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Fermented fish products-fish sauce Fish sauce is very popular as a condiment around Southeast Asia regieon also known as ngapi (Burma), nouc-man (cambodia and vietnam), nam-pla (Laos and Thailand), ketjap- ikan (indonesia) 3/14/2017 82

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Fermented fish products-fish sauce• Fish sauce is very popular as a condiment around

Southeast Asia regieon

• also known as ngapi (Burma), nouc-man (cambodia and vietnam), nam-pla (Laos and Thailand), ketjap-ikan (indonesia)

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• Mixing salt with fish (anchovies) at 1:3 ratio

• This creates environment that is suitable for starter culture (from salt and environment)

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Alcoholic beverage• Wine• Beer• Sake• Sato

Converting sugar to alcoholHowever, if the starting raw material is starch, steps for converting starch to sugar are required.

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ที่มา: senior project ของนางสาวจิราพร ผาสุข และ นางสาวพรพิมล หม่ืนอาจยิ้ม FT#3

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Sato (Thai rice wine)• rice wine = 2 processes (koji preparation and

alcoholic fermentation)

• These two processes can occur simultaneously.

• koji production: enzyme production using Aspergillus sp. / Rhizopus / Mucor (amylase, protease, lipase)

• 5-7 days at 30๐ C

• Continue mixing the bed (aeration and heat reduction)

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สาโท

• Then mix koji with cooked rice and water (to extract enzymes) and yeast

• This is called alcoholic fermentation

• Amylases in koji convert starch to fermentable sugar and fermentable sugar is then converted to alcohol

• take 7-21 days depending on temperature

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Thanut Amatayakul*

BySitthipong Chotpattarasumon, Sutchai Ruenchit, Pitiporn Ritthiruangdej and

at The 3rd International Conference on Fermentation Technology for

Value Added Agricultural Products Kosa Hotel, Khon Kaen, Thailand

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Experimental planWaxy rice

Size reduction using a food processor

Size classification using sieves No. 7, 8, 10, 12

Koji preparation Aspergillus niger TISTR 3257

for 7 days at 30oC

Measurement - Crude amylase activity - TSS - percentage of starch digestion

Alcoholic fermentationSaccharomyces cerevisiae SC90

for 7 days at 30oC

Measurement - Yeast growth - Alcohol content - 8 volatile compounds - percentage of starch digestion

Sato production

1.7-2.0 mm.

2.0-2.3 mm.

1.4-1.7 mm.

Full kernel (control)

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Figure 1. Crude amylase activity and total soluble solid of koji made from waxy rice reduced to size 1.4-1.7, 1.7-2.0, 2.0-2.3 mm and full kernel. Bars show the same letter (a, b, c or A, B) did not show significantly different (p < 0.05)

One unit of crude amylase activity was defined as the amount of reducing sugar produced per reaction time

Crude amylase activity and total soluble solid of koji

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Solid State Fermentation

Udo Ho¨ lker and Ju¨ rgen Lenz (2005) Solid-state fermentation — are there any biotechnological advantages?. Current Opinion in Microbiology, 8: 301-306

Limitation•Oxygen transfer•Heat transfer•Moisture content

LimitingEnzyme production-amylase and protease

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Volatile compounds

Concentration (mg/l)Odor

threshold (mg/L)

Odor descriptionFull kernel

2.0-2.3 mm

1.7-2.0 mm

1.4-1.7 mm

Ethyl butyrate

18.00b 54.75a 12.62b 14.54b 0.4 Strawbery, apple, banana

Ethyl decanoate

7.10b 60.19a 9.41b 6.83b 0.5 Fruity, wine

Diethyl succinate

1.25b 6.54a 1.56b 1.62b 1.8 Fruity

Phenethyl acetate

1.05b 2.98a 2.78a 1.82a,b 0.25 Fruity, green apple, banana

3-methyl-1-butanol

146.25b

235.01a

180.95b

156.20b

60 Solvent

Pentanol 5.30a 4.04b 0.70c 0.95c 0.1 Phenolic, medicinal

2-methyl-1-propanol

49.35b 86.90a 55.03b 61.05b 75 Alcohol, nail polish

Phenethyl alcohol

39.05c 61.46a 67.09a 53.40b 200 Rose, honey

Table 3 Volatile compounds in Sato made using waxy rice reduced to size 1.4-1.7 mm, 1.7-2.0 mm, 2.0-2.3 mm and full kernel

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Fatty acid ethyl and acetate esters formation

• Fatty acid ethyl esters: Condensation of acyl-CoA and higher alcohols

• Acetate esters: Condensation of Acetyl-CoA and higher alcohols

Verstrepen et al. (2003) Journal of bioscience and bioengineering, 96(2): 110-118

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Ehrlich pathwayformation of fusel alcohols by

Sacchariomyces cerevisiae

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Sake production

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Sake video

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• Use of mould originate from China• famous place: Kyoto, Kobe• Steaming sterile rice and make starch and

protein more easier for enzyme from koji to digest

• Koji (Aspergillus oryzae) = Malt• Liquefaction and saccharification• Sake mashing : Moromi (parallel

fermentation)• slowly add rice to moromi helps yeast

survival and increase ethanol to 20%3/14/2017 100

Several types of Sake

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ENOLOGY

Enology = Winemaking

British version of the word is Oenology

Wines vin

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What is wine?

Water ~ 85%Alcohol ~ 12%Everything else ~ 3%3/14/2017 107

Wineries

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Classification

• Sparkling wine (contain CO2) – champagne

• Fortified wine (added alc.): 14-24% alc.

• Red or white 9-14% alc.

• Sweet wine: reducing sugar 10 - 100 g/l

• Dry wine: reducing sugar < 10 g/l

• Called by variety of grapes

• Table wine (Natural  wine)

Vinification

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Example grape varietiesRed

• Carbernet Sauvignon

• Shiraz

• Durif

• Merlot

• Pinot Noir

• Grenache

• …

White

• Chardonnay

• Chennin Blanc

• Riesling

• Sauvignon Blanc

• Semillion

• …

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Grapes

Crushed and stemmed

Press

Juice

Fermentation

Maturation

Fining and filtration

Bottling

Saccharomyces cerevisiae

SO2

Wine vinification

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Total Soluble Solids

• Use as an indicator for maturation of the grapes

• As an approximate basis for calculating the alcohol yield

• ~ 90% of soluble solids are fermentable sugar

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How can total soluble solids be measured?

• 2 ways

• Hydrometers

• Refractometry

Brix, Baume

• % Brix represent weight of sugar in solution

(g of sucrose/ 100 g of liquid)

• 1.8 Brix = 1.0 Baume

Baume represent final expected alcohol content of wine

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Harvesting

• Hand or machine picking

Damaging skin can result in enzymatic browning, spoilage3/14/2017 115

Simple anatomy of the grape

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Pressing

Directly for white wineRed wine after maceration3/14/2017 121

Preparing juice for fermentation

• Polyphenol oxidase – browning in white wine

• Sulphur dioxide (SO2) commonly add to white wine

• Prevent browning by inactivate enzyme

• Inhibit activity of undesirable microorganisms

• 30 to 50 ppm (mg/L) or 70 to 100 ppm (highly contaminate)

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• Often used sodium or potassium metabisulphite (containing 58% SO2)

• Want grape juice 500 mL to have 30 mg/L of SO2 from potassium metabisulphite (KMBS):

• 58 g SO2 come from 100 g KMBS

• Need 0.03 g use (100*0.03)/58 = 0.0517 g/500 mL

SO2 addition

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Fermentation• Yeast

• Pure culture of Saccharomyces cerevisiae; bought in dried powder

• Activation with grape juice several hours before fermentation (108 cell / mL)

• Yeast use O2 first few hours, follow by produce CO2 and alcohol

• Adding nutrients: Diammonium phosphate (DAP)

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• C6H12O6 → 2C2H5OH + 2CO2 (1 Glucose 2 ethanol + 2 CO2)

• ~ 50% conversion

• In reality < 50% by weight3/14/2017 127

Temperature of fermentation

• Opt. temp. for yeast fermentation is between 10-15 oC (white wine)

• Red wine are fermented at higher temperature ~ 25 oC (pigments extraction)

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Stuck fermentation• premature arrest of fermentation• high level of sugar• Cause

• fermentation at too high temperature• nutritional deficiency• osmoregulation• ethanol toxicity• low temperature fermentation• too high SO2• presence of inhibitory substance

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Maturation (continued)

• Flavor: vanillin, …

• Oak: fill with water purged with SO2 for disinfection

• Wine racking (standing) : to separate wine from lees

(Lees - The solids left at the bottom of a fermentation)

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Fining of Wines(clearing of wines)

• Fining is the process of removing suspended particles from wines and will sometimes be used for softening of wines of phenolic compounds

• Common types of fining agents are Bentonite, and egg whites

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Barrels (cont.)• French barrels average $700 each

• American barrels average $300 each

• Oak species (commonly used)• Allier Limousin• American Nevers

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Oak barrel Barrel

(cask)

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Glass Air Lock

  - twin -S - Bubble

Wine Thief

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Precision Wine Hydrometers Ebulliometer

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http://www.monashscientific.com.au/EbulliometerPic.htm

Ebulliometer

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PRINCIPLEThe boiling temperature of wines is based on their alcoholic content• Note the boiling temperature of the wine to be tested • Consult a calculation disk that gives directly the alcoholic strength by volume.

calculation disk3/14/2017 141

Distillation temperature and concentration relationship

7880828486889092949698100

Alcohol ( % by volume)

Tem

pera

ture

(C)

VaporLiquid

3/14/2017 142

Color• The color of red wine is due

to red and purple anthocyanins (first come out from fermentation)--> dark red over 24 months of aging

• Old wine change from red to red/brown brick

• White wine pale (light) green/yellow, over 24 months become yellow and over 10 years become brown3/14/2017 143

Stabilization of wine• Wine stabilization involves one or more of the following:

• Removal of tartarate

• Complexing agents: Carboxylmethyl

• cellulose (CMC)

• Removal of metal ions and some tannins

• Ion exchange (cation exchange column)

• Membrane filtration

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Removal of particulate material

• Aged wine : separation of clear liquid and lees

• still contain polysaccharides, large molecular weight tannin, protein-complexed with fine tartrate crystal

• filter (1- 5 micron), filter aids (diatomaceous earth), polyvinyl polypyrolidone (PVPP, insoluble)

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Filtration• Filtration is carried out just before bottling

• Centrifugation follow by sterile membrane filtration

• Sterile filtration with

membrane 0.45 – 1.2 micron

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BottlingPurge with inert gas or nitrogen to prevent oxidation

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Closures• Corks; both synthetic and natural

• Natural cork has risk of contamination with mold

• Screw caps

• Proven to be a better seal for wine bottles than corks.

3/14/2017 148

Capsules

• Lead-based capsules very popular until it is banned to use in the late 1980’s. Now wineries use tin-aluminum or aluminum polylaminate.

• It is better to have no capsule rather than a bad one.

• Very important part of packaging, primarily for consumer appeal.

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Water

Barley

Yeast Hops

Main ingredients of beer

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Barley

Malt

germination

beta-amylase

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Color of maltLight Amber Dark

Milling to Grits

This makes the malteasier to wet at the mashing stage and aids faster extraction of the solublecomponents from the malt during the enzymic conversion.3/14/2017 160

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Adjunct cereal• To produce a more stable beer, as they contain

less protein• To produce a different flavor—maize, for example,

is said to give a fuller flavor• To produce a better beer foam due to lower fat

(lipid) levels and different proteins• To improve the ease of processing in the

brewhouse• To produce beer at lower cost

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EnzymeThe alpha enzymes break up the long

chains of starches by splitting them in half. The beta enzymes break down the starches by chopping them off a couple at a time from the ends of the chain.

The alpha enzymes are most active at 65 to 67 oC, and the beta enzymes are most active at 126 to 144 F 52 to 62 oC.

So the temperature and duration of the mash must be carefully controlled to get a good conversion.

3/14/2017 164

Wort separation• The principal objectives are to

produce bright wort and to collect the maximum amount of sugars (extract) from the residual solid materials.

• These solids are sold as animal feed, draff, or spent grains for milk and beef cattle herds.

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Wort boiling• Sterilization of the wort to eliminate all bacteria,

yeasts, and molds that could compete with the brewing yeast and possibly cause off-flavors

• Extraction of the bittering compounds from hops added early to the boil and oils and aroma compounds from late additions

• Coagulation of excess proteins and tannins to form solid particles (trub) that can be removed later. This is important for beer stability and foam

• Color and flavor formation• Removal of undesirable volatiles, such as

dimethyl sulfide, by evaporation• Concentration of the sugars by evaporation of

water3/14/2017 166

Wort cooling, aeration and yeast pitching

• The wort is cooled from almost boiling point to fermentation temperature through a heat exchanger using water as the main cooling medium.

• The temperature for fermentation is typically 8 to 13oC for lager and 14 to 17oC for ale.

• Typically, 5 to 20 million yeast cells are pitched per milliliter of wort.

• Air or oxygen is also added to the wort as an essential factor for the production of yeast membranes and, hence, new cells.

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Ageing• Chill haze formation• Clarification• Carbonation (to a limited extent)• Flavor maturation (again to a limited

extent)• Stored capacity for demand

smoothing

3/14/2017 171

Filtration• Clarification• filter press with filter aids (bentonite)• then filling• pasteurization

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