Interaction between Dairy Yeasts and Lactobacillus rhamnosus GG in Milk

第30卷　第1期2008年1月北　京　林　业　大　学　学　报JOURNA L OF BEI J I NG FORESTRY UNI VERSITYV ol.30,N o.1Jan.,2008收稿日期:200622112210http :ΠΠw w ,http :ΠΠ 基金项目:国家自然科学基金项目(30570990)、黑龙江省科技厅重点攻关项目(G B05B104)。

第一作者:王琪。

主要研究方向:植物分子生物学与基因工程。

电话:04512255190734　Email :wangqi198169@1631com 地址:150030黑龙江省哈尔滨市东北农业大学生命中心植物生物工程研究室。

责任作者:朱延明,教授。

主要研究方向:植物分子生物学与基因工程。

电话:04512255190734　E m ail :ym zhu2001@yah 地址:同上。

酵母单杂交系统在植物基因工程研究中的应用王　琪　朱延明　王冬冬(东北农业大学生命科学学院)摘要:酵母单杂交系统是由酵母双杂交系统衍生来的研究DNA 与蛋白质之间相互作用的新型系统。

该文系统阐述了单杂交系统的基本原理和技术路线,详细综述了其在植物基因工程各个领域的应用研究进展,即克隆抗渗透胁迫类转录因子基因,确定已知DNA 与蛋白质之间的相互作用,定位已证实的具有相互作用的DNA 结合结构域以及验证转录激活作用;并分析了当前该系统在植物基因工程研究中存在的问题,进而结合自己的研究对解决问题的途径进行了探讨。

关键词:酵母单杂交;植物基因工程;转录因子;转录激活结构域;DNA 结合结构域中图分类号:Q94312 文献标识码:A 文章编号:1000221522(2008)012201412207W ANG Qi ;ZH U Y an 2ming ;W ANG D ong 2dong.Application of yeast one hybrid system in plant geneengineering .Journal o f Beijing Forestry Univer sity (2008)30(1)14122147[Ch ,42ref.]C ollege of Life Science ,N ortheast Agricultural University ,Harbin ,150030,P.R.China.The yeast one hybrid system is the new type system deriving from yeast tw o hybrid system to study the interactions between DNA and protein.This paper briefly introduces the principle and technical line of this system ,and particularly summarizes the progress of this system in plant genetic engineering field ,such as cloning the transcription factor genes of osm osis resistance type ,of embry ogenesis type ,and of disease resistance type ;confirming the interaction between known DNA and protein ;making sure the localization of the confirmed DNA binding domain ;m oreover validating the transcriptional activation activity.Furtherm ore ,the authors analyzed the problems existing in this system on the research of plant genetic engineering ,as well as forecasted the development prospect according to the research.K ey words yeast one hybrid ;plant genetic engineering ;transcription factor ;transcription activation domain ;DNA binding domain 近年来,植物学的研究已经从基因组时代转向到后基因组时代。

The Fascinating World of Fermented FoodsFermentation is one of the oldest and most widespread food preservation techniques known to humanity.This ancient process,which involves the transformation of food by microorganisms,has given rise to a diverse array of flavors,textures,and nutritional benefits.From tangy sauerkraut to creamy yogurt,fermented foods are enjoyed across cultures and have become a staple in many diets.This essay delves into the fascinating world of fermented foods,exploring their history,the science behind fermentation,and the various health benefits they offer.A Brief History of FermentationFermentation has been practiced for thousands of years,with evidence of fermented foods dating back to ancient civilizations.The earliest records of fermentation can be traced to the Neolithic period,around 7000-6600BCE,when people began to ferment grains and fruits to produce alcoholic beverages.Ancient Egyptians,Greeks,and Romans also embraced fermentation,using it to make bread,wine,and cheese.Throughout history,fermentation has played a crucial role in food preservation,allowing communities to store food for extended periods. This was particularly important before the advent of refrigeration. Fermented foods also became integral to cultural and religious practices, with many traditional recipes being passed down through generations. The Science of FermentationFermentation is a metabolic process in which microorganisms such as bacteria,yeast,and molds convert sugars and other carbohydrates into alcohol,acids,and gases.This process not only preserves food but also enhances its flavor,texture,and nutritional value.There are several types of fermentation,each involving different microorganisms and resulting in distinct products:Lactic Acid Fermentation:This type of fermentation is carried out by lactic acid bacteria,which convert sugars into lactic acid.It is responsible for the tangy taste and extended shelf life of foods like yogurt, sauerkraut,kimchi,and pickles.Alcoholic Fermentation:Yeasts,particularly Saccharomyces cerevisiae, convert sugars into alcohol and carbon dioxide.This process is used to produce alcoholic beverages such as beer,wine,and sake,as well as leavened bread.Acetic Acid Fermentation:Acetic acid bacteria convert alcohol into acetic acid,resulting in the production of vinegar.This type of fermentation is used to make various types of vinegar,including apple cider vinegar and balsamic vinegar.Mold Fermentation:Certain molds,such as Aspergillus oryzae and Penicillium roqueforti,are used in the fermentation of foods like soy sauce,miso,and blue cheese.These molds contribute to the unique flavors and textures of these products.Health Benefits of Fermented FoodsFermented foods are not only delicious but also offer a range of health benefits.The fermentation process enhances the nutritional profile of foods and introduces beneficial microorganisms,known as probiotics, which support gut health.Some of the key health benefits of fermented foods include:Improved Digestion:Probiotics in fermented foods help maintain a healthy balance of gut bacteria,which is essential for proper digestion. They can alleviate symptoms of digestive disorders such as irritable bowel syndrome(IBS)and reduce bloating and gas.Enhanced Nutrient Absorption:Fermentation breaks down complex compounds in food,making nutrients more bioavailable.For example, the fermentation of dairy products increases the availability of calcium and B vitamins.Boosted Immune System:A healthy gut microbiome is closely linked to a strong immune system.Probiotics in fermented foods can enhance the body's ability to fight off infections and reduce inflammation. Reduced Risk of Chronic Diseases:Regular consumption of fermented foods has been associated with a lower risk of chronic diseases such as heart disease,diabetes,and certain cancers.The antioxidants and anti-inflammatory compounds produced during fermentation contribute to these protective effects.Mental Health Benefits:Emerging research suggests that gut health is connected to mental health through the gut-brain axis.Probiotics in fermented foods may help alleviate symptoms of anxiety and depression by promoting a healthy gut microbiome.Popular Fermented Foods Around the WorldFermented foods are enjoyed in various forms across different cultures, each with its unique flavors and traditions.Here are some popular fermented foods from around the world:Yogurt:A staple in many diets,yogurt is made by fermenting milk with lactic acid bacteria.It is known for its creamy texture and tangy flavor.Sauerkraut:This German delicacy is made by fermenting shredded cabbage with salt.The result is a tangy,crunchy,and probiotic-rich food.Kimchi:A traditional Korean dish,kimchi is made by fermenting vegetables,usually cabbage and radishes,with chili peppers,garlic, ginger,and fish sauce.It is known for its spicy and pungent flavor.Kombucha:A fermented tea beverage,kombucha is made by fermenting sweetened tea with a symbiotic culture of bacteria and yeast(SCOBY).It is fizzy,tangy,and slightly sweet.Tempeh:Originating from Indonesia,tempeh is made by fermenting soybeans with a mold called Rhizopus.It has a firm texture and nutty flavor,making it a popular plant-based protein source.Miso:A staple in Japanese cuisine,miso is a fermented soybean paste used to flavor soups,sauces,and marinades.It has a rich,umami flavor.Cheese:Various types of cheese,such as blue cheese,cheddar,and brie, are made through fermentation.The process involves the action of bacteria and molds,contributing to the distinct flavors and textures of each cheese.ConclusionThe fascinating world of fermented foods offers a rich tapestry of flavors, textures,and health benefits.From ancient preservation techniques to modern culinary delights,fermentation has played a vital role in shaping our diets and cultures.By embracing fermented foods,we can enjoy their unique tastes and reap the numerous health benefits they provide. Whether it's a spoonful of tangy yogurt,a bite of spicy kimchi,or a sip of fizzy kombucha,fermented foods continue to captivate and nourish people around the world.。

廖娟，殷智华，李嘉宇，等. 酵母发酵对米糠挥发性风味物质及营养特性的影响[J]. 食品工业科技，2023，44（22）：266−274. doi:10.13386/j.issn1002-0306.2023010169LIAO Juan, YIN Zhihua, LI Jiayu, et al. Effect of Yeast Fermentation on Volatile Flavor Substances and Nutritional Properties of Rice Bran[J]. Science and Technology of Food Industry, 2023, 44(22): 266−274. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023010169· 分析检测 ·酵母发酵对米糠挥发性风味物质及营养特性的影响廖　娟1,2，殷智华1,2，李嘉宇1,2，杨　涛1,2,*（1.中南林业科技大学食品科学与工程学院，湖南长沙 410004；2.稻谷及副产物深加工国家工程研究中心，湖南长沙 410004）摘　要：为探究酵母发酵对米糠风味以及营养特性的影响。

本研究采用顶空固相微萃取-气相色谱-质谱技术鉴定不同发酵时期（0、12、18、24、30 h ）米糠的挥发性风味物质，通过正交偏最小二乘分析确定影响米糠发酵前后风味的关键性挥发性物质，最后对发酵米糠进行了综合感官评价并比较了发酵最佳时间对其营养组成变化。

结果表明：发酵过程中米糠的挥发性物质含量和种类发生明显变化，未发酵米糠中醛类含量占比35.99%，醇类含量占比14.21%，发酵30 h 醛类含量下降到5.52%，醇类含量提高到60.87%。

其中关键性挥发性物质共15种。

在感官评价方面，关键挥发性物质与米糠香气之间有显著相关性（P <0.05或P <0.01），发酵后米糠感官评分提高，发酵18 h 的米糠感官评分最佳。

Plant-Microbe Interactions in theRhizospherePlant-microbe interactions in the rhizosphere are a crucial aspect of the soil ecosystem, playing a significant role in plant growth, nutrient uptake, andoverall soil health. The rhizosphere is the narrow region of soil that is directly influenced by the roots of plants, where a complex network of interactions occurs between the plant, soil, and various microorganisms. These interactions can beboth beneficial and detrimental, depending on the specific microorganisms involved and the environmental conditions. Understanding the dynamics of plant-microbe interactions in the rhizosphere is essential for developing sustainableagricultural practices and improving crop productivity. One of the most important aspects of plant-microbe interactions in the rhizosphere is the exchange of nutrients between the plant and the microorganisms. Plants release a variety of compounds, such as sugars, amino acids, and organic acids, into the rhizosphere through their roots. These compounds serve as an energy source for the diverse microbial community in the soil, including bacteria, fungi, and archaea. In return, the microorganisms help the plant acquire essential nutrients, such as nitrogen, phosphorus, and iron, by solubilizing and mineralizing soil nutrients, making them more available for plant uptake. This mutualistic relationship between plants and microorganisms is crucial for the overall health and productivity of the plant.In addition to nutrient exchange, plant-microbe interactions in the rhizosphere also play a vital role in plant defense against pathogens. Certain microorganismsin the rhizosphere, known as plant growth-promoting rhizobacteria (PGPR), have been shown to stimulate plant growth and enhance resistance to diseases. These beneficial microorganisms can directly inhibit the growth of plant pathogens by producing antimicrobial compounds or competing for space and resources in the rhizosphere. Furthermore, PGPR can also induce systemic resistance in plants, activating their defense mechanisms against a wide range of pathogens. Understanding the mechanisms by which PGPR confer disease resistance to plants can have significant implications for reducing the reliance on chemical pesticides in agriculture. However, not all plant-microbe interactions in the rhizosphere arebeneficial. Some microorganisms can have detrimental effects on plant health, causing diseases and reducing crop yields. For example, soil-borne pathogens, such as Fusarium and Phytophthora species, can infect plant roots and cause root rot, leading to stunted growth and wilting of the plant. These pathogenic microorganisms can outcompete beneficial microbes in the rhizosphere, disrupting the delicate balance of the soil ecosystem. Understanding the factors that contribute to the proliferation of pathogenic microorganisms in the rhizosphere is essential for developing effective strategies to manage plant diseases and maintain soil health. Moreover, the composition and diversity of the microbial community in the rhizosphere are influenced by various factors, including soil type, plant species, and environmental conditions. Different plants release different types and amounts of root exudates, which can selectively promote the growth of specific groups of microorganisms in the rhizosphere. Furthermore, the physical and chemical properties of the soil, such as pH, moisture, and organic matter content, can also have a significant impact on the structure and function of the rhizosphere microbial community. Understanding the complex interplay between these factors and their effects on plant-microbe interactions is crucial for optimizing soil management practices and promoting sustainable agriculture. In conclusion, plant-microbe interactions in the rhizosphere are a dynamic and intricate network of relationships that profoundly impact plant growth, nutrient cycling, and soil health. The exchange of nutrients, the promotion of plant defense mechanisms, and the influence of environmental factors all contribute to the complexity of these interactions. By gaining a deeper understanding of the mechanisms underlying plant-microbe interactions in the rhizosphere, we can develop innovative strategies to enhance crop productivity, reduce the reliance on chemical inputs, and promote sustainable agricultural practices. Ultimately, this knowledge can contribute to the development of a more resilient and environmentally friendly agricultural system, benefiting both farmers and the broader ecosystem.。

2020届硕士学位论文蛋白质组学解析酸面团中植物乳杆菌和酿酒酵母的互作机制作者姓名王伟指导教师张国华副教授学科专业食品工程研究方向传统酸面团的组学研究培养单位生命科学学院学习年限 2018年9月至2020年6月二〇二〇年六月山西大学2020届硕士学位论文蛋白质组学解析酸面团中植物乳杆菌和酿酒酵母的互作机制作者姓名王伟指导教师张国华副教授学科专业食品工程研究方向传统酸面团的组学研究培养单位生命科学学院学习年限2018年9月至2020年6月二〇二〇年六月Thesis for Master’ s Degree, Shanxi University, 2020Proteomics Analysis of the Interaction Mechanism Between Lactobacillus plantarum and Saccharomyces cerevisiae inSourdoughStudent Name Wei WangSupervisor Associate Prof. Guohua ZhangMajor Food EngineeringSpecialty Omics Research on TraditionalSourdoughDepartment School of Life ScienceResearch Duration 2018.09-2020.06June, 2020目录中文摘要 (I)ABSTRACT (III)第一章文献综述 (1)1.1 酸面团概述 (1)1.1.1 酸面团简介 (1)1.1.2 酸面团中的微生物群落 (1)1.1.3 酸面团发酵的优势 (2)1.1.4 酸面团的国外研究进展 (3)1.1.5 酸面团的国内研究进展 (4)1.2 酸面团中酵母菌多样性及酿酒酵母概述 (4)1.2.1 酸面团中酵母菌多样性 (5)1.2.2 酿酒酵母的生理生化特性 (10)1.2.3 酿酒酵母中的主要代谢途径 (10)1.2.4 酿酒酵母的生理功能及其在食品中的应用 (12)1.3 酸面团中乳酸菌多样性及植物乳杆菌概述 (14)1.3.1 植物乳杆菌的生化特性 (14)1.3.2 植物乳杆菌的生理功能 (14)1.3.3 植物乳杆菌在食品中的应用 (16)1.4 蛋白质组学 (17)1.4.1 蛋白质组学简介 (17)1.4.2 蛋白质组学常用的研究方法 (18)1.5 本论文的研究内容及意义 (19)第二章酸面团中微生物多样性及植物乳杆菌Sx3和酿酒酵母Sq7的研究 (20)2.1 材料和仪器 (20)2.1.1 菌株和培养基 (20)2.1.2 仪器与设备 (20)2.2 实验方法 (20)2.2.1 植物乳杆菌Sx3的培养 (20)2.2.2 酸面团中植物乳杆菌Sx3生长曲线的测定 (21)2.2.3 植物乳杆菌Sx3菌落数的测定 (21)2.2.4 植物乳杆菌Sx3发酵酸面团pH、TTA测定 (21)2.2.5 酿酒酵母Sq7的培养 (22)2.2.6 酸面团中酿酒酵母Sq7生长曲线的测定 (22)2.2.7 酿酒酵母Sq7发酵酸面团pH、TTA测定 (22)2.2.8 植物乳杆菌Sx3和酿酒酵母Sq7共发酵酸面团中菌落数、pH及TTA的测定 (22)2.2.9 高通量测序分析酸面团中细菌多样性 (23)2.2.10 高通量测序分析酸面团中真菌多样性 (27)2.3 结果和讨论 (27)2.3.1 菌落数、pH和TTA的测定结果 (27)2.3.2 不同发酵酸面团中微生物多样性 (29)2.4 结论 (30)第三章无标签定量蛋白质组学揭示酸面团中蛋白质的变化 (31)3.1 材料和仪器 (31)3.1.1 实验材料 (31)3.1.2 仪器及软件 (31)3.1.3 试剂和耗材 (32)3.2 实验方法 (32)3.2.1 蛋白质提取 (32)3.2.2 BCA法定量蛋白质浓度 (33)3.2.3 聚丙烯酰胺凝胶电泳 (33)3.2.4 还原烷基化和酶解 (33)3.2.5 肽脱盐和定量 (34)3.2.6 LC-MS/MS分析 (34)3.2.7 数据分析 (35)3.2.8 KEGG通路注释 (36)3.3 结果与讨论 (36)3.3.1 碳水化合物代谢 (36)3.3.2 氨基酸代谢 (42)3.3.3 核糖体蛋白变化 (44)3.4 结论 (47)第四章结论 (49)参考文献 (50)攻读学位期间取得的研究成果 (61)致谢 (62)个人简况及联系方式 (63)承诺书 (64)学位论文使用授权声明 (65)ContentsChinese Abstract (I)Abstract (III)Chapter 1 Review of the literature (1)1.1 Overview of sourdough (1)1.1.1 Introduction of sourdough (1)1.1.2 Microorganisms in traditional sourdough (1)1.1.3 Advantages of traditional sourdough (2)1.1.4 Research progress of foreign sourdough (3)1.1.5 Research progress of domestic sourdough (4)1.2 Diversity of yeast microbiota in sourdough and S. cerevisiae (4)1.2.1 Diversity of yeast microbiota in sourdough (5)1.2.2 Physiological and biochemical characteristics of S. cerevisiae (10)1.2.3 The main metabolic pathways of S. cerevisiae (10)1.2.4 The physiological function of S. cerevisiae and its application in food (12)1.3 Diversity of LAB microbiota in sourdough (14)1.3.1 Physiological and biochemical characteristics of L. plantarum (14)1.3.2 L. plantarum physiological function (14)1.3.3 Application of L. plantarum in food (16)1.4 Proteomics (17)1.4.1 Introduction to proteome and proteomics (17)1.4.2 Common methods for proteomics research (18)1.5 Research significance and content of this thesis (19)Chapter 2 Microbial diversity of sourdough and the study of L. plantarum Sx3 and S. cerevisiae Sq7 (20)2.1 Materials and instruments (20)2.1.1 Strains and media (20)2.1.2 Instruments (20)2.2 Methods (20)2.2.1 Incubation of L. plantarum Sx3 (20)2.2.2 Determination of Sx3 growth curve in sourdough (21)2.2.3 Determination of the number of Sx3 colonies (21)2.2.4 Determination of pH and TTA of Sx3 single culture sourdough (21)2.2.5 Incubation of S. cerevisiae Sq7 (22)2.2.6 Determination of Sq7 growth curve in sourdough (22)2.2.7 Determination of the number of colonies, pH and TTA of Sq7 single culturesourdough (22)2.2.8 Determination of the number of colonies, pH and TTA of co-cultivationsourdough (22)2.2.9 High-throughput sequencing explores bacterial diversity in sourdough (23)2.2.10 High-throughput sequencing explores fungal diversity in sourdough (27)2.3 Results and discussion (27)2.3.1 The result of colonies, pH and TTA (27)2.3.2 Microbial diversity of different sourdough (29)2.4 Conclusions (30)Chapter 3 Label-free quantitative proteomics study of protein changes in sourdough (31)3.1 Materials and instruments (31)3.1.1 Experiment material (31)3.1.2 Instruments and software (31)3.1.3 Reagents and supplies (32)3.2 Methods (32)3.2.1 Protein extraction (32)3.2.2 BCA method for quantitative protein concentration (33)3.2.3 SDS-PAGE (33)3.2.4 Reductive alkylation and enzymolysis (33)3.2.5 Peptide desalting and quantification (34)3.2.6 LC-MS/MS analysis (34)3.2.7 Data analysis (35)3.2.8 KEGG pathway notes (36)3.3 Results and discussion (36)3.3.1 Carbohydrate metabolism (36)3.3.2 Amino acid metabolism (42)3.3.3 Translation (44)3.4 Conclusions (47)Chapter 4 Conclusions (49)References (50)Research achievements (61)Aknowledgements (62)Personal profiles (63)Commitment (64)Authorization statement (65)中文摘要酸面团是一种面粉与水的混合物，是由乳酸菌、酵母、霉菌和其它微生物组成的混合发酵系统。

杨雯，胡海明，刘洪涛，等. 两株代谢咖啡酸人肠道来源菌株的筛选、鉴定及其代谢过程初探[J]. 食品工业科技，2024，45（3）：137−145. doi: 10.13386/j.issn1002-0306.2023030023YANG Wen, HU Haiming, LIU Hongtao, et al. Screening and Identification of Two Human Intestinal Strains Metabolizing Caffeic Acid and Exploring Their Metabolic Processes[J]. Science and Technology of Food Industry, 2024, 45(3): 137−145. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023030023· 生物工程 ·两株代谢咖啡酸人肠道来源菌株的筛选、鉴定及其代谢过程初探杨　雯1，胡海明2，刘洪涛2，魏晓博1，刘慧燕1, *，方海田1,*（1.宁夏大学食品科学与工程学院，宁夏食品微生物应用技术与安全控制重点实验室，宁夏银川 750021；2.湖北中医药大学基础医学院，湖北武汉 430065）摘　要：为了探索咖啡酸的体内代谢过程，本研究从人体肠道粪便中分离筛选出2株能代谢咖啡酸的菌株，通过细胞形态、16S rDNA 序列和系统发育树分析了其菌株特征，并研究了两株菌株代谢咖啡酸过程中的细菌总数和pH 变化，最后采用薄层层析法和高效液相色谱法对代谢产物进行确定，阐明咖啡酸代谢过程。

结果表明，2株菌分别为腐生葡萄球菌（Staphylococcus xylosus ）和奇异变形杆菌（Proteus mirabilis ）。

腐生葡萄球菌代谢咖啡酸含量从0.539 mg/mL 在6 h 后降至0.087 mg/mL ，12 h 后完全被代谢。

146/Enzyme-Modified Fats/Monographs FCC VWater Determine as directed under Water Determination, Appendix IIB.Packaging and Storage Store in well-closed containers. Enzyme-Modified FatsDESCRIPTIONEnzyme-Modified Fats occur as light to medium tan liquids, pastes,or powders with a strong fatty acid odor and flavor. They are produced by enzyme lipolysis of fats obtained from milk,refined beef fat,or steam-rendered chicken fat,using suitable food-grade enzymes.Enzyme-modified milkfat may be prepared from milk,concentrated milk,dry whole milk, cream,concentrated cream(s),dry cream,butter,butter oil, dried butter,or anhydrous milkfat.For enzyme-modified milk-fat,optional dairy ingredients such as skim milk,concentrated skim milk,nonfat dry milk,buttermilk,concentrated butter-milk,dried buttermilk,liquid whey,concentrated whey,and dried whey may be used to adjust the concentration of the flavors.Fat emulsions are reacted with suitable food-grade enzymes under controlled conditions to increase the flavor components.Thermoprocessing is then used to destroy the enzyme activity and provide acceptable microbiological qual-ity.Suitable preservatives,emulsifiers,buffers,stabilizers, and antioxidants as well as sodium chloride may be added. The resulting product is concentrated or dried.Function Flavoring agent.REQUIREMENTSLabeling Indicate the Acid Value.Identification A sample has a very strong fatty acid odor. Acid Value Not less than98.0%and not more than102.0% of the labeled value.Lead Not more than1mg/kg.Loss on Drying Not more than4.0%for the dry product. Microbial Limits:Aerobic Plate Count Not more than10,000CFU per gram.Coliforms Not more than10CFU per gram. Salmonella Negative in25g.Staphylococcal Enterotoxins Negative in1g. Staphylococcus aureus Not more than100CFU per gram.Yeasts and Molds Not more than10CFU per gram.TESTSAcid Value Determine as directed in Method II under Acid Value,Appendix VII,using a5-g sample.Lead Determine as directed for Method II in the Atomic Absorption Spectrophotometric Graphite Furnace Method un-der Lead Limit Test,Appendix IIIB.Loss on Drying Determine as directed under Loss on Dry-ing,Appendix IIC,drying a sample at105°for48h. Microbial Limits(Note:Current methods for the following tests may be found online at</~ebam/ bam-toc.html>):Aerobic Plate CountColiformsSalmonellaStaphylococcal EnterotoxinsStaphylococcus aureusYeasts and MoldsPackaging and Storage Store in tight containers in a cool place.Enzyme PreparationsDESCRIPTIONEnzyme Preparations used in food processing are derived from animal,plant,or microbial sources(see Classification, below).They may consist of whole cells,parts of cells,or cell-free extracts of the source used,and they may contain one active component or,more commonly,a mixture of several,as well as food-grade diluents,preservatives,antioxidants,and other substances consistent with good manufacturing prac-tices.The individual preparations usually are named according to the substance to which they are applied,such as Protease or Amylase.Traditional names such as Malt,Pepsin,and Rennet also are used,however.The color of the preparations—which may be liquid,semi-liquid,or dry—may vary from virtually colorless to dark brown.The active components consist of the biologically active proteins,which are sometimes conjugated with metals, carbohydrates,and/or lipids.Known molecular weights of the active components range from approximately12,000to several hundred thousand.The activity of enzyme preparations is measured according to the reaction catalyzed by individual enzymes(see below) and is usually expressed in activity units per unit weight of the preparation.In commercial practice(but not for FoodFCC V Monographs/Enzyme Preparations/147Chemicals Codex purposes),the activity of the product is sometimes also given as the quantity of the preparation to be added to a given quantity of food to achieve the desired effect. Additional information relating to the nomenclature and the sources from which the active components are derived is provided under Enzyme Assays,Appendix V.Function Enzyme(see discussion under Classification, below).CLASSIFICATIONAnimal-Derived PreparationsCatalase,Bovine Liver Produced as partially purified liq-uid or powdered extracts from bovine liver.Major active principle:catalase.Typical application:used in the manufac-ture of certain cheeses.Chymotrypsin Obtained from purified extracts of bovine or porcine pancreatic tissue.Produced as white to tan,amorphous powders soluble in water,but practically insoluble in alcohol, in chloroform,and in ether.Major active principle:chymotryp-sin.Typical application:used in the hydrolysis of protein. Lipase,Animal Obtained from the edible forestomach tis-sue of calves,kids,or lambs;and from animal pancreatic tissue.Produced as purified edible tissue preparations or as aqueous extracts dispersible in water,but insoluble in alcohol. Major active principle:lipase.Typical applications:used in the manufacture of cheese and in the modification of lipids. Lysozyme Obtained from extracts of purified chicken egg whites.Generally prepared and used in the hydrochloride form as a white powder.Major active principle:lysozyme.Typical application:used as an antimicrobial in food processing. Pancreatin Obtained from porcine or bovine(ox)pancreatic tissue.Produced as a white to tan,water-soluble powder. Major active principles:(1)␣-amylase;(2)protease;and(3) lipase.Typical applications:used in the preparation of pre-cooked cereals,infant foods,and protein hydrolysates. Pepsin Obtained from the glandular layer of hog stomach. Produced as a white to light tan,water-soluble powder;amber paste;or clear,amber to brown,aqueous liquids.Major active principle:pepsin.Typical applications:used in the preparation of fishmeal and other protein hydrolysates and in the clotting of milk in manufacture of cheese(in combination with rennet). Phospholipase A2Obtained from porcine pancreatic tissue. Produced as a white to tan powder or pale to dark yellow liquid.Major active principle:phospholipase A2.Typical ap-plication:used in the hydrolysis of lecithins.Rennet,Bovine Aqueous extracts made from the fourth stomach of bovines.Produced as a clear,amber to dark brown liquid or a white to tan powder.Major active principle:prote-ase(pepsin).Typical application:used in the manufacture of cheese.Similar preparations may be made from the fourth stomach of sheep or goats.Rennet,Calf Aqueous extracts made from the fourth stom-ach of calves.Produced as a clear,amber to dark brown liquid or a white to tan powder.Major active principle:protease (chymosin).Typical application:used in the manufacture of cheese.Similar preparations may be made from the fourth stomach of lambs or kids.Trypsin Obtained from purified extracts of porcine or bo-vine pancreas.Produced as white to tan,amorphous powders soluble in water,but practically insoluble in alcohol,in chloro-form,and in ether.Major active principle:trypsin.Typical applications:used in baking,in the tenderizing of meat,and in the production of protein hydrolysates.Plant-Derived PreparationsAmylase Obtained from extraction of ungerminated barley. Produced as a clear,amber to dark brown liquid or a white to tan powder.Major active principle:␤-amylase.Typical applications:used in the production of alcoholic beverages and sugar syrups.Bromelain The purified proteolytic substance derived from the pineapples Ananas comosus and Ananas bracteatus L. (Fam.Bromeliaceae).Produced as a white to light tan,amor-phous powder soluble in water(the solution is usually color-less to light yellow and somewhat opalescent),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:bromelain.Typical applications:used in the chill-proofing of beer,in the tenderizing of meat,in the preparation of precooked cereals,in the production of protein hydroly-sates,and in baking.Ficin The purified proteolytic substance derived from the latex of Ficus sp.(Fam.Moraceae),which include a variety of tropical fig trees.Produced as a white to off white powder completely soluble in water.(Liquid fig latex concentrates are light to dark brown.)Major active principle:ficin.Typical applications:used in the chillproofing of beer,in the tenderiz-ing of meat,and in the conditioning of dough in baking. Malt The product of the controlled germination of barley. Produced as a clear amber to dark brown liquid preparations or as a white to tan powder.Major active principles:(1)␣-amylase and(2)␤-amylase.Typical applications:used in baking;used in the manufacture of alcoholic beverages and of syrups.Papain The purified proteolytic substance derived from the fruit of the papaya Carica papaya L.(Fam.Caricaceae).Pro-duced as a white to light tan,amorphous powder or a liquid soluble in water(the solution is usually colorless or light yellow and somewhat opalescent),but practically insoluble in alcohol,in chloroform,and in ether.Major active principles: (1)papain and(2)chymopapain.Typical applications:used in the chillproofing of beer,in the tenderizing of meat,in the148/Enzyme Preparations/Monographs FCC Vpreparation of precooked cereals,and in the production of protein hydrolysates.Microbially Derived Preparations␣-Acetolactatedecarboxylase(Bacillus subtilis containing a Bacillus brevis gene)Produced as a brown liquid by con-trolled fermentation using the modified Bacillus subtilis.Solu-ble in water(the solution is usually a light yellow to brown). Major active principle:decarboxylase.Typical application: used in the preparation of beer.Aminopeptidase,Leucine(Aspergillus niger var.,Aspergil-lus oryzae var.,and other microbial species)Produced as a light tan to brown powder or as a brown liquid by controlled fermentation using Aspergillus niger var.,Aspergillus oryzae var.,or other microbial species.The powder is soluble in water(the solution is usually light yellow to brown).Major active principles:(1)aminopeptidase,(2)protease,and(3) carboxypeptidase activities in varying amounts.Typical appli-cations:used in the preparation of protein hydrolysates and in the development of flavors in processed foods. Carbohydrase(Aspergillus niger var.,including Aspergillus aculeatus)Produced as an off white to tan powder or a tan to dark brown liquid by controlled fermentation using Aspergillus niger var.(including Aspergillus aculeatus).Solu-ble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol,in chloroform, and in ether.Major active principles:(1)␣-amylase,(2)pec-tinase(a mixture of enzymes,including pectin depolymerase, pectin methyl esterase,pectin lyase,and pectate lyase),(3) cellulase,(4)glucoamylase(amyloglucosidase),(5)amylo-1,6-glucosidase,(6)hemicellulase(a mixture of enzymes, including poly(galacturonate)hydrolase,arabinosidase, mannosidase,mannanase,and xylanase),(7)lactase,(8)␤-glucanase,(9)␤-D-glucosidase,(10)pentosanase,and(11)␣-galactosidase.Typical applications:used in the preparation of starch syrups and dextrose,alcohol,beer,ale,fruit juices, chocolate syrups,bakery products,liquid coffee,wine,dairy products,cereals,and spice and flavor extracts. Carbohydrase(Aspergillus oryzae var.)Produced as an off white to tan,amorphous powder or a liquid by controlled fermentation using Aspergillus oryzae var.Soluble in water (the solution is usually light yellow to dark brown),but practi-cally insoluble in alcohol,in chloroform,and in ether.Major active principles:(1)␣-amylase,(2)glucoamylase(amyloglu-cosidase),and(3)lactase.Typical applications:used in the preparation of starch syrups,alcohol,beer,ale,bakery prod-ucts,and dairy products.Carbohydrase(Bacillus acidopullulyticus)Produced as an off white to brown,amorphous powder or a liquid by con-trolled fermentation using Bacillus acidopullulyticus.Soluble in water(the solution is usually light yellow to dark brown), but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:pullulanase.Typical applica-tions:used in the hydrolysis of amylopectins and other branched polysaccharides.Carbohydrase(Bacillus stearothermophilus)Produced as an off white to tan powder or a light yellow to dark brown liquid by controlled fermentation using Bacillus stearother-mophilus.Soluble in water,but practically insoluble in alco-hol,in ether,and in chloroform.Major active principle:␣-amylase.Typical applications:used in the preparation of starch syrups,alcohol,beer,dextrose,and bakery products.Carbohydrase(Candida pseudotropicalis)Produced as an off white to tan,amorphous powder or a liquid by controlled fermentation using Candida pseudotropicalis.Soluble in water(the solution is usually light yellow to dark brown)but insoluble in alcohol,in chloroform,and in ether.Major active principle:lactase.Typical applications:used in the manufac-ture of candy and ice cream and in the modification of dairy products.Carbohydrase(Kluyveromyces marxianus ctis)Pro-duced as an off white to tan,amorphous powder or a liquid by controlled fermentation using Kluyveromyces marxianus ctis.Soluble in water(the solution is usually light yellow to dark brown),but insoluble in alcohol,in chloroform, and in ether.Major active principle:lactase.Typical applica-tions:used in the manufacture of candy and ice cream and in the modification of dairy products.Carbohydrase(Mortierella vinaceae var.raffinoseuti-lizer)Produced as an off white to tan powder or as pellets by controlled fermentation using Mortierella vinaceae var. raffinoseutilizer.Soluble in water(pellets may be insoluble in water),but practically insoluble in alcohol,in chloroform, and in ether.Major active principle:␣-galactosidase.Typical application:used in the production of sugar from sugar beets. Carbohydrase(Rhizopus niveus)Produced as an off white to brown,amorphous powder or a liquid by controlled fermen-tation using Rhizopus niveus.Soluble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principles: (1)␣-amylase and(2)glucoamylase.Typical application:used in the hydrolysis of starch.Carbohydrase(Rhizopus oryzae var.)Produced as a pow-der or a liquid by controlled fermentation using Rhizopus oryzae var.Soluble in water,but practically insoluble in alco-hol,in chloroform,and in ether.Major active principles:(1)␣-amylase,(2)pectinase,and(3)glucoamylase(amylogluco-sidase).Typical applications:used in the preparation of starch syrups and fruit juices,vegetable purees,and juices and in the manufacture of cheese.Carbohydrase(Saccharomyces species)Produced as a white to tan,amorphous powder by controlled fermentation using a number of species of Saccharomyces traditionally used in the manufacture of food.Soluble in water(the solution is usually light yellow),but practically insoluble in alcohol, in chloroform,and in ether.Major active principles:(1)in-vertase and(2)lactase.Typical applications:used in the man-ufacture of candy and ice cream and in the modification of dairy products.FCC V Monographs/Enzyme Preparations/149Carbohydrase[(Trichoderma longibrachiatum var.)(for-merly reesei)]Produced as an off white to tan,amorphous powder or as a liquid by controlled fermentation using Tri-choderma longibrachiatum var.Soluble in water(the solution is usually tan to brown),but practically insoluble in alcohol, in chloroform,and in ether.Major active principles:(1)cellu-lase,(2)␤-glucanase,(3)␤-D-glucosidase,(4)hemicellulase, and(5)pentosanase.Typical applications:used in the prepara-tion of fruit juices,wine,vegetable oils,beer,and baked goods.Carbohydrase(Bacillus subtilis containing a Bacillus mega-terium␣-amylase gene)Produced as an off white to brown, amorphous powder or liquid by controlled fermentation using the modified Bacillus subtilis.Soluble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:␣-amylase.Typical applications:used in the preparation of starch syrups,alcohol,beer,and dextrose.Carbohydrase(Bacillus subtilis containing a Bacillus stearo-thermophilus␣-amylase gene)Produced as an off white to brown,amorphous powder or a liquid by controlled fermenta-tion using the modified Bacillus subtilis.Soluble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:maltogenic amylase.Typical applications:used in the preparation of starch syrups,dextrose,alcohol,beer,and baked goods.Carbohydrase and Protease,Mixed(Bacillus licheniformis var.)Produced as an off white to brown,amorphous powder or as a liquid by controlled fermentation using Bacillus licheni-formis var.Soluble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol, in chloroform,and in ether.Major active principles:(1)␣-amylase and(2)protease.Typical applications:used in the preparation of starch syrups,alcohol,beer,dextrose,fishmeal, and protein hydrolysates.Carbohydrase and Protease,Mixed(Bacillus subtilis var. including Bacillus amyloliquefaciens)Produced as an off white to tan,amorphous powder or as a liquid by controlled fermentation using Bacillus subtilis var.Soluble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principles:(1)␣-amylase,(2)␤-glucanase,(3)protease,and (4)pentosanase.Typical applications:used in the preparation of starch syrups,alcohol,beer,dextrose,bakery products,and fishmeal;in the tenderizing of meat;and in the preparation of protein hydrolysates.Catalase(Aspergillus niger var.)Produced as an off white to tan,amorphous powder or as a liquid by controlled fermen-tation using Aspergillus niger var.Soluble in water(the solu-tion is usually tan to brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle: catalase.Typical applications:used in the manufacture of cheese,egg products,and soft drinks.Catalase(Micrococcus lysodeikticus)Produced by con-trolled fermentation using Micrococcus lysodeikticus.Soluble in water(the solution is usually light yellow to dark brown), but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:catalase.Typical application: used in the manufacture of cheese,egg products,and soft drinks.Chymosin(Aspergillus niger var.awamori,Escherichia coli K-12,and Kluyveromyces marxianus,each microorganism containing a calf prochymosin gene)Produced as a white to tan,amorphous powder or as a light yellow to brown liquid by controlled fermentation using the above-named genetically modified microorganisms.The powder is soluble in water, but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:chymosin.Typical application: used in the manufacture of cheese and in the preparation of milk-based desserts.Glucose Isomerase(Actinoplanes missouriensis,Bacillus co-agulans,Streptomyces olivaceus,Streptomyces olivochromo-genes,Microbacterium arborescens,Streptomyces rubigino-sus var.,or Streptomyces murinus)Produced as an off white to tan,brown,or pink,amorphous powder,granules,or liquid by controlled fermentation using any of the above-named organisms.The products may be soluble in water,but practi-cally insoluble in alcohol,in chloroform,and in ether;or if immobilized,may be insoluble in water and partially soluble in alcohol,in chloroform,and in ether.Major active principle: glucose(or xylose)isomerase.Typical applications:used in the manufacture of high-fructose corn syrup and other fructose starch syrups.Glucose Oxidase(Aspergillus niger var.)Produced as a yellow to brown solution or as a yellow to tan or off white powder by controlled fermentation using Aspergillus niger var.Soluble in water(the solution is usually light yellow to brown),but practically insoluble in alcohol,in chloroform, and in ether.Major active principles:(1)glucose oxidase and (2)catalase.Typical applications:used in the removal of sugar from liquid eggs and in the deoxygenation of citrus beverages.Lipase(Aspergillus niger var.)Produced as an off white to tan,amorphous powder by controlled fermentation using Aspergillus niger var.Soluble in water(the solution is usually light yellow),but practically insoluble in alcohol,in chloro-form,and in ether.Major active principle:lipase.Typical application:used in the hydrolysis of lipids(e.g.,fish oil concentrates and cereal-derived lipids).Lipase(Aspergillus oryzae var.)Produced as an off white to tan,amorphous powder or a liquid by controlled fermentation using Aspergillus oryzae var.Soluble in water(the solution is usually light yellow),but practically insoluble in alcohol, in chloroform,and in ether.Major active principle:lipase. Typical applications:used in the hydrolysis of lipids(e.g., fish oil concentrates)and in the manufacture of cheese and cheese flavors.150/Enzyme Preparations/Monographs FCC VLipase(Candida rugosa;formerly Candida cylindra-cea)Produced as an off white to tan powder by controlled fermentation using Candida rugosa.Soluble in water,but practically insoluble in alcohol,in chloroform,and in ether. Major active principle:lipase.Typical applications:used in the hydrolysis of lipids,in the manufacture of dairy products and confectionery goods,and in the development of flavor in processed foods.Lipase[Rhizomucor(Mucor)miehei]Produced as an off white to tan powder or as a liquid by controlled fermentation using Rhizomucor miehei.Soluble in water(the solution is usually light yellow to dark brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle: lipase.Typical applications:used in the hydrolysis of lipids, in the manufacture of cheese,and in the removal of haze in fruit juices.Phytase(Aspergillus niger var.)Produced as an off white to brown powder or as a tan to dark brown liquid by controlled fermentation using Aspergillus niger var.Soluble in water, but practically insoluble in alcohol,in chloroform,and in ether.Major active principles:(1)3-phytase and(2)acid phosphatase.Typical applications:used in the production of soy protein isolate and in the removal of phytic acid from plant materials.Protease(Aspergillus niger var.)Produced by controlled fermentation using Aspergillus niger var.The purified enzyme occurs as an off white to tan,amorphous powder.Soluble in water(the solution is usually light yellow),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:protease.Typical application:used in the produc-tion of protein hydrolysates.Protease(Aspergillus oryzae var.)Produced by controlled fermentation using Aspergillus oryzae var.The purified en-zyme occurs as an off white to tan,amorphous powder.Soluble in water(the solution is usually light yellow),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:protease.Typical applications:used in the chill-proofing of beer,in the production of bakery products,in the tenderizing of meat,in the production of protein hydrolysates, and in the development of flavor in processed foods. Rennet,Microbial(nonpathogenic strain of Bacillus ce-reus)Produced as a white to tan,amorphous powder or a light yellow to dark brown liquid by controlled fermentation using Bacillus cereus.Soluble in water,but practically insolu-ble in alcohol,in chloroform,and in ether.Major active princi-ple:protease.Typical application:used in the manufacture of cheese.Rennet,Microbial(Endothia parasitica)Produced as an off white to tan,amorphous powder or as a liquid by controlled fermentation using nonpathogenic strains of Endothia parasit-ica.The powder is soluble in water(the solution is usually tan to dark brown),but practically insoluble in alcohol,in chloroform,and in ether.Major active principle:protease. Typical application:used in the manufacture of cheese.Rennet,Microbial[Rhizomucor(Mucor)sp.]Produced as a white to tan,amorphous powder by controlled fermentation using Rhizomucor miehei,or pusillus var.Lindt.The powder is soluble in water(the solution is usually light yellow),but practically insoluble in alcohol,in chloroform,and in ether. Major active principle:protease.Typical application:used in the manufacture of cheese.Transglutaminase(Streptoverticillium mobaraense var.) Produced as an off white to weak yellow-brown,amorphous powder by controlled fermentation using Streptoverticillium mobaraense var.Soluble in water but practically insoluble in alcohol,in chloroform,and in ether.Major active principle: transglutaminase.Typical applications:used in the processing of meat,poultry,and seafood;production of yogurt,certain cheeses,and frozen desserts;and manufacture of pasta prod-ucts and noodles,baked goods,meat analogs,ready-to-eat cereals,and other grain-based foods.REACTIONS CATALYZEDNote:The reactions catalyzed by any given active com-ponent are essentially the same,regardless of the sourcefrom which that component is derived.␣-Acetolactatedecarboxylase Decarboxylation of␣-aceto-lactate to acetoin.Aminopeptidase,Leucine Hydrolysis of N-terminal amino acid,which is preferably leucine,but may be other amino acids,from proteins and oligopeptides,yielding free amino acids and oligopeptides of lower molecular weight.␣-Amylase Endohydrolysis of␣-1,4-glucan bonds in poly-saccharides(starch,glycogen,etc.),yielding dextrins and ol-igo-and monosaccharides.␤-Amylase Hydrolysis of␣-1,4-glucan bonds in polysac-charides(starch,glycogen,etc.),yielding maltose and beta-limit dextrins.Bromelain Hydrolysis of polypeptides,amides,and esters (especially at bonds involving basic amino acids,leucine,or glycine),yielding peptides of lower molecular weight. Catalase2H2O2→O2+2H2O.Cellulase Hydrolysis of␤-1,4-glucan bonds in such poly-saccharides as cellulose,yielding␤-dextrins.Chymosin(calf and fermentation derived)Cleaves a single bond in kappa casein.Ficin Hydrolysis of polypeptides,amides,and esters(espe-cially at bonds involving basic amino acids,leucine,or gly-cine),yielding peptides of lower molecular weight.␣-Galactosidase Hydrolysis of terminal nonreducing␣-D-galactose residues in␣-D-galactosides.␤-Glucanase Hydrolysis of␤-1,3-and␤-1,4-linkages in␤-D-glucans,yielding oligosaccharides and glucose.FCC V Monographs/Enzyme Preparations/151Glucoamylase(amyloglucosidase)Hydrolysis of terminal ␣-1,4-and␣-1,6-glucan bonds in polysaccharides(starch, glycogen,etc.),yielding glucose(dextrose).Glucose Isomerase(xylose isomerase)Isomerization of glucose to fructose,and xylose to xylulose.Glucose Oxidase␤-D-glucose+O2→D-glucono-␦-lactone +H2O2.␤-D-Glucosidase Hydrolysis of terminal,nonreducing␤-D-glucose residues with the release of␤-D-glucose. Hemicellulase Hydrolysis of␤-1,4-glucans,␣-L-arabino-sides,␤-D-mannosides,1,3-␤-D-xylans,and other polysaccha-rides,yielding polysaccharides of lower molecular weight. Invertase(␤-fructofuranosidase)Hydrolysis of sucrose to a mixture of glucose and fructose(invert sugar).Lactase(␤-galactosidase)Hydrolysis of lactose to a mixture of glucose and galactose.Lysozyme Hydrolysis of cell-wall polysaccharides of vari-ous bacterial species leading to the breakdown of the cell wall most often in Gram-positive bacteria.Maltogenic Amylase Hydrolysis of␣-1,4-glucan bonds. Lipase Hydrolysis of triglycerides of simple fatty acids, yielding mono-and diglycerides,glycerol,and free fatty acids. Pancreatin␣-Amylase Hydrolysis of␣-1,4-glucan bonds. Protease Hydrolysis of proteins and polypepticles. Lipase Hydrolysis of triglycerides of simple fatty acids. PectinasePectate lyase Hydrolysis of pectate to oligosaccharides. Pectin depolymerase Hydrolysis of1,4-galacturonide bonds.Pectin lyase Hydrolysis of oligosaccharides formed by pectate lyase.Pectinesterase Demethylation of pectin.Pepsin Hydrolysis of polypeptides,including those with bonds adjacent to aromatic or dicarboxylic L-amino acid resi-dues,yielding peptides of lower molecular weight. Phospholipase A2Hydrolysis of lecithins and phosphatidyl-choline,producing fatty acid anions.Phytase3-Phytase myo-Inositol hexakisphosphate+H2O→1,2,4, 5,6-pentakisphosphate+orthophosphate.Acid Phosphatase Orthophosphate monoester+H2O→an alcohol+orthophosphate.Protease(generic)Hydrolysis of polypeptides,yielding peptides of lower molecular weight.Pullulanase Hydrolysis of1,6-␣-D-glycosidic bonds on amylopectin and glycogen and in␣-and␤-limit dextrins, yielding linear polysaccharides.Rennet(bovine and calf)Hydrolysis of polypeptides;speci-ficity may be similar to pepsin.Transglutaminase Binding of proteins.Trypsin Hydrolysis of polypeptides,amides,and esters at bonds involving the carboxyl groups of L-arginine and L-lysine,yielding peptides of lower molecular weight. GENERAL REQUIREMENTSEnzyme preparations are produced in accordance with good manufacturing practices.Regardless of the source of deriva-tion,they should cause no increase in the total microbial count in the treated food over the level accepted for the respective food.Animal tissues used to produce enzymes must comply with the applicable U.S.meat inspection requirements and must be handled in accordance with good hygienic practices. Plant material used to produce enzymes or culture media used to grow microorganisms consist of components that leave no residues harmful to health in the finished food under normal conditions of use.Preparations derived from microbial sources are produced by methods and under culture conditions that ensure a con-trolled fermentation,thus preventing the introduction of mi-croorganisms that could be the source of toxic materials and other undesirable substances.The carriers,diluents,and processing aids used to produce the enzyme preparations shall be substances that are accept-able for general use in foods,including water and substances that are insoluble in foods but removed from the foods after processing.Although limits have not been established for mycotoxins, appropriate measures should be taken to ensure that the prod-ucts do not contain such contaminants.ADDITIONAL REQUIREMENTSAssay Not less than85.0%and not more than115.0%of the declared units of enzyme activity.Lead Not more than5mg/kg.Microbial Limits:Coliforms Not more than30CFU per gram. Salmonella Negative in25g.TESTSAssay The following procedures,which are included under Enzyme Assays,Appendix V,are provided for application as necessary in determining compliance with the declared。

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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