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BS3068 industrial Microbiology > Penrose > Flashcards

Penrose Flashcards

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1
Q

What factors decide whether a process is microbial or non microbial

A

Economics and chemical feasibility

Economics cost:

  • Raw materials
  • Labour and equipment maintenance
  • Factory overheads
  • Operating costs
  • Quality control
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2
Q

What can be meant by the word fermentation?

A

–Any process involving the mass culture of micro-organisms, both aerobic and anaerobic
- Any biological process that occurs in the absence of O2
- The production of alcoholic beverages
Food spoilage
- An energy yielding process whereby organic molecules serve as both electron donors and electron accepters.

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3
Q

What is a fermentor

A
  • A fermenter is a bioreactor, or vessel for growth
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4
Q

What is upstream processing (USP) in microbial product production

A
  • Producer microbe development
  • Optimise Fermentation medium
  • The Fermentation
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5
Q

What is downstream processing (USP) in microbial product production - starting with the lowest cost first

A 
All preparative processes
following fermentation:
 - Cell harvesting & extraction
 - Product purification
-  Waste product disposal
-  Quality control
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6
Q

Why are MO used in industry

A
  • Ease of cultivation: high surface area:volume ratio, and fast growth rates
  • Metabolic diversity: growth on cheap substrates (often waste)
  • Adaptability to changes in environment
  • Ease of genetic manipulation
  • Ability to synthesise stereo-specific compounds
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7
Q

Name what is needed for USP media formation

A
  • Appropriate for organism and industrial process
  • Microbe nutritional needs
  • Bioprocess requirements
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8
Q

Name microbe nutritional needs

A
  • Carbon source - energy and anabolic metabolism
  • Nitrogen source - protein synthesis
  • Minerals - trace metals, salts
  • Additional nutrient requirements - auxotrophies: vitamins, amino acids, also growth factors etc
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9
Q

Name the bioprocess requirements

A
  • Water
  • Precursors
  • Inducers (e.g. induction expression recombinant protein)
  • Metabolic inhibitors
  • Cell permeability modifiers (facilitate release of non-secreted product)
  • Maintenance factors (e.g. antibiotic to maintain plasmid)
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10
Q

Name USP media carbon sources

A
  • Corn starch
  • Molasses
  • Malt extract
  • Starch and Dextrins
  • Sulphite waste liquor
  • Cellulose
  • Whey
  • Alkanes and alcohols
  • Fats and Oils
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11
Q

Name USP cheap nitrogen sources

A
  • Waste nitrogen sources such as urea
  • Ammonia gas
  • Fish meal
  • Corn steep liquor
  • Yeast extracts
  • Peptones
  • Soya bean meal
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12
Q

What factors need to be considered when scaling up USP fermentation

A
  • Media formulation
  • Aerobic
  • Anaerobic
  • Aseptic or non-aseptic
  • Stirred or non-stirred
  • Chemical and physical condition control
  • aeration
  • temperature
  • heat transfer
  • mass transfer: transfer of media nutrients to fermenter organism
  • control of pH
  • anti-foaming agents
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13
Q

What are the characteristics of industrial MO in USP

A
  • Genetically stable (but also amenable to genetic manipulation)
  • Efficient production of target molecule (and good understanding of biosynthetic pathway)
  • Limited/no need for vitamins/additional growth factors
    Can utilise a wide range of cheap carbon sources
  • Safe, non-pathogenic and does not produce toxic compounds
  • Product is readily harvested: extracellular - from the
  • fermentation medium
  • intracellular – cells break easily
  • Minimum production of unwanted by-products
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14
Q

Name 4 generally regarded as safe (GRAS) bacteria and their use

A
  • Bacillus subtilis: Enzyme production
  • Lactobacillus bulgaricus: Dairy fermentations
  • Lactococcus lactis:
    (Cheese/yoghurts)
  • Leuconostoc oenos
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15
Q

Name 4 generally regarded as safe (GRAS) yeasts and their use

A
  • Candida utilis
  • Kluyveromyces marxianus
  • Kluyveromyces lactis
  • Saccharomyces cerevisiae: Brewing, wine, bread, fuel ethanol
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16
Q

Name 4 generally regarded as safe (GRAS) filamentous fungi and their use

A
  • Aspergillus niger: Citric acid production
  • Aspergillus oryzae: Soy sauce production
  • Mucor javanicus
  • Penicillium roqueforti: Cheese production
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17
Q

Why do we do strain improvement?

A
  • Save money and increase productivity
  • Rapid growth
  • Genetic stability
  • Non-toxicity
  • Utilise cheaper substrates
  • Elimination compounds which may interfere with down stream processing
  • Catabolite de-repression
  • Permeability alterations (to improve product export rates)
  • Metabolite resistance
  • Production of additional products: additional enzymes, compounds to inhibit contaminant microbes, heterologous (recombinant) proteins
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18
Q

What are the natural methods of strain improvement

A

Natural recombination:

  • Conjugation
  • Transformation
  • Transduction
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19
Q

Describe the methods of mutagenesis for stain improvaement

A
  • X-ray/Gamma ray, UV radiation
  • Chemical, eg. NGS, nitrogen mustards
  • Genetic engineering (most frequently used)
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20
Q

What is downstream processing (DSP)?

A

All the processes following initial strain isolation, strain improvements and fermentation. Usually most expensive part of production process.

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21
Q

What is GOOD DSP

A

Efficient, reproducible and maximal recovery of the target product (cells, protein, chemical) to a required specification (yield, biological activity, purity), with the minimum of cost.

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22
Q

Describe the two methods for primary recovery of target protein

A

Method used will depend on product’s stability, toxicity, whether product is extracellular or intracellular, level of purity required:

  • Extracellular: remove cells, isolate product directly from culture medium, purify
  • Intracellular: permeabilise cells during fermentation, isolate, purify or harvest cells, lyse (break open) cells, fractionate cell extract, isolate, purify
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23
Q

Describe purification in DSP

A
  • Influenced by level of purity required.
  • May be simple adsorption, and concentration or require more involved separation technique e.g. ion exchange, gel filtration, hydrophobic interaction chromatography.
  • Product packaging
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24
Q

Describe quality and safety standards of DSP

A
  • Quality must be consistent between batches
    SOP (Standard Operating Procedure/Protocol): programme of standardised in house USP and DSP processes which will produce target molecule of predetermined specification and quality.
  • Product must be free from hazardous/undesirable contaminants
  • Product/manufacturing process must meet Regulatory requirements
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25
Q

What is the relationship between number of DSP steps and cost

A

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

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26
Q

What are the microbial product types (volume:value)

A

high volume/low value: amino acids, vitamins, food & beverages

  • low volume/high valuepharmaceuticals e.g. recombinant insulin, Factor VIII
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27
Q

What are the types of metabolites from microbial products

A
  • 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).
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28
Q

What are the 3 biggest primary metabolites and their world market worth

A
  • Amino acids: $3000 million
  • Monosodium glutamate: $915 million
  • L-Lycine-HCL: $600 million
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29
Q

What are the 3 biggest secondary metabolites and their world market worth

A
  • Antibiotics: $28,000 million
  • Cephalosporins: $11,000 million
  • Penicillins G and V: 4400 million
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30
Q

What is the relevance of carbohydrate metabolism in microbial energy generation

A
  • Most of a microbial cell’s energy is produced from the oxidation of carbohydrate.
  • Glucose is most commonly used carbohydrate, metabolisable by most organisms.
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31
Q

What are the two major types of glucose catabolism?

A

Respiration and fermentation

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32
Q

Describe the respiration reaction for glucose

A
  • Aerobic process in which glucose is completely broken down to CO2 and H2O.
  • C6H12O6 + 6O2 6CO2 + 6H2O + electron –> 36 ATP
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33
Q

Describe the fermention reaction for glucose

A
  • 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
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34
Q

What is the chemical code for lactate

A

C3H6O3

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35
Q

What is the chemical code for ethanol

A

C2H6O

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36
Q

Describe glycolysis under aerobic conditions

A 
- Pyruvate decarboxylated 
via PDH >>acetyl CoA 
>> condensation with
 oxaloacetate >> citrate
and TCA cycle
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37
Q

Describe what happens to electrons in fermentation

A

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

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38
Q

Compared to respiration, how is fermentation energetically?

A

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

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39
Q

Chemical fermentation reactions for producing ethanol and lactate

A

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

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40
Q

What is the net yield of alcoholic fermentation

A

2 ATP
2 NADH
per glucose

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41
Q

Describe the process of alcoholic fermentation

A

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+)

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42
Q

Describe the process of lactic acid fermentation

A

*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

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43
Q

What is the net yield from lactic acid fermentation

A

Net yield
2 ATP
2 NADH
per glucose

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44
Q

Which organisms are involved in alcoholic fermentation?

A

Microbes:Yeast, some

Bacteria (Zymomonas)

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45
Q

What processes use alcoholic fermentation

A

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

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46
Q

Which organisms are involved in lactic acid fermentation?

A

Lactic acid bacteria

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47
Q

What processes use lactic acid fermentation

A

Cheese making
Yoghurts
Pickles,
Fish/Soy sauce

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48
Q

What are the alternatives to glycolysis (EMP)?

A
  • Pentose phosphate pathway
  • Entner-Doudoroff
  • Phosphoketolose (Warburg Dickens)
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49
Q

What is meant by gycolysis EMP

A

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

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50
Q

Describe basic outline of the pentose phosphate pathway

A
  • 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.
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51
Q

What organisms use the pentose phosphate pathway

A

Bacillus subtilis, E. coli, Leuconostoc, yeast

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52
Q

What does the Entner-Doudoroff pathway yield

A
  • used by microbes lacking EMP.

- Pathway yields: one ATP, 1 NADPH, 1 NADH, 2 pyruvate/glucose molecule.

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53
Q

What organisms use the Entner-Doudoroff pathway?

A

Zymomonas, Pseudomonas, Xanthomonas, Rhizobium, Agrobacterium

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54
Q

Basic outline of phosphoketolose pathway

A
  • Warburg Dickens pathway
  • One ATP and two NADH/glucose. Allows catabolism pentoses and production pentoses from hexoses for nucleic acid formation.
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55
Q

What organisms are involved in the phosphoketolose pathway

A

Lactobacillus, Leuconostoc

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56
Q

What are the absolute minimum you should remember about the glucose pathways

A
  • 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)
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57
Q

Name 2 main yeast beverage fermentations

A
  • Beer

- Wine

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58
Q

Name 2 main yeast food fermentations

A
  • Bread

- Vinegar

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59
Q

Name a non food/drink use of yeast fermentations

A
  • Fuel ethanol production from plant biomass
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60
Q

What are the base ingredients and MO involved in 5 main beverage fermentations

A
  • 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)
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61
Q

Give an overview of alcoholic beverages and vinegar production

A
  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).
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62
Q

How long have people been making booze

A

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

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63
Q

Name the main type of brewers yeast

A
  • Sacharomyces cerevisiae

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

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64
Q

What special characteristics do brewers yeasts have?

A
  • Faster fermentation rates
  • Higher tolerances to alcohol
  • Other metabolic capabilities conferring a specific advantageous factor
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65
Q

What kind of gene copy numbers does yeast have

A

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

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66
Q

What challenges face genetic engineering of yeasts

A

Genome complexity causes challenge

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67
Q

What 2 types of brewers fermenting yeasts are there?

A

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

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68
Q

Characteristics of top fermenting yeasts

A
  • 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
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69
Q

Characteristics of bottom fermenting yeasts

A
  • 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
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70
Q

Difference between ale and lager ferementation

A
  • Genome composition, particularly, genes involved in carbohydrate metabolism, thermotolerance, ethanol sensitivity
  • ferment wort in different ways, producing different flavour spectrums
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71
Q

Describe the brewing of beer (overview and 6 stages)

A
  • 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.
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72
Q

Describe the malting process for beer

A
  • 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
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73
Q

Describe the mashing and wort prep process for beer

A
  • 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.
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74
Q

What is wort carbon and nitrogen composition important for

A
  • Colour
  • Flavour development
  • ALSO FLAVOUR IS EFFECTED BY WORT ADJUNCTS AND HOPS TOO!
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75
Q

Yeast nutrient requirements

A
  • Carbon and energy source
  • Nitrogen source (amino acids and peptides)
  • Minerals
  • Growth factors (vitamins such as biotin and B complex)
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76
Q

Describe carbon utilisation in wort

A
  • 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.
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77
Q

Why are amino acids important for yeast

A
  • important for yeast protein synthesis and growth
  • Also, have major influence on beer flavour due to conversion to flavour
    compounds
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78
Q

Why is oxygen important in yeast in beer production

A

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

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79
Q

Why is the malting and mashing of grains required

A

Brewers yeast cannot ferment starch or higher C dextrins

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80
Q

Describe boiling the hops in beer production

A
  • 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.
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81
Q

What is the latin name for common hops

A

Humulus lupulus

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82
Q

Describe fermentation in beer production

A
  • 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).
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83
Q

How is beer fermentation monitored

A

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)
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84
Q

What is the pH of beer

A

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

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85
Q

What effects beer flavour

A

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.

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86
Q

Describe finishing in beer production

A

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.

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87
Q

Describe packaging in beer production

A

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.

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88
Q

What stresses are yeast under during ethanolic fermentation?

A
  • 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
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89
Q

What is the effect off ethanol stress for yeast

A

Reduced growth, viability and metabolism

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90
Q

How do we know ethanolic stress exists in yeasts

A

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

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91
Q

How many genes are involved in yeast alcohol tolerance?

A

100s

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92
Q

What can go wrong in beer formation

A
  • 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.
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93
Q

Why is beer environment quite hostile to growth

A
  • 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
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94
Q

What are the future plans for beer production?

A
  • 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
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95
Q

Name a killer yeast toxin in beer production

A

Zymocins

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96
Q

Name and anti-microbial peptide for lactic acid bacteria contamination in beer production

A

Nisin (bacteriocin)

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97
Q

What are the barriers to improving brewers yeast

A
  • 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
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98
Q

How many litres of wine are produced each year

A

12,000,000,000

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99
Q

Which species of grape is used for wine production?

A

Vitis vinifera

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100
Q

How do varieties of developed grape differ - how many are used

A
  • 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
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101
Q

Name 8 chief grape varieties

A

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

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102
Q

Other than grapes, what can be made into wine?

A
  • 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
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103
Q

Name the 6 main wine producers in the world

A
  • US
  • Spain
  • Australia
  • Austria
  • France
  • Italy
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104
Q

Why are grapes ideal for wine production?

A
  • 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.
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105
Q

What portion of fruit is fermented to give rise to: white, red, dry and sweet wines?

A

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

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106
Q

How do you give rise to sweet wines?

A

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

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107
Q

What yeasts are used in wine making?

A
  • 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.
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108
Q

Describe primary wine fermentation

A
  • 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+)
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109
Q

What factors influence wine fermentation

A
  • 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
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110
Q

What are the effects of sulpher dioxide treatment in wine production

A
  • Represses endogeneous yeast activity
  • Preservative against microbial contamination
  • Anti-oxidant
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111
Q

Give an overview of the wine fermentation process

A
  • 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
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112
Q

Why are there secondary wine fermentations

A
  • 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)
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113
Q

What contaminants are in one ton of California wine grapes

A
  • Seven pounds of dirt, one mouse nest, 147 bees, 98 wasps, 1,014 earwigs, 1833 ants, 10899 leafhoppers and 1.5kg of bird droppings
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114
Q

What pre-fermentation problems are involved in wine production

A
  • 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)
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115
Q

What post-fermentation problems are involved in wine production

A
  • 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)
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116
Q

What are desirable fermentation properties in wine production

A
  • 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
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117
Q

Desirable technological properties for wine production

A
  • 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
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118
Q

What are the main ingredients of bread?

A

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.

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119
Q

What is added to bread as an energy source for yeast?

A

Sugar

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120
Q

Describe the recipe of bread

A
  • 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)
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121
Q

What makes bread rise

A
  • Decarboxylation

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

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122
Q

What future applications/improvements could there be to bakers yeast

A
  • 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)
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123
Q

Describe vinegar production reactions

A

Ethanol to acetaldehyde (NAD+ to NADH)

Acetaldehyde to acetic acid (NAD+ to NADH)

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124
Q

Where does the word vinegar come from?

A

French: vin-wine and aigre - sour

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125
Q

6 main varieties of vinegar

A 
Malt
Wine, red or white (includes balsamic)
Cider
Sherry
Rice wine
Distilled (malt)
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126
Q

What family do vinegar production microbes belong to

A

Acetobacteriaceae

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127
Q

What are acetic acid bacteria?

A
  • 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).
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128
Q

Describe Acetobacter and the TCA cycle

A

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

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129
Q

Describe Gluconobacter and the TCA cycle

A

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

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130
Q

What are the two stages of vinegar production

A

Primary alcoholic fermentation

Secondary conversion of ethanol to ethanoic (acetic) acid

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131
Q

Describe the Orleans process

A
  • 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
132
Q

What are the problems with the Orleans process

A

Method was very slow, and results uncertain.

Still used for a few speciality vinegars.

133
Q

Name the two current vinegar production methods

A

Trickling and Submerged processes

134
Q

Describe the trickling method

A
  • 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.
135
Q

What is the yield of the trickling method

A

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

136
Q

Describe the submerged method vinegar

A
  • 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.
137
Q

What is the yield of the submerged method vinegar

A

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

138
Q

Describe vinegar processing and flavour

A

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.

139
Q

Describe balsamic vinegar production

A
  • : 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
140
Q

What can go wrong in vinegar production

A
  • 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.
141
Q

How do microbial fermentations preserve food?

A

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

142
Q

What processes can be done along with microbial fermentations to preserve foods?

A
  • Reduction in water content of food (drying/add salt)

- Presence added/natural preservatives (nitrate – nitrite)

143
Q

Describe gari and microbial fermentation’s role in preparation

A

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.

144
Q

What is the composition of milk

A
  • Water:solids (87:13)
  • Lactose:fat:protein:ash/minerals (37:30:27:6)
  • Casein:Whey proteins (80:20)
145
Q

Name the raw ingredients, fermenting organism and country commonly produced in for these products:
Cheese, yogurt, kefir, taette, tarhana

A
  • 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
146
Q

How big is the world cheese market

A

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

147
Q

Name the generic characteristics of lactic acid bacteria

A
  • 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.
148
Q

What temperatures can some LAB survive

A

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

149
Q

Describe thermophilic thermotolerance

A

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

150
Q

Describe mesophilic thermotolerance

A

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

151
Q

Describe psychrophilic thermotolerance

A

Psychrophiles (psychrophilic) -thrive at low temps.

Prefer 10-20°C but will slowly metabolise at even 0°C

152
Q

What is energy production in LAB

A

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

153
Q

What are the two methods of lactose production

A
  • 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.
154
Q

Describe the preteolytic activity of LAB

A
  • 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
155
Q

What encode important metabolic functions in fermentations w/ LAB

A

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

156
Q

Overview of cheese production

A
  • 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
157
Q

Describe the first stage of cheese production

A
  • 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
158
Q

Describe the second stage of cheese production

A
  • 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.
159
Q

What are the two classes of cheese

A

Unripened and ripened

160
Q

Describe unripened cheese

A
  • 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.
161
Q

Describe ripened cheese

A
  • Cheeses are pressed and dried curds (sometimes cooked) subjected to second round of microbial processing, during which flavour is developed.
162
Q

What are starter cultures in cheese production

A
  • 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)
163
Q

Describe preparation of milk for cheese production

A

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

164
Q

Describe coagulation of milk proteins for cheese production

A
  • 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.
165
Q

Describe whey separation for cheese production

A
  • Curd chopped, pressed and heated to expel fluid – whey.

- Amount of residual moisture determines if cheese is soft or hard

166
Q

Describe processing of curd for cheese production

A

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

167
Q

Describe ripening of cheese for cheese production

A
  • 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
168
Q

What are the 5 basic steps of common variety cheese making

A
  1. preparation of milk
  2. coagulation of proteins
  3. whey separation
  4. processing of curd
  5. ripening of the cheese
169
Q

Describe the role of rennet in cheese production

A

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.

170
Q

What ripening agents are used in cheese ripening

A 
Bacteria and enzymes indigenous to the milk 
Lactic acid bacteria starter culture 
Rennet 
Microbial lipases 
Added bacteria, moulds or yeasts 
Environmental microbial contaminants
171
Q

How long is cheese ripened for

A

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

172
Q

How does blue cheese occur

A

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

173
Q

How are different flavour spectrums of cheeses made

A
  • 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
174
Q

What degradative processes take place during cheese ripening?

A
  • Lactose to lactic acid (diacetyl/proprionate) via glycolysis
  • Fat to fatty acids via lypolysis
  • Casein to amino acids via proteolysis
175
Q

Giving the example of Gouda, what happens to cheese flavours over time

A
  • Volitile flavour component production increases, we know this from mass spectrograph evidence
176
Q

How are Swiss-type cheeses made

A

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’.

177
Q

What are the commonly occurring problems in cheese making

A
  • 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.

178
Q

Name 3 LAB dairy food preservation techniques

A
  • Creation of anaerobic environments by CO2 production
  • Competitive exclusion of microbial/fungal pathogens
  • Production of multiple anti-microbial compounds
179
Q

What are the problems of low and high molecular weight cheese preservation

A

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)

180
Q

What is a bacteriocin

A

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

181
Q

What produces bacteriocin

A

Lactococcus lactis

182
Q

What are the functions of bacteriocin

A
  • ?? maybe we are not suuurreee??
  • growth regulation, communication, ecological
    advantage over competitors.
  • Chromosomally encoded
  • Lantibiotic – peptide antibiotic containing lanthionine
183
Q

What is bacteriocin primary metabolite

A
  • 34 amino acid, 3500Da hydrophobic cationic (+vely charged) pentacyclic peptide.
  • Unusual amino acids.
184
Q

Mechanism of action for bacteriocins

A

Inserts into lipid bilayer

forming pores. Kills rapidly via dissipation ATP/ions/proton gradient

185
Q

Describe Nisin (cheese production)

A

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

186
Q

Describe nisin anti-microbial activity against Gram +ve pathogens

A

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.

187
Q

Name 2 clinical applications of Nisin

A

Treat and prevent bovine mastitis

Wound dressings (active against S. aureus)

188
Q

What do lactococcin and pediocin do

A

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

189
Q

What do propionicin do

A

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

190
Q

What do natamycin do?

A
  • 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.
191
Q

Describe two current recent cheese developments

A

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)

192
Q

Name 3 ongoing cheese developments in various stages

A

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)

193
Q

What is the starter culture for yogurts in Europe and the US

A

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

194
Q

What type of milk is yogurt made from

A

Pasteurised skimmed, semi-skimmed or whole milk.

195
Q

Describe yogurt fermentation

A

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.

196
Q

Describe how buttermilk is made

A

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.

197
Q

How is sour cream made

A

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.

198
Q

What are probiotics?

A
  • 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.
199
Q

Name some probiotic LAB

A

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

200
Q

What are prebiotics (synbiotics)

A
  • 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.
201
Q

Name some prebiotic oligosaccharides

A

Fructo-oligosaccharides, inulin, lactulose and galacto-oligosaccharides

202
Q

Where do prebiotics come from

A

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

203
Q

Name the 4 anti-infective roles of probiotics

A

Production of inhibitory substances.

Blocking of adhesion sites.

Competition for nutrients.

Stimulation of immunity.

204
Q

What inhibitory substances do LAB produce

A
  • 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
205
Q

Ways in which probiotics may be useful (3)

A
  • Blocking of adhesion sites
  • Competition for nutrients otherwise consumed by pathogens
  • Stimulation of immunity
206
Q

Summary of anti-infective role of probiotics

A

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.

207
Q

Name 4 main flavourings or nutritional supplement (SCGL)

A

Soy sauce – traditional flavouring agent

Citric – organic acid

Glutamic – amino acid – flavouring agent

Lysine – amino acid – nutritional supplement

208
Q

Give examples of primary metabolites

A 
Products of growth processes, 
	e.g. amino acids, 
	vitamins, 
	nucleotides, 
	ethanol, 
	lactate, 
	enzymes
    cellular biomass
209
Q

Give examples of secondary metabolites

A 
Produced from pathways not required for growth: 
	e.g. antibiotics, 
	toxins, 
	pigments, 
	exopolysaccharides 
	(xanthans).
210
Q

Give some generic soy sauce facts plz

A
  • 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.
211
Q

Name the two main microbes involved in soy sauce production

A

Aspergillus oryzae (fungus)

LAB/yeast (Pediococcus, Tetragenococcus/Candida/Saccharomyces)

212
Q

What is soy sauce

A

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.

213
Q

Name a briefly describe the 4 main types of soy sauce

A
  • 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.
214
Q

What are the stages of soy sauce production

A

Koji culturing

Moromi fermentation

215
Q

Describe koji culturing

A

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.

216
Q

Describe moromi fermentation

A

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

217
Q

What is citric acid production

A

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.

218
Q

Describe the MO involved in citric acid production

A

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
219
Q

What is the world market for citric acid

A

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.

220
Q

Which process gives rise to citric acid

A

TCA cycle

221
Q

How can citric acid production be increased

A

Inhibiting aconitase activity that would convert citrate

222
Q

Describe aconitase and how it can be inhibited

A
  • 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
223
Q

Describe the industrial production of citrate by Aspergillus niger

A
  • 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.
224
Q

What production medium and conditions are used for production of citrate by Aspergillus niger?

A
  • Chelex treated to remove Fe, [Cu]
  • Low in nitrogen to minimise biomass production
  • Fermentation at 30 degrees Celsius, pH falls as citric acid produced
225
Q

What is the yield of citric acid industrial production

A
  • 0.7-0.9g citric acid/g glucose

- 18.0kg citric acid/m^3 vol/day

226
Q

Describe the downstream processing (purification) of citric acid

A
  • 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.
227
Q

How is production of amino acids regulated

A

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.

228
Q

What strategies are used to increase amino acid production?

A
  • 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
229
Q

What is the effect of combination of amino acid production increase strategies

A

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

230
Q

How are modfications to amino acid production made?

A

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

231
Q

Why is L-Glutamate acid produced

A
  • 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.
232
Q

What is the world market value for L-Glutamic acid production

A

> 800,000 tons, $915 million

233
Q

What MO produce glutamate

A
  • Principally Arthrobacter genera:
    Brevibacterium, Corynebacterium glutamicum main L-glutamate producers.
  • Gram+ve, biotin-requiring microbes with
    v. high glutamate dehydrogenase (GDH) activity
234
Q

What is the reaction to produce L-glutamate

A

Alpha-ketoglutarate + NH4+ +NAD(P)H + H+ —GHD—> L-glutamate + NAD(P)+ + H2O

  • L-glutamate is produced from alpha-ketoglutarate, a TCA cycle intermediate
235
Q

How can glutamic acid production be increased in C. glutamicum

A
  • 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.

236
Q

Describe industrial production of glutamic acid

A
  • 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.
237
Q

How can glutamic acid leave cells

A

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.

238
Q

What is the yield of L-glutamate

A

Yield: 1 g L-glutamate/1.4 g glucose

80 g/litre culture medium

239
Q

Describe the downstream processing (purification) of L-glutamic acid (and MSP prep)

A
  • 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.
240
Q

What are the toxicity concerns of MSG

A
  • 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

241
Q

Name the essential amino acids

A 
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
242
Q

Name the non-essential amino acids

A 
Alanine
Arginine
Asparagine
Aspartic Acid
Cysteine
Glutamic Acid
Glutamine
Glycine
Proline
Serine
Tyrosine
243
Q

What is the need for L-lysine

A
  • 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
244
Q

What organisms are involved in L-Lysine production

A

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

245
Q

What process gives rise to L-lysine

A

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

246
Q

Describe production of C. glutamincum lysine over-producers

A

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.

247
Q

How to increase lysine production

A
  1. Block side reactions : remove homoserine dehydrogenase (HS):
    auxotrophic for threonine isoleucine & methionine

Wt HS
~5 g/l –>15-30 g/l

  1. 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.

248
Q

Describe industrial production of L-lysine

A

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.

249
Q

What is the industrial yield of L-lysine

A

Yield: ~30-35 % conversion rate, 1 g L-lysine/ 3g glucose

40-45 g/200g molasses

250
Q

Describe the downstream processing (purification) of L-lysine

A
  • 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.
251
Q

Describe the genus Xanthomonas

A

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

252
Q

What does X. campestris pv. campestris do

A

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.

253
Q

What is the function of xantham exopolysaccharide

A

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.

254
Q

Describe the market for xantham gum

A
  • 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).
255
Q

What is Xantham gum used for

A

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

256
Q

What are the physical properties of xantham

A
  • 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
257
Q

Describe the structure of X. campestris Xanthan

A

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

258
Q

Describe xanthams interactions with locust bean/guar gums

A

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).

259
Q

What are the clinical used of xantham

A

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
260
Q

Describe the industrial production of xantham gum

A

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.

261
Q

Describe the fermentation medium for xantham gum production

A

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.

262
Q

What is the yield of xantham gum

A

Typical yield: ~ 50 g/litre

263
Q

What microbes are used directly as foods

A 
Microbes themselves can be used as a nutritional supplement to human diets or animal feeds.
Mushrooms
Yeast 
Algae (Spirulina)
Bacteria
264
Q

What is single cell protein and how is it used

A

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

265
Q

What are the three main commercial reasons for using single cell protein?

A
  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.
266
Q

Describe the rapid growth rate benefit of single cell protein

A

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.

267
Q

Describe the cheap raw materials benefit of single cell protein

A

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.

268
Q

Describe the high feed efficiency benefit of single cell protein

A

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.

269
Q

What are the 3 main stages of single cell protein production

A

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

270
Q

What is required in media preparation in single cell protein production?

A

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

271
Q

What happens in fermentation in single cell protein production?

A

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

272
Q

What happens in separation and downstream processing in single cell protein production?

A

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

273
Q

Describe the Bel process of SCP

A

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

274
Q

Describe the Symba process of SCP

A

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.

275
Q

Describe the Pekilo process of SCP

A

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.

276
Q

Describe the Pruteen process of SCP

A

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.

277
Q

Name 4 processes of single cell protein production

A
  • Bel
  • Symba
  • Pekilo
  • Pruteen
278
Q

Describe the market for

A

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.

279
Q

What is the yield of quorn

A

Yield: 15/20 g biomass/litre

280
Q

Why is quorn used as substitue, how is it “meaty”?

A
  • 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.

281
Q

What are the health and public acceptabilities of single cell proteins

A

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.

282
Q

What are the future prospects of single cell proteins

A

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.

283
Q

Numerically, what is the world market for industrial enzymes

A

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

284
Q

How long have isolated enzymes been used in industry

A

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

285
Q

Give an overview of industrial enzymes

A

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).

286
Q

Which GRAS MOs are usually involved in industrial enzyme production

A

Bacillus subtilis
Aspergillus niger
Aspergillus oryzae

287
Q

What are the large scale (or “bulk”) applications for enzymes

A

*Not usually significantly purified (mixture) so low cost

Detergents 
Starch
Drinks
Textiles
Animal feed
Baking
Pulp and paper 
Leather
288
Q

What are the main applications for speciality enzymes

A
  • Usually purified, increases cost

Enzymes in analytics (immuno diagnostics)
Enzymes in personal care products
Enzymes in DNA-based technology

289
Q

What are the main applications for enzymes used in chemical production

A
  • Usually purified, increases costs
Chirally pure amino acids and aspartame
Rare sugars
Semi synthetic antibiotics
Lipase based reactions
Enzymatic oligosaccharide synthesis
290
Q

Name the top 7 used of bulk-manufactured microbial enzymes

A

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

291
Q

How are enzymes suited to their purpose industrially

A
  • 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).
292
Q

How are industrial enzymes developed

A

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

293
Q

How large are the growth parameters for industrially importnant MOs?

A
  • Fairly narrow
  • This means that operating
    range of enzymes produced
    from them is similarly narrow.
  • However, some industrial
    processes operate outside these
    ranges.
294
Q

Name 7 main types of extremophiles

A 
Thermophiles - temp hot
Psychrophiles - cold
Acidophiles - acid
Alkalophiles - base
Halophiles- salts
Barophiles - high pressure
Xerophiles - low water
295
Q

What are extremophiles and where are they found

A

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.

296
Q

Why do extromophiles have applications in industrial enzyme production

A

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

297
Q

What was the significance of he discovery of Taq polymerase

A

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

298
Q

What was the significance of he discovery of Cellulase 103

A

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.

299
Q

What are the two goals of recombinant proteins?

A

1) Make copies of cloned genes

2) Express protein product of expressed genes

300
Q

Name two applications of copies of cloned genes from recombinant proteins

A
  • Gene for pest/insecicide resistance inserted into plants

- Gene confers ability of bacteria to clean up toxic waste

301
Q

Name two applications of expressed protein product from recombinant proteins

A
  • Amylase, cellulase and other enzymes prepare farics for clothing manufacture
  • Recombinant human growth hormone to treat stunted growth
302
Q

Name some applications of recombinant DNA technology

A 
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
303
Q

Define recombinant DNA

A

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.

304
Q

Define a recombinant protein

A

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

305
Q

Define expression system

A

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)

306
Q

Define heterologous protein

A

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

307
Q

Why is recombinant better over non-recombinant

A
  • 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).
308
Q

What 6 overall types of proteins are produced in microoganisms

A 
Hormones
Blood clotting factors/related 
Monoclonal antibodies (Mabs)
Interferons
Immunisation agents 
Research enzymes
309
Q

What hormones are produced using recombinant techniques

A
  • Insulin
  • Human thyroid stimulating hormone
  • Growth hormone
310
Q

What blood clotting factors/related proteins are produced using recombinant techniques

A
  • Coagulation factor VIII : haemophilia A.
  • Coagulation factor IX: haemophilia B.
  • Erythropoietin (EPO)
311
Q

What monoclonal antibodies (Mabs) are produced using recombinant techniques

A

Anti-cancer

Anti-iflammatory

312
Q

What interferons agents are produced using recombinant techniques

A
  • Interferon-(alpha)-2a: chronic hepatitis C.

- Gamma interferon: hepatitis B, C, herpes and viral Enteritis.

313
Q

What research enzymes

agents are produced using recombinant techniques

A
  • Restriction endonucleases

- Taq polymerase

314
Q

What immunisation agents are produced using recombinant techniques

A
  • Hepatitis B vaccine: a non-infectious subunit vaccine derived from Hepatitis B surface antigen.
315
Q

Why are microbes used for recombinant protein production

A
  • 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.
316
Q

What alternative methods of recombinant protein production are there?

A

Cultured insect / mammalian cells and transgenic animals, plants

317
Q

Which microbes are favoured for recombinant protein production

A

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

318
Q

Why is Eschericia coli used in microial recombinant protein production

A
  • 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.
319
Q

What are the components of all heterologous protein expression system

A
  • 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.
320
Q

What are the universal features of expression vectors

A
  • self replication (or integrative)
  • selectable antibiotic marker
  • accommodates insertion of foreign DNA
  • contains sequences that enable expression of foreign genes.
321
Q

What are the features of plasmid vectors

A
  • 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.
322
Q

What are the features of viral vectors

A
  • can accommodate large fragments of foreign DNA
  • used for gene therapy- e.g. Adenovirus and Vaccinia
  • often designed to integrate within host genome
323
Q

What is the name of the plasmid from which most expression vectors are derived

A

E. coli pBR322

324
Q

What are the important features of a prokaryotic (E. coli) expression vector

A
  • 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.
325
Q

What makes a strong promotor?

A

Obtained from genes expressed at high levels

Strong promoters can sustain a high rate of transcription

Must be regulatable (expression using by induction)

BS3068 industrial Microbiology (1 decks)

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