Food FermentationBy

Thanut Amatayakul

BOT323 Industrial MicrobiologySrinakharinwirot University

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Definition• Fermentation

– process of converting organic substancesะ •Anaerobic-alcoholic fermentation/lactic acid

fermentation•Aerobic-acetic acid fermentation•no microbes (enzyme)-starch hydrolysis

• Food Fermentation– process for food production through

fermentation2/28/2017 2

Fermented food • May involve using microorganism• or just enzyme• involve biochimical changes• safer, more nutrition, tastier

– production of organic acids/volatiles/alcohol/used up available nutrient for contaminated microorganism

• Initially, not intended: cheese/yogurt/wine/beer/bread/soy sauce etc.

• art more than science2/28/2017 3

Food Fermentation• Sequence of occurance

– start with bacteria (lactic acid bacteria) producing acids and antimicrobial substances protecting harmful bacteria

– follow by yeast– then Fungi

• fungi grow best under aerobic condition

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Microorganisms in Food Fermentation

added or natural for large scale : commonly added pured culturecontrollable and getting reliable outcome (food)

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Microbial metabolism– Phototrophs: light as energy source/CO2 as c-source– Lithotrophs: oxidation of inorganics– Chemotrophs: energy come from oxidation of

chemicals– Organotrophs: energy come from

oxidation of organic molecules• amino acids, peptides, proteins• sugars, polysaccharide, starch, cellulose• lipid• vitamin

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Microbial and nutrients• Large molecule of nutrients must be

digested by extracellulase enzyme then transport inside a cell.– Passive transport (not good-depeding

on external environment)– permease andะ Active transport (use

energy)

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extracellular enzymeEnzyme Major sources Application in foods

-amylase Aspergillus, Baceillus Syrup production, baking brewing-amylase Bacillus, Streptomyces, Rhizopus Production of high maltose syrup, brewing

Glucoamylase Aspergillus, Rhizopus Production of glucose syrups, baking, brewing, wine making

Glucose isomerase Arthrobacter, Streptomyces Production of high fructose syrupPullulanase Klebsiella, Bacillus Starch (amylopectin) degradationInvertase Kluyvermyces, Saccharomyces Production of invert sugar, production of soft

centered chocolateGlucose oxidase Aspergillus, Penicillium Removal of oxygen in various productsPectinase Aspergillus, Penicillium Fruit juice and wine production, coffee bean

fermentation-glucanase Bacillus, Penicillium, Trichoderma Brewing, fruit juice, olive processing

Pentosanase Cryptococcus, Trichosporon Baking, brewingProteinase Aspergillus, Bacillus, Rhizomucor,

LactococcusBaking, brewing, meat tenderization, cheese

Catalase Micrococcus, Corynbacterium, Aspergillus

Cheese

Lipase Aspergillus, Bacillus, Rhizopus, Rhodotorula

Dairy and meat products

Urease Lactobacillus WineTannase Aspergillus Brewing

-galactosidase Aspergillus, Bacillus, Escherichia, Kluyvermyces

Removal of lactose2/28/2017 8

Metabolic events• coupling between catabolism and

anabolismBreakdownProteins to Amino Acids, Starch to Glucose

SynthesisAmino Acids to Proteins, Glucose to Starch

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Catabolisms• Catabolism of sugar : Embden-Meyerhof-Parnas (EMP)

Dihydroxyacetone phosphateGlucose (C6)

Glucose-6-phosphate (C6)

Fructose-6-phosphate (C6)

Fructose-1,6-phosphate (C6)

Glyceraldehyde 3-phosphate (2C3)

1,3 Diphosphoglyceric acid (2C3)

3 Phosphoglyceric acid (2C3)

2 Phosphoglyceric acid (2C3)

Phospho-enolpyruvic acid (2C3)

Pyruvic acid (2C3)

2 ADP2 ATP

H2O

2 ADP2 ATP

2-Phosphate2 NAD

2 NADH + 2H+ATPADP

ATPADP

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Catabolism• Entner-Denduroff pathway

Glucose 6-phosphate

6-Phosphogluconolactone6-Phosphogluconate

2-Keto-3-deoxy 6-phosphogluconate

Pyruvate Glyceraldehyde 3-phosphate

EMP pathway

NADP

NADPH

H2O

H2O

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Catabolism• Kreb’s cycle /

Tricarboxylic acid cycle– give NADH and CO2/

occur when there is plenty of oxygen

– getting energy by transfer of H+ through membrane of mitochondria (eukaryotes) or plasma membrane (prokaryotes)

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Pyruvate

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Anabolism

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Flux-Metabolite

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Food FermentationNatural microorganism

creating suitable growth condition for microorganism

SaltSugarAir

Example: fermented vegetable, fish sauce

Pured Starter culture

Must eliminate normal floraadd pure starter culture at 1-5%

creating suitable growth conditionfor starter culture such as temperature

pH2/28/2017 17

Elimination of contaminated MO.

• Heat (steam, hot water)• Chemicals

– (SO2 )เ ช ่น ใ น ก ร ะบ ว น ก า ร ท ำ ไ ว น ์– NaCl– remove oxygen

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Easier is the key!

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Starter culture Lactic acid producing bacteria-ส ร ้า ง ก ร ด เ ป ็น หล ัก Homofermentative LAB Heterofermentative LAB Saccharomyces cerevisiae-แ อ ล ก อ อ อ ล ์ Ethanol producing yeast Flavor producing microorganisms Bacteria, yeasts, molds Acetic acid producing bacteria-ส ร ้า ง ก ร ด Acetobacter

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LAB fermentation- Anaerobic glycolysis for glucose utilization- Glucose … 2 Pyruvate … 2 Lactate- Net energy yield: 2 mole ATP/mole glucose- Lactococcus, Streptococcus,Pediococcus, Lactobacillus(some)‏

-Hexose monophosphate or pentose phosphate pathway -Glucose … Lactic acid (~50%) + Ethanol + co2 -Can also form acetate -Net energy yield: 1 mole ATP/mole glucose -Leuconostoc, Lactobacillus(some)‏ -Some heterofermentative Lactobacillus

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Saccharomyces

Glycolysis = produce 4 and use 2 = 2 ATP / NADHAfter glycolysis 1. alcoholic fermentation, 2. respiration, 3. glyceropyruvic fermentation

In alcoholic fermentation, pyruvate is first decarboxylated (pyruvate decarboxylase) to acetaldehyde that then serves as electron acceptor, giving rise to ethanol (Alcohol dehydrogenase).

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– 1 Glucose 2 ethanol + 2 CO2

– ~ 50% conversion

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Regulation of sugar utilizing metabolic pathways

• Low concentration of sugar: air enhance yeast cell proliferation (biomass) more than producing ethanol: respiration (Pasteur’s effect)‏

• High concentration of sugar: leading to fermentation > 9 g/L (sugar) air or no air (Crabtree’s effect)‏

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Types of cultivation

1. Submerged cultivation

2. Surface cultivation

3. Solid cultivation

Nata de coco

koji

(fermentation)

liquid

liquid

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Processes of Some Fermented Foods

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Yoghurt• 1. Yoghurt:

– Streptococcus thermophilus ะ Lactobacillus delbrueckii spp. bulgaricus equally add

– symbiotic– ferment at 42oC– Set or stirred / natural / fruit

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Yoghurt starter• S. thermophilus

– +G– Ferment lactose and

sucrose– Weak proteolytic activity– Usually combined with >

proteolytic lactobacilli– Some produce EPS

-gal: use only glucose leave galactose

• L. debrueckii spp. bulgaricus– +G– Homofermentative– D(-) lactate– use only glucose leave

galactose– High level of protease activity

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Growth curve

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Probiotics

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Probiotics• Live micro-organisms (single or

mixed culture) which when administered in adequate number confer a health benefit on the host (FAO/WHO 2001)

• Functional foods:

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Benefits from ingestion of probiotics

• Improving the intestine microbial balance• Producing lactase (lactose intolerance)• Strengthening the immune system• Reducing risk of colon cancer in human• Aiding in treatment of food allergies• Lowering blood cholesterol levels• Reducing blood pressure• Treatment of diarrhoea

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Probiotics• New evidence inactive MO. Can

provide health benefit• 2 requirement for MO. to be classified

as probiotics– Safety– Specific health claim

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• The suggested concentration for probiotic bacteria is 106 CFU/g of a product (throughout shelf-life of product)

• Human origin• Mostly found in gut, specifically

large intestine• Approximately 30-50% of gut

content are bacteria• Some 500 species2/28/2017 40

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Role of wheat starch• adjust moisture content 60% -> 45%• food for mold• source of glutamic acid

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• Bacillus, Pediococcus, Micrococcus producing lactic acid decreasing pH resulting in decreasing neutral and alkaline protease activity

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Yeast also produce flavor and alcohol

Bread Fermentation

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Yeast Cultures• S. cerevisiae, or bakers’ yeast• Properties and characteristics for bread making

– Gassing power– Flavor development– Stable to drying– Stable during storage– Easy to dispense– Ethanol – cryotolerant

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Yeast Cultures• Industrial production

– Scale up (Fig. 8-4) • Growth medium

– Molasses or another inexpensive source of sugar and various ammonium salts

• Other yeast nutrients– Ammonium phosphate– Magnesium sulfate– Calcium sulfate, trace minerals (zinc, iron)

• Cell mass production required conditions– O2 level– Temp (30C)– pH (4.0-5.0)– continuous

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Yeast cultures• Commercially available

– Yeast cream• Used directly, highly perishable

– Yeast cake• Yeast cream through filtration press or vac. filter• Refrigeration required, shelflife a few week• Metabolically active, quick fermentation

– Dry active yeast• Home bread making, small business operation• Last 6 months or longer• Require hydration, not as active

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General Manufacturing Principles

Weigh and mix ingredients

dough Fermented dough

Portioned and

shapedbake Cool

slice pack

fermentation

fermentation

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Fermentation• Lag phase usually• Bakers’ yeast facultative metabolism

(Fig. 8-6)– Aerobic (via TCA cycle)– Anaerobic glycolytic fermentation

pathway•Glucose inhibit TCA enzymes•CO2

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Sugar metabolism by bakers’ yeast

• Carbohydrate sources– Starch– Sugars (glucose and maltose)

• Transport and utilization– Sequential use

•Regulation-glucose represses enzymes involved in maltose transportation

•Maltose represses invertase expression•Mutants available

– Sugar transport (Fig 8-7)– Glycolysis2/28/2017 58

Fermentation• End products

– CO2– Other compounds

• Various acids and organic compound by yeasts• By LAB• Flavor and rheology of the dough

• Factors affecting growth– Temp-hold at 25-28C instead of the optimal growth temp

36-39C to minimize microbail contamination, and maintain yeast activity

– Relative humidity 70-80%

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Glucose

Glucose 6-phosphate

Fructose 6-phosphate

Fructose 1, 6 phosphate

DGAPDihydroxyacetone

PGALGlyceraldehyde

3-phosphate

PEPPhosphenopyruvate

PyruvateOxaloacetate

Respiration Chain

TCA Cycle

CO2

CO2Lactic acid Acetyl CoA

+36 ATP

Ethanol

CO2+2 ATP

+2 ATP2/28/2017 60

Microbiology of breadmaking

• Conventional breadmaking– S. cerevisiae– Bacteria

•Commercial baker’s yeast about 5% contaminating lactic acid bacteria

– If LAB deliberated added, can lower pH to below 4.0 and cause distinctive sour but appealing flavor, better preserved

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Cheese making• made from milk • concentrate 6-10 times• coagulation or protein

expelling whey • Processes:

– acidification:– coagulation:– cooking:– salting:– dehydration or syneresis:– moulding:– shaping: – maturation:– storage

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Cheese flavor formation

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Saukraut• fermented cabbage

• salt help draw liquid and nutrient out from cell of cabbage: 4-5% sugar with 2.5% glucose and 2% fructose

• using 2% salt

• anaerobic fermentation at 18oC

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Saukraut• -Organisms produce lactic acid, acetic acid,• ethanol and CO2• First: Leuconostoc mesenteroides• -Initiates growth more rapidly than other organisms,• produces acid and lowers pH rapidly• -CO2 replaces O2• -Produces polysaccharides• Second: Lactobacillus plantarum• -High acid production• -Utilizes polysaccharides produced by Leuconostoc• mesenteroides

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Kimchisoak half cabbage in brine (10%) overnightwashed and drainedmixing chopped minor ingredients (garlic, red pepper, green onion, ginger) and stuffing between leaf of cabbgePacked in earth jar with stone on top to keep cabbage under juiceLeu. mesenteroids predominant first and Lactobacillus ssp. laterfinal pH 4.5

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Fermented meat products• Microorganisms

– Pediococcus pentosaceus– Lactobacillus

• Preservation– Lactic acid production (low pH)‏– Salt and other additives/drying– Low water activity

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