HarrisonFDST 4030/6030Microbiology of Fermented Foods 1

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Microbiology Microbiology ofof

Fermented Fermented FoodsFoods

Microorganisms in Food and Factors Affecting Them

Hungate (1962)What kinds of organisms occur?What activities do they perform and how are these activities interrelated?In what number does each kind of organism occur?What is the magnitude of its activity, and what factors influence this magnitude?

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Homeostasis – e.g., pH

Intracellular pH maintained within narrow range

Done by pumping protons outCell expends energy to maintain homeostasis

Shift of energy from biosynthesis to maintenance of homeostasis inhibits growthIf energy needs exceed energy production the cell dies

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Microbial Physiology and Metabolism

Glycolytic PathwaysEmbden-Meyerhof-Parnas PathwayEntner-Doudoroff PathwayHeterofermentative CatabolismHomofermentative Catabolism

Tricarboxylic Acid (TCA) CycleElectron Transport Chain

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Microbial Physiology and Metabolism -Aerobes

Use the electron transport system Use molecular O2 as the terminal electron acceptor during oxidative phosphorylation

Electrons travel down the chain and protons are pumped out forming a proton gradient –drives ATP productionAerobic bacteria

Oxidizes glucose to CO2

O2 is reduced to water38 ATP produced per glucose molecule

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In Aerobes Only

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Microbial Physiology and Metabolism -Anaerobes

Lack electron transport systemReduces internal compound through fermentation 1-2 ATP produced

Use sulfur and nitrogen compounds as terminal electron acceptors in “anaerobic respiration” or

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Microbial Physiology and Metabolism -Anaerobes

Have fermentative metabolismOxidizes carbohydrates in absence of an external electron acceptor Terminal electron acceptor is an organic product from the degradation of the carbohydrate

e.g., pyruvic acid is reduced to lactic acid

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Fermentation

Purpose Microbial standpoint

Obtain energy for growth and reproductionHuman standpoint

Produce desirable flavor &/or aroma traitsProduct preservationCreate a variety of products

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Chemical Alterations during Fermentation

Production of desirable end-productsBalance of different end-products and intermediate productsLactic, acetic, propionic acids, CO2, ethanol, flavor cpds

What is produced – depends on:Which m/o are present Which enzymes are active

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Fermentation Practices

Consider factors affecting growth ofDesired m/oUndesired m/o

Can influence: Rate of reactions Final products produced

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Inoculation Types

Natural fermentationsNatural microflora present on raw product

Controlled fermentationKnown culture“Starter cultures”

Single or mixed strains

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Fermentative microorganisms

Variety – bacteria, yeasts, moldsPossible to use purified enzyme

If only 1 enzymatic reaction is involvede.g., Immobilized enzymatic fermentationCan be faster, better control over rate of conversion

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Fermentation Pathways

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Types of Pathways

Homofermentative fermentation>90% of end product is lactic acidEmbden-Meyerhof-Parnas Pathway

Aldolase and hexose isomerase present1 glucose molecule 2 lactic acid moleculesAdvantages

For microbe – 2 ATPs producedFor humans – lactic acid produced

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Types of Pathways

Heterofermentative fermentationHexose Monophosphate Pathway (or Pentose Pathway)

Phosphoketolase present 1 glucose 1 lactic acid + 1 CO2 + 1 ethanol Advantages

For microbe – 1 ATP producedFor humans – lactic acid, CO2, ethanol produced; more flavor and aroma compounds than homofermentation

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Types of Pathways

Alcoholic fermentation by yeasts1 glucose 2 CO2 + 2 ethanol Advantages

For microbe –2 ATPs1 & 2 C-cpds produced

For humans – CO2, ethanol produced

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Genetic Modifications

Modify microbee.g., Dairy starters

Most plasmid related traitsPrimarily: Lactococcus, Streptococcus, LactobacillusDesirable traits:

Phage resistanceLactose fermenting abilityProteolytic activityCitrate useNisin production

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Genetic Modifications

Production of food grade yeast Single cell proteins

Immobilized enzymesProduction of enzymes

e.g., Microbial production of rennin Production of proteins

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Fermented Foods

Fermented Dairy ProductsExamples covered next

Fermented Meat ProductsCovered in the “Microbiology of Meat, Poultry, Fish and Shellfish” section

Fermented Vegetables and FruitsWide variety of final products

Covered some in lab – e.g., sauerkrautOther vegetable types – e.g., pickles – following sectionBeer and wine – following sectionOther products covered in handout

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Buttermilk

True buttermilk - What remains after cream is churned into butterCultured buttermilk - commercial product

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Cultured Buttermilk

Milk - skim, reconstituted NFDMHeat

Destroys unwanted m/o Improves body

Starter culture addedForm curd –

Lactococcus lactis subsp. lactis Lactococcus lactis subsp. cremoris

Flavor: diacetyl, acetoin via citric acid fermentation Leuconostoc mesenteroides subsp. cremoris Lactococcus lactis subsp.lactis biovar diacetilactisLeuconostoc mesenteroides subsp. dextranicum

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Cultured Buttermilk

Incubation to TA of 0.8-0.9% lactic acid

Break-up curdCoolPackage Distribute

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Acidophilus Milk

Desire - to ingest viable bacteria Lactobacillus acidophilus - easily overgrown so milk is heat treated to kill other m/oIncubate

to 0.6-0.7% lactic acidAcid produced with no aroma

Cool, etc.

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Acidophilus Milk

Sweet acidophilus milk Concentrate of L. acidophilus added to cold milk“sweet” - no acid produced

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Acidophilus Milk

Health benefitsL. acidophilus implants in intestinesPossible benefits

Pathogen inhibitorCarcinogen suppressionEnzyme source

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Yogurt

Milk - 12-15% milk solids-not fatHeat then cool Inoculate

Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus - 1:1 ratio

Incubate - 45oC for 3-5 hto TA of 0.85-0.9% lactic acid Final level attained after packaging

Cool, etc.

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Kefir

1% ethanol, 1% lactic acidMicroorganisms - varies

Lactobacillus brevis, Lactobacillus delbrueckiisubsp. bulgaricus,Torulopsis, etc. Kefir grains

Mass of fermenting microbes Used to seed next batch

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Basic Cheesemaking

Ripened

Salted

Whey expelled

Milk coagulated

Acid produced

Lactic acid bacteria added

Milk

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Cheese - Processing

MilkInitial microbiota Pasteurized or unpasteurized

Heat may result in soft, fragile curdsHeat destroys natural enzymes in milk Heat eliminates pathogens

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Cheese - Processing

Curd formation Temperature

Hold after starters are added Acid produced

Rennet added to ppt. casein

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Cheese - Processing

Starter culturesLactic acid - from 1 or more strains

Promotes curd formationDestroys, retards or prevents growth of spoilage and pathogenic m/oInfluences enzymatic changes - greater activity at reduced pH

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Cheese - Processing

Starter culturesLactic acid producers requiring moderate curd cook

Up to 40oCSingle or mixed strains

Lactococcus lactis subsp. lactisLactococcus lactis subsp. cremorisLactococcus lactis subsp. lactis biovar diacetilactis

Thermophilic lactic acid producersCurd cook 45-54oCSingle or mixed strains

Streptococcus thermophilusLactobacillus delbrueckii subsp. bulgaricus

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Cheese - Processing

Starter culturesOthers

Propionbacterium shermaniiPenicillium roquefortiPencillium camemberti

Addition of starters May be added to vat, rubbed on surfaces of wheels, sprayed, etc.

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Cheese - Processing

Shrinking of curdCut-up curdHeat, stir, and measure %TA of wheyExpel wheySalt

Enhances flavor Helps control spoilage m/o growth

Forms and pressure

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Cheese - Processing

Ripening (Curing)Flavor and body characteristics developEnzymatic changes of fat, protein, etc. enhance flavorTime varies according to variety and temperature

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Milk

Whey (93% water, 5% lactose)

Firm Curd (Coagulated casein)

Unripened Cheese

Soft Cheese (>39% water) Surface Growth

Hard CheeseInterior Bacterial Growth

Semihard Cheese

Cream Cheese

Lactococcus lactis or L. cremoris (<38oC)Streptococcus thermophilus &

Lactobacillus sp. (50oC)Rennin

Salt added

Cottage Cheese

Cream

Ripening – temperatures, incubation times, organisms vary depending on the cheese

Bacteria or fungi growsDegradation and conversion of the curd

Cheesemaking

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Cheese - Processing

ProblemsInadequate acid formation

Bad starterResidual antibioticResidual sanitizerBacteriophages

Rotate starters to minimize

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Cheese - Spoilage

Putrid, unclean, yeasty, fermented, rancid flavors developYeasts, molds, anaerobic sporeformers

Mold on surfacesPrevent by waxing and vacuum packaging

“gassy” - Clostridium spp.

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Cheese - Safety Issues

Variety of pathogens possiblebrucellosis, botulism, shigellosis, salmonellosis, staphylococcal food poisoning, listeriosis, etc.Most common problems

Soft cheese Improperly processed

Amine formation – histamine

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Fermented Vegetables

Preservation principlesReduce activities of native enzymes

PectinolyticOxidativve

Inhibit undesirable m/oFactors that can be manipulated

Anaerobic conditionsNaClAcidTemperature – 18-27oC

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Microorganisms Involved

Normal microbiotaPrimary ones:

Leuconstoc mesenteroidesLactobacillus plantarumPediococcus cerevisiaeLactobacillus brevis

What role does each serve?

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General Flow Sheet for Fermented Vegetables

Raw material Remove damaged and spoiled material Clean/Wash

Cored/PeeledBlanched/CookedPierced/Shred

Place in FermenterAdd Water Addition of Sugars, Starter Cultures, NaCl

Fermentation

Unpacked

ContainersFilled

Fresh FermentedProducts

Blached

Canned Pasteurized

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Pickle Fermentation - Salt Stock Procedure

Raw cucumbers placed in wooden brine tanks5% NaCl added

Inhibits undesirable microbes and Extracts water and water soluble substances -e.g., sugars which are then fermented to lactic acid

Over 6-9 weeks NaCl increased to 15.9%

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Pickle Fermentation - Salt Stock Procedure

Microorganisms involved in the fermentationLeuconstoc mesenteroidesLactobacillus plantarum - Most importantPediococcus cerevisiae - Important Lactobacillus brevis - Undesirable; Gas production Enterococcus faecalis

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Pickle Fermentation - Salt Stock Procedure

Spoilage Economic lossBloaters - Gas trapped in cucumbers Caused by Enterobacter spp., lactobacilli, pediococciSoftening – Caused by pectolytic organisms, e.g., Bacillus, Fusarium, Penicillium, Phoma, Cladosporium, Alternaria, Mucor, Asperigillus,others Off-colors, etc

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Pickle Fermentation - Controlled Fermentation

ProcedureCucumbers placed into chlorinated brine with 6-7% NaCl Brine acidified with acetic acidSodium acetate addedInoculated with P. cerevisiae and L. plantarum or just L. plantarum10-12 day fermentationFinal pickle pH ~4.0

AdvantagesReduces economic lossMore uniform pickleFaster than salt stock

Spoilage possible – Bloaters, Softening, Off-colors

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Changes During Pickle Fermentation

(Jay, 2000)

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Sauerkraut Fermentation

Starter culture - natural microbiota Procedure

Raw cabbage2.25-2.5% NaCl

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Changes during Sauerkraut Fermentation

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Flow Chart for Brewing

Malting

Milling, Mashing

Wort Boiling

Fermentation

Post-fermentation Treatments

Conversion of barley starch to fermentable sugars and protein to free amino acids

Hot water extraction of sugars, amino acids, and other yeast nutrients and enzymes – Yields sweet wort

Boiling with hops to extract aroma and bitter compounds; Heated to sterilize – Yields hopped wort

S. cerevisiae fermentation of sugars to ethanol & CO2

Maturation, clarification, packaging, pasteurizationHarrisonFDST 4030/6030

Typical Brewing Changes

(Doyle et al, 2002)

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Flow Chart for Red Wine Making

Grapes Harvested Grapes Crushed (SO2 added optional) Add Starters (optional)

Maceration and Partial Fermentation

(color and tannin extraction)

Pressing(removal of skins)

Fermentation Completion;Racking Off Yeast Lees

WineMalolactic

Fermentation(optional)

Aged (in oak barrels, etc.)

Fined, Clarified, Packaged

Final Product –Red Wine

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Flow Chart for WhiteWhiteWhite Wine Making

Grapes Harvested Grapes Crushed (SO2 added optional)

Pre-fermentaton; Settling

Pressing(removal of skins)

Add Starters(optional)

Fermentation;Racking Off Yeast Lees

Aged (optional)

Fined, Clarified, Packaged

Final Product –White Wine

White Wine Malolactic Fermentation (optional)

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Grapes are emptied into binsand then enter the destemmermachine.

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Grapes go to the destemmer-sorting machine

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Rotating grape press

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Temperature controlled

fermentation vats

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Spoilage Microorganisms of Beer and Wine

Beer Spoilage - YeastsSaccharomycesTorulasporaZygosaccharomycesHanseniasporaDekkeraDebaryomycesPichia

Wine Spoilage – BacteriaMicrococcusBacillusPediococcusStreptococcusLactobacillusAcetobacterGluconobacterZymomonasMegaspheraPectinatusObesumbacterium