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Industrial fermentationIndustrial fermentationProduction with microbesProduction with microbes
Industrial microbiologyIndustrial microbiology
The use of microbes in the productionThe use of microbes in the production
Start from an ancient timeStart from an ancient time
Large scale production Large scale production ––
Early 20Early 20thth century century –– beverage industry, vinegar, beverage industry, vinegar,
baking yeast, citric and lactic acidbaking yeast, citric and lactic acid
Late 20Late 20thth century century –– development of development of
biotechnology from the antibiotic industrialbiotechnology from the antibiotic industrial
Production with microbesProduction with microbes
Fermentation industryFermentation industry
Ability of microbes to produce Ability of microbes to produce ‘‘usefuluseful’’ productproduct
Usually a naturally produce substances for Usually a naturally produce substances for growth, and maintenance.growth, and maintenance.
Metabolites Metabolites
Primary metabolite Primary metabolite –– produce during growthproduce during growth
Secondary metabolite Secondary metabolite –– produce after growth produce after growth stagestage
Substrate
Product
Cell
Time
Substrate
Product
Cell
Time
Primary metabolite
Secondary metabolite
Trophophase Idiophase
Production with microbesProduction with microbes
Interdisciplinary areaInterdisciplinary areaMicrobiologistsMicrobiologists……..of course..of course
Molecular biologistsMolecular biologists
Chemists/biochemistsChemists/biochemists
Engineers Engineers –– hardware, softwarehardware, software
Lawyers Lawyers –– patent and permission patent and permission
InvestorsInvestors
Management team!Management team!
ProductsProductsI. Foods and foods relatedI. Foods and foods related
-- Fermented meat, Cheeses and milk products, Fermented meat, Cheeses and milk products, mushrooms, bakermushrooms, baker’’s yeast, coffee, pickles, single s yeast, coffee, pickles, single cellcell--proteins, vinegar, amino acids, vitamins, proteins, vinegar, amino acids, vitamins, alcoholic beveragesalcoholic beverages
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ProductsProductsII. Organic acidsII. Organic acids
-- Citric acids, Itaconic acidCitric acids, Itaconic acid
III. Enzymes and microbial transformationIII. Enzymes and microbial transformation-- Commercial enzyme, sterol conversionCommercial enzyme, sterol conversion
IV. Engineered microbesIV. Engineered microbes-- Insulin, human growth factorInsulin, human growth factor
Production with microbesProduction with microbesSteps in Industrial fermentationSteps in Industrial fermentation
Isolation of microbes that produce your interested Isolation of microbes that produce your interested productproduct
Screen for the best producing strain: naturally or Screen for the best producing strain: naturally or mutation.mutation.
Optimize production condition (growth Optimize production condition (growth ––basically) basically) in labin lab
Scale up from lab scale (up to 10 L) to industrail Scale up from lab scale (up to 10 L) to industrail scale (>10,000 L) (raw material?)scale (>10,000 L) (raw material?)
Food and food related productsFood and food related products
Whole cells productsWhole cells productsIncluding Including –– mushrooms and Singlemushrooms and Single--cell protein cell protein (SCP)(SCP)
Mushrooms Mushrooms –– fruiting body of mold!fruiting body of mold!Highly consumed every year Highly consumed every year –– high nutrition.high nutrition.Grow on decaying organic matter in soil or wood.Grow on decaying organic matter in soil or wood.
Stalk and cap = compact myceliumStalk and cap = compact myceliumGills = site of reproductive sporesGills = site of reproductive spores
E.g. E.g. Agaricus bisporusAgaricus bisporus (botton mushroom), (botton mushroom), Lentinus edulusLentinus edulus (Shitake mushroom)(Shitake mushroom)
Agaricus bisporus
Shitake mushroom Oyster mushroom Straw mushroom
Food and food related productsFood and food related products
SingleSingle--cell protein cell protein –– yeast, and cyanobacteriayeast, and cyanobacteria
Alternative food sources Alternative food sources –– mostly used now as mostly used now as
supplementary diet e.g. yeast is a source for B supplementary diet e.g. yeast is a source for B
Vitamins.Vitamins.
Organisms: yeast, Organisms: yeast, Spirulina Spirulina
Growing biomass and freeze dryGrowing biomass and freeze dry
Raw material from other industrial, farmingRaw material from other industrial, farming
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Food and food related productsFood and food related productsBakerBaker’’s yeasts yeast
Same process as in single cell protein Same process as in single cell protein
Food and food related productsFood and food related products
Cheese productionCheese productionCan produce from any milk.Can produce from any milk.4 phases: coagulation, separation, shaping 4 phases: coagulation, separation, shaping and ripeningand ripening
Coagulation: rennin enzyme or acids precipitate Coagulation: rennin enzyme or acids precipitate caseincasein
From calvesFrom calves’’s stomachs, engineered bacterias stomachs, engineered bacteriaAcid production by lactic acid bacteriaAcid production by lactic acid bacteriaCurd entrap bacteria, fat globules and other Curd entrap bacteria, fat globules and other material.material.
Cheese productionCheese productionAdditives (salt and herbs) may be added after Additives (salt and herbs) may be added after seperation or shaping.seperation or shaping.
Additives and shape effect property of cheese Additives and shape effect property of cheese productproduct
Ripening Ripening –– add flavor and lookadd flavor and look
Natural or inoculation of microbes e.g. Natural or inoculation of microbes e.g. Penicillium Penicillium roquefortii (roquefortii (spores are usually added into blue spores are usually added into blue cheese), and cheese), and Penicillium camembertiPenicillium camemberti is added to the is added to the surface of a cheese known as Camenbertsurface of a cheese known as Camenbert
Blue cheese
Brie
Gorgonzola
Swiss
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Food and food related productsFood and food related products
Yogurt and other fermented milk productsYogurt and other fermented milk productsYogurt, kefir, buttermilk, sourcreamYogurt, kefir, buttermilk, sourcreamYogurt usually use mixture of 2 cultures:Yogurt usually use mixture of 2 cultures:
Streptococcus thermophilusStreptococcus thermophilus: ini. acid production: ini. acid productionLactobacillus bulgaricusLactobacillus bulgaricus
0.5 0.5 -- 1.0% lactic acid 1.0% lactic acid Fermented at 28Fermented at 28--32 32 °°C C Taste due to acetaldehyde, diacetyl, and acetoin Taste due to acetaldehyde, diacetyl, and acetoin Product need to contains live culturesProduct need to contains live cultures
A mixed lactic acid and alcoholic fermentation.
Streptococcus lactis, Lactobacillus bulgaricus, yeasts
Kefir
This milk product is used for its medicinal therapeutic value.
Lactobacillus acidophilus
Acidophilus milk
Made with skimmed or partly skimmed pasteurized milk.
Streptococcus sp. Leuconostoc sp.
Cultured buttermilk
Cream is inoculated and leave to develop acidity
Streptococcus sp. Leuconostoc sp.
Sour cream
DescriptionMicroorganismsFermented Product
Some Foods Produced from Fermented Milk.
Industrial production of yogurt
Kefir Buttermilk
Food and food related productsFood and food related productsAlcoholic beveragesAlcoholic beverages
Accidental discovered from ancient human Accidental discovered from ancient human Saccharomyces cerevisiaeSaccharomyces cerevisiaeFermentation of sugar in fruit, grains and otherFermentation of sugar in fruit, grains and otherIn case of other materials In case of other materials ––need to convert need to convert polysaccharide to fermentable sugarpolysaccharide to fermentable sugarRaw materials are regionalRaw materials are regional…….variety of products.variety of products
Wine Wine –– grapes, fruitsgrapes, fruitsBeer Beer –– cereal grain cereal grain –– barley, sorgum, wheatbarley, sorgum, wheatOther Other –– rice wine (rice), teguila (agave cactus), vodka rice wine (rice), teguila (agave cactus), vodka (corn), mead (honey).(corn), mead (honey).
Tequila
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Food and food related productsFood and food related products
Fermented meatFermented meatUse to preserve foodUse to preserve food
E.g. salami, summer sausage, cured ham E.g. salami, summer sausage, cured ham
Thailand Thailand –– naam (fermented pork), fermented naam (fermented pork), fermented sausage, fermented fish, fish saucesausage, fermented fish, fish sauce……etcetc
Main organism Main organism –– Pediococcus serevisiae, Pediococcus serevisiae, Lactobacillus plantarum, Lactobacillus plantarum, other lactic acid bacteriaother lactic acid bacteria
Production of lactic acid to change pH of food Production of lactic acid to change pH of food
Production of probiotics in some strainsProduction of probiotics in some strains
Food and food related productsFood and food related productsFermented fruits and vegetablesFermented fruits and vegetables
Found around the worldFound around the world
Hundreds if not thousand type of productsHundreds if not thousand type of products
With bacteria, yeast and moldWith bacteria, yeast and mold
Depends mainly on native microbes associate with Depends mainly on native microbes associate with the plants.the plants.
Most included mixed culture fermentationMost included mixed culture fermentation
Conditioning by human to favor growth of Conditioning by human to favor growth of beneficial microbes beneficial microbes -- saltingsalting
Rhizopus oligosporus, Rhizopus oryzae, Lactobacillus delbrueckii, Saccharomyces rouxii
Soy sauce
Leuconostoc mesenteroides, Saccharomyces marscianus, Flavobacterium spp., Fusarium spp.
Coffee
Lactobacillus mesenteroids, Lactobacillus plantarum
Sauerkraut
Pediococcus cerevisiae, Lactobacillus plantarum
Pickles
Leuconostoc mesenteroides, Candida sp.Cocoa products
OrganismsProducts
Food and food related productsFood and food related productsFlavoring agents: amino acids and nucleotidesFlavoring agents: amino acids and nucleotides
Monosodium glutamate, inosinic acid Monosodium glutamate, inosinic acid –– flavor flavor enhancersenhancersOriginally use seaweed, fishOriginally use seaweed, fishProduce by enzymatic hydrolysis of yeast RNA Produce by enzymatic hydrolysis of yeast RNA ((Candida utilis)Candida utilis)Now direct fermentation = monosodium glutamate Now direct fermentation = monosodium glutamate and 5and 5’’--IMP (inosine monophosphate)IMP (inosine monophosphate)Brevibacterium ammoniagenesBrevibacterium ammoniagenes produce inosine produce inosine and then chemically phosphorylatedand then chemically phosphorylated
Improve flavor of fruit juicesAspartateAlanine
Antioxidant in powdered milkL-tryptophaneL-histidine
Added to plant derived food to inprove nutritional value
L-lysine L-methionineL-threonineL-tryptophane
Low-caloric sweetenerL-aspartyl-l-phenylalanineMethyl ester (aspartame)
Flavor enhancerSodium glutamate
Infusion solutions for surgery patientsMixed amino acid
UtilizationAmino acids
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Acetate75Brevibacterium flavum
L-lysine
Glucose>100Corynebacterium glutamicum
L-glutamic
Sucrose55Escherichia coli K12
L-threonine
Glucose39CorynebacteriumL-lysine
C-sourceApprox. yield (g/L)
OrganismAmino acid
Corynebacterium glutamicumCorynebacterium glutamicum E. coliE. coli
NonNon--food productsfood products
Enzyme productionEnzyme productionProduce in large scale around the worldProduce in large scale around the worldMajor producer Major producer –– Novozyme (44%), Genencor Novozyme (44%), Genencor Intl, USA (18%)Intl, USA (18%)
Cheese productionMicrococcusLipases
Fruit juice clarificationAspergillusPectinase
Cheese manufactureAlcaligenes,Aspergillus,Candida
Rennin
Starch liquefaction, brewing, baking, feed, detergents
BacillusBacterial amylases
Fructose syrup productionAspergillusGlucoamylase
Antitumor agentEscherichia,Serratia
Asparaginase
SweetenersBacillus, StreptomycesGlucose isomerase
Semisynthetic penicillinsEscherichiaPenicillin acylase
DetergentsBacillus, StreptomycesBacterial proteases
UseOrganismsEnzyme
NonNon--food productfood product1. Vitamins1. Vitamins 2. Antibiotics2. Antibiotics
3. Alkaloids3. Alkaloids 4. Steroids4. Steroids
5. Non5. Non--Steroid Hormones/cell regulators (cytokines):Steroid Hormones/cell regulators (cytokines):
1. Epidermal growth factor 2. Proinsulin1. Epidermal growth factor 2. Proinsulin
3. Insulin 4. Human 3. Insulin 4. Human growth hormonegrowth hormone
5. Somatostatin 5. Somatostatin 6. Interferons6. Interferons
7. Platelet7. Platelet--derived growth factorderived growth factor
8. Fibroblast growth factor 9. Tumor Necrosis Fa8. Fibroblast growth factor 9. Tumor Necrosis Factorctor
10. Other cytokines are coming on line all the time10. Other cytokines are coming on line all the time
NonNon--food productfood product
6. Blood coagulating factor XIII6. Blood coagulating factor XIII
7. The restriction enzymes7. The restriction enzymes
8. Other enzymes (e.g. ligase, DNA polymerases 8. Other enzymes (e.g. ligase, DNA polymerases
etc.)etc.)
9. Solvents9. Solvents
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How do the microbes produce these How do the microbes produce these products?products?
Genetically modified microorganismsGenetically modified microorganisms
Clone genes that encode protein of interest into Clone genes that encode protein of interest into
vectors and express this genes in microbes used in vectors and express this genes in microbes used in
production.production.
Able to produce high amount of compounds than in Able to produce high amount of compounds than in
original organisms with ease of manipulation.original organisms with ease of manipulation.
Microbes able to utilize waste from other process as Microbes able to utilize waste from other process as
a raw material for fermentation.a raw material for fermentation.
AntibioticsAntibioticsPenicillin by Penicillin by Penicillium chrysogenumPenicillium chrysogenum
Now most of the antibiotics were discover from soil Now most of the antibiotics were discover from soil microorganisms especially in microorganisms especially in Streptomyces spp.Streptomyces spp.
Engineered genes into production strains.Engineered genes into production strains.
Modification of existing antibiotics.Modification of existing antibiotics.
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Streptomycin from Streptomyces griseus
AlkaloidsAlkaloidsNitrogen containing organic compounds that Nitrogen containing organic compounds that derived from plants derived from plants Have medicinal purposes:Have medicinal purposes:
Atropine Atropine –– dilates pupils of the eyes (some muscle dilates pupils of the eyes (some muscle disease)disease)Morphine and codeine Morphine and codeine –– relief of painrelief of painCocaine Cocaine –– local anestheticlocal anestheticQuinine, caffeine, nicotine, strychnine, serotonin , and Quinine, caffeine, nicotine, strychnine, serotonin , and LSDLSD
Atropineserotonin
SteroidsSteroidsTypes of products Types of products include cortisone include cortisone and reproductive and reproductive hormones hormones
Human proteinsHuman proteins
Biotech DrugsBiotech DrugsMainly recombinants proteins and monoclonal Mainly recombinants proteins and monoclonal antibodies.antibodies.Highly active fields of biotechHighly active fields of biotechLots of new products each year Lots of new products each year
For list of product approve in US For list of product approve in US –– www.bio.orgwww.bio.org
Timeline in drug developmentTimeline in drug development