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 ProcessStarch 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 rawmaterialsV arious Asian countries with various lo

cal namesB 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 formulations

Flour 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

riceMix with

herbs/spices and loog-pang

powderAdd water,

mixShape 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 ReferenceReferenceAmylomyces rouxiiAmylomyces rouxii Chatisatien, 1977,Chatisatien, 1977, Rhizopus Rhizopus spp.spp. Pichayanglura andPichayanglura and AspergillusAspergillus Kulprecha, 1977;Kulprecha, 1977; MucorMucor Chaownsungket, 1978;Chaownsungket, 1978; PenicilliumPenicillium Pongpoon, 1980;Pongpoon, 1980;

Lotong, 1998Lotong, 1998

Mold s in loog-pang

Molds in fermentation starters

StarterStarter Mold MoldChinese yeast cakeChinese yeast cake Amylomyces, RhizopusAmylomyces, RhizopusRagiRagi Amylomyces, Rhizopus,Amylomyces, Rhizopus, Mucor,Mucor,

Aspergillus, Aspergillus, FusariumFusariumMurchaMurcha 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 SSttaarrtteerrssSSaacccchhaarroommyyccooppssiiss

ffiibbuulliiggeerraalloooogg--ppaanngg,, mmaannyy ssttaarrtteerrccaakkeess

PPiicchhiiaa aannoommaallaa lloooogg--ppaanngg,, bbuubboodd,, mmuurrcchhaaCCaannddiiddaa sspppp.. rraaggiiSSaacccchhaarroommyycceess 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 y

easts - - 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

. - - l oog pang kao mag: 38 samples - loog pang lao: 1 9 samples

Most sampl es obt ai ned f r omcent r a l part and northeast part of Thailand.

Isolation of moldsSpread 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 identificationMold 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

3127

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 2200II ssssaattcchheennkkiiaa oorriieennttaalliiss 3 33PPiicchhiiaa aannoommaallaa 1 77PPiicchhiiaa bbuurrttoonniiii 2 44PPiicchhiiaa ffaabbiiaanniiii 1 33PPiicchhiiaa hheeiimmiiii - 11PPiicchhiiaa mmeexxiiccaannaa - 22

YYeeaasstt ssppeecciieess LLoooogg -ppaannggkkaaoo--mmaagg

lloooogg--ppaannggllaaoo

Pichia spp.

Identification results (.)

YYeeaasstt ssppeecciieess LLoooogg ppaannggkkaaoo--mmaagg

lloooogg--ppaannggllaaoo

CCaannddiiddaa ggllaabbaarrttaa - 33CCaannddiiddaa rrhhaaggiiii 1 33RRhhooddoottoorruullaa pphhiillyyllaa 1 -SSaacccchhaarroommyycceess cceerreevviissiiaaee - 1TToorruullaassppoorraa ddeellbbrruueecckkiiii 1 -TToorruullaassppoorraa gglloobboossaa 2 -TTrriicchhoossppoorroonn aassaahhiiii - 1

Summary - -of yeasts in loog pang kaomag

YYeeaasstt NNoo.. ooffssaammpplleess

SS.. ff iibbuulliiggeerraa 2233SS.. ff iibbuulliiggeerraa ++ TT.. gglloobboossaa 22SS.. ff iibbuulliiggeerraa ++ CC.. rrhhaaggiiii 11SS.. ff iibbuulliiggeerraa ++ PP.. aannoommaallaa 11SS.. ff iibbuulliiggeerraa ++ PP.. ffaabbiiaanniiii 11SS.. ff iibbuulliiggeerraa ++ RR.. pphhiillyyllaa 11SS.. ff iibbuulliiggeerraa ++ PP.. bbuurrttoonniiii ++ II .. oorriieennttaalliiss 11II .. oorriieennttaalliiss 11TT.. ddeellbbrruueecckkiiii 11

Total 38 samples

7

Summary - of yeasts in loog pang lao

P.. aannoommaallaa ++ SS.. cceerreevviissiiaaee 11SS.. ff iibbuulliiggeerraa ++ TT.. gglloobboossaa ++ TT.. aassaahhiiii 11SS.. ff iibbuulliiggeerraa ++ CC.. rrhhaaggiiii ++ PP.. bbuurr tto

Yeast NNoo.. ooffssaammpplleess

SS.. ff iibbuulliiggeerraa 77SS.. ff iibbuulliiggeerraa ++ CC.. ggllaabbrraattaa 22SS.. ff iibbuulliiggeerraa ++ PP.. aannoommaallaa 22SS.. ff iibbuulliiggeerraa ++

onniiii 11SS.. 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 11PP.. aannoommaallaa ++ PP.. bbuurr ttoonniiii ++ PP.. mmeexxiiccaannaa ++

II .. oorr iieennttaalliiss11

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 diameterS trong activity: ratio > 1Low activity: ratio < 1No activity: ratio = 0

Amylolytic activity of moldsMold genera No. of isolate had amylolytic

activityLP-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 yeastsAll 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 nn n ,48

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

nnnn nn nnn remaining nnnnnnn produce d ethyl alcohol 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 Pichi

a anomala , Issatchenkia orientalis and Tor ulaspora 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 , which 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 in traditional Sath o production.

Sel ect i on of t he effi ci ent mi cr ob es that play important roles in Sath o p

roduction. Then use them as mixture of pure cultures in fresh form or dry fo -rm as loog pang.

Research require for the improv ement of Sato quality

Selection of raw materials for each varie ty of Sath o, including control the quality of raw materials.

Production process control. Management of finishing

process, e.g. clarification, pasteurizatio n, etc.

Etc.

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