department of microbiology, faculty of science, kasetsart university microbial technology in satho...
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Department of Microbiology, Faculty of Science,
Kasetsart University
Microbial Technology in Satho Production
Savitree Limtong
Satho Definition (สาโท)
Satho is an alcoholic beverage that produces from rice by fermentation (no distillation), its ethyl alcohol content must not more than 15 % by volume. In Satho production rice is converted to ethyl alcohol by pure culture of molds and yeasts or loog-pang. To increase alcohol concentration sugar may be added to prolong alcohol fermentation. (Thai Community Product Standard, Ministry of Industry)
It is a kind of rice wine. Other names are Lao-tho or Nam-khao
Marketed Sath o in Thailand
Marketed Sath o in Thailand
Traditional Satho Production
-Ground loog pang-0102( . . %)
(Chaownsungket, 1978, 2003; Lotong, 1998)
-1Incubate 3 days
Polish glutinous rice
Wash to remove slimy matter
Wash and steep overnight
Drain
Steam
Drain
Mix and put in container
Add water
- Ferment 1 2 weeks
Filter to remove solid
Satho
Traditional Satho Production (cont.)
2 processesSaccharification processFermentation process
By microorganisms in loog-pang.
Saccharification Process
Starch in glutinous rice/rice is hydrolyzed to sugars.Amylolytic microorganisms.
Amylolytic molds: Amylomyces rouxii and Rhizopus spp.Amylolytic yeast: Saccharomycopsis fibuligera
During molds growth lactic acid is produced and pH of sugar syrup is reduced to flavor ethyl alcohol fermentation by yeasts.
Fermentation Process
Sugars are fermented to ethyl alcohol.
Ethyl alcohol fermenting yeasts:Saccharomyces cerevisiae (main
producer)Amylolytic molds and yeast:Amylomyces rouxii and Rhizopus spp.;Saccharomycopsis fibuligera
Some yeast species produce some flavor compounds of Satho.
Satho
(Photograph by Savitree Limotng and Somporn Sintara)
- Loog pang ( Chinese yeast cake)
Thai term for dry form of “fermentatio n starter” for production of traditional
fermented products from starchy rawmaterials
V arious Asian countries with various lo cal names
B anh men: Vietnam Chu: ChinaKoji: Japan Nuruk: KoreaMurcha: India R agi: IndonesiaR agi tapai: Malaysia Bubod: Philippines
Fermentation starter
The mixed cultures of molds, yeasts and bacteria grown on rice or other c
ereals (and some native herbs)
(Batra and Millner, 1974; Saono, 1982; 1Lotong, 998 and Thanh et al., 1999).
-Loog pang
- 3 kinds of Loog pang- - Loog pang kao mag
(alcoholic sweetened rice)- Loog pang lao (rice wine)- - - - Loog pang num som sai chu
(vinegar)
- Loog pang production
Various formulationsFlour or crushed rice /glutinous rice
various herbs / spices (garlic, ginger, galanga, pepper, long pepper, clove, licorice root, red onion, cardamom)water
-loog pang of the previous batch (source of microorganisms)
rice hull or rice bran
Loog-pang production
(Chaownsungket, 1978, 2002; Lotong, 1998)
Place on bamboo tray
Sprinkle over the balls with loog-pang
powder
Incubate at room temp. for 2-5 days
Dry in sun light
Loog-pangFlour or crushed rice/glutinous
rice
Mix with herbs/spices and
loog-pang powder
Add water, mix
Shape into small ball (2-6 cm diameter)
-Loog pang
(Photograph by Savitree Limtong and Somporn Sintara)
Good quality of loog-pang:- Light- Creamy white in color- Smooth texture with porous inside- No acid smell
M - icroorganisms in loog pang and their role
Amylolytic molds and yeast:P roduction of amylolytic enzymes and st
arch hydrolysis (main role)Production of small amount of ethyl alcoholP roduction of acids
Alcohol fermenting yeasts:P roduction ethyl alcohol from sugar
Bacteria:P roduction of acids
Mold Mold ReferenceReference
Amylomyces rouxiiAmylomyces rouxii Chatisatien, 1977,Chatisatien, 1977, Rhizopus Rhizopus spp.spp. Pichayanglura andPichayanglura and AspergillusAspergillus Kulprecha, 1977;Kulprecha, 1977; MucorMucor Chaownsungket, Chaownsungket, 1978;1978; PenicilliumPenicillium Pongpoon, 1980;Pongpoon, 1980;
Lotong, 1998Lotong, 1998
Mold s in loog-pang
Molds in fermentation starters
StarterStarter Mold Mold
Chinese yeast cakeChinese yeast cake Amylomyces, RhizopusAmylomyces, RhizopusRagiRagi Amylomyces, Rhizopus,Amylomyces, Rhizopus,
Mucor,Mucor, Aspergillus, Aspergillus, FusariumFusarium
MurchaMurcha Mucor, RhizopusMucor, RhizopusBanh MenBanh Men Amylomyces, Rhizopus,Amylomyces, Rhizopus,
MucorMucorBurbodBurbod Mucor, Mucor, RhizopusRhizopus
(Saono et al., 1974; Kozaki, 1976; Batra and Millner, 1976; Hadisepoetro et al., 1979; Tamang and Sarkar, 1995; Lee and Fujio, 1999)
Morphology of molds in loog-pang
Rhizopus sp.
(Photo from IT)
Morphology of molds in loog-pang
Mucor sp.
sporangium
(Photo from IT)
Phylum Zygomycota, Class Zygomycetes, Order Mucorales, Family Mucoraceae
Morphology of molds in loog-pang
Aspergillus sp.
(Photo from IT)
Phylum Ascomycota
Yeasts in fermentation starters
1977(Chatisantien, ; Chaown , 1978; , 1972; Saono, 1982; Tamang and Sarkar, 1995; 199Lotong, 8; Lee and Fujio, 1999; Thanh et al., 1999)
YYeeaasstt ssppeecciieess SSttaarrtteerrss
SSaacccchhaarroommyyccooppssiissffiibbuulliiggeerraa
lloooogg--ppaanngg,, mmaannyy ssttaarrtteerrccaakkeess
PPiicchhiiaa aannoommaallaa lloooogg--ppaanngg,, bbuubboodd,, mmuurrcchhaaCCaannddiiddaa sspppp.. rraaggii
SSaacccchhaarroommyycceess sspppp.. lloooogg--ppaanngg,, bbaannhh mmeenn,,bbuubboodd,, rraaggii
-Morphology of yeasts in loog pang
Saccharomyces cerevisiae
(Photo from Barnett et al., 2000)
Study on Molds and Yeasts -in Loog pang
Objective
To isolate and identified molds and yeasts - - from Loog pang kao mag and L
- oog pang lao.
To preliminary investigate amylolytic activity of molds and yeasts.
To preliminary investigate ethyl alco hol fermenting ability of yeasts.
- Collection of loog pang samples
- Samples were collected during 1997 1999 from many provinces in Thailand
.
- - loog pang kao mag: 38 samples - loog pang lao: 1 9 samples
Most sampl es obt ai ned f r omcent r a l par t and nor t heast par t of Thai l
and.
Isolation of molds
Spread plate technique using PDA.Collection of mold pellets from enrichment acidified YM broth (pH 37. ). Collection of mold pellets from enrichment acidified YM broth containing 30% glucose.Preparation of products and collection of rice grains that cover with different kind of molds.
Loog-pang kao-mag was used to p reparedalcoholic sweeten rice (kao-mag).
-Loog pang lao was used to p reparedSatho (lao).
Alcoholic sweeten rice (kao-mag) produced by loog-pang kao-mag
(Photograph by Somporn Sintara)
Satho produced by loog-pang lao
(Photograph bySomporn Sintara)
Isolation of yeasts
Enrichment in acidified YM broth (pH 37. ) Enrichment in acidified YM broth containing 30% glucose.Preparation of products and isolation from the products:
Loog-pang kao-mag was used to prepar ed alcoholic sweeten rice (kao-mag).
-Loog pang lao was used to p repared Satho (lao).
Result of mold and yeasts isolationLoog-pang No. of sample Number (isolate)
Mold Yeast
Loog-pang 38 91 43kao-mag
Loog-pang lao 19 3548
Total 57 126 91
Identification of molds
Morphological characteristics and use the taxonomic keys in many monographs written by:
Arx (1978)Ingold (1978)Samson and Pitt (1990)Alexopoulos et al. (1996)Hanlin (1998a)Hanlin (1998b)
Results of molds identification
Mold Loog-pangkao-mag
Lao
Amylomyces 31 12Rhizopus 27 13Actinomucor 5 5Aspergillus 9 -A. niger group 13 1Mucor 2 5Monascus 2 -Penicillium 1 -Unidentified isolate 1 -
Summary of molds in loog-pang kao-magMold Number of sample
Amylomyces 4Rhizopus 3Amylomyces + Rhizopus 9Amylomyces + Rhizopus + 12
other 1 or 2 generaAmylomyces + another genus 6Actinomucor + Rhizopus 2Aspergillus +A. niger group + Rhizopus 1Aspergillus +A. niger group + Mucor 1
MonascusTotal 38 samples
31
27
Summary of molds in loog-pang lao.Mold Number of sample
Rhizopus 3Rhizopus + Amylomyces 8Rhizopus + Actinomucor 3Rhizopus + A. niger group 1Amylomyces 2Actinomucor 1Actinomucor +A. niger group + Mucor 1
Total 19 samples
15
Identification of yeasts
Standard methods as described by Y arrow (1998).
Morphology, including ascospore formationP hysiological characteristicsB iochemical characteristics
Assimilation of carbon compounds were test ed by API 2 0 C AuxTM (Bimerieux, France).
Taxonomic key “TheYeasts: A Taxonomic Study” 4th edition. K urtzman
and Fell, 1998.
Results of yeasts identification
SSaacccchhaarroommyycceess ff iibbuulliiggeerraa 31 2200
II ssssaattcchheennkkiiaa oorriieennttaalliiss 3 33
PPiicchhiiaa aannoommaallaa 1 77
PPiicchhiiaa bbuurrttoonniiii 2 44
PPiicchhiiaa ffaabbiiaanniiii 1 33
PPiicchhiiaa hheeiimmiiii - 11
PPiicchhiiaa mmeexxiiccaannaa - 22
YYeeaasstt ssppeecciieess LLoooogg -ppaannggkkaaoo--mmaagg
lloooogg--ppaannggllaaoo
Pichia spp.
Identification results (.)
YYeeaasstt ssppeecciieess LLoooogg ppaannggkkaaoo--mmaagg
lloooogg--ppaannggllaaoo
CCaannddiiddaa ggllaabbaarrttaa - 33
CCaannddiiddaa rrhhaaggiiii 1 33
RRhhooddoottoorruullaa pphhiillyyllaa 1 -SSaacccchhaarroommyycceess cceerreevviissiiaaee - 1
TToorruullaassppoorraa ddeellbbrruueecckkiiii 1 -TToorruullaassppoorraa gglloobboossaa 2 -TTrriicchhoossppoorroonn aassaahhiiii - 1
Summary - -of yeasts in loog pang kaomag
YYeeaassttNNoo.. ooff
ssaammpplleess
SS.. ff iibbuulliiggeerraa 2233
SS.. ff iibbuulliiggeerraa ++ TT.. gglloobboossaa 22
SS.. ff iibbuulliiggeerraa ++ CC.. rrhhaaggiiii 11
SS.. ff iibbuulliiggeerraa ++ PP.. aannoommaallaa 11
SS.. ff iibbuulliiggeerraa ++ PP.. ffaabbiiaanniiii 11
SS.. ff iibbuulliiggeerraa ++ RR.. pphhiillyyllaa 11
SS.. ff iibbuulliiggeerraa ++ PP.. bbuurrttoonniiii ++ II .. oorriieennttaalliiss 11
II .. oorriieennttaalliiss 11
TT.. ddeellbbrruueecckkiiii 11
Total 38 samples
7
Summary - of yeasts in loog pang lao
P.. aannoommaallaa ++ SS.. cceerreevviissiiaaee 11
SS.. ff iibbuulliiggeerraa ++ TT.. gglloobboossaa ++ TT.. aassaahhiiii 11
SS.. ff iibbuulliiggeerraa ++ CC.. rrhhaaggiiii ++ PP.. bbuurr tto
Yeast NNoo.. ooffssaammpplleess
SS.. ff iibbuulliiggeerraa 77
SS.. ff iibbuulliiggeerraa ++ CC.. ggllaabbrraattaa 22
SS.. ff iibbuulliiggeerraa ++ PP.. aannoommaallaa 22
SS.. ff iibbuulliiggeerraa ++
onniiii 11
SS.. ff iibbuulliiggeerraa ++ PP.. aannoommaallaa ++ PP.. ffaabbiiaanniiii 11
S. fibuligera ++ CC.. rrhhaaggiiii ++ PP.. mmeexxiiccaannaa ++II .. oorr iieennttaalliiss
11
SS.. ff iibbuulliiggeerraa ++ CC.. ggllaabbrraattaa ++ PP.. ffaabbiiaanniiii ++II .. oorr iieennttaalliiss
11
P.. aannoommaalaa ++ PP.. hheeiimmiiii 11
PP.. aannoommaallaa ++ PP.. bbuurr ttoonniiii ++ PP.. mmeexxiiccaannaa ++II .. oorr iieennttaalliiss
11
Total 19 samples
10
2
Determination of amylolytic activity
Clear zone Clear zone
(Photograph by Somporn
Sintara)
Amylolytic activity; express as c lear zone diameter / colony diameter
S trong activity: ratio > 1Low activity: ratio < 1No activity: ratio = 0
Amylolytic activity of molds
Mold genera No. of isolate had amylolytic activity
LP-kao-mag LP-Lao
Amylomyces 9, 21 (31) 3, 9 (12)Rhizopus 14, 11 (27) 15 (15)Actinomucor 1, 4 (5) 2, 5 (5)Aspergillus 9 (13) -A. niger group 9 (9) 1 (1)Mucor 2 (2) 5 (5)Monascus 2 (2) -Penicillium 1 (1) -Unidentified isolate 0 (1) -Number in pink = strong amylolytic activity
Amylolytic activity of yeasts
All isolates of Saccharomycopsis fibuligera revealed strong amylolytic activity (1.9 -3.3) , clear zone diameter /colony dia
meter), except YKM025. Some other yeast species showed relati
vely low activities (ratio as 1). C. rhagii, C. glabrata, I. orientalis, P. heimii, P. mexicana, R. philaya, S. cerevisiae, T. delbreckii and T. globosa did
not have activity.
Ethyl alcohol fermentation
18% glucose medium, shaking flask cultivation 48atroomtemperature, h
Saccharomycopsis fibuligera most isolates produced low ethyl alcohol
(less than 2%v/v)11 isolates produce -d 2 .0 3.0 %v/v 3 isolates produced between 3.04-3.7%v/v
Most of the remaining species produced et hyl al cohol less than 4%v/v
Ethyl alcohol fermentation (cont.)
High ethyl alcohol fermenting yeast (4.1-5.3 %v/v) were found in 7 isolates of Pich
ia anomala , Issatchenkia orientalis and T orulaspora globosa.
The isolates that produced exceptionally
high ethyl alcohol (6 %v/v) were Torulaspora globosa 03YKM 2 Issatchenkia orientalis 036
Pichai burtonii YKM034 Pichia burtonii nL048
Ethyl alcohol fermentation (cont.)
Saccharomyces cerevisiae produced ethyl alcohol at 4.7%v/v. 2 isolates, i.e., Rhodotorula philyla and Trichosporon asahii could not ferment.
Conclusions
1. Most samples of loog-pang kao-mag and loog-pang lao contained Amylolysis and Rhizopus and almost all of them had amylolytic activity. This character related to their roles in starch hydrolysis during Satho production.
Conclusions (cont.)
2. Most samples of loog-pang kao-mag and loog-pang lao comprised of Sacc
haromycopsis fibuligera , whi ch showed strong amylolytic activity. Its character related to the role of this yeast in starch hydrolysis during product formation.
Conclusions (cont.)
3. The results of this work indicated that Saccharomyces cerevisiae might not be the main ethyl alcohol producer but other yeast species could also be the major players, e.g.Torulaspora globosa, Issatchenkia orientalis, Pichia burtonii and Pichia anomala. However, it might be the result of using old samples of loog-pang and unproper technique for isolation of S. cerevisiae .
Research require for the impro vement of Sath o quality
Confi r mat i on r ol es of mi cr oor gani sms i n t r adi t i onal Sath o pr oduct i o
n. Sel ect i on of t he effi ci ent mi cr ob
es that play important roles in Sath o production. Then use them as mixtur
e of pure cultures in fresh form or dry -form as loog pang.
Research require for the improv ement of Sato quality
Selection of raw materials for each vari ety of Sath o, including control the qual
ity of raw materials. Production process control.
Management of finishing process, e.g. clarification, pasteurizati
on, etc.Etc.
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