Fundamental Food Microbiology

<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN" "http://www.w3c.org/TR/1999/REC-html401-19991224/loose.dtd">  <xHTML><HEAD><TITLE>Untitled</TITLE>  </HEAD> <xBODY style="FONT-SIZE: 14px; FONT-FAMILY: Arial,sans-serif" text=black vLink=purple aLink=red link=blue bgColor=white> 교과서: Fundamental Food Microbiology <UL> Section 1. Introduction to microbes in foods(chapter 1∼4) section 2. Microbial growth in food (chapter 5∼8) section 3. Beneficial uses of microorganisms in food (chapter 9∼16) section 4. Microbial food spoilage (chapter 17∼21) 필요하다고 판단되는 기타 자료</UL> Chapter 1. History and Development of Food Microbiology         I. IntroductionAll foods harbor one or more types of microorganisms except a few sterile foods.         Some of them have desirable roles(fermentation)         Others cause food spoilage & food-borne diseases 미생물에 의한 유익한 역할을 연구하고 통제가 필요할 때는 통제하기 위해         -Isolation of them in pure culture         -Study their characteristics         II. 미생물의 발견 1658  Kircher saw minute living worms in putrid meat & milk.  Magnification power was so low that he could not have seen bacteria 1664 R. Hooke described the structure of molds 1675(6) A. van Leeuwenhoek with x300 magnification power.  sketched morphological characteristics of bacteria.  described some of them to be motile. 1838  Ehrenberg proposed bacterial classification and introduced the term "bacteria" for the first time.         III. Where are they coming from? 썩는 고기에서 구데기가 자연적으로 생겨난다--생물의 자연발생설(spontaneous generation, abiogenesis) 1665 Redi 그러나 파리가 접근하지 못하게 하면 구데기가 생기지 않는다.  (생물속생설)Biogenesis주장 1749 Needham  the appearance of animalcules in boiled meat (spontaneous generation) 1765 Spallanzani disproved Needham's theory (이 때쯤에 생물은 산소를 필요로 한다는 Lavoisier가 발표를 하였고 이를 근거로 끓여서 바로 뚜껑을 닫은 sealed flask에서는 산소가 없거나 부족하여 spontaneous generation이 불가능하였다고 Spallanzani의 설을 묵살.  Needham은 과가열하여 영양소가 파괴되었기 때문이라고 반박 1830 Schultze by passing air through acid 1838 Schwann by passing air through red hot tube 1854 Schroeder by passing air through cotton-----모두 boiled infusion에서 미생물이 번식하지 않음을 확인하고 산소부족때문이 아님을 보여줌. 1864 Louis Pasteur swan necked flask 이용하여 abiogenesis 격퇴 1870 John Tyndall dust-free environment에서 boiled infusion은 부패되지 않음을 실험성공         IV. What are their functions? 1837 Schwann, 1843 Helmholtz 부패나 발효는 공기중의 미생물 때문이다. 1875 Pasteur 포도주의 발효, 산패는 모두 미생물 때문이다.  우유나 고기가 부패되는 것, 광견병과 같은 질병도 모두 미생물 때문이다. 1880s-1890s Robert Koch (1843∼1910) anthrax, tuberculosis, cholera 균 순수배양성공, 기타 많은 공헌         V. Early development in Food Microbiology (prior to 1900) 8000∼2000BC 일정 season에 과잉생산된 식품이 부패되는 것을 방지하기 위해 drying, smoking, salting, low temp., baking, spices, honey 등을 이용하여 저장. Schwann과 Helmholtz는 미생물에 의해 식품등이 부패되는 것을 알았지만 그 기작은 알지 못했으나 나중에 Pasteur가 이는 미생물의 대사에 의해 대사물이 축적되기 때문이라고 설명. 1888 Gartner 식중독을 일으킨 식품에서 salmonell균 순수분리 1894 Denys S. aureus가 식중독을 일으킨다는 사실을 확증 1860 Pasteur yeast가 알콜발효하고 Acetobacter aceti가 포도주를 시게 한다.  그래서 undesirable 미생물을 선택적으로 죽이기 위해 pasteurization을 고안 (145oF/30min) 1873 Lister 우유산패세균인 Lactococcus lactis를 serial dilution 법에 의해 순수분리 1878 Cienkowski 설탕에 slime을 만드는 Leuconostoc mesenteroides를 순수분리         VI. Important M/O in foods A. Foodborne diseases: 식중독미생물이 생산과 소비단계에서 오염 B. Food spoilage: all foods harbor microorganisms C. Food bioprocessing: Fermented food D. Food biopreservation: antimicrobial metabolites E. Probiotics: live cells of bacteria       Chapter II. Characteristics of predominant microorganisms in food         I. Introduction 미생물은 식품에 부패를 일으키고 또는 새로운 식품 및 식품재료의 생산에 관여하기도 하며 식중독을 유발하기 때문에 관심을 가져야 한다.  (그러나 virus는 식품을 매개로 질병을 유발할 수 는 있으나 식품에서 번식하지 못한다). Bacteria: rapid growth & ubiquitous presence, most important in food spoilage and foodborne diseases, and also used to control microorganisms in foods. 본 장에서는 classification & nomenclature와 characterization of microorganisms in food를 다룬다.         II. Characterization of microorganisms Living cells---procaryotic(before nucleus)---bacteria               eucaryotic (with nucleus)---fungi (yeasts & molds)               (Virus는 living cell로 보지 않는다) 분류는 division(문), class(강), order(목), family(과), genera(속), species(종)의 체계로 분류되며 E. coli를 예로 들면 Protophyta(문), Schizomycetes(강), Eubacteriales(목), Enterobacteriaceae(과), Escherichia(속), coli(종)이다. * Eucaryotes는 같은 genus에서는 interbreed할 수 있다. *A strain is the descendant of a single colony(single cell).  Among the strains in a species, one is assigned as the type strain to be used as reference strain. 미생물의 분류를 위하여 효모와 곰팡이는 morphology, reproduction, biochemical nature of the macromolecules, metabolic patterns를 비교하고, 세균은 Gram stain, protein profile, a.a sequence of specific proteins, base composition, nucleic acid hybridization(If their DNA have 90% or more homology, they are considered the same), nucleotide base sequence(Sequences in 16S RNA are compared, because the nucleotide sequence in 16S RNA is most conserved), %G+C (If 2 strains differ by 10% or more, they are most likely not related).  기타 morphological, physiological, biochemical test의 결과 90%이상 같으면 같은 세균으로 본다.         III. Nomenclature 속.종의 2기명법을 쓴다.  미생물 분류의 예.   Reading: A closer look at language of microbiology [ASM News 65(5), 1999]         IV. Morphology and structure of m/o in foods A. Eucaryotic cells are generally much larger than procaryotic cells. B. Bacterial cells (cocci, bacilli, curved, G- or G+)    G-: outer membrane, middle membrane, inner membrane (see Fig. 2.2 p.18)    The resistance of G- bacteria to many enzymes (lysozyme), hydrophobic molecules(SDS, bile salts), and penicillin is due to barrier property of OM.  LPS molecules also have antigenic properties. C. Viruses         V. Important microorganisms in Food A. Mold   Low pH, low water activity, high osmotic pressure에 세균보다 잘 번식, 부패, mycotoxin 생산, 식품발효 등에 이용 A. flavus; aflatoxin 생산 A. oryzae; α-amylase 생산/ 고지 곰팡이로 이용 A. niger; citric acid 생산 & β-galactosidase생산 Geotrichum candidum; yeast-like colony/ 식품가공기계표면 및 dairy products에서 잘 번식 Mucor;자연에 광범위하게 분포 Penicillium camembertii,  P. requefortii; 치즈숙성 Rhizopus; 과일 채소 부패/ amylase 생산 B. Important yeasts Saccharomyces cerevisiae; 알콜 및 빵 발효 Pichia; forms pellicles in beer, wine, brine  예, P. membranefaciens Rhodotorula: pigmenting of meat, fish, sauerkraut.  세포내 지방 축적 Torulopsis: spoill milk due to ability to ferment lactose, flavor production in soy sauce Candida; rancidity in butter & dairy products, 예, C. lipolytica C. Virus Bacteriophage.  No bactereiophage of Pediococcus is yet known D. Important bacterial genera see Table 2.1 on p. 21 see p. 22-30       Chapter 3. Sources of microorganisms in food         I. Introduction The internal tissues of healthy plants and animals are essentially sterile. Yet raw and processed foods contain different types of molds, yeasts and bacteria.  이러한 미생물들은 천연적으로 식품에 있는 것도 있고 또 접촉에 의해 외부로부터 오염되는 것도 있다.   외부 오염원: 공기, 흙, sewage, water, human, ingredients, equipments, packages and insects         II. Predominant microorganisms in different sources A. plants 자연에서 오염됨 식품중에는 천연 natural animicrobial compounds를 가진 것도 있다.  식품재료를 수확하여 저장, 유통시키는과정중에도 미생물이 오염될 수 잇다. 기공전에 미생물수를 감소시키기 위해서는 재배하는 동안 비료의 선택이 중요하고(treated sewage를 이용), 수확시에 상처가 나지 않도록 하고, 표면은 깨끗한 물로 세척하고, 가공전까지 저온에 저장한다. B. Animals, Birds, fish and shellfish 식용동물이나 조류는 소화관, 호흡기, 배뇨기, 유분비기관 및 피부, 발굽, 털, 날개 등에 미생물이 있는 데 그람당 1010정도의 세균이 있을 수 잇다. 보통 동물은 Salmonella, pathogenic E. coli, Campylobacter jejuni, Yersinia enterocolitica와 Listeria monocytogenes를 가지고 있으나 증상은 나타내지 않는다.  산란계는 난관(ovary)에 Salmonella enterocolitica를 가지고 있는 경우 노란자가 이 세균에 의해 오염될 수 있다. 또 축산물의 경우 동물의 표면이 변에 의해 오염되며, 오염된 물이나 사료를 쓰게 되면 식품의 오염이 불가피하다. 그리고 fishs아 shellfish는 Vibrio parahaemolytics, V. vulnificaus 및 V. cholerae 등의 오염이 가능하다. C. Air 공기중의 미생물은 먼지에 부착되어 잇다.  먼지에서 번식하지는 않으나 일시적으로 존재하는 상태라고 본다.  먼지속의 미생물수는 습도, 먼지의 크기 및 먼지의 양, 온도, 공기의 흐름속도, 존재 미생물의 건조내성 등이 먼지의 미생물수에 영향을 준다. 일반적으로 건조공기중에 먼지가 적으면서 온도가 높으면 미생물의 수가 적다. 공기중에는 Bacillus, Clostridium, 공팡이의 포자, G+세균 및 효모가 많다. 주변에 병원성균의 오염원이 잇으면 병원균도 오염될 가능성이 크다. 따라서 공기를 통한 오염을 줄이려면 공기중에 먼지가 적어야 하며(여과를 하던지), 실내공기압을 높이던지, 숩도를 낯추고, UV 등 등을 설치한다. D. Soil 농산물이나 축산물에는 여러 가지 흙유래의 미생물이 있게 된다.  대표적인 세균으로는 Enterobacter, Pseudomonas, Proteus, Micrococcus, Enterococcus, Bacillus, Clostridium 등이다. E. Sewage 오수를 농작물의 비료로 쓰게 되면 오염이 우려된다.  Sewage를 비료로 쓰고자 하면 잘 처리(Treatment)하여 병원성균을 사멸시키고 써야 한다. F. Water 물은 재배, 가공, 저장(어름), 가공, 기구의 세척 등에 사용되므로 가공식품의 미생물적 품질에 큰 영향을 미친다.  가공 세척 등에 사용되는 물은 염소소독을 한 것으로 사용하여야 한다.  음용수에 병원균이나 콜리폼은 없겠으나 Pseudomonas, Alkaligenes, Flavobacterium 등은 흔히 존재하고 처리를 잘못하면 병원균이나 부패미생물이 있게 된다. G. Human 가공공장이나 단체급식소에서 일하는 사람은 식중독세균을 포함하는 병원균을 식품에 전파시키기 쉬운데 그 주요 이유는 손세척을 잘하지 않았을 때, 미적 및 개인위생이 부적절할 때, 옷이나 머리가 불결할 때이다.  특히 손이나 얼굴 등의 피부에 상처가 있거나 감염이 있을 때, 그리고 감기, sore throat, 간염 등을 앓고 있을 때 문제가 된다. H. Food ingredients 미생물학적으로 청결히 처리된 것을 사용하여야 한다. I. Equipment 여러 가지 가공조작을 위해 가공기계를 사용하게 되는 데 기계에는 주변환경(수분, 영양상태, 온도 등)과 사용시간에 따라 미생물이 번식하게 되는데, 처음에는 수가 적었더라도 많은 수로 번식할 수 있다.  특히 작은 부품이나 세척작용이 쉽게 이루어질 수 없는 부분에서는 여러 가지 미생물이 번식하며 가공하는 식품을 지속적으로 오염시키 우려가 있다. J. 기타 파리, vermin, 조류, 쥐 등이 병원균을 전파할 수 있으므로 잘 차단하여야 한다.                   Chapter 4. Significance of microorganisms in food         I. Introduction 자연계에는 여러 종류의 미생물이 있으나 일반적으로 천연식품에는 몇몇 종류의 미생물이 있다.  이들 미생물은 원래 ecological niche이기 때문에 있을 수도 있고 생산과 소비단계에서 외부로부터 오염된 것도 있다.  미생물의 수도 가공하는 동안 내외부조건에 따라 다르게 나타난다. 가장 숫자가 많은 미생물은 주어진 조건이 그 미생물의 번식에 가장 적합한 미생물이다.         II. Raw and ready-to-eat meat products 도축한 육류/가금류의 고기는 표피, 털 내장 등으로부터 세균이 오염되게 되며 기타, 도축등의 기구, 사료, 물, 흙, 사람등으로부터도 오염가능하다.  Salmonella, yersinia enterocolitica, Campylobacter jejuni, E. coli, C. perfringens, S. aureus 등이 적은 수이지만 나타날 수 있다.  조류육은 특히 Salmonella에 의해 오염되기 쉽다. 도축한 고기는 보통 -1∼5oC에 저장하기 때문에 저온균의 번식에 유의해야 한다.  저온성 pathogen인 Listeria monocytogenes와 Yersinia enterocolitica가 중요한 병원균이고  ground meat에는 104-5/g, Salmonella가 1/25g 정도 된다.         III. Raw and pasteurized milk Healthy udder: Micrococcus, Streptococcus, Corynebacterium <103/ml Mastitis cattle: Streptococcus agalactiae, S. aureus, coliforms, Pseudomonas Pathogens in milk: Salmonella, Listeria monocytogenes, Y. enterocolitica, C. jejuni Equipment: G- rods(Pseudomonas, Alcaligenes, Flavobacterium), G+(Micrococcus, Enterococcus) Grade A milk: 총균수 ≤20000/ml, coliform ≤10/ml Pasteurized milk: Thermodurics(Micrococcus, some Enterococcus, Streptococcus, some lactobacilli [L. viridescens])         IV. Shell eggs & liquid egg 계란은 환경으로부터 오염이 가능하고 미생물의 수는 107정도된다.  그 중에 Pseudomonas, Alcaligens, Proteus, Citrobacter, E. coli, Enterobacter, Enterococcus, Micrococcus, bacillus 등이 있다.  닭의 변으로부터 Salmonella 오염이 가능하고 닭의 ovary로부터 S. enteritidis가 오염될 수 있다.  G-세균은 난각의 구멍을 통해 계란안으로 들어갈 수 있는 데 특히 물에 젖었을 때 더욱 그렇다. 계란 자체는 항미생물작용을 갖는 데 lysozyme, conalbumin(binds iron), alkaline pH(8.0-9.0).  그러나 난황은 pH가 7정도이기 때문에 미생물의 번식이 쉽다.         V. Fish & shellfish 특히 조개류는 filter에 의해 먹기 때문에 많은 물을 통과시키므로 세균과 virus를 함유한다.  특히 유의해야 할 것은 Vibrio parahaemolyticus, V. vulnificaus, C. botulinum 등이다.         VI. Vegetables, fruits& nuts 동물과 접하거나 오염된 용수를 사용하였을 때는 Listeria monocytogenes, shigella, Salmonell, Campylobacter, C. botulinum, C. perfringens가 오염될 수 있다.         VII. Cereals, starches & gums 가공하지 않은 것에는 총균수 ∼104/g, coliforms ∼102/g, yeasts & molds ∼103/g         VII. Canned foods B. stearothermophilus, B. coagulans, C. thermosaccharolyticum, D. nigrificans 등이 부패를 일으키기 쉽고 C. botulinum에 의한 식중독이 위험. 위의 세균은 ≤30oC에 저장하면 포자가 발아를 하지 않아 부패가 되지 않으나 ≥40oC이상에 저장하면 부패된다.         VIII. Sugars and confectionary Lactobacillus, Leuconostoc, Bacillus와 Clostridium의 포자         X. Soft drinks and fruit juices pH가 2.5∼4.0, 단 과일주스는 >4.5.  따라서 효모, 곰팡이, 젖산균, 식초산균 등이 주요 부패균이다.         XI. Mayonaise 등 pH3.5∼4.0. Yeasts and molds, 젖산균(L. fructivorans)         XII. Spices and condiments Bacillus, Clostridium의 포자.  때로는 Salmonella와 S. aureus가 존재.       Chapter 5. Microbial growth characteristics         I. Microbial reproduction A. Binary fission   Bacteria: binary fission, asexual reproduction   Yesats: budding( asexual reproduction) & ascospore (sexual reproduction)   Molds: spores( asexual reproduction) & spores (sexual reproduction) B. Generation time doubling time for the entire population V. parahaemolyticus는 under optimal conditions have as low as 10∼12 min Generation time calculation:    C. Optimum growth temp, pH, Aw, OR potential, nutrients D. Growth curve see Fig. 5.2 on 57         II. Microbial growth A. Mixed population    mixed population vs pure culture B. Sequence of growth    predominant type이 바뀐다. C. Symbiotic growth D. Synergistic growth E. Antagonistic growth       Chapter 6. Factors influencing microbial growth         I. Intrinsic factors or food environment 식품자체의 환경: nutrients, growth factors, antimicrobials, Aw, pH, OR potential---이러한 요인들이 개별적으로 또는 관련을 맺고 미생물의 번식을 촉진시키거나 저해한다. A. Nutrients and growth Nutrients:CHO, proteins, lipid, minerals, vitamins.  Water(생화학반응에 필수적이지만) is not considered a nutrient.  식품에는 위의 5대 영양소를 모두 갖고 있지만 그 함량은 식품마다 다르다. 식품에 번식하는 미생물은 영양요구성이 제각각 다르다.  세균이 요구성이 가장 크다.  미생물은 macromolecules를 이용할 수 있는 능력이 있는 데 곰팡이가 그 능력이 가장 우수하다. 1. CHO in food Monosaccharides: hexoses and pentoses Disaccharides: lactose, sucrose, maltose Oligosaccharides: raffinose, stachyose Polysaccharides: starch, glycogen, cellulose, inulin, hemicellulose, dextran, pectin, gums Lactose is only in milk Glycogen in animal tissues, esp. in liver Pentoses, most oligosaccharides & polysaccharides are naturally present in foods of plant origin 모든 미생물은 glucose를 이용할 수 있으나 기타 CHO의 이용은 미생물에 따라 다르다.  그 이유는 mono- disaccharides는 transpot mechanism의 존재여부, 거대분자의 이용은 분해효소의 생산여부 2. Proteins in food 단백질의 여러 가지 형태; simple proteins, peptide, NPN(amino acids, urea, ammonia, creatine, trimethylamine 등) 많은 미생물은 albumin과 같은 수용성단백질은 쉽게 분해하지만 collagen과 같은 불용성단백질은 잘 분해하지 못한다.  단백질은 분해하여 a.a.나 peptide의 형태로 흡수하고, peptide는 세포내에서 a.a.로 분해되어 cellular components, energy and various byproducts(NH3, H2S)를 만든다. 3. Lipids in food Free fatty acids, glycerides, phospholipids, waxes and sterols Pseudomonas, Achromobacter, Alcaligenes는 lipase를 생산하고 세포가 분해되었을 때 그 효소로 말미아마 spoilage (예, rancidity), desirable flavors (예, cheese ripening), beneficial (L. acidophilus--metabolize cholesterol) 4. Minerals & Vitaamins in food 미생물은 번식에 인, Ca, Mg, 철, S, K 등을 필요로 한다. 대부분의 미생물은 B vitamins의 합성가능/ 배지나 식품에 포함되어 있기도 한다. Molds, yeasts & G- 세균은 일반 식품에서 잘 번식하나, 일부 G+세균 특히 젖산균중에서 Lactobacilli는 영양요구성(특히, B vitamins)이 높아 잘 번식하지 못하는 수가 있다. B. Growth factors & inhibitors in food Food can have some factors that either stimulate growth or adversely affect the growth of microorganisms. C. Aw and growth 1. Principle Aw: Biological function에 이용될 수 잇는 물로서 소위 free water의 측정치를 의미한다. Bound water: necessary to hydrate the hydrophilic molecules and dissolve the solutes, and is not available dor biological functions.  It does not contribute to Aw.  0< Aw = Po/P < 1 2. Aw of food (0.1∼0.99) Cereals, crackers, sugar, salt, dry milk: 0.10-0.20 Noodles, honey, chocolate, dried egg: <0.60 Jam, jelly, dried fruits, nuts, parmesan cheese: 0.60-0.85 Fermented sausage, dry cured meat, sweetened condensed milk, maple syrup: 0.85-0.93 Evaporated milk, tomato paste, bread, fruit juices, salted fish, sausage: 0.93-0.98 Fresh meat, fish, fruits, vegetables, milk, egg: 0.98-0.99 3. Aw and microbial growth Free water: transport of nutrients, removal of waste material, xarry out enzymatic reaction, synthesize cellular materials, other biochemical reactions, such as hydrolysis of a polymer. D. pH and growth 1. Principle 0 < pH: -log[H+] <14 2. pH of foods 3. pH and microbial growth pH affect microbial growth & viability of microbial cells E. Redox potential 1. Principle Oxidation-reduction other than O2, NO3, SO4 can accept electrons 2. Redox potential of food it is influenced by its chemical composition, process treatment, storage condition (air). Fresh foods of plant and animal origin are in a reduced state due to the presence of reducing substances, such as ascorbic acid, reducing sugars, the -SH groups.  Following stoppage of respiration, O2 will diffuse inside and change redox potential.. Processing such as heating, can increase of decrease reducing compounds.  A food stored in air will have a larger Eh than when it is stored under vacuum or in modified gas. 3.  Redox potential and microbial growth see text         II. Extrinsic factors Storage conditions (temp, RH, gas) A. Temp. and growth 1. Principle 10oC up---catalytic rate of enzyme doubles 2. Food and temp 3. Microbial growth and viability       Chapter 7. Microbial sporulation and sublethal injury         I. Microbial sporulation and germination Molds, yeasts and bacteria: are capable of forming spores. Molds: form spores sexually and asexually/a method of reproduction Yeasts: form spores sexually/a method of reproduction Bacteria: form spores asexually/not a method of reproduction/ survival in unfavorable condition Compared to bacterial spores, mold and yeast spores are less resistant to environmental stress. A. Mold spores Perfect molds: sexual and asexual spores/ sexual reproduction si very rarely observed. imperfect molds: asexual spores (conidiospore, sporangiospore, arthrospore) only B. Yeast spores Ascomycetes-Hemiascomycetes  -- true yeasts forming sexual spores Non-sporulating yeasts -- false yeasts C. Bacterial spores G+; Bacillus, Clostridium, Sporolactobacillus, Sporosarcina, Thermoactinomyces G-; Desulfotomaculum  Bacillus, Clostridium, Desulfotomaculum은 식품의 부패나 식중독 등에 중요하다. Bacterial spores= endospores and one/cell Position: terminal. central, off-central Surface of spore is negatively charged and hydrophobic Spores as compared to vegetative cells are much more resistant to physical and chemical antimcirobial treatments, because the specific structure of the spores is guite different from that of the vegetative cells from which they are formed (see Fig. 7.1 on 75) ◆ The protoplasm core: contain important cellular components such as DNA, RNA, enzymes, DPN, divalent cations, and very little water. ◆ Inner membrane: Forerunner of cell membrane ◆ Germ cell wall:  Forerunner of cell wall ◆ Cortex: peptide, glycan, outer fore-spore membrane ◆ Spore coat: protein layers that provide resistant to spores ◆ During germination and outgrowth, the cortex is hydrolyzed and the outer fore-spore membrane and spore coat are removed by the emerging cell. Spores are metabolically inactive(dormant)  see Fig. 7.2 on 76 D. Sporulation condition: Nutrient availability (C,N,P), growth temp., pH Triggering compound is formed when nutrition depletion and other unfavorable condition occur.  Adenine bistriphosphate could be the triggering comp., as it is synthesized by spore-formers under the conditions of carbon or P depletion. E. Dormancy Spores are formed in such a manner as to remain viable in unfavorable condition Dormancy can be ended by a series of biochemical reactions involved in spore activation, germination, outgrowth and growth.   Superdormancy F. Activation Spore activation prior to germination is accompanied by reorganization of macromolecules in the spores. Activation Methods: sublethal heat treatment, radiation, treatment w/ oxidizing and reducing agents, exposure to extreme pH and sonication. Heat treatments probably accelerate the germination process by increasing the permeablility of spore structures to germinating agents for the macromolecular reorganization.  This process can be reversible. G. Germination Several structural and functional events occur during this stage.  The dormant stage is irreversibly terminated. Structural chages are: hydration of core, excretion of Ca + DPN, and loss of resistance and refractile property. Functional changes are: initiation of metabolic activity, activation of specific proteases and cortex-lytic enzymes and release of cortex ltic products.  This is a metabolically degradative process. Germination can be initiated by many factors: pH, Temp., high pressure, lysozymes, nutrients (aminoacids, CHO), calcium-DPN, and others.  It is inhibited by D-alanine, ethanol, EDTA, NaCl(high conc.), NO2, sorbate. H. Outgrowth Outgrowth constitutes the biosynthetic and repair processes.  Swelling occurs due to hydration and nutrient uptake, repair and synthesis of RNA, proteins, materials for membrane and cell wall, dissociation of coats, cell elongation, and RNA replication.  The factors that can enhance the process include nutrients, pH & temp. I. importance of spore in food Bacillus, Clostridium & Desulfotomaculum  → associated with food spoilage and foodborne diseases.  Superdormancy.         III. Microbial injury and repair A. Sublethal injury Microbial cells and spores exposed to different physical and chemical sublethal treatments(stresses) suffer injury that is reversible in nature Stresses: low heat, low temp., low Aw, radiation, high hydrostatic pressure, electric pulse, low pH, preservatives, sanitizer and heated microbiological mesia(>48oC)  see Table 7.1 on 79 G- bacteria are more susceptable to injury than G+ bacteria. B. Manifestation of bacterial sublethal injury Sublethal stress--three physiologically different subpopulations. ① the uninjured (normal) ② reversibly injure (injured) ③ irreversibly injured (dead) Their relative percentages vary greatly and are dependent upon the species and strains, nature of suspending media, nature and duration of a stress, methods of detection.   see Table 7.2 for normal, injured and dead cell differentiation C. Sites and nature of injury Structural and functional components damaged by sublethal stresses: cell wall(OM), IM, rRNA, DNA plus some enzymes. Freezing & drying: cell wall(OM) & IM are more evidently damaged. sublethal heating: rRNA radiation: DNA   see 2nd para on 81 D. Repair of reversible injury Bacterial cells can repair injury in a suitable environment and become similar to the normal cells.  Cell repair well in a medium rich in metabolyzable C & N, and vitamins.  Supplementation with catalase and pyruvate enhances repair.  (see text on 82)  Reading: J. Food Protection 42: 362 Flowers and Ordal, Current methods to detect stressed staphylococci) E. Injury in yeasts and molds F. Importance of injured microorganisms in food 적당한 환경에서 번식하여 부패나 식중독을 일으킬 수 있으나, 미생물 실험해보면 없는 것으로 나타나기 때문에 위험할 수 잇다.    1. detection    2. Enhancing the shelf=life of foods    3. Enhancing the viability of starter cultures       Chapter 8. Microbiological metabolism of food components         I. Respiration and fermentation during growth The energy generating metabolic pathways also produce many metabolic products that the microbial cells either use for the synthesis of cellular components or release into the environment. [see diagram on 88]         II. Metabolism of food CHO; CHOs are transported and metabolized A. Degradation of polysaccharides B. Degradation of disaccharides [in microbial cells] C. Degradation of monosaccharides    metabolized by 5 major pathways.   Many microorgani는 have more than 1 pathways.    EMP, HMS shunt, E-D pathway, phosphoketolase pathway-----pyruvate의 대사방법이 다르다 D. Fermentation    Energy generation by substrate level phosphorylation---several different pathways are used for the metabolism of pyruvate.         1. EM pathway [used by homolactic, Bacillus, yeasts]--pyruvate to lactate or ethanol         2. HMS shunt [used by heterolactic, Pseudomonas]--acetyl P to acetate, pyruvate to lactate         3. E-D pathway [used by Pseudomonas]--pyruvate to ethanol         4. Pentose phosphoketolase pathway--acetyl P to ethanol or acetate, pyruvate to lactate         5. Hexose phosphoketolase pathway [Bifidus]--acetate, and pyruvate to lactate         6. Some specific pathways                 a. mixed acid pathway pyruvate to lactate, succinate, formate                 b. propionic acid pathway                 c. butyrate, butanol, acetone[Clostridium]                 d. diacetyl, acetoin, butanediol fermentation [Enterobacter] E. Anaerobic respiration sulfate and nitrate as e- acceptors F. Aerobic respiration: TCA cycle G. Synthesis of polymers         III. Metabolism of food proteins; Microorganisms transport amino acids and small peptides(6-8) into the cell         A. Aerobic respiration (decay)2         B. Fermentation (putrefaction)--NH4, H2, CO2, H2S         IV. Metabolism of lipids; fatty acids through acetyl CoA       Chapter 9. Microorganisms used in food fermentations         I.  Introduction Microorganisms used in foods         : actively growing cells[yogurt]; They and their metabolites have no detrimental effect on the health of humans         : non-growing cells= increase the shelf-life of raw milk         : Microbial byproducts=lactic acid, acetic acid, a.a., bacteriocin; microorganisms have to be regulatory approved and should be safe.         : cellular components of microorganisms SCP, dextran, enzymes; microorganisms have to be regulatory approved and should be safe. These microorganism, byproducts, cellular components ahve to be safe, food-grade and approved by regulatory agencies.         II. Microbiology of fermented foods 세계적으로 약 2000종의 발효식품이 있다.  They are mostly fermented by natural microflora [natural fermentation] and some are by pure cultures [pure culture fermentation or controlled fermentation]         III. Lactic acid culture [10 genera 정도가 이용되며 이들 중 반 정도는 최근에 명명됨] Lactic acid bacterial genera Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, [Enterococcus, Aerococcus, Vagococcus, Teragococcus, Carnobacterium] 최근에 명명된 속들 예 Lactococcus-originally Group N Streptococcus    Enterococcus-originally Group D Streptococcus    Vagococcus-motile, but other characteristics are not distinguishable from Lactococcus    Tetragenococcus-Pediococcus halophilus 하나가 여기에 편입됨    Carnobacterium-Obligately heterofermentative, originally Lactobacillus에 속했었다.         IV. Genus Lactococcus  Lactococcus속에는 몇 종이 잇으나 L. lactis 한 종만 dairy fermentation에 쓰여진다. L. lactis는 L(+) lactic acid를 생산하며 pH는 4.5까지 내릴 수 있다. 0.5 ∼ 1.0μm이며 pairs and in short chains로 나타난다. non-motile and facultative anaerobic to microaerophilic. L. lactis의 subsp. 중 lactis는 40℃ 번식                     cremoris는  40℃, 4% NaCl 번식불가                     diacetylactis는 많은 CO2생산, diacetyl production from citrate         V. Genus Streptococcus S. thermophilus만 starter로 쓰인다, Pairs and in long chains, 37-40℃에 번식하나 52℃에는 번식불가.  pH는 4.0까지 낮춘다. does not ferment galactose and sucrose, survive at 60℃ for 30min.         VI. Genus Leuconostoc grows well between 20-30℃ with a range of 1-37℃, pH 4.5-5.0, 6 species known L. mesenteroides has 3 subsp. They are mesenteroides, dextranicum, cremoris.         VII. genus Pediococcus form tetrads or pairs.  Single cells or chains are absent.  pH 3.6.  Lactose not fermented.         VIII. Genus Lactobacillus Group 1: Obligately homofermentative [L. delbruckii, L. leichmanii, L. acidophilus, L. helveticus] Group 2: Facultatively heterofermentative (depending on CHO and amount) [L. casei, L. palntarum, L. sake, L. curvatus] Group 3. Obligately heterofermentative [L. fermentum, L. kefir, L. brevis, L. sanfrancisco, L. reuteri] L. curvatus and sake are capable of growing at low temp(2-4℃, and are associated with fermentation of vegetable and meat products.         IX. Genus Bifidobacterium (과거에는 Lactobacillus에 속했음. acetic acid와 Lactic acid 생산)         X. Genus Propionibacterium         XI. Genus Acetobacter A. aceti가대표적. obligately aerobic. oxidize ethanol to acetic acid         XII. Yeasts S, cerevisiae: bread leavening, alcohol fermentation, enzyme(invertase), flavor C. utilis: SCP Kluyvermyces marximus: hydrolyze lactose.  spoilage of some dairy products.         XIII. Molds A. oryzae: fermentation of some oriental foods, sake, soy sauce, miso A. niger; citric acid, gluconic acid, enzyme P. roquefortii: roquefort cheese ripening        Chapter 10. Biochemistry of some beneficial traits         I. Introduction Beneficial microorganism metabolize some of the components in the starting material (such as milk and meat) to produce energy and cellular materials and to multiply----end-products are formed and excreted as by-products--some of the by-products impart unique characteristics (texture and flavor).         II. Transport         III. Transport system         A. PEP-PTS system for lactose transport in L. lactis high energy P of PEP  --→Enz I, HPr, Fac III, Enz II lac and to lactose [Fac III, Enz II lac are specific for lactose].  Lactose is transported into cytoplasm as lactose-P         B. Permease system for lactose transport in L. acidophilus Lactose + H+ (OUTSIDE) --→ Lactose + H+ (inside)         IV. CHO available inside the cells for metabolism  -- →mono and disaccharides         V. Homolactic fermentation  see Table 10.1 on 115         VI. Heterolactic fermentation of CHO Leuconostoc and Group II Lactobacilli lack fructose diphosphate aldolase, but have glucose-P dehydrogenase and xylulose phosphoketolase         VII. Metabolism of pentose Leuconostoc and Group II Lactobacilli---→ferment pentoses by the pentose phosphate pathway as they have phosphoketolase.  This enzyme is inducible in Group II Lactobacilli only when a pentose is present.  In this case no CO2 is produced.         VIII. Hexose fermentation by Bifidobacterium         IX. Diacetyl production from citrate Diacetyl is important in fermented dairy products for its pleasing aroma.  Many LAB can produce it from pyruvate, generated from CHO metabolism.  L. lactis subsp. acetylactis can produce large amount of it from citrate.         X, XI, XII.  amino acids and small peptides are actively transported.  Many starter cultures metabolize lipids poorly.       Chapter 11. Genetics of some beneficial traits         I. Introduction Since the 1950s, it has been recognized that many important characteristics in dairy starter culture are unstable.  A Lactococcus lactis once able to ferment while growing in milk (producing lactic acid and coagulating the milk) was found no longer ferment lactose and became commercially not useful. 이러한 예가 다른 traits에서도 나타난다. ① hydrolyze proteins ② ability to utilize citrate (for diacetyl production) ③ loss of resistance to bacteriophage ④ loss of hydrolysis of sucrose The 1960s   Genetic basis of instability of the trait began to be known. The 1970s   기타 많은 연구가 진척되었다.         II. Plasmids and plasmid linked traits in starter cultures Chromosomal DNA: carries genetic codes for vital functions of a cell (key enzymes in EMP or HMS) Plasmid and transposons: carries genetic codes only for non-vital functions (ability to hydrolyze a larger proteins)---→plasmid를 가진 것이 가지지 않은 것에 비해 유리한 고지를 점령함. A. Important characteristics of bactertial plasmids ·ds circular and self replication 1<to<100kb ·many not be present in all species or all strains of the species ·A strain can have more than 1 type of plasmid ·A plasmid: more than 1 copy in a cell ·A copy number of a plasmid can be decreased or increased by manipulation or can be spontaneously lost ·Two types of plasmid in a cell may be incompatable, resulting in the loss of one ·Plasmid can be transferred from one cell to another spontaneously or by manipulation. ·Plasmid transf er can occur either only between closely related strains or between widely related strains from different species of genera ·A plasmid can be cryptic (ie, not known to carry genetic code for a known trait) B. Some characteristics of small (about 10kb) and large (>10∼150kb) plasmids ·copy #  small plasmids---multiple copy (10∼40/cell)           large plasmids---low copy # (1/cell) ·amplification small plasmids amplified to a high #                large plamids can not be amplified ·conjugal transfer  small plasmids; non-conjugative                     large plasmids; conjugative ·Stability; small plamids are unstable            large plasmids are usually stable C. Presence of plasmids in some starter culturres Lactococcus: most strains have 2-10 or more types of plasmids Str. thermophilus: a few strains carry plasmids Leuconostoc: most strains have 1-10 or more types Pediococcus: 0-3, not much studied Lactobacillus; L. acidophilus: rarely, at most a few, L. plantarum; 2-7 plasmids D. Phenotype assignment to a plasmid Loss of lactose fermentability=due to loss of a plamid that codes genes necessary for lactose hydrolysis (also for lactose transfer) E. Plasmid linked traits in starter culture bacteria Lac+, pro+, cit+, bac+, phager, 녗+, mal+, mu+, R/M system         III. Gene transfer methods in starter cultures A. Transduction -- through bacteriophage B. Conjugation -- plasmids transfer C. Transformation -- ds DNA piece D. Protoplast fusion E. Electroporation F. Gene cloning       Chapter 12. Starter cultures and bacteriophage         I. Introduction Starter cultures: selected strains of food-grade microorganisms of known and stable metabolic activities and other characteristics that are used to produce fermented foods of desirable appearance, body, texture and flavor.         II. History Before 1950s; undefined culture, small scale production Since 1950s; larger scale production, single dry culture, ---- needs large facilities and manpower             To overcome phage problems ① rotation of strains ② multiple strain culture ③ improved media 1960s; frozen culture concentrate & phage inhibitory media 1970s; different types of frozen concentrate culture for direct inoculation & freeze-dried concentrated culture (viability problem).         III. Concentrated cultures 발효식품 제조시 적당한 속도로 발효시키기 위해 106-7cells/ml or g을 접종한다. In the conventional process, a bulk culture of 108-9cells/ml needs to be inoculated at about 1% level to the raw material.  In suitable media we can obtain ∼1010/ml--→harvested by centrifugation/resuspended in a liquid at a level of 1012-13/ml--→360ml of frozen concentrate in DVS is added to 5000 gallons of milk to get 106-7cells/ml. The suspending medium contains cryoprotective agent to reduce cell death and injury during freezing/thawing --→The culture is frozen at -78 or -196℃/transported/stored at -20℃ or below.  Just before inoculation, a container is thawed in warm potable water(45℃) and added to the raw material.         IV. Starter culture problems A. Strain antagonism In mixed culture, when a starter culture contains two or more strains, dominance of one over the other(s) can change the culture profile.  Dominance can result from optimum growing environment or production of inhibitory metabolites.  The culture producers test the compatibility between the desirable strains to avoid strain antagonism. B. Loss of desired trait A strain carrying a plasmid-linked trait can lose the trait during subculturing and under some conditions. C. Cell death and injury D. Inhibitors in raw materials The milk may contain either antibiotics, given to the animals to treat infections like mastitis, or sanitizers. E. Bacteriophages widely distributed in the environment, especially in food fermentation environments. 1. Morphology: 2. Life cycle (report) adsorption(needs Ca++)  --→injection of DNA---synthesis phage materials and self assembly--→cell wall lysis to release the phage (∼200).  It takes 20∼30 min. Temperate phage: The phage DNA(prophage) is carried by the bacterial cells(lysogenic state), which is called lysogenic strain.  Prophage can be induced by a physical(uv) or chemical(mitomycin C) methods. 3. Host specificity All phage requires Ca++ for their adsorption on the cell surfaces of LAB. 4. Control methods proper sanitation use of phage sensitive media rotation of strains use of mixed strains phage resistant strains; phage sensitive strains are grown in the presence of a specific lytic phage, survivors are used.         V. Yeasts and molds        Chapter 13. Microbiology of fermented food production         I. Introduction Fermented food seemed to be originated around 7000-8000BC in the tropical areas of Mediterranean and the Indus Valley.  Currently more than 2000 different fermented foods are consumed worldwide.         II.  General Methods of production A. Raw materials Plants & animal products including milk, meat, fish, egg, different vegetables, fruit & fruit juices, grains, beans, etc. B. Microorganisms used Bacteria, yeasts and molds C. Fermentation process Natural fermentation back slopping controlled fermentation   1. Natural fermentation A product produced by natural fermentation can have some desirable aroma rsulting from the metabolites of the associated flora.   2. Back slopping Part of previous batch is added   3. Controlled fermentation 106cells/ml of a pure culture of single or mixed strains. Their is usually less chance of product failure and food-borne diseases.  However, growth of desirable secondary flora may not occur.  As a result, a product may not have some delicate flavor characteristics.         III. Fermented dairy products A. 우유;  3.2% protein, 4.8% lactose, 3.9% lipids, 0.9% mineral, 87.2% water, traces of vitamins B. Fermented milk products   1. buttermilk: Lactococcus sp   2. yogurt; S. thermophilus, L. bulgaricus   3. acidophilus milk; L. acidophilus   4. bifidus milk; bifidobacterium   5. yakult; L. casei   6. kefir; L. kefir (yeasts w/ Leuconostoc, lactobacillus, Lactococcus)   7. kumiss: L. bulgaricus + yeasts C. Microbiology of cultured buttermilk fermentation 1. Product characteristics lactic acid- pleasant acid taste diacetyl-aroma CO2- slight effervescence 2. Process 3. Starter( controlled fermentation) L. lactis subsp. lactis or cremoris for acid Leuconostoc mesenteroides subsp. cremoris for diacetyl and CO2 L. lactis subsp. lactis biovar diacetylactis is generally not used because it may produce too much acetaldehyde (causing yogurt flavor defect). 4. Growth at 72oF, there is balanced growth of the two sp., and balanced production of acid, diacetyl, and CO2. >72oF, the growth of Lactococcus sp. is favored, with more acid and less flavor.  <72oF, the growth of Leuconostoc sp. is favored, with less acid and more flavor. 5. Biochemistry sugar--→lactcate, CO2, acetate citrate--→CO2, diacetyl( -O2, acetoin=no flavor, +O2, diacetyl) + acetaldehyde For a desirable flavor, diacetyl: acetaldehyde; >3:1  to <4.5:1 6. Genetics Lac. lactis sp.   Lac+,                 should not produce slime,                 should not be very proteolytic,                 should be resistant to phages Leuconostoc sp. should be able to utilize citrate to produce more diacetyl and less acetaldehyde and ferment lactose                 should not produce slime                 should be resistant to phages The two: should not produce inhibitory compounds against each other, but can be inhibitory to undesirable organisms. 7. Microbial problems Green yogut flavor due to too much acetaldehyde Slime production id undesirable(may be produced due to contamination) Yeasts flavor; due to lactose fermenting yeast contamination Cheesy flavor; proteolytic pschrotrophs during storage D. Microbiology of yogurt fermentation 1. characteristics Yogurt flavor is due to combined effects of lactate, acetaldehyde, diacetyl, and acetate, but 90% of it due to acetaldehyde. 2. Processing Batch or continuous process a stabilizer is added to give desired gel structure.  Heating destroys vegetative mcirobes and slightly destabilize casein for good gel formation.  After fermentation, it is quickly cooled to 85oF in about 30min. to slow down starter growth and acid production, especially by Lactobacillus. 3. Starters (controlled fermentation) Normally 2 sp. are used.  L. delbruckii subsp. bulgaricus & Sterp. thermophilus 4. Growth The balanced growth of the two sp., it is fermented at 110oF(43.3oC). At this temp., both acid and flavor compounds are produced at the desired level. Above this temp., Lactobacilli will predominate(more acid less flavor) Below this temp., Stre. is favored with a product containing less acid & more flavor.             They grow in symbiotic relationship ① S. thermophilus grows rapidly in the presence of dissolved oxygen and produce formic acid & CO2 ② The anaerobic condition, formic acid & CO2 stimulates the growth of Lactobacillus, which has good protease and peptidase system, and glycine, valine, histidine, leucine and methionine are necessary for good growth of Streptococcus which lacks protease.  S. thermophilus then grows rapidly until pH drops to about 5.5, at which time the growth of Streptococcus slows down.  However, Lactobacillus continues to grow fairly rapidly until the temp. is  reduced to 85oF, following a drop in pH to 4.8.  At 40oF with about pH 4.3, both stops to grow. The two species have a synergistic effect on the growth, rate of acid production, and amount of acetaldehyde formation when grown together as compared to when grown individually.(8-10 ppm vs 25ppm of acetaldehyde) 5. Biochemistry a. Lactose metabolism  : EMP b. flavor production      Major flavor compound is acetaldehyde (25ppm), with some diacetyl + acetate. c. formate production formate is produced by S. thermophilus from pyruvate(--→formate + acetate) d. slime production (glycan) 6. Genetics Lac+, Gal+, Pro+, Phager, symbiotic and synergistic relationship, antagonistic effect, good survival to freezing and drying. 7. Microbial problems Too much acetaldehyde---green flavor Too much diacetyl---buttery flavor Too much acid---sour taste fast proteolysis/accumulation of bitter peptides---bitter taste yeast growth---fruity  flavor E. Cheeses since 7000BC 1. Unripened: soft--cottage cheese, Mozzarella cheese 2. Ripened: soft---Brie cheese            semi-solid----Gouda and Blue cheese            Hard---Cheddar and Swiss cheese F. Microbiology of cottage cheese 1. characteristics Cottage cheese is made from low-fat or skim milk unripened and has a buttery aroma due to diacetyl. 2. Processing 3. Starters (controlled fermentation) L. lactis subsp. lactis and cremoris  for acid Leuconostoc mesenteroides subsp. cremoris for diacetyl Lac. lactis subsp. lactis biovar diacetylactis not generally used because of too much CO2, which causes curd particles to float. 4. Growth/Biochemistry and genetics 5. Microbial problems ① slow growth: weak or loss of Lac+, Phage attack or less viable cells in frozen concentrate, antagonism among starter cultures. ② floatation of curd: because of too much CO2 production by  flavor producers ③ Harsh flavor: because of more acetaldehyde and less diacetyl production ④ low yield: because of partial proteolysis of casein by heat-stable proteases(Pseudomonas) ⑤ flavor loss: because of reduction of diacetyl to acetoin ⑥ spoilage because of high moisture and low acid of the product/ sorbate is  used. G. Microbiology of Cheddar cheese 1. characteristics 2. Process 3. starter 4. growth 5. Biochemistry 6. genetics 7. microbial problems bitter  flavor: accumulation of bitter peptides (1000∼13,000), rich in hydrophobic a.a. Faster starter tends to produce bitter peptides more than slow starters. Mold grow on the surface S. aureus: can grow and produce enterotoxin Biological amines(histamine, tyramine) H. Microbiology of Swiss cheese 1. characteristics 41%moisture, 43% fat, medium-sized eyes(openings) uniformly distributed. It has a sweet taste because of proline. 2. processing 3. Starters S. thermophilus for acid, Propionibacterium as secondary for eye formation, taste and  flavor. 4. growth 5. Biochemistry 6. genetics 7. Microbial problems Clostridium tyrobutyricum---rancidity & gas blowing I. Microbiology of Blue cheese 1. characteristics 46% moisture, 50^ fat, crumbly body, mottled blue color, sharp lipolytic  flavor. 2. processing 3. Starters L. lactis subsp. cremoris and lactis & Leuconostoc as primary starters Penicillium roquefortii: spores as secondary starters. mold spores (50oF at high RH) germinate quickly, produce mycelia, and spread inside to give the mottled appearance.  Their growth continues during curing.  Puncturing helps to remove CO2 and let the air in to help the growth of molds. 4. Biochemistry J. Accelarated ripening 1. curing at high temp. 2. addition of enzymes: as intracellular enzymes have an important role in curing, enzyme obtained from starter cell lysis of starter cultures is added to increase the rate of curing. 3. slurry method         IV. Fermented meat products A. Types Most fermentation probably originated in the Mediterranean nations and later spread to European countries and North America. B. Microbiology of semi-dry sausage 1. characteristics Semi-dry sausages: summer sausage, thuringer, semi-dry salami 30% fat, 20% protein, 3% mineral, 47% water. Tangy taste: lactate, aceatet, diacetyl, some breakdown products from proteolysis and lipolysis.  Spicies contribute additional  flavor. 2. Processing Nitrite/about 100ppm final concentration 3. Starters Pediococcus acidilactici for high temp & low pH Lactobacillus plantarum for low temp., and high pH P. pentosaceus for both conditions. Micrococcus & S. carnosus-- secondary microflora for desired product color  Micrococcus reduce nitrate to nitrite  S. carnosus produce catalase to destroy H2O2, which discolors the product. 4. Growth 5. Biochemistry 6. Genetics 7. Microbial problems         V. Fermented vegetable products Almost all vegetables can be fermented through natural process since they harbor many types of LAB. A. Microbiology of sauerkraut 1. characteristics 2. Processing   fine shredding: helps the sugars come out of the cell   Tight packing: create anaerobic condition, thus preventing growth of aerobes.   Salt stimulates the growth of LAB and discourages the growth of undesirables.   Natural inhibitors in cabbage also discourages the growth of G- and G+ bacteria 3. Starters and growth Raw material has a large # of undesirable organisms and a small population of LAB (<1%). Lactococcus and Leuconostoc---in high #,     Lactobacillus and pediococcus in low #. L. mesenteroides starts a rapid growth, no oxygen and low temp(18oC) When acidity is about 1%, growth of L. mesenteroides slows down. L. brevis starts to grow rapidly acidity <1.5% Ped. pentosaceus                        1.8% L. plantarum                            2.0% 4. Biochemistry Leuconostoc and L. brevis     hexose & pentose to lactate, acetate, ethanol, CO2 and diacetyl Ped. pentosaceus and L. plantarum     hexoses ---- lactic acid     pentose ---- lactic acid, acetate, ethanol 5. Genetics 6. Microbial problems        Chapter 14. Food ingredients and enzymes of microbial origin         I. Introduction Many microbial metabolites can be used as food additives for the improvement of nutritional value, flavor, color and texture.  They include proteins, essential a.a., vitamins, aroma compounds,  flavor enhancers, salty and sweet peptides, colors and organic acids. Recombinant DNA technology is effectively used.         II. Microbial proteins (SCP)         yeasts: Candida, Saccharomyces, Torulopsis         Bacteria: Methylophilus Advantages ① land shortage and environmental calamities can be overcome ② use of agricultural and industrial wastes ③ waste disposal problem/ reduce cost of production ④ microbial proteins good source of B vitamins, carotene and CHO Disadvantages ① poor in some essential a.a. like methionine ② high nucleic acid content---uric acid to cause gout & kidney stone formation.         III. Amino acids essential a.a. are produced to supplement food         IV. Vitamins B vitamins & vitamin C, D, E. Vitamin C is produced by yeast using cheese whey.         V. Flavor compounds & Flavor enhancers Flavor compounds: diacetyl(buttery), acetaldehyde(yogurt), cheese flavor, propionic acid (nutty), pyrazines(roasted nutty), terpenes(fruit or flowery) Flavor enhancers: MSG, nucleotides Lysyglycine: strong salty taste monellin, tharmatin--sweet peptides from plant sources are produced by microorganism through gene cloning. Aspartame         VI. Colors astazanthine by Phaffia carotene by Monascus         VII. Stabilizers Polysaccharides are used as stabilizers and texturizers. Many polysaccharides of plant origin are used.  Some are obtained from microbial source 예) dextran from Leuconostoc mesenteroides is used in ice cream and confectionaries.  Xanthan gum from Xanthomonas campestris.         VIII. Organic acids Lactic, acetic and propionic acids imporve taste(flavors & taste) and keeping quality (antibacterial action). Ascorbic acid: reducing agent to maintain color (to prevent loss by oxidation). Citric acid: improve taste & texture (in beverages), stabilize color in fruits, antibacterial property produced by Aspergillus niger.         IX. Microbial enzymes in food processing Use of specific enzyme(instead of microbial cells) has several advantages. ① A specific substrate is converted to a specific product ( production of different metabolites by live cells from the same substrate is avoided) ② A reaction step can be controlled and enhanced more easily ③ By using recombinant DNA technology, efficiency can be improved. Main disadvantages: If a substrate is converted to a product through many steps (e.g., glucose to lactate) microbial cells must be used. A. Enzymes used 1. α-amylase, glucoamylase, glucose isomerase (see Table 14.1 on 174)    α-amylase is used for bread making to slow down staling (starch crystallization due to loss of moisture) 2. Catalase    H2O2 removal after hydrogen peroxide pasteurization. 3. Cellulase, hemicellulase and pectinase    citrus juice extraction to increase juice yield ① The polysaccharides trap juice during processing ② increase viscosity, causing problems during juice extraction ③ cloud the juice 4,5,6,7. Invertase, lactase, lipase, protease B. Enzyme production by recombinant DNA technology   mRNA  - cDNA  -cloned in bacteria         X. Immobilized enzymes enzyme recycling A. adsorption on a solid support B. Covalent bonding C. Entrapping D. Croos linking         XI. Thermostable enzymes catalyze reactions above 60℃.  Reaction rate doubles for every 10℃ increase in temp. At high temp, the growth of contaminated microorganisms can be reduced. Thermostability: ion pairing & H-bonding on the surface &                 increases in internal hydrophobicity  increases thermostability Methods to increase thermostability ① a thermostable enzyme producing microorganism not on the list of GRAS---→ the gene is introduced into GRAS listed microorganism ② Switch one or two a.a. residues on the surface to increase ionic or H-bonding.         XII. Enzymes in food waste treatment        Chapter 15. Food biopreservation of microbial origin         I. Introduction Food-grade bacteria associated w/ food fermentation are capable of producing different types of metabolites that have antimicrobial properties (see Table 15.1 on 182).         II. Use of viable cells    Lactobacillus: lactate, bacteriocins, H2O2         III. Use of organic acid in biopreservation Acetic acid: characteristic aroma, inhibiting microbial growth Propionic acid: fungistatic, effecive in controlling growth and reducing viability of bacteria. Lactic acid: flavors enhancement, antibacterial (1-2% level)  even at pH 5.0 or above.            <5.0 bactericidal effect pKa of acetic acid 4.8        propionic acid 4.9        lactic acid 3.8: the low antibacterial effectiveness of lactic acid is thought to be due to its low pKa         IV. Use of diacetyl as biopreservative citrate---→diacetyl(intense aroma & quite volatile): antibacterial against G+ & G- G- are particularly sensitive at pH 5.0 or below.  Effective at ∼0.1-0.25%. The dicarbonyl group (-C(O)-C(O)-) reacts with arginine in enzymes and modifies this catalytic sites.         V. Hydrogen peroxide as a food preservative LAB produce and excrete hydrogen peroxide under aerobic condition to protect themselves.  Under anaerobic condition, very little hydrogen peroxide is expected to be produced. Hydrogen peroxide is permitted in refrigerated raw milk and raw liquid eggs (about 25ppm).         VI. Reuterin as a food preservative Lactobacillus reuteri, found in the gastrointestinal tract of humans & animal, reuterin(β-hydroxypropionaldehyde) in the presence of glycerol.         VII. Bacteriocins of starter: 대표적인 것은 nisin이 있다.         VIII. Yeasts       Chapter 16. Health benefits of beneficial bacteria         I. Introduction Advocate: Metchnikoff in 1907         II. Microbiology of the human GI tract Human GI tract ∼1016 microorganism/500bacterial sp/이 중에서 30∼40 종이 전체의 95%이상 차지함. Microbial levels in small intestine (jejunum and ileum)=106∼7cells/g                   large intestine (colon) 1010-11cells/g Predominant types in small intestine: Lactobacillus and Streptococcus                      large intestine: Enterobacteriaceae, Bacteroides, Fusobacterium, Clostridium, Eubacteria, Enterococcus, Bifidobacterium, Lactobacillus The intestine of a fetus in the uterus is sterile.  It is inoculated with vaginal and fecal flora from mother at birth.  이들이 소화관내에 들어가 정착한다. 1st couple of days : E. coli & Enterococcus appears in large numbers in the feces.   Breast-fed babies, large number of Bifidobacterium with lower level of E. coli and Enterococcus.   Formla-fed babies, E. coli, Enterococcus with Clostridium and Bacteroides predominate, with Bifidobacterium almost absent. Intestinal microflora         autochthonous: indigenous; capable of adhering to intestinal cells         allochthonous: transient; passing or temporarily colonizing a site where the specific indigenous types has been removed due to some factor (such as antibiotic intake)         III. Important characteristics of beneficial bacteria Lactobacillus acidophilus, L. reuteri, Bifidobacterium are in the GI tract of human as well as in animals and birds.  they are less sensitive to stomach acid than many other bacteria, and highly resistant to bile and lysozyme present in the GI tract. Under normal conditions, these bacteria metabolize lactic and acetic acids + specific inhibitory compounds. L. acidophilus produce bacteriocins, effective against closely related G+ bacteria.         IV. Beneficial effects of probiotics ·provide protection against enteric pathogens ·supplying enzymes to help metabolizing some food nutrients ·detoxifying some harmful food components and metabolites in the intestine         ·stimulating intestinal immune systems ·improving intestinal peristaltic activity A. Lactose hydrolysis 원래 lactase는 사람의 소장에서 생산됨.  그래서 lactose intolerance한 사람은 이용되지 않은 lactoserk 대장으로 가서 gas와 산을 생산---fluid accumulation, diarrhea, flatulance 젖산균은 소장에서 lactasetodtks 요구르트 젖산균은 애 not survive stomach acidity and are not normal intestinal bacteria Benefit(?): reduced amount of lactose as compared to milk + lactase from dead cells Some other lactobacillus: colonize small intestine & supply lactase B. Reducing serum cholesterol level metabolize dietary cholesterol --reduced absorption deconjugate bile salts--prevent readsorption in the liver--재활용이 되지 않으므로 간에서 cholesterol을 이용하여 bile salt 합성하는 때문에 serum cholesterol의 양이 감소한다. C. Reducing colon cancer Many of the undesirable bacteria in the colon have enzymes that can activate procarcinogens, either present in the food or produced through metabolism of undesirable bacteria.  LAB controll the growth of undesirable bacteria D. Reducing intestinal disorders E. Miscellaneous benefits         V. some aspects to consider The health benefit theory of fermented foods and beneficial bacteria is controvercial A. strain variation B. Sensitivity to stomach acid C. Viability and injury of cells D. Dose level and duration E. Induced lactase trait F. Antibacterial substances G. True sp/str H. Microbiology research       Chapter 17. Important factors in microbial food spoilage         I. Introduction Microbial food spoilage occurs as a consequence of microbial growth in a food or the release of extracellular and intracellular (following cell lysis) enzymes in the food.         II. Sequence of events ① Microorganisms get into food ② condition to grow ③ length of time for microbial cells to obtain high numbers necessary to cause the detectable changes in a food.         III. Significance of microbial types Microbial multiplication is an important components in spoilage         IV. Significance of microbial numbers To produce detectable changes in quality, microorganisms must multiply and attain certain levels, often referred to as the spoilage detection level.  Although it varies with the types of food and microorganisms, bacteri and yeasts needs to reach to about 107cells/ml(g).  Spoilage detection level can range from 106 to 108 cells/g. Spoilage associated with H2S, amines and H2O2 formation can be detected at a lower microbial load, while formation of lactic acid may be detected at a higher load.  Slime formation at 108cells/g or higher.  A food with higher initial load will spoil more rapidly than one with a low initial load. (see Fig. 17.1) Just the mere presence of 107cells/ml without growth will not immediately cause a food to lose its acceptance quality.  Bioprocessed foods, in general, contain very high numbers of microorgani는 (108∼1010).  However, under normal conditions, they are desirable types, and the fermented foods are not considered spoiled.         V. Significance of predominant microorganisms When a food is spoiled, it contains predominantly one or more types.  They may not be present initially in high numbers in the unspoiled product., e.g. beef(pH 6.0): intial microbial number ≒ 103cells/g with Pseudomonas 1%, Acinetobacter & Moraxella 11%, and others.  Aerobic storage at 2℃ for 12days, population reaches 6 x 107cells/g with Pseudomonas 99% and others 1%. Beef stored anaerobically, the population reaches 107, predominant bacteria will be Lactobacillus and /or Leuconostocs.         VI. Some important food spoilage bacteria. A. Psychrotrophic bacteria Definition; growing at 5℃ and below but multiplying rapidly at 10-25℃. 식품의 조장온도는 0℃근처지만 때로는 10℃ 또는 그 이상이 되는 수가 있다. 이때 psychrotrophic bacteria가 부패를 유발할 수 있다. 1. some important psychrotrophic aerobic spoilage bacteria Pseudomonas fluorescens, P. fragi, and other Pseudomonas sp., Acinetobacter, Moraxella, Flavobacterium 2. some important psychrotrophic facultative anaerobic spoilage bacteria Brochothrix, Lactobacillus, Leuconostoc, Enterococcus, Enterobacter, Serratia, Fafnia, Proteus 3. Some important thermoduric psychrotrophs Fac. anaerobes: B. coagulans, B. megaterium, L. viridescens Anaerobes: Clostridium            B. Thermophilic bacteria Pedio. acidilactici, S. thermophilus, Bacillus & Clostridium C. Bacteria capable of growing relatively rapidly in food at pH 4.6 or below (LAB)         VII. Food types perishable, semiperishable, non-perishable. In addition to the intrinsic parameters, extrinsic parameter (storage condition) play important roles.         VIII. Metabolism of food nutrients CHO by LAB (see Table 17.1 on 210) odor: production of volatile end-products color: pigment production or oxidation of natural color texture: break-down of pectins gas: CO2, H2, H2S Slime: dextran and confluent growth Loss of liquid: break-down of structures holding water/ lowered pH to reduce water holding capacity.         IX. Preference for utilization of food nutrients The characteristics of food spoilage differ greatly.  This is mainly due to differences in the nature and amount of a specific nutrients in a food, the type of microorganisms, and the nature of metabolism.  In general, microorganisms prefer to use CHO first, followed by NPN and poteinaceous compounds, and the lipids.  Fermentable CHO will be metabolized first.  If fermentable CHO is present in sufficient quantities, the metabolic pathway remains unchanged.  However, fermentable CHO are present in limiting quantity, microorganisms start using NPN and proteinaceous compounds Example) Yeasts growing in a fruit juice containing relatively high amount of metabolizable CHO will produce CO2 and H2O aerobically, or alcohol and CO2 (anaerobically).  Pseudomonas fluorescens, aerobically in fresh meat with limiting amount of glucose, will first metabolize it and then atart metabolizing free a.a. and other NPN.  Then produce extracellular proteases to break down meat proteins, and the lipids. In milk, lactose utilizer---use lactose         non-utilizer---NPN & proteinaceous compounds. 따라서 신선육에 포도당 등의 당을 첨가하면 단백질이 이용되지 않는데 이를 "Protein sparing effect"라고 한다.  이 방법이 실제로 이용되기도 한다.         X. Microbial growth in succession Mixed microbial sp.   predominent type grows    some other sp grow   third type grows 예) 우유 ①rapid growth of Lactococcus (pH 6.5--→4.5) ②aciduric yeasts grow and increase pH (to 5.8) ③at high pH Pseudomonas metabolize NPN and proteinaceous compounds, to increase pH further by producing basic metabolites.       Chapter 18. Spoilage of specific food groups         I. Introduction Foods contain microorganisms--→growth of some sp. dictated by microbial type, food type, food environment.         II. Fresh and ready-to-eat meat products A. Raw meat Pseudomonas Post rigor meat: rich in NPN (13mg/g of a.a. and creatine), peptides, proteins                 but low in CHO (1.3mg/g glucose & glucose-6-p) with a pH of 5.5. DFD mest has almost no CHO, a pH of 6.0 0r above. ◇Refrigerated meat (modified atmosphere, CO2 + O2) will favor facultative anaerobe Brochothrix thermosphacta, metabolizing glucose to acetic acid and acetoin, and leucine and valine to isovaleric and isobutyric acids to produce cheesy off-odor. ◇Psychrotrophic fac. anaerobes & aerobes grow in vacuum-packaged meats.  Lactobacillus curvatus and L. sake metabolize glucose to lactic acid, leucine and valine to isovaleric and isobutyric acids.  Cheesy odor when number reaches 108cells/ml.  However, when they metabolize cysteine and produce H2S, the product will have undesirable odor and color.  H2S oxidizes myoglobin to metmyoglobin, causing a green discoloration. B. Ready-to-eat meat products high heat processed: commercially sterile low heat processed: 140-150oF(60-65oC)--includes franks, bologna, ham, luncheon meat ; spores of Bacillus & Clostridium, some extremely thermoduric vegetative sp. (L. viridescens), some Enterococcus, Micrococcus can survive. Serratia liquifaciens: causes amino acid break down causing ammonia-like flavor (diaper smell). Due to the growth of hydrogen peroxide producing lactic acid bacteria, the products may have gree to gray discoloration.         III. Eggs and Egg products A. Shell eggs Egg shell & inner membrane + bacteria and mold hyphae can penetrate.  The pore size increases during storage.  The presence of moisture enhances the entrance of mobile bacteria. Egg pH 9-10, lysozyme, conabumen, avidin (binds riboflavin), protease inhibitors protect egg. green rot; causing greening of albumen (P. fluorescens) black rot; muddy discoloration of yolk (Proteus vulgaris) red rot; red pigment production (S. marcescens) fungal rot; oiled eggs by molds B. Egg products         IV. Fish. crustaceans, and mollusks A. Fish fish spoilage; autolytic enzyme (ungutted fish), unsatrurated fatty acid oxidation, microbial growth G- rods; trimethylamine oxide --→ trimethylamine B. Crustaceans C. Mollusks Pseudomonas and Vibrio break down N compounds, resulting in the production of NH3, amines and volatile fatty acids.         V. Milk and milk products A. Raw milk Those microorganisms with lactose-hydrolyzing enzymes have an advantage B. Pasteurized milk Thermoduric microorganisms survive pasteurization. Flavor defects are detectable when the population reaches ≥106cells/ml C. Liquid products D. Butter 80% fat, bacteria (Pseudomonas), yeasts(Candida), molds(Geotrichum candidum)         VI. Vegetables & fruits A. Vegetables rich in CHO (≥5%), low in protein (1-2%) hig in pH excpt tomato. Microorganisms grow more rapidly in damaged or cut vegetables. B. Fruits rich in CHO(>10%), very low in protein (≤1.0%), pH 4.1 or lower.         VII. Soft drinks, fruit juices and vegetable juices only aciduric molds, yeasts and bacteria(Lactobacillus, Leuconostoc, Acetobacter)         VIII. Cereals & their products A. Cereal grains (10-12% moisture) Aspergillus, Penicillium & Rhizopus: spoil when moisture is high. B. Refrigerated dough Heterotrophic & psychrotrophic Lactobacilli & Leuconostocs produce gas. C. Breads Bread mold: Rhizopus stolonifer (특히 starch crystallization releases moisture 때) Mucoid variant of B. subtilis---→ ropy bread D. Pastas E. Pastries(cakes, baked shells filled with custard, eream etc)         IX. Liquid sweeteners and confectionaries: osmophilic yeasts         X. Mayonaise         XI. Fermentaed foods. A. Fermented meat       Chapter 19. Food spoilage by microbial enzymes         I. Introduction A food is considered spoiled when changes are detectable and the microbial population reaches 107-9cells/ml or g.  These changes are brought about by catalytic actions of large number of microbial enzymes.  Most of the enzymes are intracellular and act on the nutrients that are transported inside the cells.   After microbial cells die normally so that intracellular and extracellular enzyme are not inactivated.  These enzymes can break down the food nutrients to cause spoilage.         II. Characteristics of heat stable enzyme of psychrotrophic bacteria Heat-stable proteinases and lipases is produced in milk by some of Pseudomonas sp. A highly active proteinases or lipases produced by a Pseudomonas strain can produce extrinsic proteolysis or lipolysis, even a level of 105-6cells/ml.  Some of these enzymes are not completely destroyed by pasteurization.  They are generally inactivated by UHT treatment.         III. Spoilage of food with heat-stable enzymes Canned apricot과 Rhizopus 곰팡이의 문제 설명.       Chapter 20.  New food spoilage bacteria in refrigerated foods         I. Microorganisms that grow in refrigerated food chilling and refrigeration= -1℃∼7℃ psychrophile optimum temp= 12∼15℃, growth range≤-5∼22℃   However, the definition of psychrotroph is not clear-cut.  optimum temp. 25∼30℃, but not grow at 35℃.  a subgroup of mesophiles, but not of a psychrophile.   Recent definition: psychrotrphs have been used for mesophilic subgroup capable of growing at 4.4℃ or below.         II. Popularity of refrigerated foods Increase in two-income families, single parent households, singles, the elderly---demand convenient foods.  People prefer refrigerated foods to harshly processed foods.         III. Microbiological problems MA packaged products: new microbial problems         IV. Incidence of spoilage in vacuum packaged refrigerated food A. Spoilage of unprocessed beef by clostridia Purge: liquid(many bacteria[108] through microspcopic observation); Leuconostoc 같지만 Clostridium 모양의 세균이 많다.  plate count했을 때 액체배지 배양시 배양불가---→혐기적 배양시 배양가능.  가열하여 다른 세균은 사멸시키고 clostridia의 포자만 생존하므로 count 가능)---Clostridium laramie로 확인됨.  이 세균은 산소에 extremely sensitive. grows optimally at 12-15℃(range -2∼22℃) B. Spoilage of roasted beef by Clostridium Spoiled sample showed gas & purge  ∼108cells/ml of Leuconostoc.  현미경관찰 Clostridium with spores are seen.  plate나 액체배지에 배양불가--위의 C. laramie와 동일 C. Spoilage of pork chops D. Spoilage of tofu by clostridia tofu during refrigerated storage; gas and purge + off-odor; large # of motile rods with terminal oval spores---C. laramie로 확인됨. E. Spoilage of unripened soft cheese by Leuconostoc 108-9cells/g of Leuconostoc; gas and liquid accumulation F. Spoilage of low heat processed meat products by Leuconostoc gas/purge production 108-9cells/ml LAB, <103cells/ml of Brochthrix thermosphacta. G. Ammonia odor in turkey rolls Strong diaper-like smell with gas and liquid accumulation Leuconostoc-like cells---L. mesenteroides Lactobacillus-like cells--L. sake G- motile rods--Serratia liquifaciens; ammonia produced (pH does not change because of added phosphate) H. Yellow discoloration of luncheon meat Hugh # of Enterococcus faecium subsp. caseoliflavus survives cooking. I. Gray discoloration of turkey luncheon meat Hydrogen peroxide produced by Lactobacillus sp. J. Pink discoloration by Hafnia alvei, Proteus vulgaris, Serratia liquefaciens K. L. see text Ground beef by Leuconostoc, Lactobacillus, Enterobacter       Chapter 21. Indicators of microbial food spoilage         I. Introduction Food spoilage by live cells and extracellular& intracellular enzymes Indicators---microbiological, chemical(microbial metabolites), sensory         II. Microbiological criteria APC   1. Refrigerated raw meat storted aerobically: psychrotrophic aerobes especially G- aerobes at 10-25℃incubation.   2.  Refrigerated raw meat storted anaerobically (vacuum-packaged): Psychrotrophic Enterobacteriaceae + psychrotrophic clostridium (C. laramie)   3. Refrigerated low-heat processed, vacuum apckaged meat products; Psychrotrophic Enterobacteriaceae + LAB         III. Chemical criteria Metabolic by-products: H2S, NH3, CO2, diacetyl, acetoin, indole production, pH change, water-holding capacity         IV. Assay of heat-stable enzymes; Pseudomonas fluorescens  </xBODY></xHTML>