成绩单也没法打，出国成绩单于是一拖再拖。我请自己忍耐一下，好吗？

P&G的第一轮考试轻松通过。一天早上P&G的电话要我去参加他们的英才见面会。于是，满心喜欢地第一次以正当的理由走进了五星级酒店。然而，拥挤的会场，无聊的 演讲，冗长的内容。四点多没有吃饭就去了酒店，去了才知道没有饮食方面的照顾，而且要至少到九点才结束。快十点的时候出来，饥肠辘辘。这就是世界五百强 的、有着上百年历史的外企。我请自己不要失望，好吗？

一天接到系里一个老师的电话，要我过去帮忙做个研究生的项目。我答应了。老师先介绍了一个内容，我首先想到的解决方法就是分子克隆。老师和研究生姐姐也认可了，可是说这样的东西系里三年内都没有人做过了。我 说我还比较熟悉一些过程，暑假在IBP做了几乎一个假期，也学了些东西。研究生姐姐说那就好，让我回去找找资料。于是找文献，结果发现跟我的大思路类似的 东西在今年就有国外的人发了文章。不知道该怎么办了。化学合成或者是诱变育种的方法我真的一点都没有接触过。我请自己不要放弃，接着努力，好吗？

食堂师傅四毛钱的米饭打的像两毛半，一碗面不知道有多少盐，咸得我只吃了不到十根（不是裤带面），收拾碗筷和残食的师傅把残食弄到了我身上，吃出小虫虫，我没说话就走了。我知道，可能我的一次投诉，就会让一个师傅下岗或者停职，至少写个检讨吧。大家都不容易，也许他或她的背后就是一个家庭，一个要供养的大学 生，一个生病的老人。我请自己不要强求，好吗？

刚洗好的衣服，掉到了地上，满是灰土。我请自己不要郁闷，再洗一遍，好吗？

出国申请的事情依旧进展缓慢，我的自卑到了无以复加的地步。我请自己自信一些，好吗？

跟MM闹别扭了。就算先是她的不对，但后来是我的错。我请自己不要冲动。对不相识的人都笑脸以对，而对MM却那么苛刻。我请自己宽容一些，好吗？

刚才同学来电话，说是Unilever的招聘宣讲会，问我去不去。我说去。我让自己不要把自己逼到一个没有退路的死胡同里，好吗？

我要自己看到自己的优点，看到未来的光明，不要总是觉得自己这也不行那也不好；我要自己从阴影中走出来，想想过去其实我实现的事情也不比没有实现的事情少多 少；我要自己相信自己能有一个美好的未来；我要自己笑一笑，不要总在伤心的时候望着窗外的天空听悲伤的歌曲；我要自己不要总是对别人宽容，而对自己或是 MM苛刻；我要自己在晚上躺在床上的时候能够想想自己的收获，不要总把焦点集中在自己的过失上；我要自己不要总是在别人有困难的时候去帮忙和安慰，而在自 己遇到困难的时候却总是一个人面对，连心里的那一扇透气的窗都紧闭不开。

好吗？

日子依然继续，不好不坏（blogcn: 2006-09-26 19:51）

因为去新加坡的师兄走后那个项目就搁置下来了，再加上相关负责老师的从这学期开始的一学期的产假，PHBHHx的纳米级颗粒的制备的项目去不了了，这让我难过了两天，毕竟认认真真地看 了很多资料。老师不冷不热的回信让我的一腔热情碰得冰冷。于是只能周一下午去找了另一位老师，老师很好，聊了一个多小时，答应我去跟着她的课题组做。其间谈到去新加坡的那个师兄，老师笑兮兮充满喜欢地说：“**说他在我的组里做了什么什么，其实基本上啥也没做，推荐信只要写得不要太那个（太夸张）就行了。 ”其实国外教授现在也知道，中国学生的推荐信基本是自己写的，然后找老师签字。。。这下好了，又得重新找资料、看文献，熟悉一个新的研究内容。。。

说到了推荐信，其实昨天早上就在跑这件事情。思来想去自己系里没有什么太牛的老师，于是就去找了大二时教过我物理化学的理学院的从日本回国的大牛（好像SCI就有50多篇~~ ）。老师倒是热情得出乎我的意料。。我去他上课的教室等他，他一进来看见我在，直接在第一排大声说：“小于，你怎么来了？”然后径直往我坐的后排走。弄得我怪不好意思的，没想到老师还记得我~~ 


后来他也答应给我写推荐信了，只是我得提供自己资料，他才能写出positive的内容。教授说，出国一定要对自己今后的发展有好处，要不然不出也罢，为了出国而出国是很dangerous的。那个dangerous吓得我一哆嗦。

给大一的小朋友们做经验交流，没想到最让全场掌声雷动的竟然是那句“真正的爱情还是很值得追求的东西，那是种很美妙的感觉”。已经有阵子没有在上百人面前讲 过话了，竟然有些紧张。于是，趁机发挥吧，“我自从离开学生会就很少有机会在这么多人面前讲话了，因此，讲你们珍惜每一个在很多人面前讲话的机会，这至少对你是一个锻炼。”

尽管暑期实习的答辩我很不满意，但依然取得了优秀的成绩。做与不做是不一样的，加上我10个打印页的报告，老师是能看出好坏的。但如何能在众人面前把握好自己表达的重点，有简有繁地表达自己的思想，的确是我应该加强的地方。这两天去了baidu和P&G的校园宣进会，明白了原来大牛都是怎样炼成的 了。只那在近千人面前的自如的谈吐，便不是我这等小辈一朝一夕可以练成的。

昨天去找院里的辅导员签P&G的推荐信，里面要填英语水平，于是我写了CET6&TEM4。MM问我为什么不写TOEFL&GRE ，我愣了一下，说它们都不是资格认证考试。是啊，那两个考试是我为了出国才考的，我提醒自己，别忘了自己那个大洋彼岸的遥远的梦。

我的生活轻闲得让保研的同学都羡慕我的随心所欲，但谁又知道我心中的苦。。。毕竟保研的人已经有了一年后的着落，考研的人依靠三个多月的奋斗至少可以让自己 问心无愧，而我想起未来等offer的日子，便一阵冷汗，我可能在别人已经开始读研究生或者开始工作后，依然过着没有着落的日子，虽然我已经做好心理准 备，但我依然无法确定地想像出那是一种怎样的煎熬。现在确定自己的申请方向也让我头疼不已。。。而面对PS，我甚至不知道自己是谁。