ADVANCE FOOD MICROBIOLOGY ITP 520

Outlines
MICROORGANISM OF FOOD
FERMENTATION AND • Introduction
PRODUCTION OF INGREDIENT • Food Fermentation
• Fermentation of ingredient

Lilis Nuraida

Department of Food Science and Technology

Bogor Agricultural University
Indonesia

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Fermentation Technology Fermentation process

• Fermentation is the chemical transformation of
organic substances into simpler compounds by the
action of enzymes produced during the growth of
desirable microorganisms such as molds, yeasts, or
bacteria.
• Fermented foods are foods produced by the action
of microorganisms (molds, yeasts or bacteria or a
mixture of them)
• Fermented foods represent on average
one-third of total food consumption.
• Natural fermentation
– Raw materials contain both desirable and associated
microorganisms
– Condition of incubation set to favor desirable microorganism
– Difficult to produce product with consistent characteristic
– Chances of product failure due to growth undesirable
microorganism and growth of pathogen are high
• Back slopping
– Some products from a successful fermentation are used as
starter
– Difficult to produce product with consistent characteristic
– Chances of product failure due to growth undesirable
microorganism and growth of pathogen are high

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 Fermentation process
• Controlled fermentation
– Raw material may be heat treated and
inoculated with a high concentartion (106
cells/ml) of known starter culture
– Incubation condition is set for the optimum
growth of the starter culture
– Product with consistent and predictable
Industrial use of microoganism
• Foods and food additives
– The food products are good sources of protein, vitamins, and other
organic nutrients.
– Microbial processes are employed for large scale production of amino
acids.
• Alcoholic beverages.
– Beer, wine, and other alcoholic beverages.
• Manufacture of various chemicals.
– Produce a variety of chemicals (various alcohols, lactic acid, acetic
acid, citric acid, gluconic acid, etc.) which are being recovered, purified
and sold.

characteristics • Therapeutic compounds.

– Antibiotics, vitamins, and steroid drugs are prominent in this category.
– Less chance of failure and growth of
• Industrial enzymes.
patogenic microorganism – A number of microbial enzymes have industrial applications, and are
produced on a large scale

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History of Fermented Foods

• Circa 5000 BC people has made wine in Babylon,
3000 BC in ancient Egypt, 2000 BC in pre-Hispanic
Mexico, and 1500 BC in Sudan
• 3000 BC milk fermentation began in Babylon
• 1500 BC people made bread
- leavened bread in ancient Egypt
• In Asia:
• A number of the most important fermentations use molds
for grain or soybean fermentation; dairy products and
other animal proteins (excepting fish) are not widely used
• 300 BC koji-making process can be traced back in China
FOOD FERMENTATION • The Chinese were probably the first to develop
vegetable fermentation
• Javanesse (Indonesia) were the first to develop
tempe, fermented soybean

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Benefit of Food Fermentation Principles of Food Fermentation Process
• Fermentation under high salt concentration
• Improved shelf life – Example: Terasi, petis, fish sauce, tauco, soy sauce
• At room temperature milk spoils in <8 hours, • Products are used as condiment or seasoning
fermented can stay up to 2 days
• Fermentation to produce specific texture:
• Milk vs dadih, yoghurt, cheese
– remedy meat using pulses as substrates, example: tempe,
• Improved nutrient and digestibility
oncom
• tempe vs soy
– Cheese (ripenned)
• vitamin B12 in traditionally fermented tempe
• Lactic acid fermentation:
• Improved taste and flavor
– Example: tempoyak, dadih, urutan, sayur asin,
• tempe vs soy, yoghurt vs milk
sauerkraut/kimchi, yoghurt, cheese, sausages
• Reduced toxicity/antinutrient
• Alcoholic fermentation:
• phytate in soy decreases during fermentation
– Example: tape, brem, tuak, wine, beer, sake
• Health functions
• Acetic acid fermentation:
• Bioactive compound in tempe, probiotic
– Example: nata de coco, vinegar
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Bacteria involve in food

The objectives of fermentation fermentation
• Lactic Starter Culture:
• Fermentation of fruits, vegetables, – Lactococcus, Streptococcus, Leuconostoc, Pediococcus,
Lactobacillus, Bifidobacteria
milk, fish, meat is to preserve and • Mostly in dairy products
modify sensory and nutritional – P. halophilus (now Tetragenococcus halophilus) involves in
salt fermentation of tauco/soy sauce
properties • Propionibacterium:
– P. freundenreichii, P. jenseni, P. thoenii and P. acidipropioni
• Fermentation of cereals and seeds is – Involve in natural fermentation of Swiss type Cheese
not as much their preservation, but • Acetobacter:
– A. aceti is used in acetic acid fermentation,
rather the modification of their sensory A. xylinum in nata de coco
and nutritional properties

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 Yeast in food fermentation
• The most important Saccharomyces cerevisiae:
– Leavening bread, producing beer, wine, distilled liquor,
industrial alcohol, producing invertase
– To flavor some food (soups): yeast extract
– Top yeast: grow very rapidly at 20 C, producing alcohol and
CO2, from clump and float
– Bottom yeast: grow better at 10 to 15 C, produce CO2 slowly,
grom slowly, settle at the bottom
• Candida utilis:
– Single Cell proteins
• Kluyveromyces marxianus:
– Capable of hydrolysing lactose, have been associated with
natural fermentation along with LAB
– Involve in alcoholic dairy fermentation: kefir
Mould in food fermentation
• Aspergillus, Penicillium, Rhizopus and Mucor:
– Have been used in food processing and to produce
additive or enzyme for use in foods
• The strains for food should not produce mycotoxin
– It is difficult to identify a non-mycotoxin producer
strain in natural fermentation
• A. oryzae is used in producing sake, soy sauce, miso
• P. roquefortii: for ripening Roquefort, Gorgonzola and
Blue Cheese
• P. camembertii: Camembert cheese
• P. caseicolum: Brie Cheese
• A. niger: to produce citric acid and gluconic acid, also for
producing pectinase and amylase (food ingredient)

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Fermented Foods: Fermented Foods:

Milk, meat an fish Cereals, legumes and tuber
Substrate Product Type of Fermentation Substrate Product Type of Fermentation
Milk Yoghurt, Cheese, sourcream, Lactic fermentation Barley Beer Alcoholic fermentation
buttermilk, Dadih, Glutinuous Sake, brem, tape Alcoholic fermentation
mold ripened cheese, Lactic fermentation with rice
secondary starter Maize pozol Lactic fermentation
kefir Lactic and yeast Soybean Nato Bacterial fermentation
fermentation
Miso, tauco, soy sauce Mold, lactic and yeast
Meat Sausages, urutan, pepperoni Lactic fermentation fermentation
Fish, Shrimp Terasi, Rusip, Belacan, Peda, Lactic and proteolitic Tempe, oncom Mold fermentation
fish sauce fermentation
Cassava tape Alcoholic fermentation
Growol, ogi Lactic fermentation

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 Fermented Foods: LACTIC FERMENTATION
Fruit and vegetables
Substrate Product Type of Fermentation
Fruit wine Alcoholic fermentation
vinegar Alcoholic and acetic
fermentation
Tempoyak, mandai Lactic fermentation
Vegetables Kimchi, sauerkraut, sawi Lactic fermentation
asin, olive, pickles
Coconut sap Wine, tuak and vinegar
Coconut water Vinegar, Nata de coco Acetic acid fermentation

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Microbiology of Yoghurt fermentation Microbiology of Cheese

• Many types of yoghurt available in the market
• Milk fermented using starter culture: Lactobacillus delbrueckii
subsp bulgaricus and Streptococcus salivarius subsp
thermophillus in ratio 1:1 (should not exceed 2:3)
• For balance growth fermentation is conducted at 43.3 C
– Symbiotic growth:
• Initially Streptococcus grows rapidly and produces formic
acid and CO2
• Anaerobic condition, formic acid and CO2 stimulate the
growth of Lactobacillus (has good proteinase and
peptidase systems) producing peptides and amino acids
such as glycine, valine, histidine etc that are necessary
for Streptococcus (lacking proteinase).
• At pH 5.5 Growth of Streptococcus slows down
• The species produce about 8 to 10 ppm acetaldehyde
when grown separately, but 25 ppm or higher in
symbiotic culture
• Cottage cheese
– Mixed strains of Lac. Lactis subsp cremoris and lactis are
predominantly used for acid
– Leuconostoc mesenteroides subsp cremoris can be
added initially (if citrate is added) for diacetyl
– Lact. Lactis subsp lactis biovar diacetylactis is used for
diacetyl but grown in cream (added to the dry curd)
• Blue Cheese:
– Lac. Lactis subsp cremoris and Leuconostoc sp as
primary starters:
• Grow until curing and produce lactate, diacetyl, acetate,
CO2 and acetaldehyde from lactose
– Penicillium roquefortii as secondary starter:
• Grows during storage at 50 F in high humidity giving the
mottled green appearance.
• Growth continue until curing

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Microbiology of Kefir
• Made by the native people of the northern
Caucasus
• Mixed fermentation of lactic and yeast
• Product contains 0.5-1.5% alcohol
• Fermented using kefir grain:
– Mesophilic lactic acid bacteria: L. lactis etc
– Aromatic lactic acid bacteria: Leuconostoc
– Yeast: Candida kefir, Saccharomyces lactis etc
– Thermophilic lactobacilli: L. brevis, L. bulgaricus
• Composition of kefir grain: lipid 4.4%, protein
34.3%, carbohydrate 45.7%, abu 12.1% and
moisture 3.5%
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Microbiology of sauerkraut
• Raw material (shredded cabbage) has a large
number of undesirable microorganism and small
population of LAB
• 2.25% salt, sugars, absence of O2 and low T
facilitate Leuconostoc mesenteroides to grow rapidly
• When the acid has reached 1% as lactate, growth of
Leuconostoc slows down, Lactobacillus brevis starts
growing until acid about 1.5%. P. pentosaceus then
takes over until 1.8% acid. Finally L. plantarum
brings the acid level up to 2%.
• Flavor of sauerkraut is combined effect of lactate,
acetate, ethanol, CO2 and diacetyl
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Microbiology of semi-dry sausages
• Summer sausage, thuringer and semi-dry salami
• Controlled fermentation:
– 106 – 107 cells/g mix (should not be mixed with salt, cure or
spices)
– Starter vary depending upon the fermentation temperature
and the desired pH
– For high T, low pH: P. acidilactici
– For low T, high pH: L. plantarum
– P. pentosaceus may be used in both condition
– Some can have both Pediococcus and Lactobacillus
– For desired color: Micrococcus sp and Staphylococcus
carnosus as secondary starter
• Natural fermentation:
– Lactobacillus sake, L. curvatus and Leuconostoc spp.
Present in raw materials are important as starter, esp. when
fermentation at low T (60 – 70 F) and the final pH 5.0
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Alcohol Fermentation
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 Alcohol Fermentation Key Microorganism
Saccharomyces Lager-type beer
• Foods: Tape carlsbergensis*)
Saccharomyces Ale-type beer
• Beverages cerevisiae*) Wine, sake
• Beers – produced by fermentation of grains or starchy
staples. Schizosaccharomyces Heavy rum
pombe
• Wines – produced by fermentation of fruit juices
• In natural fermentation involves different yeast *)Unable to break down starch or other complex carbohydrates
species such grow successively at initial. The most • Malting:
•Germinantion of barley to induce production of amylase
active fermentation stage to end of fermentation:
•Beer
Saccharomyces cerevisiae
•Amyolytic molds and yeast
• Rice wines/sake/brem – produced by fermentation of •Filamentous fungus Aspergillus oryzae (koji): sake
glutinuous rice •Mucor, Amylomuces rouxii: tape
• Spirits – produced by distillation of a wine or beer. In, sake, tape and natural fermentation of wine, lactic acid bacteria also involve
and produce lactic acid

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ACETIC ACID FERMENTATION

Reaction in acetic acid fermentation
• Vinegar is literally translated as sour wine
– It can be made from almost any fermentable carbohydrate Alcohol fermentation by yeast
source, for example fruits, vegetables, syrups and wine.
• The vinegar process
1 Glucose 2 ethanol + 2 carbondioxide
– a two stage process,
• where yeasts convert sugars into alcohol,
• followed by Acetobacter, which oxidize alcohol to Acetic acid fermentation by Acetobacter
acetic acid.

• Definition of Codex:
– Liquid produced from fermentation of ethanol from suitable
sources (e.g wine, cider). Example include cider vinegar, wine
vinegar, malt vinegar, balsamic vinegar, spirit vinegar, and fruit
(wine) vinegar

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 Vinegar starter
• The organisms involved in vinegar production
usually grow at the top of the substrate, forming a
jelly like mass.
– This mass is known as 'mother of vinegar'.
– The mother is composed of both Acetobacter
and yeasts, which work together.
• The principal bacteria are Acetobacter acetic
A. xylinum and A. ascendens.
• The main yeasts are Saccharomyces
ellipsoideus and S. cerevisiae.
Nata de coco
• Made of coconut water
– Other substrate has been used is tofu waste
liquid, but not commercialized
• Other substrate: sugar, nitrogen source,
acetic acid
• Fermentation is done in a shallow layer
• The cellulose is harvested and used as a
dessert (deacidified and mixed with
syrup)

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Fungal fermentation Tempe

• Fermented soybean
– Other legumes could also be used
• Tempe – Bean and the mold are protein source
•
• Oncom Used starter culture: laru or usar
– Main microorganisms: Rhyzopus oligosporus
• Mixed fermentation: • Process:
– Soybean are soaked overnight, seed coast are removed,
– Two stages fermentation: split into halves, boiled, dried, cooled, inoculated.
– packed into banana leaves or perforated plastic bag and
– Involve mold, yeast and lactic acid then left to ferment for 2 days
bacteria • The mold grows through to knit together the beans
and turn them into tempe.
• Kecap
– Alternative: use of dehulled soybean
• Tauco

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 Biochemical changes occuring in
Microbiology of Tempe soybean during fermentation
• Hydration/Acid fermentation
– Acid fermentation take place during soaking • Changes in lipids
• pH falls to 4.5-5.3 – Deliberating fatty acid during a 72 h at 37 C
• Does not affect the mold growth, but prevents the • Changes in carbohydrate
development of undesirable bacteria that might spoil the
bean – Principals sugar in soybean: sucrose, stachyose and
• Lactic acid bacteria may involve during this step raffinose
– Acidification the bean lengthen the fermentation time – Principal changes in carbohydrate are the rapid removal of
before ammonia is deliberated hexoses and the slow hydrolysis of stachyose
• The mold in tempe fermentation is very proteolytic; • Changes in amino acids
deamination following hydrolysis releases ammonia, – Available lysine and methionine show a decrease during
causing pH to raise. tempe fermentation
• Above pH 7, sufficient free ammonia is released to kill – pH increase to 7.1, as the result of active proteolysis and
the mold
deamination of amino acids

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Soy sauce and Tauco Microorganism of soy sauce

• Kecap: dark liquid (sweet and salty)
and tauco
• Mold fermentation:
– Sweet soy sauce with addition of palm sugar and spices
– Two type of sot sauce: – Aspergillus oryzae or soyae
• Chinese style: soybean as raw material • Koji: Mixture of soybean, cracked wheat and wheat
bran; mixture of wheat bran and soybean flour or rice
• Japanese style: mixture soybean and wheat as raw flour innoculated with mold spore.
material
– During mold fermentation, proteinases, amylses and other
• Tauco: slurry, with addiiton palm sugar and spices enzymes are produced. The flora of lactic acid bacteria are
• Two stages of fermentation: also developed
– Mold fermentation (2-3 days) • Brine fermentation:
– Brine fermentation (months) – Lactic acid fermentation by lactic acid bacteria incl.
• Tauco: brine fermentation is shorter Pediococcus (Tetragenococcus) halophillus
– Alcoholic fermentation by yeast such as S. cerevisiae or
Zygosaccharomyces soyae
• Sometime B. subtillis also grows
– Completion fermentation and aging

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 Ingredients or substances from
microorganism
• Enzymes
• Amino acid
• Flavoring
• Biogum:
– Dextran
– Xanthan
PRODUCTION OF FOOD – Etc
INGREDIENT • Organic acids (citric acid)

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Microbial Enzymes Fungal enzyme

◼ Production is fast and controllable Enzyme Source Extracell/Intra Application
◼ Can use waste as substrate (e.g mollases) a-Amylase Aspergillus E Baking
◼ Inducible Enzymes : Catalase Aspergillus I Food

• generally extracellular enzymes

Cellulase Trichoderma E Waste
Dextranase Penicillium E Food
• need inducer (i.e.substrate), substrate has to be
Glucose oxidase Aspergillus I Food
added to the medium
Lactase Aspergillus E Dairy
• easy to harvest
Lipase Rhizopus E Food
• amylase, lipase, protease, pektinase etc
Rennet Mucor miehei E Cheese
▪ Constitutive Enzymes Pectinase Aspergillus E Drinks
production is not dependent of substrate generally Protease Aspergillus E Baking
intracellular

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 Bacterial enzyme Microbial enzymes production
◼ Growing the culture in suitable media:
Enzyme Source Extracell/Intra Application
❑ pH
a-Amylase Bacillus E Starch
❑ N sources
b-Amylase Bacillus E Starch
Escherichia ❑ Inducers
Asparaginase I Health
coli ◼ Harvesting the enzymes:
Glucose isomerase Bacillus I Fructose syrup ❑ Intracellular:
Penicillin amidase Bacillus I Pharmaceutical
◼ Whole cells used as intracellular enzymes sources
Protease Bacillus E Detergent
❑ Extracellular:

◼ Cells separation

◼ Precipitation with ammonium salt and purification

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L-Glutamate Fermentation L-Glutamate Synthesis

• The most amino acid industrially produced (80% of the total • Optimum condition for
amino acid produced by fermentation) glutamate production by
C. glutamicum is low
• Used as flavor enhancer concentration of biotin in the
• Glutamate producers : medium.
• Gram positive nonmotile bacteria which requires biotin for • The best condition is when
biotin has been depleted
growth, with low amount or activity of α-ketoglutarate
• Over production of glutamate
dehydrogenase and high activity of glutamate under excessive biotin is done
dehydrogenase by addition of detergen or
• 1957 : isolation of L-glutamate producing soil bacterium (Tween 40 or 60) or β-lactam
Corynebacterium glutamicum, Kyowa Hakko Co. antibiotics (penicillin)
• Several mutants of Corynebacterium and
Brevibacterium have low isocitrate lyase activity
• Mostly used by industries :Corynebacterium,
Brevibacterium, Microbacterium , Arthrobacter
• Less used: mold and yeast

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 L-Glutamate Fermentation L-Glutamate Synthesis
• Glutamic acid is synthesized inside cells and secreted into the
medium
• Glutamic acid is crystallised to form monosodium glutamate
• Production Medium Stoichiometry :
• Main raw material is substance containing C : glucose, fructose,
maltose, sucrose, xylose, and acetic acid
• Requires N source : ammonium salts, amonia (NH3) C6H12O6 + NH3 + 1,5 O2 C4H9O4N + CO2 + 3 H2O
• Requires biotin (limiting factor, depending on C as substrate):
• when the amount is excessive, cell membrane will be (glucose) (glutamic acid)
strengthened, thus glutamic acid is held within the cell
• critical concentration : 0.5 µg/g dry cells
• in a medium with10% glucose, biotin added is 0.5 µg/l
3 C2H4O2 + NH3 + 1,5 O2 C4H9O4N + CO2 + 3 H2O
Fermentation Technique
• Glutamic acid fermentation is aerobic, requires aeration system (acetate) (glutamic acid)

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Thank you

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