Name: novel production protocol for small-scale manufacture of probiotic fermented foods
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作者承认在发酵乳生产地面上的实际支持，培训奶牛​​合作社乌干达农村东非奶业发展（EADD）项目。 CSK食品营养被确认为生产，质量控制和起动机文化的发酵能力的评估。 Lactosan GMBH＆CO，奥地利，被确认为生产提供援助。作者感谢卡琳Overkamp（TNO微生物学与系统生物学，宰斯特，荷兰）的HPLC分析，和德里克Assimwe（UIRI）记录在乌干达作者声明。这些录音的发酵食品终身项目乌干达恩德培，2016年五月在阿姆斯特丹VU大学和Micropia博物馆IDRC支持的启动会议（CIFSRF第2阶段）期间发生被确认为财政贡献和提供实验室设施制作这个视频文章中所述。作者感谢维姆面包车的Egmond酸奶细菌的显微镜。

1.鲜入门的制备为了准备新的启动器，取1升任超高温（UHT）处理的牛奶或新鲜牛奶。如果是UHT奶，继续执行步骤1.3。如果是新鲜牛奶，继续执行步骤1.1。 <ol><li>新鲜牛奶牛奶测试<ol><li>为感官试验，以检测异常或牛奶的变质，检查乳的视觉外观，检查它的气味。 </li><li>对于布上沸腾试验，牛奶的小样本转移到一汤匙。持上述热源的勺子，直到奶开始沸腾。在牛奶取下的热量和外观源勺子检测凝血的迹象，表明质量较差的牛奶。 </li><li>对于lactometer测试，补缸，烧杯玻璃或其他任何空心物体至少15厘米高的牛奶。放置在牛奶的lactometer。在30℃下印度文的28或更多的牛奶Lactometer读数吃了足够的密度的牛奶不添加水。 </li><li>对于乙醇试验中，使在蒸馏水80％的乙醇溶液。通过混合牛奶茶匙用80％的乙醇在一杯一茶匙混合牛奶等量与等量的80％的乙醇，例如。看混合物凝血检测的迹象，表明质量较差的牛奶。 </li></ol></li><li>当使用新鲜牛奶，过滤用筛或滤布（孔径0.1 - 0.5mm）的牛奶，以前在15分钟沸水杀菌。 </li><li>巴氏杀菌<ol><li>转移牛奶放入一个小锅里，用盖子封闭锅。放置锅在一个稍大的锅。填充更大的泛水，可达2厘米的小盘的边缘的下方。 </li><li>热火此设置使用的热量适当源（如电加热，燃气燃烧器，木炭炉，木生火），直到牛奶达到85°C，与实验室温度计测量。 </li><li>打开源程序的热量E要低并保持85℃温度30分钟。 </li></ol></li><li>删除与从与泛水牛奶锅，让它冷却。可选泛用冷水转移到泛加速冷却过程。不要从锅里取出盖子，除了考虑温度测量。 </li><li>让牛奶冷却到45℃，如实验室或厨房温度计测量。在这一点上，奶转移到真空烧瓶中，并通过加入种子培养物（1克）的一个分组的内容接种牛奶。 </li><li>离开接种牛奶在真空烧瓶16小时，以允许发酵发生。检查发酵乳，为具有pH计或pH试纸测得这应该是4.4或更小，的最终pH。 </li><li>发酵后，搅拌或摇动产品约5 - 10分钟，以获得平滑的质感。 </li><li>存放，直到下一个步骤。 <ol><li>对于家庭生产，倒入新鲜首发成冰块托盘，使10毫升冰块。放置在深冷冻机（-18℃）的冰块和3个月内第2继续进行。 </li><li>对于生产最多资源贫乏地区的100升的规模，继续第3节，从而保证一步3.5 1.5步完成后，开始16小时。另外，放置在冰箱（7°C）新鲜起动步1.6后5天内第3进行。 </li><li>对于生产各种非乳制品类发酵食品中的，继续进行第4部分。 </li></ol></li></ol> 2.生产在家庭层面的小批量<ol><li>用牛奶任何方便的金额（建议1 - 10升）。 </li><li>执行步骤1.1。 </li><li>执行步骤1.2。 </li><li>巴氏杀菌<ol><li>转移牛奶放入一个小锅里，用盖子封闭锅。放置锅在一个稍大的锅。填充更大的泛水，可达2厘米的小盘的边缘的下方。 </li><li>热火此设置使用的热量适当源（ 如电加热，燃气燃烧器，木炭炉，木生火），直到牛奶达到60°C，与实验室或厨房温度计测量。 </li><li>选择性:在这一点上，以5％，建议浓度添加糖（重量/体积），如用天平进行测定。与先前在15分钟沸水淹没巴氏杀菌混合勺子搅拌均匀。直至牛奶达到85°C，如用实验室或厨房温度计测量继续加热。 </li><li>打开热源为低，并保持85℃温度30分钟。 </li></ol></li><li>执行步骤1.4。 </li><li>让牛奶冷却到45℃，如实验室或厨房温度计测量。在这一点上，接种通过添加一个冰块牛奶（正如根据第1制备的）为每个升牛奶和奶转移到（一）真空烧瓶（多个）。 </li><li>离开接种牛奶保温瓶允许直至用pH计或pH试纸测定达到pH值为4.4的发酵发生。这估计需要12小时。 </li><li>执行步骤1.7。 </li><li>发酵乳转移到冰箱（7°C），并让其冷却食用前至少3小时。当根据适当的冷藏存储，该产品是最好的一个月内消耗。 </li></ol> 3.生产中的乡村环境中的资源贫乏的国家注:使用最多100升每1升新鲜牛奶首发。 <ol><li>执行步骤1.1。 </li><li>执行步骤1.2。 </li><li>巴氏杀菌: <ol><li>转移牛奶成适当大小的奶罐和用盖封闭罐。放置可以在一个大盆。填充泛水，最多可能的最高水平。 </li><li>热火这种设置使用的热量适当源（木之火是最快的，往往是最经济有效的），直到牛奶达到60°C，与实验室测量或厨房温度计。 </li><li>可选:根据不同口味偏好，每升将50g的比例加入白糖。与先前在15分钟沸水淹没巴氏杀菌混合勺子搅拌均匀。直至牛奶达到85°C，如用实验室或厨房温度计测量继续加热。 </li><li>减少热量并保持85℃温度30分钟。 </li></ol></li><li>从与泛水牛奶中取出即可，让它冷却。为了加快冷却过程中，所述罐传送到泛用冷水。不要从可去除盖子，除用于测量温度。 </li><li>让牛奶冷却到45℃，如实验室或厨房温度计测量。从冷水锅里取出即可。在这一点上，通过将完整的新鲜起动器（这永远不能过量）接种牛奶。 </li><li>环绕奶罐毯子隔离的目的和预防说唱在温度ID下降（理想情况下，温度将保持45和35℃之间）。离开接种牛奶罐以允许发生发酵，直至达到pH值为4.4。这个过程大约需要12小时。 </li><li>取下即可毯子，打开即可。 <ol><li>可选的:取出发酵乳的含水顶层，得到益生菌发酵乳（酸乳）的一平滑的纹理。 </li><li>选择性:在由10毫升的杯或匙剂量的0.1％的浓度（体积/体积）添加食品级人工香料（ 例如，草莓或香草），例如（取决于风味浓度和的优先客户）。 </li><li>可选:添加防腐剂。具有成本效益和安全的选择是0.12克/升的最大浓度使用山梨酸钾（来源:http://www.efsa.europa.eu/en/efsajournal/pub/4144建议3毫克/公斤体重/天）。 注意:用体重幼儿下面10千克应采取不铁道部Ë比最大30毫克山梨酸钾的服用剂量为250ml发酵乳，这是当对应于大约2汤匙每50 L的剂量</li><li>与先前通过在15分钟沸水浸没灭菌金属勺充分搅拌的发酵乳。 </li></ol></li><li>可选:包装的发酵乳，食用前转移到冰箱（7°C）和冷却至少3小时。 注意:如果在正常的冷藏储存，低于7°C，产品最好的两个星期内消耗掉。当根据适当的冷藏储存，并加入防腐剂3.7.3下所述的产物在4周内最好消耗。 </li></ol> 4.生产其他发酵食品本节介绍了生产比发酵乳等发酵食品的大约50 L的最大容积<ol><li>通过在升煮沸的水淹没一个巴氏杀菌纱布东15分钟。领带松散奶酪布盖在碗或盘可以通过使用绳子或橡皮筋容纳约2升。 </li><li>倒在纱布新鲜首发，避免溢出，并保持碗的底部和棉布之间足够的空间。 </li><li>放在冰箱约7°C碗8-12小时，直到大约0.5升乳清已经从酸奶放入碗中排水。 </li><li>使用乳清，使不同类型的发酵食品。实施例在下面给出。 注:所获得的发酵食品将包含乳清（牛奶）。牛奶和其衍生物引起过敏的食物和不耐受的最常见的成分之间的认可。 <ol><li>用于生产Obushera，高粱或小米基于乌干达饮料遵循22 Mukisa 等人描述的协议。 </li><li>用于生产宇治，从玉米或高粱或两者的混合物制成的肯尼亚粥，遵循歼使用的协议农业技术大面肯雅塔大学在肯尼亚和科尔特等人。15。 </li><li>对于生产Zomkom，布基纳法索基于高粱饮料，遵循Christèle 等 23科尔特等 15所描述的协议。 </li><li>对于生产Mutandabota，从猴面包树和牛奶的果实，被广泛食用南部非洲产品遵循姆波富等 24描述的过程。注意，对于此产品是在第1得到的新鲜起动，可直接使用而不首先进行步骤4.1 - 4.3。 </li></ol></li></ol>

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威尔伯特Sybesma及REMCO科尔特是Yoba生命基金会的创始人，一个不以盈利为目的的组织，由荷兰税务机关认定为公共慈善机构（PBI），旨在促进当地的生产和发酵产品在非洲消费。与Yoba种子培养制成发酵产品，包括酸奶，zomkom，obushera和mutandabota不是由基金会这样的市场，但Yoba生命根基刺激当地的生产和所有权，允许在食品行业非洲企业家的创收活动。该Yoba生命的基础分配，并通过合作伙伴和志愿者组成的网络销售与干菌种等准备使用的香袋，以促进本地生产乳制品和谷类为主所控制细菌发酵产品。

使用基本设备和这篇文章中描述了一种新的发酵食品发酵剂培养物的简单的生产协议有助于生产在家庭层面，并在资源贫乏的国家农村环境高品质的益生菌发酵乳。至关重要的是巴氏灭菌最少30分钟的牛奶，以便获得足够的减少或消除腐败微生物的和潜在的病原微生物的。此外，适当的加热导致乳清蛋白17的变性，这将随后与酪蛋白胶束25从而在发酵乳26形成交联键的关联。因此，最终产物的粘度的比较少广泛加热牛奶27增加。它也很关键，以避免在45℃以上的乳接种，因为在较高温度下的细菌将失活。 同样地，如果可用的和成本- 有效，使用UHT奶是优选的，特别是用于生产新鲜起动。首先，超高温灭菌奶是不必要的微生物几乎免费的。其次，在UHT牛奶乳清蛋白已被广泛地变性，这使得它们对细菌蛋白酶更方便，促进其降解和酸奶菌的后续生长。在冷却的过程中，蛋白质部分再折叠到其天然状态，因此重新加热之前接种为酸奶生产有利。长时间发酵的结果在pH &lt;4.​​4，这常常是不希望的发酵乳。发酵可以及时通过将发酵乳在冰箱被停止。 在非洲本地生产酸奶已被证明含有乳球菌28的显著水平如L.乳 ，这会导致生产具有苦味29-32肽的，这是通常undesirab乐31。因此，至关重要的是，发酵发生在一个温度为45和35℃之间15,28。由于温度将发酵期间在半受控环境慢慢降低，接种在45℃，允许的最高温度下进行。在发酵过程中足够的隔离是必要的，以避免在35℃以下的温度的降低。 手工生产在乡村环境的酸奶具有由腐败菌的微生物污染的风险相对较高。具体地，酵母是能够在酸奶中的酸性条件下进行传播。酵母污染可导致过多的气体的生产，无论是在孵化阶段或储存过程中，从而破坏了酸奶立即或显著缩短其货架寿命33,34。酵母水平可以通过加热灭菌和/或清洁的所有餐具的次氯酸钠和设备每个接触机智之前被最小化<html>小时牛奶或酸奶。已经发生酵母腐败特别是经过，严重和广泛的消毒和清洁要求，以消除污染。 以上概述的风险清楚地表明，在资源贫乏的国家，这种生产协议的应用的成功是由它的手动性质和与其相关联的增加的变质率的限制。食品变质的速度变得即使在缺乏冷却设施或有电力的大幅度波动的可用性较高的社区。另外，缺乏负担得起的高品质的包装材料可以限制所描述的协议的摄取。所需的设备是基本的和普遍的，但在实验室或厨房温度计是必不可少的。最后，目前只有有限的国家号码具有以产生冷冻干燥的起始培养设施。大多数资源贫乏的国家缺乏这种设施，因此依靠进口来获得这款产品。 _content"&gt;其中食品，发酵乳包括酸奶为益生菌最常见的载体。生产的发酵乳不从温度计除了需要特殊的设备，因此在上述的组合中描述第一通用益生菌起始培养13用简单的生产协议允许的重现性，控制和安全生产的益生菌发酵乳几乎任何人在任何地方，包括生产在农村地区的家庭和家庭手工业水平，资源贫乏的国家，在传统上实行天然发酵，益生菌同等水平细菌无法保证9。中和1g小袋自我稳定的发酵剂使小规模生产者成本有效地产生发酵的食物，而不需要购买大量的发酵剂打开包装后，通常丧失活力，特别是当保持在室温下和在高的相对湿度，这是据我们所知，所有其他可用的酸奶发酵剂培养的情况。此外，该被导入干燥的细菌数量，由于到在此协议中描述的预培养方法仍然很低，低于在最终产品中的益生菌培养浓度为0.1％，因为几乎所有的起始培养的局部产生。 所生产的发酵乳在本文中详细描述，可以掌握生产益生菌发酵的食物，随后可应用于发酵多种类型的其他食物（如本文中提到的）艺术的第一步。要升级基本发酵乳，一个可能的下一步是制作真正的水果发酵乳。这需要无菌果的制备保持或购买现成的水果（水果，糖和任选的一些添加剂的混合物）保持发酵35,36之后要与发酵乳混合。

本文介绍了一个生产协议，使生产营养丰富，益生菌发酵乳等益生菌发酵食品与已被证明健康的好处种子培养的帮助。该协议是强大和易于被任何人在任何地方执行，包括资源贫乏的国家。 在撒哈拉以南非洲地区，儿童死亡的21％，&lt;5岁的引起的腹泻1，其中29％是由特别是2轮状病毒引起的病因。它已被证明的发酵食品含有L的1×10 10 CFU的推荐剂量，每天消费鼠李 GG刺激肠道健康和减少轮状病毒相关性腹泻3-7的发生率和严重性。 有益细菌存在于各种传统发酵食品遍布撒哈拉以南非洲8-10。然而，这些食物几乎都是家庭PRODuced，有一个相对较短的保质期，并增加由于发酵11不受控制的本质安全风险。此外，这些传统食品的消费下降，由于城市化和饮食非洲10,12一般西化。 通用益生菌L.鼠李 yoba 2012 13，其是相同的L.鼠李 GG，原专利L.后导入鼠李糖 GG已经过期14。干燥的种子培养具有A w为约0.1，这使存活率高，在室温下至少一年的储存期之后（数据未显示）。 种子培养封装在1克部分和可用于生产最多100升的发酵乳的。当产生一个或两个批次，每天的起始培养可以帮助前夕从每周高达1000升的生产能力RY天的新鲜牛奶。 采用L的鼠李糖 yoba 2012年S.热 C106作为佐剂文化，使益生菌乳酸菌传播15是还没有被报道由其他作者独特的细菌联合。生产协议涉及在1L乳冷冻干燥起子培养（种子培养）的内容的一个包（1克）的悬浮，造成温育一段16小时之后的所谓新鲜起动。新鲜起动机随后用于一个最大的100升的乳接种。预孵育的这种方法减少使用，从而降低了生产成本相比，直接孵育如例如由戈夫16描述冷冻干燥的细菌的量。用于本文中所描述的协议的制剂保持冷冻干燥培养的优点，如货架寿命稳定性和容易储存和处理，从而产生高度řeproducible发酵。 此外，生产协议作为本文中所描述并不需要高科技设备，而不是在发达国家17使用的标准生产方法。 最后新鲜起动机可用于生产各种各样的发酵食品的原料的其它（非乳制品）接种作为成功所示Obushera，Kwete，宇治和Zomkom，所有基于谷类的传统发酵食品乌干达（前两个），肯尼亚和布基纳法索，分别为15。这仅仅是在非洲开发18发酵食品的大量的一小部分。 发酵是大分子，如淀粉，糖和蛋白质成产品已增加消化率，通过酶和/或微生物的活动的破裂。发酵是在撒哈拉以南非洲广泛使用的技术。火车带烘干和盐这醚使食品保鲜在没有降温设施或工业技术，如罐头9,19的。传统的发酵通常使用背喷溅，其中来自先前批的一小部分被添加到新的批次，以促进发酵，减少发酵失败11的概率。哈里森和汤姆金斯20描绘了广泛的基础上玉米，高粱或小米非洲传统发酵食品，和有益的作用这些食物可以减少儿童腹泻和相关的死亡率带给孩子断奶，例如。 NOUT和萨卡19描述了传统的生产，广泛消费在非洲西部的一个发酵的木薯菜。 Franz 等9提供的谷物和蔬菜的食物，含淀粉的块根作物，食品和动物蛋白质的食物归类非洲发酵的食物丰富的描述。除此之外，Narvhus和Gadaga <s达&gt; 21具体介绍了传统的生产在非洲发酵乳。 用于生产益生菌发酵乳（或其他发酵食品）尤其是在国内生产的情况下，冷冻机的可用性是有利的新鲜起动储存在冷冻部分。在资源贫乏的国家，生产的发酵乳可以很好地牛奶生产社区执行。在乌干达当地社区目前的经验表明，奶牛合作社是从他们自己的牛奶供应拿起酸奶生产出适合实体。牛奶罐的可用性是有利的，虽然它也是可能的，虽然较少优选，以在一个更大的锅制备的发酵乳大批量。此外，电力和资源贫乏的国家冷却当地的商店和亭设施与其接驳存在的可用性是在业务的拓展和减少°的优势˚F造成的损失变质。然而，如在该协议将讨论的，通过添加山梨酸钾能够保存数天的酸奶在环境温度和减少功率波动的情况下spoilages。

<table><tbody><tr><td>Yoba starter culture</td><td>Yoba for Life foundation</td><td>-</td><td></td></tr><tr><td>Milk</td><td>Obtained from local cow</td><td>-</td><td>Fresh, whole milk, free from antibiotics and obtained from a healthy cow</td></tr><tr><td>Optionally sugar</td><td>Bought at local supermarket</td><td>-</td><td>Canesuger was used, but beet sugar is also possible. Free from impurities</td></tr><tr><td>Laboratory Thermometer</td><td>Narang Scientific Industries</td><td>TM-01</td><td></td></tr><tr><td>Lactometer</td><td>Narang Scientific Industries</td><td>LM-01</td><td></td></tr><tr><td>Ethanol 96%</td><td>Sigma Aldrich</td><td>476226</td><td></td></tr><tr><td>pH paper</td><td>Macherey-Nagel GmbH &amp;amp; Co</td><td>90206</td><td></td></tr><tr><td>Cheesecloth</td><td>Beyond Gourmet</td><td>-</td><td>Any high quality food grade cheese cloth can do</td></tr><tr><td>Sieve/strainer</td><td>Cuisinart</td><td>CTG-00-3MS</td><td>Any fine mesh strainer can do, but stainless steel strainers are preferred.</td></tr><tr><td>1 L Thermos flask</td><td>Bought at local warehouse</td><td>-</td><td></td></tr><tr><td>1 L pan + lid</td><td>Bought at local warehouse</td><td>-</td><td>Stainless steel</td></tr><tr><td>2 L pan</td><td>Bought at local warehouse</td><td>-</td><td>Any heat- and waterproof material will do</td></tr><tr><td>50 L pan</td><td>Bought at local warehouse</td><td>-</td><td>Any heat- and waterproof material will do</td></tr><tr><td>Milk can</td><td>Narang Scientific Industries</td><td>MC-SS-30</td><td>Any stainless steel milkcan can do</td></tr><tr><td>Blanket</td><td>Bought at local warehouse</td><td>-</td><td>The thicker the blanket, the better the isolation</td></tr></tbody></table>

novel production protocol for small-scale manufacture of probiotic fermented foods

鼠李糖乳杆菌 yoba 2012和嗜热链球菌 C106的一种新颖的干燥细菌联合是在1升牛奶中培养。此新鲜起动器可用于生产发酵乳等发酵食品，无论在家中或在小规模在农村设置。对于新鲜起动器，1升牛奶巴氏消毒在配合到一个更大的含水泛锅，放置在热源。在此水浴，牛奶被加热并在85℃孵育30分钟。此后，将奶冷却至45℃，转移到真空烧瓶中，接种与干燥细菌和左为30℃和45℃之间的至少16小时。频繁家生产的目的，新鲜的起动器被冻成冰块，它可以用于生产高达2升的发酵乳的小体积。对于小规模生产在资源贫乏的国家，高达100升的牛奶我的巴氏杀菌的目的在奶罐进行S中的被放置在一个大的平底锅在85℃充满水，并加热在消防30分钟，随后冷却至45℃。接着，将100μL的批次中接种之前提到的1升新鲜制备的起动器。在30和45℃之间的温度下保证一种有效的发酵，牛奶可以覆盖有12小时的毛毯。用于生产非乳品发酵食品，新鲜的起动器被留在一个干酪布为12小时，并在排出截止乳清可以随后用于广泛的食品原料的接种，包括蔬菜和谷类基于食品。

表1的左侧示出了协议的第1节的示意图。这之后是在表上的右侧部2的一个广义结合图和3。 牛奶使用如本文中记录所述的制造协议的种子培养物接种发酵特性。 图1示出了新鲜起动器的发酵（如在第1节中所述）。 图2示出的发酵乳的发酵（最终产品，如在第2和3）中描述。因为在农村地区的实践严格的温度控制是困难的，测量在37℃的温度和45℃进行的。 在37℃下发酵，测试的两种小袋的酸化剖面显示在发病略有差别的约30分钟的指数酸化的，无论在生产新鲜起动和最终产品。两种培养物的最终pH值16小时后是相同的。 典型的pH值和滴度为传播L.鼠李糖和S.热菌 以下对不同的发酵食品的描述生产过程可在表2中找到。细胞计数为L.鼠李糖和S.热分别MRS琼脂培养基和LM17上确定的，通过使用常用的微生物电镀方法。 L.最终细胞计数鼠李糖 yoba 2012分别为1.7×10 7，3.7×10 9和3.3×10 9 CFU毫升-1发酵乳，分别kwete和宇治。最终pH值分别为4.3和4.2为发酵乳和kwete。初步结果表明，在加入POTAS的sium山梨酸在发酵前不产生负面影响，但即使提高了可行L.数量鼠李 yoba 2012细菌在酸奶后两周（数据未显示）的生产。 图3示出的高效液相色谱（HPLC）结合的非发酵乳的折射指数（RI）检测器相比，发酵乳的结果。结果表明，在牛奶减少乳糖的水平，而乳酸水平和半乳糖的水平增加，这是在种子培养的代谢活性的结果。 <img alt="图1" src="/files/ftp_upload/54365/54365fig1.jpg" /> 图1.发酵制成半成品的新鲜起动机的轮廓脱脂奶（1.5％脂肪，3.5％蛋白质）。1升牛奶中接种种子培养之一小袋（1克），在37℃下发酵（••••，重复），以及45℃（ - - - - ，复制）16小时。在pH值为4.4的直线（----）插入作为参考来确定何时发酵完成。 <a href="https://www.jove.com/files/ftp_upload/54365/54365fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54365/54365fig2.jpg" /> 图2.制成半成品的发酵乳（最终产品）的发酵轮廓脱脂奶（1.5％脂肪，3.5％蛋白质）。1升牛奶中接种新鲜起动机的一个2θg且在37℃下（发酵•• ••，重复），以及45℃（ - - - - ，复制）16小时。在pH 4.4的直线（----）插入作为参考，以确定在完成发酵时。<a href="https://www.jove.com/files/ftp_upload/54365/54365fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54365/54365fig3.jpg" /> 图3 的非发酵乳（黑色）和乳发酵用种子培养物（蓝色）16小时的HPLC洗脱模式。洗脱模式表明乳糖和生产半乳糖和乳酸的消耗。糖和弱酸被确定的保留时间的基础和紫外线吸光度和折射率的峰面积比对。 <a href="https://www.jove.com/files/ftp_upload/54365/54365fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54365/54365tbl1.jpg" /> 表1.种子APPLICA文化<html>化协议。 <a href="https://www.jove.com/files/ftp_upload/54365/54365tbl1large.jpg" target="_blank">请点击此处查看该表的放大版本。</a> <img alt="表2" src="/files/ftp_upload/54365/54365tbl2.jpg" /> 表2 中的各种数据的发酵食品。 鼠李糖乳杆菌 yoba 2012 的传输被从科尔特等人 15获得。发酵在37℃下进行，除非另有说明。 L的细胞计数rhamnosu小号yoba 2012年和S.热 C106接种后两个多样1和1之间×10 6×10 7 CFU毫升-1。官能mutandabota用含有完全L的起动机制备鼠李糖 yoba 2012所报告的姆波富等24。 ND，未测定。D / 54365 / 54365tbl2large.jpg"目标="_空白"&gt;请点击这里查看此表的放大版本。 补充文件，本地生产和非洲农村地区销售的益生菌酸奶。拍摄和照片拍摄于Balawoli和Namagera，乌干达，2016年五月的片段展示了价值链从牛奶接待加工，包装，销售，最后一批50升的益生菌酸奶的消费量开始，随用随制只有基本设备。 <a href="https://www.jove.com/files/ftp_upload/54365/yoba_production_in_Uganda_suppl_material.mp4" target="_blank">请点击这里下载。</a>

Watch this Scientific Journal Video about Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods at JoVE.com

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

对于益生菌发酵食品的小规模生产新生产协议

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

novel-production-protocol-for-small-scale-manufacture-probiotic

Nanyang Technological University

Research

JoVE Journal

Biology

23.6K 次观看.  Yoba for Life Foundation. A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture. 

视频: Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

科研

环境科学

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

严格厌氧/缺氧条件下微生物培养的介质制备

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

生物化学

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

优质发酵鱼产品的制备

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

生物工程

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

免疫与感染

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

乳酸菌的培养、生长和活力:质量控制视角

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...