Lect 11 : Soy Fermentation Flashcards
[SOY SAUCE]
What are the important MOs involved in soy-sauce fermentation? Name one from each type of MO
- Fungi
* Aspergillus Oryzae
* Aspergillus Sojae
- Mucor sp, Rhizopus sp - Bacteria
* Tetragenococcus Halophilus
- Lactobacillus Delbrueckii
- Leuconostoc Mesenteroides - Yeast
* Zygosacchromyces rouxii (Z. rouxii)
* Candida Versatilis
- Torulopsis sp, Hansenula sp
[SOY SAUCE]
All bacteria involved in soy sauce ferm is LAB. True or False?
True
[SOY SAUCE] soybean ratio
In Japanese soy sauce, what is usually used for ferm, other than soy beans? How does the proportion of this component and soy bean affect its flavor?
Wheat.
A higher ratio of wheat to soy beans gives rise to ‘white soy sauce’, which has a lighter taste / flavor
[SOY SAUCE]
State the general flow of soy sauce fermentation
- Pre-processing of raw materials
- soy beans : defatted, soaked for 16h, steamed/autoclaved for 1h
- wheat : roasted and crushed
- after that, both wheat and soybeans are cooled to 25°C and mixed in 1: 1 ratio - Inoculate tane koji / A. oryzae / A. sojae
- Incubate at 28-32°C for 3 days
- Fermentation #1 - Mix koji with salt brine (23%) in a ratio of 1:1.5 to make moromi/mas
- Ferment the mash/moromi
- Fermentation #2 : this is the MAIN fermentation - Press (filter) soy sauce
- Pasteurise (and re-filter)
- Bottle
[SOY SAUCE] - Heat treatment of raw materials
How are the soybeans treated and why? [2]
- They are defatted, or else there will be separation of fat from sauce in product which is undesirable
- They are steamed (wet heat) for easy proteolysis of proteins (which generates flavor due to break down of proteins into a.acids)
[SOY SAUCE] - Heat treatment of raw materials
Why is it important to not over-steam the soy beans?
Overcooking can cause over-denaturation of proteins. Since 3d structure of proteins changes too much, cannot fit into active sites of proteins + enzymes can no longer break down –> less proteolysis–> less generation of aromatic compounds
.
.
.
(enzymes are proteins and may denature too)
[SOY SAUCE] - Heat treatment of raw materials
What are the 3 purposes of roasting wheat at 150-160°C ?
- Gelatinises starch in wheat for easy hydrolysis by fungi (mould)
- gelatinisation : causes starch granules to swell and rupture - Enhance digestibility of starch
- due to breakingdown of starch granules during gelatinisation - Evaporate water for easy breaking by crushing
[SOY SAUCE] - Fermentation stage 1
What are the 2 fermentation stages in soy sauce ferm?
- Koji fermentation – solid state fermentation
- Mash / moromi fermentation – liquid-state fermentation
[SOY SAUCE] - Fermentation stage 1
What is the main goal of koji fermentation?
To maximise enzyme production – amylase (starch in wheat), protease (protein in soybean), lipase, cellulase etc
[SOY SAUCE] - Fermentation stage 1
What MO is koji starter culture ? Name the specific strains [2]
Mould
- Aspergillus Oryzae – more commonly used
- Aspergillus Sojae
[SOY SAUCE] - Fermentation stage 1
What is tane koji? How is it prepared and used?
Which country is tane koji used in?
It is prepared by culturing mould spores on rice, which is then used as a starter culture to inoculate into different types of substrates for ferm
(e.g. substrates : soybeans, rice –> produce soy sauce, sake,,)
Country : Japan
[SOY SAUCE] - Fermentation stage 1
What is the function of koji (starter culture)?
It is a source of enzymes (may contain LAB, yeast) to convert non-fermentable substrates (solid raw material) into metabolisable products which can then be fermented by bacteria and yeasts
[SOY SAUCE] - Fermentation stage 1
What are the steps involved in fermenting the raw materials with koji? [3]
Step 1 : inoculation
step 2 & 3 : where to put materials and conditions to leave the materials?
- Inoculate steamed soybean and wheat with tane koji OR A.Oryzae OR A. Sojae
- tane koji : It typically consists of Aspergillus oryzae (commonly known as koji mold) and various yeasts. - Spread the mixture to depth of 30-40cm on a larger PEFORATED stainless steel plate (usually rectangular but can be round)
- perforated : with holes –> allow air to pass - Aerate for 2-3 days with 30°C temperature
[SOY SAUCE] - Fermentation stage 1
What is the water activity of the fermenting mixture after the koji fermentation
0.6-0.7
[SOY SAUCE] - Fermentation stage 1
What are the conditions needed for koji fermentation? [2]
- Oxygen as moulds are AEROBIC
- Forced aeration should have controlled relative humidity
- to remove CO2 and other unwanted volatiles to satisfy O2 requirements of moulds
(high relative humidity favour mould growth)
[SOY SAUCE] - Fermentation stage 2
What is used for moromi/mash fermentation?
Brine (salt) solution– 23%
[SOY SAUCE] - Fermentation stage 2
What are the MOs involved in moromi fermentation? Name the main strains
- Bacteria, LAB – Tetragenococcus Halophilus (salt-loving)
- Yeasts – Zygosacchromyces rouxii and Candida Versatilis
[SOY SAUCE] - Fermentation stage 2
What is the purpose of the LAB, Tetragenococcus halophilus?
It produces lactate, thus reducing pH to 5.5 or lower to inhibit the action of spoilage MOs
[SOY SAUCE] - Fermentation stage 2
What is the main role of yeasts in moromi ferm?
To convert simple sugars from the wheat into volatiles which adds to the aroma of soy sauce
- phenolic compounds : woody/spicy/smokey/colve-like aroma
- furanones : sweet and caramel aroma
[SOY SAUCE] - Fermentation stage 2
The LAB and yeast in mormomi ferm are always added artifically. True or False?
False, they can be naturally present / added
[SOY SAUCE] - Fermentation stage 2
What volatiles do Zygosacchromyces rouxii and Candida Versatilis / Candida Etchellis produce?
Zygosacchromyces rouxii : Alcohol
Candida Versatilis / Candida Etchellis : phenolic compounds
[SOY SAUCE] - Fermentation
Why is soy sauce fermentation not aspetic ? (i.e. why can’t it be too clean?)
Because several MOs like LAB and yeast are needed for moromi ferm. If aspetic conditions are used from koji making, the naturally present LAB and yeast may die. –> thats why soy sauce is only pasteurised at the end when MOs are no longer needed
[SOY SAUCE] - Post-processing : Pressing
How is pressing conducted on day 1/2/3 of post-fermentation to obtain soy sauce?
Day 1 : free-run –> soy sauce allowed to flow freely and drip out of cloth on its own weight
- have best quality and nicest aroma
Day 2 : Moromi that is wrapped in cloth is squeezed under low pressure
Day 3 : More pressure is added so that there is minimal liquid left
- poorest quality –> more tannins and phenolic substance squeezed out from solid residue, making soy sauce taste bitter
[SOY SAUCE] - Post-processing : Pasteurization
Why is pasteurization carried out (i.e. what are the benefits)? [3]
What is 1 problem with pastuerization?
◡̈ Kills enzymes and microbes, extending shelf life
◡̈ Causes Maillard reaction which causes desirable browning in product
◡̈ Concentrates flavor compounds (mAillard rxn also produces volatile compounds)
ᴖ̈ Accelerates the precipitation of protein complexes –> undesirable, unappealing to consumers
- heat causes proteins to denature, reducing their solubility thus they aggregate and precipitate and settle to the bottom
[SOY SAUCE] - Flavour
How does soy sauce taste? [2]
- Salty–> due to high salt conc (from brining)
- Savoury / umami –> due to glutamic acid, MSG present (from break down of proteins)
[SOY SAUCE] - Aroma
Why is the aroma of soy sauce so complex?
There are 200 volatile flavour compounds present in soy suace
[SOY SAUCE] - Flavour
Which 2 yeasts produce the soy sauce-like taste in soy sauce?
- Zygosacchromyces Rouxii
- Candida Versatilis
[SOY SAUCE] - Flavour
WHat are the 2 important volatile compounds that is present in soy sauce and what aromas do they have?
- Furanones : sweet-caramel-like flavour
- HEMF ( 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone) –> gives characteristic flavour of Japanese soy sauce
- HMF - Phenolic compounds : spicy, woody, smoky aroma
- 4-ethylguaiacol, 4-ethylphenol
[SOY SAUCE] - Debate
How are non-brewed (non-fermented) soy sauce made and what is a problem with artificially made soy sauce?
- Main step is to boil soybeans with hydrochloric acid for 15-20h, rapidly cooled, neutralised, filtered, coloured, sweetened, salted and refined
- Undesirable carcinogenic compounds are formed
- 3-MCPD (3 monochloropropane-1,2,diol)
- 1,3-DCP (1,3-Dichlorpropane-2-ol)
[TEMPEH]
What is the main MO involved in tempeh making and state the specific name.
Fungi (mould)
- The species is Rhizopus Oligosporus
[TEMPEH]
In tempeh making, a pure starter culture is commonly used. However, a mixed culture can also be used, which consists of which moulds?`
Rhizopus Oligosporus, R. Oryzae, R.Stolonifer etc
[TEMPEH]
What are some properties of the mould, Rhizopus oligosporus?
[6]
Hints :
1,2. What does it digest as energy source?
3. What can it not ferment?
4. What flavour does it produce?
- High lipolytic activity
- High proteolytic activity –> release of ammonia afer 48-72h (ALKALINE FERM)
- Inability to ferment sucrose
- Produces typical tempeh flavour
- Strong antioxidant activity
- Rapid growth at 37°C
[TEMPEH]
What are the main steps in tempeh making? [8]
[Clean, sort, dry]
1.Wash and boil for 30 min
2. Drain, de-hull and rinse
3. wash and SOAK for 16-24h at 22-26°C
4. Boil for 30-60 min (second boiling)
5. Drain and cool
6. Inoculate with starter culture (Rhizopus Oligosporus)
7. Pack into trays and incubate at 25-30°C, 70-85% R.H. for 24-48h
8. Store at 4°C / freeze
[TEMPEH]
In tempeh making, what is the purpose of soaking the soy beans after the first boiling ?
Main purpose : Restrict growth of pathogens and undesirable MO
- Promotes the growth of endogenous LAB, which produces lactic acid and reduces pH to 4.5-5.0
[TEMPEH]
In tempeh making, what is an alternative method, apart from soaking the soy beans after the first boiling ?
Direct acidification of soy beans with lactic /acetic acid
[TEMPEH]
Why is tempeh making considered an alkaline fermentation?
Rhizopus oligosporus deanimates fatty acids as an energy source to -
- produce AMMONIA and
- CAUSES pH TO RISE FROM 5.0 TO 7.0
[TEMPEH]
In tempeh making, other MOs may be involved other than mould. Name an example if applicable
Bacteria
- Klebsiella pneumoniae
- Citrobacter freundii
Yeast
[TEMPEH]
Tempeh has nutritional aspects to it, and one of it is that tempeh has increased ____ content, especially ____ __
vitamin, vitamin B12
[TEMPEH]
why tempeh production cannot occur in too clean an environment, especially when a nutritional tempeh containing vit b12 is wanted?
Vit B12 is not synthesized by fungi
- It is only synthesised by bacteria found in the environment (adventitious bacteria = foreign, non-native) , thus if you want enhanced vit b12 content, cannot be in a too clean environment.
[TEMPEH]
What are the adventitious bacteria names that are involved in vitamin B12 synthesis in Tempeh? [2]
- Klebsiella pneumoniae
- Citrobacter freundi
[TEMPEH]
It is safe to store tempeh for a relatively long time under the right conditions. Why?
The mould in tempeh, Rhizopus oligosporus, does not produce mycotoxins
[TEMPEH]
What are some important volatile compounds in tempeh? [9]
(i don’t think need to memorise?? idk)
Carbonyl compounds(aldehyde + ketone)
1. 2-methylpropanal
2. Hexanal
3. (Z)-4-heptenal
4. 3-(methylthio)propanal
5. (E,E)-2,4-decadienal
6. 1-octen-3-one
Non-carbonyl
7. 2-acetyl-1-pyrroline
8. Dimethyl trisulfide
9. 2-ethyl-3,5-dimethylpyrazine
[INTRO- SOYBEAN PASTE]
How is soybean paste fermentation different from soy sauce fermentation? [2]
All the same steps, except:
1. brining is with dry salt instead of brine solution
2. No pressing of liquids out(i.e. no filtering)
[INTRO- SOYBEAN PASTE]
What is used in koji culture for miso?
What are the 3 kinds of koji that can be used for soybean paste fermentation in general?
Miso : Cereal mixture – barley/rice koji
Koji for soybean paste
1. Rice koji
2. Barley koji
3. Soybean koji
- chosen based on type of substrate used for paste
[INTRO- SOYBEAN PASTE]
What are the main groups of MO in soybean paste ferm? Name one from each category
Same as soy sauce
Bacteria
- Lactobacillus Delbrueckii, Tetragenococcus halophilus. pediococcus acidilactici
Yeast
- zygosacchromyces rouxii
- candida versatilis
[INTRO- DOUCHI]
Douchi is a fermented product originating in China. What is Douchi and what is it used as?
Fermented black whole soybeans. It is used as a condiment (like chilli/salt, to add flavour to food)
[INTRO- DOUCHI]
The koji step (inoculating koji into beans)for Douchi fermentation is required. True or False?
True
[INTRO- DOUCHI]
What MOs make up the koji for Douchi?
A. oryzae/A. sojae + yeast + LAB + other bacteria (e.g. Bacilli)
[INTRO- DOUCHI]
What is the main differences between making of douchi and soybean paste?
For Douchi fermentation :
-Need to wash koji
- Fermentation after koji step is anaerobic (soybean paste : may be a mix of both aerobic and anaerboc)
[INTRO- NATTO]
Which MO is involved in natto fermentation? Give specific name
Bacillus subtilis var natto
[INTRO- NATTO]
What are 4 characteristics of Bacillus subtilis var natto?
Hints:
1. What does it digest as food source
2. Condition for survival (O2)
3. Spore forming?
4. Need koji? Why?
- Strong lipolytic, amylolytic, and MOST IMPORTANTLY, proteolytic (deanimation of a.acid into ammonia, ALKALNE FERM)
- Facultatively anaerobic
- Spore former
- No need koji for enzyme production, as extracellular enzymes produced by the bacteria itself
[INTRO- NATTO]
Natto fermentation is considered alkaline fermentation. True or False?
True.
Bacteria in natto ferm is strongly proteolytic, releases ammonia and fatty acids
[INTRO- STINKY TOFU]
What MO is involved in stinky tofu?
Bacteria. But species are undefined as it is natural fermentation / backslopping
[INTRO- STINKY TOFU]
Is stinky tofu fermentation considered alkaline fermentation? Explain with stating final pH of product.
Yes. There is strong proteolysis, causing pH of product to increase to pH 7.5.
[INTRO- STINKY TOFU]
What are the products of stinky tofu fermentation?
- Ammonia(Due to strong proteolysis and deanimation of a.acids)
- Fatty acid release (also from breakdown of lipids)
