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Microbial Technology in Satho Production

Microbial Technology in Satho Production. Savitree Limtong. Department of Microbiology, Faculty of Science, Kasetsart University. Satho Definition ( สาโท ).

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Microbial Technology in Satho Production

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  1. Microbial Technology in Satho Production • Savitree Limtong Department of Microbiology, Faculty of Science, Kasetsart University

  2. 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 concentrationsugar may be added to prolong alcohol fermentation.(ThaiCommunity Product Standard, Ministry of Industry) • It is a kind of rice wine. • Other names are Lao-tho or Nam-khao

  3. Marketed Satho in Thailand

  4. Marketed Satho in Thailand

  5. Satho Polish glutinous rice Wash and steep overnight Filter to remove solid Drain Steam Ferment 1-2 weeks Wash to remove slimy matter Add water Ground loog-pang (0.1-0.2%) Drain Incubate 1-3 days Mix and put in container Traditional Satho Production (Chaownsungket, 1978, 2003; Lotong, 1998)

  6. Traditional Satho Production(cont.) • 2 processes • Saccharification process • Fermentation process • Bymicroorganisms in loog-pang.

  7. Saccharification Process • Starch in glutinous rice/rice is hydrolyzed to sugars. • Amylolytic microorganisms. • Amylolytic molds: Amylomycesrouxii 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.

  8. Fermentation Process • Sugars are fermented to ethyl alcohol. • Ethyl alcohol fermenting yeasts: Saccharomyces cerevisiae (main producer) • Amylolytic molds and yeast: Amylomycesrouxii and Rhizopus spp.; Saccharomycopsis fibuligera • Some yeast species produce some flavor compounds of Satho.

  9. Satho (Photograph by Savitree Limotng and Somporn Sintara)

  10. Loog-pang (Chinese yeast cake) • Thai term for dry form of“fermentation starter” for production of traditional fermented products from starchy raw materials • Various Asian countries with various local names • Banh men: VietnamChu: China • Koji: JapanNuruk: Korea • Murcha: IndiaRagi: Indonesia • Ragi tapai: MalaysiaBubod: Philippines

  11. Fermentation starter The mixed cultures of molds, yeasts and bacteria grown on rice or other cereals (and some native herbs) (Batra and Millner, 1974; Saono, 1982; Lotong, 1998 and Thanh et al., 1999).

  12. 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)

  13. 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

  14. Flour or crushed rice/glutinous rice Loog-pang Dry in sun light Mix with herbs/spices and loog-pang powder Incubate at room temp. for 2-5 days Sprinkle over the balls with loog-pang powder Add water, mix Shape into small ball (2-6 cm diameter) Place on bamboo tray Loog-pang production (Chaownsungket, 1978, 2002; Lotong, 1998)

  15. Loog-pang • Good quality of loog-pang: • Light • Creamy white in color • Smooth texture with porous inside • No acid smell (Photograph by Savitree Limtong and Somporn Sintara)

  16. Microorganisms in loog-pang and their role • Amylolytic molds and yeast: • Production of amylolytic enzymes and starch hydrolysis(main role) • Production of small amount of ethyl alcohol • Production of acids • Alcohol fermenting yeasts: • Production ethyl alcohol from sugar • Bacteria: • Production of acids

  17. Molds in loog-pang Mold Reference Amylomyces rouxiiChatisatien, 1977,Rhizopus spp. Pichayanglura andAspergillusKulprecha, 1977;MucorChaownsungket, 1978;PenicilliumPongpoon, 1980; Lotong, 1998

  18. Molds in fermentation starters Starter Mold Chinese yeast cake Amylomyces, Rhizopus Ragi Amylomyces, Rhizopus,Mucor, Aspergillus, Fusarium Murcha Mucor, Rhizopus Banh Men Amylomyces, Rhizopus,Mucor Burbod Mucor, Rhizopus (Saono et al., 1974; Kozaki, 1976; Batra and Millner, 1976; Hadisepoetro et al., 1979; Tamang and Sarkar, 1995; Lee and Fujio, 1999)

  19. Morphology of molds in loog-pang Rhizopus sp. (Photo from IT)

  20. sporangium Mucor sp. Morphology of molds in loog-pang Phylum Zygomycota, Class Zygomycetes, Order Mucorales, Family Mucoraceae (Photo from IT)

  21. Aspergillus sp. Morphology of molds in loog-pang Phylum Ascomycota (Photo from IT)

  22. Y e a s t s p e c i e s S t a r t e r s Y e a s t s p e c i e s S t a r t e r s l o o g - p a n g , m a n y s t a r t e r S a c c h a r o m y c o p s i s l o o g - p a n g , m a n y s t a r t e r S a c c h a r o m y c o p s i s c a k e s f i b u l i g e r a c a k e s f i b u l i g e r a g , b u b o d , m u r c h a g , b u b o d , m u r c h a l o o g - p a n P i c h i a a n o m a l a l o o g - p a n P i c h i a a n o m a l a s p p . r a g i C a n d i d a s p p . r a g i C a n d i d a s p p . l o o g - p a n g , b a n h m e n , S a c c h a r o m y c e s s p p . l o o g - p a n g , b a n h m e n , S a c c h a r o m y c e s b u b o d , r a g i b u b o d , r a g i Yeasts in fermentation starters (Chatisantien, 1977; Chaownsungket, 1978; Dijen, 1972; Saono, 1982; Tamang and Sarkar, 1995; Lotong, 1998; Lee and Fujio, 1999; Thanh et al., 1999)

  23. Saccharomyces cerevisiae Morphology of yeasts in loog-pang (Photo from Barnett et al., 2000)

  24. Study on Molds and Yeasts in Loog-pang

  25. Objective • To isolate and identified molds and yeastsfrom Loog-pang kao-mag and Loog-pang lao. • To preliminary investigate amylolytic activity of molds and yeasts. • To preliminary investigate ethyl alcohol fermenting abilityof yeasts.

  26. 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: 19 samples Most samples obtained from central part and northeast part of Thailand.

  27. Isolation of molds • Spread plate technique using PDA. • Collection of mold pellets from enrichment acidified YM broth (pH 3.7). • Collection of mold pellets fromenrichment 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 prepared alcoholic sweeten rice (kao-mag). • Loog-pang lao was used to prepared Satho (lao).

  28. Alcoholic sweeten rice (kao-mag) produced by loog-pang kao-mag (Photograph by Somporn Sintara)

  29. Satho produced by loog-pang lao (Photograph by Somporn Sintara)

  30. Isolation of yeasts • Enrichment in acidifiedYM broth (pH 3.7) • Enrichment in acidifiedYM broth containing 30% glucose. • Preparation of products and isolation from the products: • Loog-pang kao-mag was used to prepared alcoholic sweeten rice (kao-mag). • Loog-pang lao was used to prepared Satho (lao).

  31. Result of mold and yeasts isolation Loog-pang No. of sample Number (isolate) Mold Yeast Loog-pang 38 91 43 kao-mag Loog-panglao 1935 48 Total 57 12691

  32. Identificationof 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)

  33. Resultsof molds identification Mold Loog-pang kao-mag Lao Amylomyces 31 12 Rhizopus 27 13 Actinomucor 5 5 Aspergillus 9 - A. niger group 13 1 Mucor 2 5 Monascus 2 - Penicillium 1 - Unidentified isolate 1 -

  34. 31 27 Summary of molds in loog-pang kao-mag Mold Number of sample Amylomyces 4 Rhizopus3 Amylomyces + Rhizopus 9 Amylomyces + Rhizopus+12 other 1 or 2 genera Amylomyces +another genus 6 Actinomucor +Rhizopus2 Aspergillus+A. niger group+Rhizopus1 Aspergillus+A. niger group+Mucor 1 Monascus Total 38 samples

  35. 15 Summary of molds in loog-pang lao. Mold Number of sample Rhizopus 3 Rhizopus +Amylomyces8 Rhizopus + Actinomucor 3 Rhizopus+ A. niger group 1 Amylomyces 2 Actinomucor 1 Actinomucor +A. niger group + Mucor 1 Total 19 samples

  36. Identificationof yeasts • Standard methods as described byYarrow (1998). • Morphology, including ascospore formation • Physiological characteristics • Biochemical characteristics • Assimilation of carbon compounds were tested by API 20 C AuxTM (Bimerieux, France). • Taxonomic key“The Yeasts: ATaxonomic Study” 4th edition. Kurtzman and Fell, 1998.

  37. Y e a s t s p e c i e s L o o g - p a n g l o o g - p a n g Y e a s t s p e c i e s L o o g p a n g l o o g - p a n g k a o - m a g l a o k a o - m a g l a o S a c c h a r o m y c e s f i b u l i g e r a 31 2 0 S a c c h a r o m y c e s f i b u l i g e r a 2 0 I s s a t c h e n k i a o r i e n t a l i s 3 3 I s s a t c h e n k i a o r i e n t a l i s 3 P i c h i a a n o m a l a 1 7 P i c h i a a n o m a l a 7 P i c h i a b u r t o n i i 2 4 P i c h i a b u r t o n i i 4 Pichia spp. P i c h i a f a b i a n i i 1 3 P i c h i a f a b i a n i i 3 P i c h i a h e i m i i - 1 P i c h i a h e i m i i 1 P i c h i a m e x i c a n a - 2 P i c h i a m e x i c a n a 2 Resultsof yeasts identification

  38. Y e a s t s p e c i e s L o o g p a n g l o o g - p a n g Y e a s t s p e c i e s L o o g p a n g l o o g - p a n g k a o - m a g l a o k a o - m a g l a o C a n d i d a g l a b a r t a - 3 C a n d i d a g l a b a r t a 3 C a n d i d a r h a g i i 1 3 C a n d i d a r h a g i i 3 R h o d o t o r u l a p h i l y l a 1 - R h o d o t o r u l a p h i l y l a S a c c h a r o m y c e s c e r e v i s i a e - 1 S a c c h a r o m y c e s c e r e v i s i a e T o r u l a s p o r a d e l b r u e c k i i 1 - T o r u l a s p o r a d e l b r u e c k i i T o r u l a s p o r a g l o b o s a 2 - T o r u l a s p o r a g l o b o s a T r i c h o s p o r o n a s a h i i - 1 T r i c h o s p o r o n a s a h i i Identification results (cont.)

  39. e r a + P . a n o m a l a 1 e r a + P . a n o m a l a 1 S . f i b u l i g e r a + P . f a b i a n i i 1 S . f i b u l i g e r a + P . f a b i a n i i 1 S . f i b u l i g e r a + R . p h i l y l a 1 S . f i b u l i g e r a + R . p h i l y l a 1 S . f i b u l i g e r a + P . b u r t o n i i S . f i b u l i g e r a + P . b u r t o n i i Summary of yeasts in loog-pang kao-mag N o . o f N o . o f Y e a s t Y e a s t s a m p l e s s a m p l e s S . f i b u l i g e r a 2 3 S . f i b u l i g e r a 2 3 S . f i b u l i g e r a + T . g l o b o s a 2 S . f i b u l i g e r a + T . g l o b o s a 2 S . f i b u l i g e r a + C . r h a g i i 1 S . f i b u l i g e r a + C . r h a g i i 1 S . f i b u l i g S . f i b u l i g 7 + I . o r i e n t a l i s 1 + I . o r i e n t a l i s 1 I . o r i e n t a l i s 1 I . o r i e n t a l i s 1 T . d e l b r u e c k i i 1 T . d e l b r u e c k i i 1 Total 38 samples

  40. . a n o m a l a + S . c e r e v i s i a e 1 P . a n o m a l a + S . c e r e v i s i a e 1 S . f i b u l i g e r a + T . g l o b o s a + T . a s a h i i 1 S . f i b u l i g e r a + T . g l o b o s a + T . a s a h i i 1 10 S . f i b u l i g e r a + C . r h a g i i + P . b u r t o n i i 1 S . f i b u l i g e r a + C . r h a g i i + P . b u r t o n i i 1 S . f i b u l i g e r a + P . a n o m a l a + P . f a b i a n i i 1 S . f i b u l i g e r a + P . a n o m a l a + P . f a b i a n i i 1 S. fibuligera + C . r h a g i i + P . m e x i c a n a + 1 + C . r h a g i i + P . m e x i c a n a + 1 I . o r i e n t a l i s I . o r i e n t a l i s S . f i b u l i g e r a + C . g l a b r a t a + P . f a b i a n i i + 1 S . f i b u l i g e r a + C . g l a b r a t a + P . f a b i a n i i + 1 I . o r i e n t a l i s I . o r i e n t a l i s . a n o m a a + P . h e i m i i 1 P . a n o m a l a + P . h e i m i i 1 2 P . a n o m a l a + P . b u r t o n i i + P . m P . a n o m a l a + P . b u r t o n i i + P . m e Summary of yeasts in loog-pang lao N o . o f N o . o f Yeast s a m p l e s s a m p l e s S . f i b u l i g e r a 7 S . f i b u l i g e r a 7 S . f i b u l i g e r a + C . g l a b r a t a 2 S . f i b u l i g e r a + C . g l a b r a t a 2 S . f i b u l i g e r a + P . a n o m a l a 2 S . f i b u l i g e r a + P . a n o m a l a 2 S . f i b u l i g e r a + S . f i b u l i g e r a + 1 e x i c a n a + 1 x i c a n a + I . o r i e n t a l i s I . o r i e n t a l i s Total 19 samples

  41. Clear zone Determination of amylolytic activity Amylolytic activity;express as clear zone diameter/colony diameter Strong activity: ratio > 1 Low activity: ratio <1 No activity:ratio= 0 (Photograph by Somporn Sintara)

  42. Amylolytic activityof molds Mold genera No. of isolate had amylolytic activity LP-kao-magLP-Lao Amylomyces9, 21 (31) 3, 9(12) Rhizopus14, 11 (27) 15 (15) Actinomucor1, 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

  43. Amylolytic activity of yeasts • All isolates of Saccharomycopsis fibuligera revealed strong amylolytic activity (1.9-3.3), clear zone diameter /colony diameter), except YKM025. • Some other yeast species showed relatively low activities (ratio as 1). • C. rhagii, C. glabrata, I. orientalis, P. heimii, P. mexicana, R. philaya, S. cerevisiae, T. delbreckiiandT. globosa did not have activity.

  44. Ethyl alcohol fermentation • 18% glucose medium, shaking flask cultivationat room temperature, 48 h • Saccharomycopsis fibuligera • most isolates produced low ethyl alcohol(less than 2%v/v) • 11 isolates produced 2.0-3.0 %v/v • 3 isolates produced between 3.04-3.7%v/v • Most of the remaining species produced ethyl alcohol less than 4%v/v

  45. Ethyl alcohol fermentation (cont.) • High ethyl alcohol fermenting yeast (4.1-5.3 %v/v) were found in 7 isolates of Pichia anomala, Issatchenkia orientalis and Torulaspora globosa. • The isolates that produced exceptionally high ethyl alcohol (6 %v/v) were • Torulaspora globosa YKM032 • Issatchenkia orientalis YMK036 • Pichai burtoniiYKM034 • Pichia burtonii YL048

  46. 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.

  47. Conclusions 1. Most samples of loog-pang kao-mag and loog-pang lao contained Amylolysisand Rhizopusand almost all of them had amylolytic activity. This character related to their roles in starch hydrolysis during Satho production.

  48. Conclusions (cont.) 2. Most samples of loog-pang kao-mag and loog-pang lao comprised of Saccharomycopsis fibuligera, which showed strong amylolytic activity. Its character related to the role of this yeast in starch hydrolysis during product formation.

  49. 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 .

  50. Research require for the improvement of Satho quality • Confirmation roles of microorganisms in traditional Satho production. • Selection of the efficient microbes that play important roles in Satho production. Then use them as mixture of pure cultures in fresh form or dry form as loog-pang.

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