Indonesian Soy Sauce (Kecap)

8/6/2019 Indonesian Soy Sauce (Kecap)

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INDONESIAN SOY SAUCE (KECAP)FERMENTATION

SardjonoDepartment of Food and Agricultural Product Processing TechnologyFaculty of Agricultural TechnologyGadjah Mada UniversityYOGYAKARTA, INDONESIA


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HEALTH BENEFIT OF JAPANESE FERMENTED SOYSAUCE

http:// www. Raysahelian.com/soysauce.html

Promote digestion

Antimicrobial activity againts : Staphylococcus aureus, Shigellaflexneri, Vibrio cholera, Salmonella enteridis, Esherichiacoli

Antihypertensive effects

Inhibit benzopyrene induce neoplasia

Antioxidant activities, anticarcynogenic effects

Inhibit histidine decarboxylase anti alergy


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KECAP IS LIQUID SEASONING, BROWN

BLACK IN COLOR, PRODUCED FROM

FERMENTED SOYBEAN, WITH ADDITION OF

COCONUT SUGAR

PRODUCED BY TWO STEP OF FERMENTATION :

1. MOLD FERMENTATION ( solid substrate fermentation,with different of mold used ) produced KOJI

DEGRADATION OF POTEIN

2. BRINE FERMENTATION , (22% NaCl solution ,natural

selective microorganisms: lactic acid bacteria and

halophyilic yeast )

produced MOROMIPROTEIN DEGRADATION AND EXTRACTION, FLAVOR

DEVELOPMENT


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KOJI FERMENTATION

Degradation by extracellularmold enzymes

Extraction : enzymatic, physical?Flavor development :

microbiological? Chemical?

FERMENTATION TIME ??

MOROMI FERMENTATION

KEY OF PROCESS FOR

SOY SAUCE FACTORY


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SIMPLYFIED PROCESS FLOW SHEET OF KECAP

PRODUCTION

soybean

COOKINGMOLD FERMENTATION

DRYING THE KOJI

BRINE FERMENTATION

SEPARATION OF BEANCOOKING / BOILING

starter or wildfermentation

Coconut sugar

NaCl solutIon (22%)

kecap

soybean

koji

moromi

OPTIONAL


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KOJI MOROMI


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BRINE FERMENTATION (MOROMI)BRINE FERMENTATION (MOROMI)

Salt concentration, pH

and temperature

enzymes activity,population ofmicroorganism


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KoikuchiKoikuchi--shoyushoyu

(representative fermented soy sauce in Japan)(representative fermented soy sauce in Japan)

Soybean orDefatted soybean roasted wheat (50%)

cooked

KOJI FERMENTATION starter

lactic acid bacteria MOROMIyeast FERMENTATION

(pH, temp)

PRESSED cake

PASTEURIZED

koikuchi-shoyu


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KOJI FERMENTATION(traditional method)

Small factory Medium / big factory2 4 DAYS FERMENTATION

Un-control condition


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MYCOFLORA AND AFLATOXIN IN KOJI FROM SEVERALTRADITIONAL FACTORIES (Sardjono, 1985)

Spesies jamur 1 2 3 4 5 6 7 8 9 10

Aspergillus

A. flavus/parasiticus 34 20 50 50 30 50 22 16 18 16

A. niger var. niger 0 0 0 0 0 0 0 0 0 0A. versicolor 0 14 2 0 0 0 0 0 0 0

A. alliaceus 32 0 0 0 0 0 0 0 0 0

A. fumigatus 0 0 0 0 50 0 0 28 0 0

A. sojae 0 32 0 32 30 58 0 38 36 30

A. oryzae 0 0 58 0 0 0 100 0 38 30

Eurotium

E. rubrum 0 30 0 30 40 38 58 54 62 50E. chevalieri 60 32 30 42 32 20 0 46 42 54

E. amstelodami 0 46 40 32 32 0 46 0 0 0

Fusarium

F. moniliforme 38 48 0 32 36 0 0 0 0 0

F. chlamydosporum 0 0 36 0 32 0 0 0 0 0

F. poae 42 0 0 36 0 0 0 0 0 0

PenicilliumP. echinulatum 1 4 0 0 0 0 0 0 0 0

P. melinii 0 0 0 2 0 0 0 0 0 0

Genera lainnya

Rhizopus oryzae 20 48 30 48 48 70 20 68 62 100

Curvularia lunata 0 0 12 0 0 0 0 0 0 0

AFB1 (ppb) 14 15 ND 36 16 13 ND 30 ND ND


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Aspergillus sojae

Aspergillus oryzae

COMMON MOLD USED IN SOY SAUCE FERMENTATION


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Aspergillus flavus

Aspergillus oryzae


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Aspergillus oryzae

Aspergillus sojae


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konidia

fialida

metula

vesiklua

BISERIATE UNISERIATE

GeneraAspergillus


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Aspergillus flavus

Medium Skim Milk Agar Medium AFPA

Aflatoxigenic test

Clear zone

Orange inreverse color

Proteolytic test

Simple method for selection of mold strain

Pepton 10.0 g/l

Yeast extract 20,0Ferri Amm. Sitrat 0,5

Dichloran 0,002

Agar 15,0

pH : 6,2 + 0,2


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IMPORTANT FACTORS IN KOJI FERMENTATIONIMPORTANT FACTORS IN KOJI FERMENTATION

1. Softness of beans promotes the fungal growth andpenetration of mold important to protein digestibility and formolnitrogen production

2. Molds used for koji productionAspergillus oryzae : produce amylase and protease enzymesAspergillus sojae : produce more complex of protease enzymes

and produce glutaminase3. Fermentation conditions :Relative Humidity (affect water activity of bean)

important to fungal mycelia growth andprevention on spore formationOxygen important to fungal growth and affecting thespore formationTemperature important for production and activities of enzymes


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The role ofThe role of AspergillusAspergillusoryzaeoryzae andand AspergillusAspergillussojaesojae

Aspergillus oryzae : amylase and protease enzymes

sugars important to Lactic acid bacteriaand yeast in brine fermentationAspergillus sojae : more complex of protease enzyme andproduce glutaminaseBeans contained a lot of glutamine and glutamic acid as amino acid

residue

easily change glutaminaseglutamic acid

pyroglutamic acid meaningless as flavorcomponent


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ENZYMES PRODUCED DURING KOJI FERMENTATIONENZYMES PRODUCED DURING KOJI FERMENTATION

ProteinaseAlkaline (the highest proportion and activity) Optimum pH : 10.5Semi alkaline 8.3Neutral I 7.0Neutral II 6.0

Acid I 3.2Acid II 3.0

Acid III 3.0

Acid carboxypeptidasesAcid carboxypeptidase (I, II, III and IV ) 3 4

Leucine aminopeptidase(Enzyme I, II, III, IV, V, VI, VII and Arylamidase) 5.0 8.5


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CEREAL PROTEIN

PEPTIDES

AMINO ACIDS

GLUTAMINE PYROGLUTAMIC ACID

GLUTAMIC ACID

ALKALINE PROTEINASE

SEMI ALKALINE PROTEINASE

NEUTRAL PROTEINASE I AND IIACID PROTEINASE I-III

ACIDCARBOXYPEPTIDASES I IV

LEUCINE AMINOPEPTIDASES I - VII

GLUTAMINASE((Aspergillus sojae)


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THE DIFFERENCES BETWEEN MOROMI FERMETATION OFTHE DIFFERENCES BETWEEN MOROMI FERMETATION OFJAPANESE SHOYU AND KECAPJAPANESE SHOYU AND KECAP

JAPANESE SHOYU KECAPMoromi are put inside Moromi are put outside

( no sun light ) ( direct sun light )

Low temp. in the beginning higher temperaturefermentation

slower Lactic bact growth faster Lactic bactgrowth

slower pH change faster pH change

higher protease activity lower protease activity

higher formol nitrogen lower formol nitrogen

No pellicle yeast pellicle yeast( because Lack of sugars)

not necessary sun light sun light inhibit their growth

Pellicle forming yeast cause off-flavor in Japanese shoyu


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Advantage and disadvantaged drying ofAdvantage and disadvantaged drying of kojikoji

DRYING and CRUMBLING OF KOJIDRYING and CRUMBLING OF KOJI

Removed someRemoved some DecreasedDecreased glutaminaseglutaminase Protease andProtease and LeucineLeucineVolatile compoundVolatile compound activityactivity amino peptidaseamino peptidasediminished stronglydiminished strongly

diminished production ofdiminished production of

glutamicglutamic acid ammonia,acid ammonia, formolformol nitrogennitrogen

lower buffering capacitylower buffering capacity

Inhibitory effectsInhibitory effectsonon microbmicrob. growth. growthIn brine fermentation?In brine fermentation? Fermentation not moreFermentation not more allowedallowed

affecting the production ofaffecting the production ofmoromimoromiflavorflavor


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Whole soybean : black/yellow?

RAW MATERIAL :

Defatted soybean

DIFFERENT CHARACTERISTIC OF FINAL PRODUCT?

Different in : 1. Composition oil in final product?Amino acid in final product?

2. Physical structure solid substratefermentation?


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R3

R1

R2

H OH

OCH3

CH=CHCOOH

R1 R2 R3

H H -O-CH(COOH)-CH(OH)COOH

OH H -O-CH(COOH)-CH(OH)COOH

OH OH -O-CH(COOH)-CH(OH)COOH

H H H(Daidzein)

(Isoflavon derivatives)



Ferulic acid

DEFFERENTIATION OF NON-VOLATILE COMPOUND OF SOY SAUCEFROM WHOLE AND DEFATTED SOYBEAN


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KOJI FERMENTATIONKOJI FERMENTATION

Solid Substrate FermentationSolid Substrate Fermentation

Principles?Principles?

Design of Bioreactor?


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Environmental change during Solid SubstrateEnvironmental change during Solid SubstrateFermentation and their effectsFermentation and their effects

waterMold growthProduct formationSpore formationMycotoxin/pathogen

heat

Mold growth

Product formationEnzyme activitiesMycotoxin

pH Enzyme activitiespathogen

O2, CO2

Mold growthProduct formationMycotoxin

Biomass determination:Viable count methodMetabolic activities

OCR and CPRlost of solidproduct formation

Biomass componentN and proteinDNAGlucosamineErgosterol

Other phenomena

Growth kinetics and the model of growth in SSF?


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AERATIONHEAT

REMOVAL

HUMIDIFY( aw)

agitation

OCR and CPRTransfer of O2 into interparticlesDiffusion of O2 substrate particlesTransfer of CO2 into interparticles

Remove by conduction

Remove by convectionJacket system (externally?)Chilled water

Chilled water?

Fungi are susceptible for shear process

Mixing frequency depend on void fraction

Handling of

substrate

pH?

PROCESS OPERATION IN

Solid Substrate Fermentation REACTOR


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A. oryzae, soybean

0

0.001

0.002

0.003

0.004

0.005

1 2 3 4 5 6 7 8

Fermentation time (days)

OCR,C

PR

(u

mol/s/mg)

0

24

6

8

10

12

Gluc

osamine(mg

)

OCR(umol/s/mg) CPR(umol/s/mg) Gluc(mg)

Sardjono, et al., 1998


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A. oryzae, soybean

0

2

4

6

8

10

12

14

1 2 3 4 5 6 7 8Fermentation time (day)

OCR

,CPR(mmol/

mginitialdrymatter)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.140.16

0.18

Drym

atterloss

(g/ginitialdrymatter)

OCR(mmol/mg)CPR(mmol/mg)Dry matter loss(g/g initial dry matter)



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Glucosamine, wateractivity and sporeconcentration



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GlucosamineGlucosamine and enzymes activitiesand enzymes activities



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KOJI FERMENTATION(traditional method)

Small factory Medium / big factory

2 4 DAYS FERMENTATION

Un-control condition



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THE SURFACE OF DRIED KOJITHE SURFACE OF DRIED KOJI(After crumbling the(After crumbling the kojikoji to remove spore and mycelia)to remove spore and mycelia)

FRACTURED / CRACKED


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Batch type with rectangular perforated plateBatch type with rectangular perforated plate

Solid Substrate Fermentation equipmentSolid Substrate Fermentation equipment


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Materials turn over?

Heat, CO2 removal only

on the surface of substrate

Limited aeration

Batch type with rectangular perforated plateBatch type with rectangular perforated plate

Solid Substrate Fermentation equipmentSolid Substrate Fermentation equipment


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BRINE FERMENTATION (MOROMI)BRINE FERMENTATION (MOROMI)

NaCl concentration, pHand temperature

enzymes activity,population ofmicroorganism 5 6 MONTH FERMENTATION


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MOROMI FERMENTATIONMOROMI FERMENTATION

Continuing the activity of koji enzymes:

pH of moromi , temperatureExtraction of protein from koji:

salt concentration, time of fermentation

Flavor development / formation:

pH, temperature microbial activityBacillus died ( 2 months) ???

Lactic acid bacteria at first, followed by salt tolerantyeast

( pH 4 - 5 )Detoxification of mycotoxins, if any

cyclopiazonic acid ( 3 months), AFB1?


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SALT TOLERANT LACTIC ACID BACTERIA ANDSALT TOLERANT LACTIC ACID BACTERIA AND

OSMOPHYLIC YEASTOSMOPHYLIC YEAST

Lactobacillussp

Saccharomyces ruoxii

MOROMI

Pediococcussp


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What must be happened during moromi fermentation?

Prepare the condition for the activity of koji enzymes

The growth of microorganisms which play an important roleduring brine fermentation

Extraction of soluble protein from the fermented bean (koji)

To prevent the pH from declining too quickly as a result of

sudden growth of Lactic acid bacteria

A rapid drop in pH obstructs the action of proteolyitic enzymesand glutaminase enzyme of koji decreasing the soluble

protein and glutamic acid in final products

pH and salt concentration the growth of Lactic acidbacteria and yeast which are important during brine

fermentation


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Aspartic Acid

Glutamic Acid

Serin

HistidinGlysin

Arginin

Alanin

Tirosin

Metionin

Valin

FenilalaninIsoleusin

leusin

Lysin

43,722

79,110

68,703

22,63927,294

32,038

10,006

26,45

25,08452

24,06604

38,2510842,85296

32,61771

63,9268

AMINO ACIDS ppm

Umami taste

Mengalamitransaminase

oleh bakteri

asam laktatmenjadikomponenflavor moromi

AMINOA

CID

S

IN

MOROMI


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COMPOUNDS

Propylene glycol

1-Propanol, 2-methyl-

2-Pentanol

1-Butanol, 3-methyl-

1-Butanol

2-Octanol

1-Octen-3-ol

Acetic acid

Propanoic acid

Propanoic acid, 2-methyl

Butanoic acid

Butanoic acid, 3-methyl-

Butanoic acid, 2-methyl-

Phenol, 2-methoxy-

Phenol

2(3H)-Furanone, dihydro-

2-furanmethanol

Furfural

Benzaldehyde

pyrones

Maltol

unknown

unknown

unknown

unknown

unknown

unknown

unknown

unknown

furans

aldehyde

unknown compounds

alcohols

acids

phenols

furanones

odorant

flowery

sweet, caramel like

condiment-like

sour

sweet, caramel-like

sour, spoiled like

sweet sour

green-like

stale (apek)

astringent, stale (ape

cereal like

mushroom-like

spoiled-like

soysauce like

rancid

roasted potato

nutty

condiment like

cheesy

KO

MPONEN

FL

AVOR

MORO

MI

YANGB

ERHASILDIDETEK

SI


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3-methyl -1-butanol and 1- propanol-3-methyl, might attribute to a powerfullmeaty and soy sauce odor and flavor at high dilution.

Furan methanol, a powerfull aromatic compound at very low threshold value.

2-methyl propionic acid also produce stronger flavor (Zhang Yanfang and TaoWenji, 2009).

Rich in amino acid able to get more flavor compound through transamination bytransaminase enzyme of Lactic Acid bacteria (Mengjin Liu et al., 2008).

SENYAWA YANG MEMBERIKAN KONTRIBUSI PADA

FLAVOR PENTING PADA MOROMI


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Indonesian Soy Sauce (Kecap)