Penrose Flashcards

Question

What is the relationship between number of DSP steps and cost

Answer 1

The more DSP steps in obtaining the product, greater the losses, and greater the expense.

Answer 2

high volume/low value: amino acids, vitamins, food & beverages - low volume/high valuepharmaceuticals e.g. recombinant insulin, Factor VIII

Answer 3

- Primary metabolites: products of growth processes, e.g. amino acids, vitamins, nucleotides, ethanol, lactate, enzymes cellular biomass - Secondary metabolites: produced from pathways not required for growth: e.g. antibiotics, toxins, pigments, exopolysaccharides (xanthans, polyhydroxy alkanoates).

Answer 4

- Amino acids: $3000 million - Monosodium glutamate: $915 million - L-Lycine-HCL: $600 million

Answer 5

- Antibiotics: $28,000 million - Cephalosporins: $11,000 million - Penicillins G and V: 4400 million

Answer 6

- Most of a microbial cell's energy is produced from the oxidation of carbohydrate. - Glucose is most commonly used carbohydrate, metabolisable by most organisms.

Answer 7

Respiration and fermentation

Answer 8

- Aerobic process in which glucose is completely broken down to CO2 and H2O. - C6H12O6 + 6O2 6CO2 + 6H2O + electron --> 36 ATP

Answer 9

- Anaerobic process in which glucose is partially broken down to various products, principally ethanol and lactate - C6H12O6 ---> 2 ethanol + 2 CO2 + 2 ATP C6H12O6 ---> 2 lactate+ 2 ATP

Answer 10

``` - Pyruvate decarboxylated via PDH >>acetyl CoA >> condensation with oxaloacetate >> citrate and TCA cycle ```

Answer 11

In a fermentation, electrons are passed from a donor molecule to an acceptor, via an electron acceptor (usually NAD). Donor and acceptor can both be an organic molecule. ATP generation via substrate level phosphorylation only

Answer 12

Fermentation is energetically wasteful as glucose is only partially oxidised, principally to ethanol and lactate.

Answer 13

C6H12O6+ 2ADP + Pi + 2 NAD+ ---> 2 ethanol + 2 CO2 + 2 NADH + 2H+ + 2 ATP C6H12O6 + 2ADP + Pi + 2 NAD+ ---> 2 lactate + 2 NADH + 2H+ + 2 ATP

Answer 14

2 ATP 2 NADH per glucose

Answer 15

1) Glucose becomes 2 pyruvate (2 ADP -> 2 ATP, 2 NAD+ -> 2 NADH) 2) 2 Pyruvate is decarboxylated (-2CO2) to become 2 acetylaldehyde 3) 2 acetylaldehyde become 2 ethanol (2NADH -> 2NAD+)

Answer 16

*Homolactic fermentation 1) Glucose -> 2 pyruvate (2 ADP -> 2 ATP, 2 NAD+ -> 2 NADH) 2) 2 pyruvate to 2 lactate (2NADH -> 2NAD+) No decarboxylation No intermediate: pyruvate directly accepts electrons from NADH

Answer 17

Net yield 2 ATP 2 NADH per glucose

Answer 18

Microbes:Yeast, some | Bacteria (Zymomonas)

Answer 19

Beer, wine, vinegar (initial stage) Bread making Fuel ethanol

Answer 20

Lactic acid bacteria

Answer 21

Cheese making Yoghurts Pickles, Fish/Soy sauce

Answer 22

- Pentose phosphate pathway - Entner-Doudoroff - Phosphoketolose (Warburg Dickens)

Answer 23

EMP refers to the people who discovered this method of glycolysis - Embden, Meyerhof and Parnas - it is the most common method

Answer 24

- Pentose phosphate pathway primarily an anabolic pathway that utilises the 6 carbons of glucose to generate 5 carbon sugars and to generate reducing power (NADPH) - 1 ATP, 12 NADPH molecules/glucose. - Operates simultaneously with glycolysis.

Answer 25

Bacillus subtilis, E. coli, Leuconostoc, yeast

Answer 26

- used by microbes lacking EMP. | - Pathway yields: one ATP, 1 NADPH, 1 NADH, 2 pyruvate/glucose molecule.

Answer 27

Zymomonas, Pseudomonas, Xanthomonas, Rhizobium, Agrobacterium

Answer 28

- Warburg Dickens pathway - One ATP and two NADH/glucose. Allows catabolism pentoses and production pentoses from hexoses for nucleic acid formation.

Answer 29

Lactobacillus, Leuconostoc

Answer 30

- First step of all reactions is Glucose to 2 pyruvate which gives 2ATP and 2 NADH - Three options then: Aerobic conditions:citric acid cycle (4CO2 and 4H2O and 26 ATP) Anaerobic conditions: Alcohol fermentation (2 ethanol + CO2+ 2 ATP +2NADH) and Lactic acid fermentation (2 lactate + 2 ATP +2NADH)

Answer 31

- Beer | - Wine

Answer 32

- Bread | - Vinegar

Answer 33

- Fuel ethanol production from plant biomass

Answer 34

- Beer: Barley, other cereals: Saccharomyces cerevisiae (worldwide) - Bourbon/whiskey: Corn, rye: Saccharomyces cerevisiae (US) - Wine: Grapes, other fruit: Saccharomyces ellipsoideus (worldwide) - Cider: Apples, other: Saccharomyces spp. (Worldwide) - Sake: Rice: Saccharomyces saki (Japan)

Answer 35

1. Carbohydrates obtained from grains, potatoes, or molasses are fermented by yeasts to produce ethanol in the production of beer, ale, and distilled spirits such as whiskey. 2. The sugars in fruits such as grapes are fermented by yeasts to produce wines. 3. Acetobacter and Gluconobacter oxidize ethanol to acetic acid (vinegar).

Answer 36

Idek bro but maybe from like 15,000 BC people were doing alcoholic/LA fermentation of fruits and grains

Answer 37

- Sacharomyces cerevisiae | - But 600 yeast strains IDed and used in industrial use/research

Answer 38

- Faster fermentation rates - Higher tolerances to alcohol - Other metabolic capabilities conferring a specific advantageous factor

Answer 39

They are diploid, polyploid or aneuploid (several copies of gene), therefore less likely to acquire mutation during fermentation process.

Answer 40

Genome complexity causes challenge

Answer 41

Top fermenting yeast like Sacharomyces cerevisiae and bottom fermenting yeast like S. carlsbergenisis and S. pastorianus

Answer 42

- S. cerevisiae - e.g - Flocculate (FlO gene) and float to surface - Used in brewing beers and stouts - Unable to ferment melobiose disaccharide) - Fermentation temps: 15-24ºC Able to grow at 37ºC

Answer 43

- Flocculate and sediment to bottom - Used in brewing lagers - Can ferment melobiose disaccharide - Fermentation temps:5-14ºC - Cold adapted, and often unable to grow at 37ºC

Answer 44

- Genome composition, particularly, genes involved in carbohydrate metabolism, thermotolerance, ethanol sensitivity - ferment wort in different ways, producing different flavour spectrums

Answer 45

- Beer is a fermentation of barley (or other grains) and hops by yeast. - The grain starch is broken down into glucose & other sugars and then fermented to ethanol. - The finish product is aged and then packaged for distribution and consumption. 6 stage process: ``` Malting Mashing and wort preparation Boiling with hops Fermentation Finishing Packaging ``` - From start to finish is typically 4-10 weeks. - Beer has a shelf life of around 6 months.

Answer 46

- Partial germination of cereal grain (e.g.barley) for 6-9 days. - Grain germinates and produces amylases and proteases. - Amylase provides sugar for the yeast fermentation, proteases solubilise compounds in the grain important for the quality of the beer. - Germinated malt is then dried, sometimes roasted, then crushed

Answer 47

- Mashing solubilise starch and other flavours in the grain and extracts flavours and preservatives. - Malt is suspended in water mixed with boiled malt adjuncts (now termed mash). - Mash is then incubated at 65-70°C for ~ 2hrs to allow the amylase to break down starch to glucose. - Temp then raised to above 75°C to inactive malt enzymes; mash allowed to settle.

Answer 48

- Colour - Flavour development - ALSO FLAVOUR IS EFFECTED BY WORT ADJUNCTS AND HOPS TOO!

Answer 49

- Carbon and energy source - Nitrogen source (amino acids and peptides) - Minerals - Growth factors (vitamins such as biotin and B complex)

Answer 50

- Fermentation of mostly maltose and malto triose (converted by ntracellular alpha-glucosidase to glucose) - Sucrose is extracellularly converted to glucose and fructose via a cell-surface invertase.

Answer 51

- important for yeast protein synthesis and growth - Also, have major influence on beer flavour due to conversion to flavour compounds

Answer 52

Fermentation may be anaerobic, but some O2 is still needed for sterol (cell membrane lipid) synthesis.

Answer 53

Brewers yeast cannot ferment starch or higher C dextrins

Answer 54

- Hops and wort are combined and boiled for 2.5 hours. The liquid is removed and ready for fermentation. Boiling with hops serves several purposes - - Concentration - Sterilisation, killing many microbes that might spoil the beer - Further inactivation of enzymes in the mash. - Solubilisation of important flavour/anti-microbial compounds in the hops and mash. Some of these add to the flavour of the beer while others, especially from the hops, have antimicrobial qualities which help preserve the beer.

Answer 55

Humulus lupulus

Answer 56

- Fermentation begins by adding the brewers yeast Saccharomyces carlsbergensis (or others) to the wort. The starter culture is usually obtained from a previous batch of beer and is added at a very high concentration (500 grams per 120 litres). Fermentation is at a low temperature between 3.3 and 14°C for 8 to 14 days, during which the glucose in wort is converted to ethanol and CO2. - Other compounds in the wort are also fermented to add to the characteristic flavour of beer (termed adjuncts).

Answer 57

Fermentation progress monitored by measuring metabolite production or substrate consumption: - CO2 evolved - Ethanol production - Heat generated - Reduction in specific gravity (relative density of an aqueous solution)

Answer 58

Beer has a pH of around 4, 3-8 % alcohol,and a complex and often unique flavour spectrum.

Answer 59

Ethanol: from metabolism of wort sugars via glycolysis Glycerol from DHAP – glycerol-3- phosphate (sweet flavour, smoother beer texture) Acids produced from both aerobic and anerobic metabolism wort sugars: acetic, lactic, succinic (cause drop in pH from ~5.2 to ~4) Acidity helps to preserve the beer Organoleptic (flavour and aroma) qualities not due to the ethanol, but to volatile compounds from hops (humulones) and yeast metabolites (aldehydes, higher C alcohols, diketones, esters, sulphur compounds) Flavouring/colouring agents are also added via wort adjuncts or during beer maturation process.

Answer 60

The fermented wort (green beer) is aged at 0 °C for a period of weeks or months depending on the brewer. At this time the yeast settle to the bottom of the vessel, bitter flavours are mellowed and other compounds are formed that enhance flavour.

Answer 61

This can involve filtering, pasteurisation, carbonation to 0.45 to 0.52% CO2, and clarification (removal of protein hazes using proteases). All of these processes depend upon the type of beer being made and each brewery will specialize the fermentation, aging and finishing of their beer.

Answer 62

- Osmotic stress from concentrated wort sugars (high gravity) - Oxidative stress - Low/high temperature - Ethanol (up to 8% for ales/lagers, 14% for wines) - Heat shock proteins (Hsps) highly induced

Answer 63

Reduced growth, viability and metabolism

Answer 64

Microarray analysis combined with mutant phenotypic analysis shows multiple stress response systems active during fermentation

Answer 65

- Microbial contamination: Wort is a rich nutritional source and subject to contamination by range microbes e.g. coliforms, acetic acid bacteria, lactic acid bacteria. - Lactic acid bacteria Lactobacillus and Pediococcus spp an occasional problem.

Answer 66

- pH 3.5-4.5 - very little oxygen - anti-microbial compounds from hops (tannic acid, catechins, other polyphenolates) - low levels readily utilisable energy sources - relatively high ethanol concentration

Answer 67

- Continuous beer production using immobilised yeast - Lower alcohol/calorie beer [achievable but see Nevoigt (2002)] - Ferment dextrins and pentoses - Directly degrade starch to fermentable sugars - Higher alcohol tolerance - Produce anti-microbials against most common wild yeast contaminants: Zymocins – killer yeast toxins, Nisin – bacteriocin (anti-microbial peptide) for lactic acid bacteria contamination

Answer 68

Nisin (bacteriocin)

Answer 69

- Serious Governmental, legal barriers and Consumer distrust of GMOs in traditional beverages - Organoleptic qualities of beers and lagers critically important: changing gene expression can cause deleterious flavour changes - More than a single strain may be involved in fermentation

Answer 70

12,000,000,000

Answer 71

Vitis vinifera

Answer 72

- 4000 varieties - Differ in: size, colour, shape of the berry, juice composition, ripening time, and resistance to disease - Only about a dozen are commonly used for wine making

Answer 73

Riesling, Chardonnay, Cabernet, Sauvignon, Pinot Noir, Gewurztraminer, Sauvignon Blanc, and Muscat grapes

Answer 74

- Juice from almost any fruit with a reasonable sugar content can be turned into wine e.g. plum, peach, rhubarb. - Vegetables can be used too, e.g. Parsnip, Beetroot, Dandelion

Answer 75

- US - Spain - Australia - Austria - France - Italy

Answer 76

- High sugar content of grapes, when ripe – up to 160/240 g/litre: This results in high ethanol levels (up to 14 %) - Rich in amino acids & other nutrients needed for yeast growth and metabolism. - Naturally acidic – grape juice has a pH of ~2.8-3.8. - Rich in pleasant organoleptic compounds.

Answer 77

White - clarified juice only Red - whole macerated fruit Dry - all sugar fermented to alcohol Sweet - sugar only partially fermented

Answer 78

Fermentation stopped when correct degree of sweetness reached by removing yeast, cooling/heating or addition of metabolic inhibitor such as SO2

Answer 79

- Saccharomyces apiculati, or wild yeast, indigenous to many fruits, including grapes. Sometimes used as starter in wine production. Less tolerant to ethanol levels, and can produce undesirable flavours. - Saccharomyces cerevisiae, (mostly used in beer and bread making). - Saccharomyces elipsoideus, true wine yeast mostly indigenous to grapes. Different strains of this cultivated yeast indigenous to different regions, with different fermentation characteristics. Has much higher tolerance of ethanol and SO2 than wild yeast.

Answer 80

- Alcoholic fermentation - Glucose to 2 pyruvate (2 ADP to 2 ATP and 2 NAD+ to 2 NADH) - 2 pyruvate to 2 acetylaldehyde via decarboxylation (-2CO2) - 2 acetylaldehyde to 2 ethanol (2 NADH to 2 NAD+)

Answer 81

- Starter yeast strain used, or yeast spp in natural microflora - Temperature - pH - Initial sugar concentration in grape must - Nutritional quality of grapes: B vitamins, nitrogen etc

Answer 82

- Represses endogeneous yeast activity - Preservative against microbial contamination - Anti-oxidant

Answer 83

- Pressing and juice extraction - Elimination of contaminants: wild yeast (SO2) - Additional starter yeast - Primary alcoholic fermentation - Remove excess yeast - Secondary fermentations: Malo-lactic fermentation using lactic acid bacteria such as Leuconostoc oenos, Second yeast fermentation to give carbonation - Remove excess yeast - Development of final wine bouquet (in barrel - red/ bottle - white) - Bottling, final maturation

Answer 84

- Additional yeast alcoholic fermentations to increase carbonation (sparkling wines) - Malic acid - lactic acide conversion: carried out to reduce acidity/astringent taste using Leuconostoc oenos - Leuconostoc treatment also adds flacour, and reduces likelihood of subsequent microbial contamination (via production bacteriocins)

Answer 85

- Seven pounds of dirt, one mouse nest, 147 bees, 98 wasps, 1,014 earwigs, 1833 ants, 10899 leafhoppers and 1.5kg of bird droppings

Answer 86

- Mould on grapes (Noble rot, caused by Botrytis) – off flavours. - Contamination by wild yeast (Schizosaccharomyces, Brettanomyces, Mycoderma), causing spontaneous fermentation and off flavours. (hence SO2 treatment)

Answer 87

- Spoilage by microbial acetic acid producers (Acetobacter) - Lactic acid bacteria (Lactobacillus, Leuconostoc and Pediococcus) - Chemical oxidation - Cork taint: chemical leaching from cork/drying out of cork allowing entry of air bourn microbial contaminants (e.g fungi)

Answer 88

- Low sulphide/DMS/thiol formation - Zymocidal (killer) properties - Low volatile acidity production - Genetic marking - Low higher alcohol production - Proteolytic activity - Liberation of glycosylated flavour precursors - Low nitrogen demand - High glycerol production - Metabolic properties with health implications (bacteriocins) - Hydrolytic activity - Low biogenic amine formation (histamine) -Modified esterase activity

Answer 89

- Rapid initiation of fermentation - High genetic stability - High fermentation efficiency - High sulphite tolerance - High ethanol tolerance - High osmotolerance - Low foam formation - Low temperature optimum - Flocculation properties (dropping to the bottom of a fermentor - easier to separate) - Moderate biomass production - Easily sedimented - Flavour characteristics - Resistance to desiccation

Answer 90

Bread consists of flour, water, salt, sugar and yeast, usually Saccharomyces cerevisiae, though natural microflora + lactic acid bacteria are sometimes also used to leaven bread – sourdough. Yeast cannot metabolise starch, so added sugar provided as energy source.

Answer 91

- Ingredients mixed and kneaded well – this results in glutens - wheat flour proteins associating to form long molecular ‘strings’. Gluten binds bread together, allowing formation of a dough. - Dough traps CO2 produced during fermentation, and bread rises due to pressure of CO2 build up. - Yeast also modify gluten, helping to promote even expansion, as well as adding flavour + nutrients (the yeast themselves + various metabolites)

Answer 92

- Decarboxylation | - As sugars are metabolised, CO2 & alcohol are released into the bread dough, making it rise.

Answer 93

- Utilisation starch as carbon source add amylase genes - Cryo-resistance (frozen bread doughs) increase production of cryoprotectant ‘antifreeze’ disaccharide sugar – trehalose - Osmotolerance (maintenance of viability during drying plus increased metabolic efficiency of yeast in sweet doughs) modify osmotic shock response system - Express flour modifying hydrolyases (e.g. flour improving enzymes – amylases, proteases, lipases normally obtained from filamentous fungi)

Answer 94

Ethanol to acetaldehyde (NAD+ to NADH) | Acetaldehyde to acetic acid (NAD+ to NADH)

Answer 95

French: vin-wine and aigre - sour

Answer 96

``` Malt Wine, red or white (includes balsamic) Cider Sherry Rice wine Distilled (malt) ```

Answer 97

Acetobacteriaceae

Answer 98

- Any kind of bacteria that produce acetic acid by oxidation of ethanol All can grow at low pH (<5). - Strictly aerobic, very high O2 demand. - Main genera Acetobacter and Gluconobacter - Gram-negative rods, found naturally on plant material, often in association with yeast (esp. fermented fruits).

Answer 99

peroxidans – full TCA cycle, therefore can oxidise acetic acid to CO2 + H2O

Answer 100

suboxydans – incomplete TCA cycle, therefore usually oxidises ethanol to acetic acid only.

Answer 101

Primary alcoholic fermentation | Secondary conversion of ethanol to ethanoic (acetic) acid

Answer 102

- Open vat - 14th Century, in Orleans, France - Vinegar from previous batch used to inoculate fresh batch - Acetic acid bacteria growing as bio film on liquid surface, inoculate, increasing acetic acid production, harvest

Answer 103

Method was very slow, and results uncertain. | Still used for a few speciality vinegars.

Answer 104

Trickling and Submerged processes

Answer 105

- 1820s - Circulation of alcoholic juice over films of acetic acid bacteria attached to inert supports such as beechwood/other wood shavings, charcoal. - Forced air is pumped up through the shavings via a high pressure pump. - Oxidation of alcohol is very exothermic, so generator has to be cooled. - Process takes about 3 days.

Answer 106

85 % conversion ethanol to acetic acid 10-15 kg acetic acid/m3 vol generator/day Final acetic acid concn: 10-12%

Answer 107

- Method developed in late 1940s. - Stainless steel bioreactor, operating at very high stir rates: 1450-1750 rpm creating very fine air bubbles and high level of medium aeration. - Specially selected strains acetic acid bacteria tolerant of suspension culture. Highly sensitive to O2 and ethanol levels. - Operated semi-continuously: Starting medium is 7-10 % acetic acid and 5 % ethanol. One batch will take 24-48 hours to ferment after which the fermenter is half emptied and new feedstock medium added for another run. - Cells inoculated at high density, and increase in biomass only slightly during the bioconversion.

Answer 108

90 % conversion ethanol to acetic acid 50-60 kg acetic acid/m3 vol generator/day Final acetic acid concn: 14 %

Answer 109

After bioconversion, vinegar diluted (4 %), clarified and filtered. Flavouring due also to organoleptic compounds present within original alcoholic substrate. Colouring may be added, or de-colourisation using ferricyanide bleaching.

Answer 110

- : thick (resin like) and aromatic vinegars made from concentrated grape must. - Processed using the Orleans method, aged in a succession of different wood barrels (chestnut, ash tree, cherry, mulberry, juniper and oak). - For official designation as Balsamic, vinegar must be at least 6 years old, and some is aged much longer (10-12 years). - Expensive – vintage Balsamics market at ~£70-£100/250 ml bottle

Answer 111

- Mostly bacteriophage infection - Microbial infection can also be a problem: Lactic acid bacteria can cause sliminess. Acetic acid bacteria can oxidise the acetic acid. - Metal ions forming insoluble salts with the acetic acid can cause off flavours and cloudiness.

Answer 112

Add variety, improve palatability of food staples Increase nutrient value Changes in pH, principally acidification (lactic, acetic acids) Creation of anaerobic environments Production of multiple anti-microbial compounds such as bacteriocins (peptide antibiotics), ethanol, H2O2. Reduction in toxicity of certain food staples

Answer 113

- Reduction in water content of food (drying/add salt) | - Presence added/natural preservatives (nitrate – nitrite)

Answer 114

Prepared from Cassava, starchy root from member of Yucca family ``` Carbohydrate staple (along with yams, Sweet potatoes, rice, etc) in tropical countries where wheat/potato culture unfeasible. ``` Toxic in raw state due to high cyanide (HCN) content HCN reduced by soaking sliced root. Cassava soaked root slices cooked directly, or starch extract (starch paste) turned into tapioca flour Microbial fermentation starch via Rhizopus fungus and lactic acid bacteria reduces toxicity further (acidification) & creates a fermented gruel more nutritious & flavoursome than starch paste alone.

Answer 115

- Water:solids (87:13) - Lactose:fat:protein:ash/minerals (37:30:27:6) - Casein:Whey proteins (80:20)

Answer 116

- Cheese: milk, lactic acid bacteria/fungi, worldwide - Yoghurt: milk, Streptococcus lactis, Lactobacillus bulgaricus, worldwide - Kefir: milk, Streptococcus lactis, Lactobacillus bulgaricus, Asia - Taette: milk, S. lactis var. taette, Scandinavia - Tarhana: wheat meal and yogurt, lactic acid bacteria, Turkey

Answer 117

10, 000,000,000 kg/annum From 50, 000,000,000 litres milk 5-10 litres of milk are required to make 1 kg of cheese. Made in every part of the world >4000 different varieties

Answer 118

- All produce lactic acid. - Non-motile Gram+ve bacteria. - Non-spore forming. - Cocci or rods - Lactococcus or Lactobacilli. - Micro-aerophilic. - Mesophilic - cold reduces growth & viability: lactic acid production is inhibited at <20oC; optimum growth temp is - 30-35 oC. - Ubiquitous contaminants of milk. - Production of bacteriocins (e.g. nisin). - Resistance to bacteriocins.

Answer 119

Some LAB can be moderately thermophilic - survive up to 55 oC.

Answer 120

Thermophiles (thermophilic) – tolerant of high temp. range from 50°C to 100°C, but prefer temps between 50°-70°C.

Answer 121

Mesophiles (mesophilic) – prefer 30-40°C & tolerate of temps of 20°C to ~50°C.

Answer 122

Psychrophiles (psychrophilic) -thrive at low temps. | Prefer 10-20°C but will slowly metabolise at even 0°C

Answer 123

Lack functional haem based electro-transport system and complete Krebs cycle Energy production therefore mostly by substrate level phosphorylation - fermentation of lactose

Answer 124

- homofermentative: lactic acid only product - heterofermentative: lactic acid main but ethanol + others also possible: CO2, acetic/proprionic acid, acetaldehyde - principal flavour of yoghurt, diacetyl - an important buttery flavour compound.

Answer 125

- Generally weak proteolytic activity - LAB auxotrophic for many amino acids - prefer protein rich media. - Metabolism of milk proteins/peptides/amino acids important for cheese flavour development

Answer 126

Plasmids - lactic acid production: plasmid instability can lead to increased fermentation times.

Answer 127

- Cheese is dehydrated milk curd and concentrated milk fat - Formed from the coagulation of milk proteins, separated from the whey, pressed or moulded into a solid mass and matured via action of microbial enzymes. - 5-10 litres of milk are required to make 1 kg of cheese. - First stage: milk protein coagulation (curdling) - Second stage: maturation, or flavour ripening

Answer 128

- Milk protein coagulation (curdling) - Milk protein casein (4 different kinds casein in milk) is curdled (precipitated) through addition of acid, action by lactic acid bacteria, and the enzyme rennet (chymosin). - Curd is separated from the liquid portion of milk, called whey

Answer 129

- Maturation, or flavour ripening - Hard cheeses (cheddars) are produced by lactic acid/other bacteria growing in the interior of the curd. - Semi-soft cheeses (Camembert) are ripened by bacteria/fungi growing on the surface (e.g. Penicillium cambertii) - Soft cheeses are also ripened by Penicillium spp growing on the cheese surface.

Answer 130

Unripened and ripened

Answer 131

- Curd more or less used directly e.g. Cottage cheese – curd generated from Streptococcus cremoris and Leuconostoc cremoris fermentation of skimmed milk. - Cream cheese – same microbes as for cottage cheese, except whole milk or single cream used.

Answer 132

- Cheeses are pressed and dried curds (sometimes cooked) subjected to second round of microbial processing, during which flavour is developed.

Answer 133

- Balanced mixtures of selected micro-organisms. - Fermentation faster and more controlled Consistency of aroma and flavour - Minimise bacterial contamination of culture. - Usually a mixture of lactic acid bacteria (LAB): Lactococcus lactis, L. cremoris, & Streptococcus thermophilusvmain starter cultures, added at high cell density (108-9 cfu/g)

Answer 134

Can be from any mammal, but usually cow, sheep, goat, buffalo. Chemical & microbiological quality checked. Maybe pasteurised.

Answer 135

- Principally casein - via acidification (lactic acid) and action of proteolytic enzymes from LAB plus any added proteases (rennet, also called chymosin). - Coagulation traps milk fat and moisture.

Answer 136

- Curd chopped, pressed and heated to expel fluid – whey. | - Amount of residual moisture determines if cheese is soft or hard

Answer 137

Addition of salt, heating, addition flavouring agents, more rennet, microbial ripening cultures. Cheese is shaped, and stored.

Answer 138

- Very complex, and still largely uncharacterised. - ~25-35 % of insoluble curd proteins converted into soluble peptides and amino acids - the major flavouring elements in mature cheese. - Flavour development due to both microbial fermentation and action added enzymes

Answer 139

1. preparation of milk 2. coagulation of proteins 3. whey separation 4. processing of curd 5. ripening of the cheese

Answer 140

LAB protease production of relatively minor importance during initial cheese curd formation, therefore additional proteases (rennet) used Rennet is a general term used to describe a variety of enzymatic coagulants (proteases) used in cheese making. They can be of animal, plant or microbial origin (bacteria or fungi). One of the best known and widely used coagulants for cheese making is rennin, an aspartyl protease which is extracted from the fourth stomach, or abomasum, of a young calf. For economic (and social acceptability) reasons calf rennet became scarce, which led to investigation of other sources of milk-coagulating enzymes, e.g.moulds, (Endothia parasitica, Mucor miehei) and bacteria (Bacillus subtilis, Bacillus cereus). Rennet possesses another important function in addition to coagulating milk. As the cheese matures, rennet hydrolyses casein into peptides. This is important for developing desirable flavour compounds and preventing bitterness in cheese. Now, recombinant rennet used – cheeses made with it are termed ‘vegetarian’ cheeses.

Answer 141

``` Bacteria and enzymes indigenous to the milk Lactic acid bacteria starter culture Rennet Microbial lipases Added bacteria, moulds or yeasts Environmental microbial contaminants ```

Answer 142

FROM MONTHS TO 1-2 YEARS - ACCORDING TO CHEESE VARIETY

Answer 143

Holes made in cheese to aerate it - allowing growth of Penicillum mould

Answer 144

- Organoleptic qualities individual cheeses consequence of microbial metabolism of milk carbohydrates, fats and proteins/peptides/amino acids - Choice of starter and secondary cultures (bacteria, yeast, filamentous fungi), and on local cheese microflora - Released microbial metabolites (secreted or via autolysis of starter or secondary cultures) - Chemical interactions between milk components and microbial metabolites - Storage environment and duration of ripening

Answer 145

- Lactose to lactic acid (diacetyl/proprionate) via glycolysis - Fat to fatty acids via lypolysis - Casein to amino acids via proteolysis

Answer 146

- Volitile flavour component production increases, we know this from mass spectrograph evidence

Answer 147

Made using propionic acid producing bacteria (PAB) secondary cultures PAB are Gram positive short rod-shaped bacteria which metabolise lactate to proprionate and CO2 3 Lactate --> 2 Propionate+Acetate+CO2+H2O. PAB grow in many cheese varieties during ripening, and are the characteristic microflora associated with Swiss-type cheeses such as Emmental, Gruyère, Appenzell and Comté. Excreted proprionate, and proline released during autolysis of PAB, gives Swiss cheese its characteristic sweet, nutty flavour. CO2 causes the characteristic ‘holes’.

Answer 148

- Slow lactic acid production at start of fermentation is an ongoing problem. ``` Can be due to: Bacteriophage infection (major problem) esp. for Streptococcus spp. ``` Loss of desirable phenotypes due to plasmid instability from continued successive sub-culturing (back slopping). Microbial contamination: endogenous microbes Staph. aureus, Listeria monocytogenes – particular problem since it can grow at refrigeration temperatures.

Answer 149

- Creation of anaerobic environments by CO2 production - Competitive exclusion of microbial/fungal pathogens - Production of multiple anti-microbial compounds

Answer 150

Low molecular weight: - Lactic, proprionic and acetic acids: cause acidification (pH<5) affecting growth of Gram-ve enteric pathogens - H2O2 (LAB often lack catalase): causes free radical damage of Pseudomonas spp. - Diacetyl (formed during transformation citrate): active against - Gram-ve enterics, Aeromonas, some Gram-+ve Bacillus spp. - Reuterin (formed anaerobically from glycerol by Lactobacillus spp. ): active against many enteric pathogens, Clostridium, Staphylococcus, some fungi: Candida and protozoa: Trypanosoma High molecular weight: - Bacteriocins (peptide antibiotics)

Answer 151

narrow spectrum anti-microbial peptide (only inhibit species closely related to the bacteriocin producer).

Answer 152

Lactococcus lactis

Answer 153

- ?? maybe we are not suuurreee?? - growth regulation, communication, ecological advantage over competitors. - Chromosomally encoded - Lantibiotic – peptide antibiotic containing lanthionine

Answer 154

- 34 amino acid, 3500Da hydrophobic cationic (+vely charged) pentacyclic peptide. - Unusual amino acids.

Answer 155

Inserts into lipid bilayer | forming pores. Kills rapidly via dissipation ATP/ions/proton gradient

Answer 156

LAB bacteriocin used to control food spoilage organisms in both heat-processed and low pH foods (dairy products, egg products, sauces (mayonnaise), saurkraut, canned foods, some wines). First isolated in 1928, granted WHO approval as a food preservative in 1969, and FDA registration as a food additive in 1988. E assignment E234 Very low toxicity to humans, LD50 7g/kg. GRAS status food preservative Heat stable, activity greatest at acidic pH Effective at very low concentrations

Answer 157

Includes Listeria, Clostridia, Bacillus, Enterococcus, Staphylococcus. e.g. Bacillus sporothermidans – thermophilic, spore forming pathogen problematic in UHT-treated foods. Little activity against Gram-ves and fungi.

Answer 158

Treat and prevent bovine mastitis Wound dressings (active against S. aureus)

Answer 159

Producer organisms have GRAS status but bacteriocins currently not widely in commercial use

Answer 160

Bacteriocin from freeze-dried culture of Propionibacterium freudenreichii ssp. Shermannii used in production of MicroGard food storage and packaging

Answer 161

- Polyene antifungal produced by Streptomyces spp. - Commercial use as surface anti-mycotic for cut/sliced foods such as cold meats and cheese. - Clinical applications in treatment of fungal keratitis, particularly infections caused by Aspergillus spp.

Answer 162

Recombinant calf rennet (chymosin) approved by FDA. Bovine gene expressed in GRAS microbes such as yeast Now used in 70% of U.S. cheese In UK, chymosin-produced cheeses are called "vegetarian cheese” (vegetarian rennet)

Answer 163

Low fat, full flavour cheeses - fat-moisture balance important for flavour, texture and melting characteristics Pro-biotic cheeses Long(er) life cheeses - bacteriocin (nisin) supplemented (via nisin-over-expressing starter cultures or as additive) to inhibit growth of spoilage organisms (increased shelf life processed cheeses/cheese spreads)

Answer 164

1:1 ratio of Streptococcus thermophilus (provides lactic acid) and Lactobacillus bulgaricus (provides diacetyl and other flavours/aromas).

Answer 165

Pasteurised skimmed, semi-skimmed or whole milk.

Answer 166

Lactose present in the milk is converted by high cell density LAB innoculum to lactic acid which decreases the pH to ~ 5.3 (12-24 hr, 30-35 oC). Casein becomes destabilized and irreversibly coagulates to form a gel. LAB exopolysaccharide also contributes to yoghurt texture.

Answer 167

Butter milk is made from semi-skimmed milk. Fermented by Streptococcus lactis (lactic acid producer) plus Leuconostoc citrovorum which convert lactic acid to aldehydes and ketones which gives it its flavour and aroma. Popular milk drink with the lactose intolerant – much of lactose is removed during fermentation.

Answer 168

Sour cream is produced by the same bacteria as buttermilk, Streptococcus lactis (lactic acid producer) plus Leuconostoc citrovorum which convert lactic acid to aldehydes and ketones which gives it its flavour and aroma, but the starting milk product is pasteurised light cream. LAB less numerous than in buttermilk.

Answer 169

- Probiotic - Greek "for life". - Mono- or mixed cultures of live micro-organisms which, when applied to animal or man, beneficially affect the host by improving the properties of the indigenous micro flora. - Common terms for probiotics are "friendly", "beneficial" or "healthy" bacteria. - Probiotic bacteria generally lactic acid bacteria used in the production of yoghurt, fermented milk products and dietary supplements. - Consumed by man for thousands of years, but health benefits only recently being appreciated.

Answer 170

Lactobacillus acidophilus, L. casei, L. bulgaricus, L. plantarum , L. salivarius, L. rhamnosus, L. reuteri, Bifidobacterium bifidum, B. longum, B. infantis and S. thermophilus.

Answer 171

- Prebiotics: functional food ingredients which manipulate the composition of colonic microflora in order to improve health. - Non-host digestible oligosaccharides which promote growth & colonisation of intestinal bifidobacteria - Not destroyed during cooking - Doses of 4-20 g/day efficacious - Synbiotic: simultaneous administration of both probiotic and prebiotic.

Answer 172

Fructo-oligosaccharides, inulin, lactulose and galacto-oligosaccharides

Answer 173

Obtained from fruit, vegetables. e.g. bananas, asparagus, garlic, wheat, tomatoes, Jerusalem artichoke, onions and chicory

Answer 174

Production of inhibitory substances. Blocking of adhesion sites. Competition for nutrients. Stimulation of immunity.

Answer 175

- Inhibitory to Gram +ve and -ve - Organic acids (lactate, acetate) (acidify medium) - CO2 (anaerobic environment) - H2O2 (oxidises -SH groups causing denaturing of enzymes or peroxidation of membrane lipids --> increased membrane permeability) - Bacteriocins: ribosomally synthesized antimicrobial proteins or peptides, may undergo post-translational modification - Inhibitory factors may reduce not only numbers of viable pathogenic organisms but may inhibit pathogen metabolism & toxin production

Answer 176

- Blocking of adhesion sites - Competition for nutrients otherwise consumed by pathogens - Stimulation of immunity

Answer 177

Production of inhibitory substances. - LAB produce a variety of substances inhibitory to both G+ve & G-ve bacteria. - Include organic acids, hydrogen peroxide and bacteriocins. - Inhibitory factors may reduce not only numbers of viable pathogenic organisms but may inhibit pathogen metabolism & toxin production. Blocking of adhesion sites. - Competitive inhibition for bacterial adhesion sites on intestinal epithelial surfaces. Competition for nutrients. - Probiotics may utilise nutrients otherwise consumed by pathogens. Stimulation of immunity. - Underlying mechanism(s) immune stimulation poorly understood but specific LAB cell wall components or cell layers may act as adjuvants and increase humoral immune response.

Answer 178

Soy sauce – traditional flavouring agent Citric – organic acid Glutamic – amino acid – flavouring agent Lysine – amino acid – nutritional supplement

Answer 179

``` Products of growth processes, e.g. amino acids, vitamins, nucleotides, ethanol, lactate, enzymes cellular biomass ```

Answer 180

``` Produced from pathways not required for growth: e.g. antibiotics, toxins, pigments, exopolysaccharides (xanthans). ```

Answer 181

- First made over 3000 years ago in China - World production (2002): 5,000,000,000 litres - Important general purpose savoury food flavouring agent & condiment, particularly in the South East Asia. - Now, used world-wide.

Answer 182

Aspergillus oryzae (fungus) LAB/yeast (Pediococcus, Tetragenococcus/Candida/Saccharomyces)

Answer 183

Soy sauce is a dark brown liquid made from soybeans that have undergone two types of fermentation processing. Soy sauces have a salty, sharp taste, but are lower in sodium than traditional table salt. Rich in mono-sodium glutamate, which enhances the flavour intensity of savoury foods.

Answer 184

- Shoyu is a blend of soybeans and wheat. - Tamari is made only from soybeans. - Teriyaki sauce can be thicker than other types of soy sauce and includes other ingredients such as sugar, vinegar and spices. - Worcestershire sauce is made from soy sauce by adding spices and further fermentation.

Answer 185

Koji culturing | Moromi fermentation

Answer 186

Fungal digestion and mobilisation of soy bean storage carbohydrates and proteins: 1. Aerobic fungal digestion of beans & wheat releases fermentable sugars and dextrins – 2-3 days in large, aerated vats, 25-30 oC. 2. Salt (24 % brine) added to extract soluble sugars & nutrients, and inhibit growth spoilage microbes.

Answer 187

LAB/yeast fermentation of sugars, proteins and amino acids: 3. Anaerobic homolactic acid fermentation by lactic acid bacteria such as Pediococcus halophilus & ethanol fermentation by Pichia or Saccharomyces. 6-9 months fermentation in vat to allow flavour development, ambient temps. Liquid soy sauce filtered, pasteurised & bottled

Answer 188

World market 400,000 tons, $1.4 billion Citric acid is a colorless, crystalline acid with a sweet tart flavour. Found in fruit juices such as lemon, lime, and pineapple. Originally extracted from lemon juice, but in 1920s, Pfizer produced citric acid using Aspergillus niger. This was the first aerobic industrial fermentation process. Now, citric acid almost exclusively produced by A. niger fermentation of carbohydrates. Main producers are Western Europe, China and USA.

Answer 189

Main producer of citric acid is the filamentous fungus Aspergillus niger - Obligate aerobe. - Deuteromycete. - Commonly found in soil, decaying matter, can cause plant wilts. - Excretes large amounts of citric acid into medium to scavenge for Fe (siderophore) - GRAS

Answer 190

Used as a flavouring in beverages (soft drinks), confectionary, and other sweet foods. Pharmaceutical products. Metal chelator, used in to maintain metals in solution for electroplating, metal cleaning. Detergent uses.

Answer 191

Inhibiting aconitase activity that would convert citrate

Answer 192

- Aconitase is an iron sulphur centre enzyme. - It requires Fe2+ for activity. - Citric acid production is therefore increased by growing A. niger on low Fe-containing media, or by adding Fe-antagonists, such as Cu

Answer 193

- Obligate aerobe, therefore high aeration important. - Grown initially in surface culture on solid growth substrate in large vats, but now submerged batch cultured in corrosion resistant, stainless steel/steel lined with glass fermenters of 20 000-90 000 litre capacity. - Can produce citric acid from starch, starch hydrolysates, sucrose, sugar cane molasses, sugar beet molasses. For optimum citric acid production, medium must have a minimum of 140g/litre fermentable sugar (increased activity glycolytic enzymes). - Inoculum for fermenter is produced on solid growth medium, and formed into pellets.

Answer 194

- Chelex treated to remove Fe, [Cu] - Low in nitrogen to minimise biomass production - Fermentation at 30 degrees Celsius, pH falls as citric acid produced

Answer 195

- 0.7-0.9g citric acid/g glucose | - 18.0kg citric acid/m^3 vol/day

Answer 196

- Fungal mycelium removed. - Culture supernatant filtered. - Heated with lime (Ca), Ca citrate precipitates. - Sulphuric acid added, which forms Ca sulphate, liberating the citric acid. - Dilute citric acid de-colourised with activated charcoal, and evaporated to produce white crystals.

Answer 197

Amino acids are primary metabolites whose production in vivo is tightly regulated, often via feedback (end product) inhibition of rate-limiting enzyme in synthetic pathway.

Answer 198

- Manipulate regulation of enzyme activity to remove feed back inhibition - Increase the production of anabolic enzymes. - Block pathway(s) that lead to unwanted by products - Block pathway(s) that result in the degradation of the product molecule - Limit the ability of the producer to make the precursor only - Modification of culture medium also important

Answer 199

Amino acid synthetic pathways over-lap, so above strategies frequently result in over-producer strains with various amino acid auxotrophies Combination(s) of above strategies used in over-producing glutamate, lysine and other amino acids

Answer 200

Initially, most modfications to amino acid production made using classical chemical/irradiation mutagenesis, however now recombinant DNA technology much more important.

Answer 201

- Amino acid with main use is as a savoury food flavour enhancer – soups, sauces, snackfoods. - Mono-sodium glutamate - MSG - Extensive use in oriental/processed food cooking. - ‘E number’ E621.

Answer 202

>800,000 tons, $915 million

Answer 203

- Principally Arthrobacter genera: Brevibacterium, Corynebacterium glutamicum main L-glutamate producers. - Gram+ve, biotin-requiring microbes with v. high glutamate dehydrogenase (GDH) activity

Answer 204

Alpha-ketoglutarate + NH4+ +NAD(P)H + H+ ---GHD---> L-glutamate + NAD(P)+ + H2O - L-glutamate is produced from alpha-ketoglutarate, a TCA cycle intermediate

Answer 205

- Increase the production of anabolic enzymes. Increase synthesis of isocitrate dehydrogenase (IDH) - Block pathway(s) that result in the degradation of the product molecule Remove a-ketoglutarate dehydrogenase (a-KGDH) - [Strategies also used in over-producing other amino acids, such as lysine] -Initially, most modifications to L-glutamate production made using classical mutagenesis shot gun approaches, however now over-production via more precise recombinant DNA technology.

Answer 206

- Corynebacteria nutritionally quite fastidious, Vitamins, amino acids, purine/pyrimidine supplementation necessary. - Cane or beet molasses growth substrate, preferred carbon source glucose or sucrose. - Nitrogen source is limited to avoid inhibition GDH activity. - Fermentation is aerobic, batch, carried out in 450,000 litre fermenter, at 30-37 oC, for about 40 hrs. L-glutamate produced about 20 hours into fermentation.

Answer 207

Glutamic acid is not secreted, so medium is adapted to make bacteria ‘leaky’ – limit biotin, restrict membrane lipid biosynthesis by adding antagonists (e.g. C16-C18 unsat. fatty acids) or surfactant detergents.

Answer 208

Yield: 1 g L-glutamate/1.4 g glucose | 80 g/litre culture medium

Answer 209

- Bacterial cells removed. - Culture supernatant filtered. - Medium acidified with HCl, glutamate crystallises out of solution. - Crystals filtered, washed. - Monosodium glutamate (MSG) is prepared by adding NaOH to the glutamate crystals, which initially re-dissolve, then re-crystallise.

Answer 210

- Very widespread use in pre-prepared processed savoury foods - MSG has GRAS status, but increasing questions about safeness of current daily RDAs Chinese food syndrome: - Symptoms (within 1-2 hrs of chinese meal): headache, flushing, sweating, facial swelling, numbness, chest pain,abnormal heart rhythm - Cause: acute hypersensitivity reaction to MSG

Answer 211

``` Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine ```

Answer 212

``` Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamic Acid Glutamine Glycine Proline Serine Tyrosine ```

Answer 213

- World market >300,000 tons, $600 million - Essential amino acid not manufactured by the mammalian body - Only source of L-Lysine is through diet or supplementation. - Promote muscle growth, absorption of calcium, collagen formation, and production of metabolic enzymes. - High requirement by high wheat and corn intake vegetarians, important animal feed additive (cattle, sheep) - Some L-lysine made by chemical synthesis

Answer 214

L-lysine producers principally from Arthrobacter genera: Brevibacterium flavum, Corynebacterium glutamicum - Gram+ve, biotin-requiring microbes

Answer 215

L-lysine is produced from oxaloacetate, a TCA cycle intermediate.

Answer 216

High yields of lysine were obtained by modifying regulatory signals that controlled lysine synthesis: Blocking side reactions Preventing feedback inhibition Classical mutagenesis and screening methodology used to isolate original over-producers, now genetic engineering used.

Answer 217

1. Block side reactions : remove homoserine dehydrogenase (HS): auxotrophic for threonine isoleucine & methionine Wt HS ~5 g/l -->15-30 g/l 2. Remove aspartokinase (APK) feed back control mechanism: Wt APK APK/HS ~5 g/l --> ~35 g/l --> >60 g/l AEC growth inhibitory analogue of lysine. Mutagenise and plate on AEC media. Colonies which grow are resistant to AEC have mutation in aspartokinase lysine binding site.

Answer 218

C. glutamicum overproducer mutants defective in homoserine dehydrogenase convert all aspartate semialdehyde to lysine, so supplementation with threonine, methionine & isoleucine necessary. Cane or beet molasses usual growth substrate, usually supplemented with soya bean hydrolysate, or yeast extract; other CHOs, inc. acetate, ethanol sometimes used. Fermentation is aerobic, batch, with high initial inoculum (~1-1.5 mg/ml, ~10^8 cfu/ml) to minimise lag phase, at around 28 oC, for about 60 hrs. Final cell density is typically around 15 mg/ml (~10^9 cfu/ml). L-lysine produced thoughout growth, but mainly during exponential phase, tailing off during stationary phase. Specific transporter for lysine, so directly exported into growth medium.

Answer 219

Yield: ~30-35 % conversion rate, 1 g L-lysine/ 3g glucose | 40-45 g/200g molasses

Answer 220

- Bacterial cells removed. - Culture supernatant filtered. - Medium acidified with HCl, L-lysine (positively charged) adsorbed onto cation exchange column. - L-lysine eluted from column with ammonia solution (NH4OH). - Eluted L-lysine acidified with HCl. L-lysine crystallises as L-lysine hydrochloride.

Answer 221

Small, motile, flagellated Gram-ve rods, aerobic, produce yellow pigments and typically parasitise plants

Answer 222

Causes brown/black rotting lesions (back rot) on brassica (cabbage and cauliflower) leaves. However, most noted for the fact that it produces copious amounts of the exopolysaccharide (EPS) biopolymer xanthan.

Answer 223

Thought to retain water & prevent dehydration, faciliate binding cells to surfaces & each other, act as a barrier to phage attachment & infection, harmful chemicals (for animal pathogens avoidance also of the immune system). Xanthan is a secondary metabolite.

Answer 224

- World market $408 million /year, market,cost is $20-25/kg. - Annual world wide production of ~30,000 tons. - Many micro-organisms capable of synthesising EPS, e.g. EPS from LAB important in texture of yoghurt. - However, Xanthan only bacterial EPS produced industrially on a large scale. - Xanthan gum discovered 50 years ago in Illinois (USA). - Gained FDA recognition as a GRAS additive in 1969. - E number E415 (designated as ‘thickener’). - Used in wide variety applications, food (thickening agent), adhesive, emulsifier, petroleum industry (lubricant for oil drills).

Answer 225

Used in wide variety applications, food (thickening agent), adhesive, emulsifier, petroleum industry (lubricant for oil drills).

Answer 226

- non-toxic - water soluble - stable over a wide range temps & pH (acid/base & high salt compatible) - high viscosity of dilute solutions, stable to freeze thawing. - pseudoplastic: ie increase in shearing pressure causes a decrease in viscosity. However, viscosity restored when shearing ceases

Answer 227

High mol wt rigid, helical hetero-polysaccharide with a primary structure of repeated penta-saccharide units (D-glucose units, D-mannose & one glucuronic acid, molar ratio 2:2:1). Xanthan main chain consists of b-D-glucose units linked at 1 to 4 positions. Chemical structure of the main chain is similar to that of cellulose. Proportion of side chain mannoses linked to acetic & pyruvic acid, conferring a net negative charge. Considerable diversity in structure between strains, and growth media

Answer 228

Xanthan interacts with galactomannan gums (e.g. locust bean gum, guar gum), so that the viscosity of a mixture of these polymers is increased synergistically (and the amount of polymer needed substantially reduced).

Answer 229

Xanthan is well tolerated by the human immune system, active at very low concentrations, and has GRAS status for food applications. Its hydrogel properties, alone and in conjunction with other exopolysaccharides, and the way the human digestive system processes xanthan have led to investigations of its utility in drug formulations, and in substitutes for body fluids such as saliva - Time release drug delivery

Answer 230

X. campestris pv. Campestris main producer. Xanthan production during fermentation increases viscosity of medium, causing O2 & nutrient transfer problems. Aerobic, fed batch culture usually used, but possible to grow continuously, provided nitrogen is limited. 50,000-200,000 litre paddle stir fermenters. Starter culture is grown in rich media, to allow high initial inoculum.

Answer 231

usually a carbon:nitrogen ratio of 10:1 glucose or sucrose preferred, at 3-4 % v/v nitrogen: casein or soya hydrolysate, corn steep liquor, yeast extract or urea. trace salts and buffering with phosphate to pH 7.0 (gum production markedly influenced by pH) Culture normally grown at 28-30 oC, for about 3 days. Xanthan produced during late exponential, and into stationary phase.

Answer 232

Typical yield: ~ 50 g/litre

Answer 233

``` Microbes themselves can be used as a nutritional supplement to human diets or animal feeds. Mushrooms Yeast Algae (Spirulina) Bacteria ```

Answer 234

Single cell protein is composed of cells or protein extracted from micro-organisms grown in large quantities. The idea of using SCP as animal feed or human food is not new; yeast has been used as food protein since the beginning of the century. However, in the past 15 years, there has been a dramatic increase in research on SCP and in the construction of large-scale plants for its production, especially for the production of yeast

Answer 235

1. The rapid growth rate of micro-organisms and high productivity 2. Will grow on cheap raw materials. 3. High feed efficiency. 4. Process occupies little land. 5. Production is independent of seasonal and climactic changes. 6. SCP has consistent production quality. 7. Strain selection and development straightforward, and amenable to improvement.

Answer 236

Doubling time of bacteria can be between 0.3-2 hours For yeasts 1-3 hours, for algae 2-6 hours Filamentous fungi 4-12 hours. Mammalian cells - often days.

Answer 237

Including waste carbon sources such as whey or sugar beet molasses (yeast), lignocelluosic (plant/woody waste) (mushrooms) methanol (Methylophilus methylotrophus), or even CO2 (in the case of algae such as Spirulina or Chlorella.) Conversion waste to SCP removes environmental pollutant & produces a useful nutrient source.

Answer 238

Feed efficiency is expressed as g protein produced / kg feed consumed. Fungi, algae and bacteria have a feed efficiency which is many times higher than protein production by cattle or pigs.

Answer 239

1) Media preparation 2) Fermentation 3) Separation and downstream processing

Answer 240

May require pre-treatment if waste product with toxic potential.

Answer 241

Usually continuous culture, at close to microbe’s (mew)max

Answer 242

Cells separated from spent medium via filtration/centrifugation. Often heat shocked to reduce nucleic acid levels, washed, pasteurised, dehydrated & packed

Answer 243

Utilises waste from dairy industry – whey - to grow lactose-using yeast. Operated by Bel Industries in France. Product, Protibel, is eaten by humans & animals.

Answer 244

Developed in Sweden. Utilises waste from potato processing to grow yeast SCP for animal feed supplement. The substrate is predominantly starch, so two symbiotic yeasts are used: Sacchromycopsis fibulgeriaI, which produces the amylases needed to degrade the starch, and Candida utilis which provides biomass . Process operates in 2 stages: S. fibulgeriaI is grown in a small fermenter on the sterilised potato waste. It degrades the starch, and the resulting broth pumped into a larger fermenter of 300 000 litre capacity containing C. utilis which dominates the fermentation, & usually constitutes 90 % of final SCP product.

Answer 245

Utilises spent sulphite liquor from wood processing industry (which contains both monosaccharides & acetic acid) to grow a filamentous fungus, Paecilomyces variotii. Supplements of other carbon sources (molasses, whey, hydrolysed plant biomass) also used. Fermentation is operated continuously, producing about 10,000 tonnes of SCP/year, for animal feed.

Answer 246

Popular in 1970s when oil prices were low, and conventional animal feedstock prices high. Initially operated by ICI. Alkanes used as carbon source (methane, reduced to methanol) to produce a SCP feedstock for chickens, pigs & veal calves. Methylophilus methylotrophus – obligate aerobe isolated from activated sludge. Grown on methanol or ammonia substrates. SCP production process largely a failure for ICI for economic reasons (oil prices rose, soya feed stock prices fell). However, Pruteen notable for size & sophistication of fermenter technology.

Answer 247

- Bel - Symba - Pekilo - Pruteen

Answer 248

RHM started mycoprotein research in 1964 to investigate potential for F. graminearum as SCP. Took 20 years for UK MAF approval as human food protein. Current production ~1000 tonnes/year. Produced using fermentation facilities of defunct Pruteen SCP plant. Aerobic fermentation in 40,000 litre fermenter. O2 levels strictly controlled to prevent anaerobic metabolism and production of ‘off flavour’ metabolites. High grade corn syrup made from corn, wheat or potato starch as carbon source, with biotin and mineral supplements. Ammonia used as N source, and to control pH. Fermentation is continuous, at 30 oC, pH 6.0. Fungal biomass divides every 4-5 hr.

Answer 249

Yield: 15/20 g biomass/litre

Answer 250

- Quorn has little flavour - Microfilamentous structure of the fungus can be partially modified to resemble the texture of meat (chicken) . - Quorn producer fungus has too high a nucleic acid content (10 %), so RNA of the biomass is reduced by a heat shock at 64 oC for 30 mins. This renders the fungi unviable, & induce RNAses which degrade RNA to nucleotides, which diffuse out of the cell. Nucleic acid content reduced to <2%. -Mycelium harvested by vacuum filtration. Filter cake formed is a mass of interwoven fungal hyphae which can be frozen as sheets, granulated or powdered.

Answer 251

Most SCPs are not used for human consumption – used as animal feed. Exceptions are yeast extract and Quorn. Nucleic acid content some SCPs can be a problem for humans: digestion leads to high levels of purine compounds, which in turn causes elevations in uric acid – this may crystallise in joints/tissues causing gall stones, kidney stones or gout. Filamentous fungi SCP could be contaminated with hepatotoxic aflatoxins. Microbes could also absorb toxic/carcinogenic compounds from growth substrates. Allergic responses or digestive problems also a consideration.

Answer 252

SCP has to compete with soybean and fishmeal for the animal feed supplement market, with both these products at present heavily subsidized within the European Community agricultural market. Despite several marketing efforts SCP has never been accepted by the UK consumer as dietary food - with the exception of Quorn mycoprotein. Potentially, the facility to produce SCP would be of major use for generating food for the population of tropical countries or arid regions such as Middle East and Africa. In these countries there are a number of problems with the traditional food production: food tends to be high in carbohydrate, low in protein high percentage of land may be unsuitable for use in agriculture. Unfortunately, most of these countries cannot afford the initial investment required to undertake projects to generate SCP.

Answer 253

~5000 tonnes/year, $1,500,000,000

Answer 254

Isolated enzymes (proteases) were first used in detergents in 1914, their protein nature proven in 1926 and their large-scale microbial production started in 1960s

Answer 255

Industrial enzyme business is steadily growing due to improved production technologies, engineered enzyme properties and new application fields. Majority of bulk enzymes produced by fermenter culture of GRAS-status microbes. Usually the production organism (& often individual enzyme) have been genetically engineered for maximal productivity and optimised enzyme properties. Large volume industrial enzymes are usually not purified but sold as concentrated liquids or granulated dry products. However, enzymes used in special applications like diagnostics or DNA-technology need to be highly purified. Purified enzymes have found many applications in fine chemical industry (stereo-specific synthesis or stereo-specific chemical conversions).

Answer 256

Bacillus subtilis Aspergillus niger Aspergillus oryzae

Answer 257

*Not usually significantly purified (mixture) so low cost ``` Detergents Starch Drinks Textiles Animal feed Baking Pulp and paper Leather ```

Answer 258

* Usually purified, increases cost Enzymes in analytics (immuno diagnostics) Enzymes in personal care products Enzymes in DNA-based technology

Answer 259

* Usually purified, increases costs ``` Chirally pure amino acids and aspartame Rare sugars Semi synthetic antibiotics Lipase based reactions Enzymatic oligosaccharide synthesis ```

Answer 260

34% - Detergents (proteases, lipases) 14% - Dairy processes (proteases: rennets) 11% - textiles (Cellulases) 7% - Beverages and brewing (amylases, proteases) 7% - Animal feed 5% - Amylases

Answer 261

- Criteria used in the selection of an industrial enzyme include specificity, reaction rate, pH and temperature optima and stability, effect of inhibitors and affinity for substrates. e. g. enzymes used in paper industry should not contain cellulose-degrading activity as a side activity because this activity would damage the cellulose fibres. - Enzymes used in animal feed industry must be thermo tolerant to survive in the hot extrusion process used in animal feed manufacturing. The same enzymes would have maximal activity at the body temperature of the animal. - Enzymes used in industrial applications must usually be tolerant against heavy metals & have no need for cofactors. - Should be maximally active already in the presence of low substrate concentration (ie not allosterically regulated).

Answer 262

Selection of an enzyme Selection of a production strain Construction of an overproducing strain by genetic engineering Optimisation of culture medium and production conditions Optimisation of recovery process (and purification if needed) Formulation of a stable enzyme product (packaging) Protein engineering – ‘designer’ enzymes

Answer 263

- Fairly narrow - This means that operating range of enzymes produced from them is similarly narrow. - However, some industrial processes operate outside these ranges.

Answer 264

``` Thermophiles - temp hot Psychrophiles - cold Acidophiles - acid Alkalophiles - base Halophiles- salts Barophiles - high pressure Xerophiles - low water ```

Answer 265

Extremophiles are microbes that can live and reproduce in harsh environments. Mostly Archaea. In the past 40 years they have been found in hot springs, volcanic areas, the deep sea, in the Antarctic biotopes and in other geothermal sites, all places earlier believed too severe to support life.

Answer 266

More recently, search for novel species was fuelled by industry because it was realised that the ability of these microbes to survive in such extreme conditions was strictly related to special features, which mainly consisted of novel enzymes and biochemical pathways. However, to date, there are only two major examples of actual applications of these biocatalysts that have reached the market: Taq polymerase, isolated from Thermus aquaticus & Cellulase 103 was isolated from bacteria living in soda lakes

Answer 267

new frontiers in molecular biology by becoming the key element of the polymerase chain reaction (PCR).

Answer 268

Isolated from bacteria living in soda lakes, & breaks down the microscopic fuzz of cellulose fibres that traps dirt on the surface of cotton textile, without harming the natural fabric. This biocatalyst was introduced in 1997 by Genencor International (Rochester, NY, USA) as a novel detergent agent that helps to keep cotton fabric looking `as new' even after thousands of washing cycles.

Answer 269

1) Make copies of cloned genes | 2) Express protein product of expressed genes

Answer 270

- Gene for pest/insecicide resistance inserted into plants | - Gene confers ability of bacteria to clean up toxic waste

Answer 271

- Amylase, cellulase and other enzymes prepare farics for clothing manufacture - Recombinant human growth hormone to treat stunted growth

Answer 272

``` Site-directed mutagenesis Diagnosis of human genetic diseases Microbial diagnostics DNA fingerprinting Transgenic plants and animals Gene therapy Gene banks - conservation Molecular evolution and archaeology ```

Answer 273

Recombinant DNA is made by splicing a foreign DNA fragment into a small replicating molecule (such as a plasmid), which will then amplify the fragment along with itself.

Answer 274

Any protein produced by any kind of cellular machinery derived from a recombinant DNA expression system (in vivo - whole organisms)

Answer 275

Combination of a vector (plasmid, viral) which controls expression and the cellular machinery (host – bacterium, yeast, animal etc) used to produce the protein (which can be self – homologous - or foreign - heterologous)

Answer 276

Recombinant protein not naturally or normally expressed by particular tissue or cell type (derived from recombinant DNA).

Answer 277

- Much easier to increase levels of protein of interest without severely disrupting cellular function (expression of recombinant protein can be tightly regulated) - Safer source - eg. venom, microial toxins, viral capsid proteins - More specific - targeted modification of protein sequence to obtain improved properties (protein engineering).

Answer 278

``` Hormones Blood clotting factors/related Monoclonal antibodies (Mabs) Interferons Immunisation agents Research enzymes ```

Answer 279

- Insulin - Human thyroid stimulating hormone - Growth hormone

Answer 280

- Coagulation factor VIII : haemophilia A. - Coagulation factor IX: haemophilia B. - Erythropoietin (EPO)

Answer 281

Anti-cancer | Anti-iflammatory

Answer 282

- Interferon-(alpha)-2a: chronic hepatitis C. | - Gamma interferon: hepatitis B, C, herpes and viral Enteritis.

Answer 283

- Restriction endonucleases | - Taq polymerase

Answer 284

- Hepatitis B vaccine: a non-infectious subunit vaccine derived from Hepatitis B surface antigen.

Answer 285

- Rapid growth, easy culture on simple, inexpensive substrates. - Straightforward production scale-up (fermenter). - Very amenable to genetic manipulation. - Can secrete recombinant protein into culture medium, facilitating product recovery and purification.

Answer 286

Cultured insect / mammalian cells and transgenic animals, plants

Answer 287

GRAS favoured (B. subtilis, Sacharomyces, Aspergillus [niger /oryzae]), though these are not always the most efficient producers of recombinant proteins.

Answer 288

- Biology, physiology, genetics and plasmids very well understood. - Under optimal conditions, 10-50 % of the total cellular protein can be the protein product of the cloned gene - Non-pathogenic strains (K12 principally) used.

Answer 289

- cDNA or the gene that encodes the target heterologous protein. - DNA (expression) vector (usually a plasmid carrying the cDNA or gene) which contains promoter and other signals to direct transcription. - Cellular components that translate the cloned gene, and carry out any post-translational modifications: Bacteria, yeast, cultured eukaryotic cell lines, animals and plants.

Answer 290

- self replication (or integrative) - selectable antibiotic marker - accommodates insertion of foreign DNA - contains sequences that enable expression of foreign genes.

Answer 291

- small (~2-8 kb) and circular - contain origin of replication (Ori) which allows them to replicate autonomously. - copy number (average no of plasmids/cell) varies from a few to several hundred depending on the origin of replication.

Answer 292

- can accommodate large fragments of foreign DNA - used for gene therapy- e.g. Adenovirus and Vaccinia - often designed to integrate within host genome

Answer 293

E. coli pBR322

Answer 294

- An origin of replication (ORI). This is the site where bacterial DNA polymerases bind and start the process of copying the plasmid. This is essential for the plasmid to be passed on to it's daughter cells. - An antibiotic resistance gene. This allows reomoval of non-transformants. Tetracycline and ampicillin resistance are commonly used. - A suitable promoter to drive expression of the inserted gene. - A multiple cloning site (MCS), where the gene is inserted. Usually contain recognition sites for several restriction enzymes, allowing flexibility in the choice of enzymes. - A transcription terminator, to stop transcription after the gene. - Ribosome binding site (Shine Dalgarno) to allow translation - A stop codon to terminate translation. - Safety features.

Answer 295

Obtained from genes expressed at high levels Strong promoters can sustain a high rate of transcription Must be regulatable (expression using by induction)