utilization of yeasts in local industries · utilization of yeasts in local industries jaroon...

Utilization of Yeasts in Local Industries

Jaroon KUMNUANTA

Panorama Hotel, Mae Hong Son, THAILAND

e-mail : [email protected]

In Thailand, alcohol and baker’s yeast production are the large scaleindustry that employ pure yeast culture in the process. In thefuture, there is a potential to employ yeasts in other industriessuch as single cell protein production and production of novelproducts from the genetically engineered yeasts. Up to now, thelocal industry and academicians are attempting to develop newtechnology for local fermentation industries.

Liberalization of alcoholic beverage production from the monopolyconcession scheme in the past to the new market driven scheme allows thesmall and medium size industries to enter into this business country wide.At early stage, the low quality alcoholic beverages were produced, but in ashort period of time the quality was improved. The good and acceptablequality of wine is available in the market now and gradually the local wineindustry will be established. Since the volume of local consumption ofalcoholic beverage is high, there is the room for local fruit wines whichaim for the local consumption to gain more market share. Good winesfrom locally produced grapes are available and in the near future thevineyard will expand to other provinces. Varieties of fruit wines areproduced by small firms and a number of them are acceptable to localconsumers such as mangosteen wine, rosella wine, longan wine, litchewine, pineapple wine and a number of herbal wines etc. Recently thevolume of imported wine was decreased which indicated that acceptabilityof consumers is encouraging for the locally produced wines.

The high temperature and high humidity is optimal for the growth ofbacteria and carried over from fruits to the juice which often

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inhibit the fermentation of yeast or impart the sour flavor and offodor to the wine. Acid producing bacteria such as lactic acidbacteria also produce antimicrobial substances which is toxic towine yeast and often cause stuck fermentation. A special yeaststrain which is not sensitive to the bioactive substances and acidtolerance is desirable for the sound fermentation. It could beused as mixed culture together with the famous wine yeast strainsto ensure the completion of fermentation. Native alcoholicbeverages such as toddy wine needs the local yeast strain whichimpart special odor to the toddy wine. The selected strain shouldpossess the floculating or sedimenting characteristics to facilitatethe clarification of wine.

Rice wine is another famous local wine which the fermentation process isdifferent from the fruit wine. Special strains of molds and yeasts arerequired for this purpose. Traditionally, the seed culture called“Loogpang” which is the cultures of mold, yeast and bacteria aremaintained by transferring the mixed microbial cultures and herbs fromthe original to the successive ones. Unlike the japanese sake whichemploy the strain of Aspergillus oryzae in the process, the amylolyticfungi of local rice wine is mainly Rhizopus, Mucor and Amylomycesspecies. To make good rice wine at larger scale, special strains ofamylolytic fungi and yeast are required. Many research have been done atlaboratory scale but the commercial scale production need to be assessed.Equipment for the large scale koji preparation is the essential technologyto develop. The growth of fungi in Mucoraceae is sensitive to agitation,shaking or rotation, therefore, special design of bioreactor to control theaeration, humidity and temperature is required. Selection of special yeastand mold strains which impart desirable aroma and flavor are still requiredeven though many good strains are available from the culture collection.

Industrial alcohol fermentation is produced from molasses which is thecheap raw material. Despite the long history of alcohol production, theindustry has some difficulties during summer due to the high temperatureand high rate of bacteria contamination in the fermentor. Majority of themare lactic acid bacteria which produce bioactive substances and acids toxic

to yeast and resulted in stuck fermentation. Special strain of yeastresistant to bioactive substance, acid tolerance and high temperaturetolerance is required. There were some research carried out along this linewith certain degree of success but application of the selected strains in theproduction scale need to be verified.

The well established technology which has good potential for newinvestment is the single cell protein (SCP) production. So far the slopwaste from alcohol factory have enough carbon source for SCP productionand economically feasible for the investment. The demand for animalfeed is increasing annually and we have to import soy bean for oil andanimal feed to meet the local consumption. SCP could contribute to partof the protein and vitamins requirement for animal feed industry.

Soysauce fermentation is another interesting industry that has beenprogress dramatically in the past 10 years. Pure culture of mold isemployed by big factories and the koji fermentation process is developedlocally. However, little has been done on the yeast and aromadevelopment during aging which is rather complex. The original methodaged the soysauce in the small jar exposed to sunlight for over 6 months.The complexity and aroma development in the traditional process has notbeen elucidated. The yeast Saccharomyces rouxii play an important rolefor the flavor and odour development together with the aging technic ofeach factory. Analysis of aromatic compounds would be necessary forcharacterization and improvement of the aging process.

Palmyra palm cake is produced by employing the natural flora of yeastpresent in palmyra palm fruit. The dough is made from the rice flourmixed with the juice extracted from the palm fruit. Unique aroma ofpalmyra palm cake is derived from the native yeast flora. So far, there isno in depth study on this topic.

In conclusion, utilization of yeasts in local industry is increasinglyimportant and need more attention from researchers and industrialists tocollaborate. There is a good opportunity to invest in the yeast relatedbusiness in the near future.

Rice Wine in Southeast Asia Countries : Thailand, Laos,

Vietnam and Myanmar

Puspita LISDIYANTI1 and Michio KOZAKI2

1Research Associate

2Professor Emeritus at Department of Applied Biology andChemistry, Tokyo University of Agriculture, 1-1-1 Sakuragaoka,Setagaya-ku, Tokyo 156-0054, JAPAN

The traditional alcohol beverages produced in Southeast Asia are broadlyclassified into (1) rice and cereal wine, (2) palm wine, and (3) distilledspirit from rice, cereal, or palm wine. Rice wine is said to originate fromYunnan-Guizhou provinces of China. Thus, the brewing methods of ricewine and drinking customs is suggested to spread as concentric cycle fromthis area into the surrounding areas. Basically, the common method ofmaking rice wine is as follows: powdered starter (ragi, lakpaeng, bubod,men, mochikouji) is spread on steamed rice, ferment them in a wide-mouthed jar for saccarifying process, and transfer into narrow-mouthed jarfor alcohol fermentation. In some places, rice chaff is applied. When ricechaff is added during fermentation process, long fine bamboo straw isusually used when drink the product.

Although, Southeast Asia lies in the tropical zone, which rich in manyvarieties of fruits, fruit wine could not be found in this area. Palm winefrom the sap of palm trees is considered as the fruity wine in SoutheastAsia.

Nowadays, the distilled spirit from rice or palm wine is a more populartraditional alcohol beverage than its origin (rice or palm wine) inSoutheast Asia area. From our survey in the north part of Vietnam, Laos,and Myanmar, rice wine has been switched to distilled spirit of rice or

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palm wine. Unique and traditional distillation method still be used in thisarea.

1. Brewing alcohol beverage in Southeast Asia is originfrom Yunnan-Guizhou provinces of China

It is the common knowledge that on the basis of making process, alcoholicbeverages are divided into three broad categories; brewing alcoholbeverages (wine, beer, sake, etc) distilled spirit (wisky, brandy, vodka,tequila, etc), and mixed of brewing and distilled spirit (liquor, vermont,etc). Furthermore, brewing alcohol beverages could be divided into twocategories on the basis of fermentation processes; single fermentationprocess (wine) and combination fermentation process (beer and sake).Wine can therefore be made by the direct fermentation of the raw material,while the production of beer or sake requires the hydrolysis of starch toyield sugars fermentable by yeast, as a preliminary step.

In Southeast Asia (SEA) area, it is assumed that we could found manykinds of fermented fruits like wine, because this area is abundant in manykinds of fruits that contained high sugar concentration. However, in thereality, except of palm wine and banana wine, it is difficult to findalcoholic beverages made from fruit juices in this area. In Luzon Island,the Philippines, we could find basi, a wine from press juice of sugar cane.However, basi was only produced in the certain area, and did not wide-spread consumed in Asia continental and island area. From our survey onthe study of alcohol beverages in Southeast Asia, we found many fruitsliquor (dipping the fruits in distilled alcohol), but we do not foundfermented fruit juices. Our survey is similar with the investigation of Dr.Naomichi Ishige, staff of National Museum of Ethnology, Osaka, Japan onthe study on the comparison of food culture of SEA peoples. He reportedthat there is no full-scale production of fermented fruit juices in SoutheastAsia.

Even, there are no full-scale of fermented fruit juices production inSoutheast Asia, as living on rice people, from long time ago, there arefermented cereals and fermented rice in the area.

From Nepal to Sikkim area (the North of India), chan (brewing alcoholicbeverage) has been made among farm household. The processing of chanis as followed: directly ferment the steamed millet or corn with starterwithout water addition. This kind of solid fermentation process is called astsubu sake (grain wine). It is assumed that tsubu sake was the oldestprocess of alcoholic beverages making and the oldest method of solidfermentation. In addition, because this area famous by their cerealsproduction, the cereals wine is the famous product of this area.

However, the route of spreading of rice wine is unknown. The origin ofrice was suggested from the area of the middle of Chan Jiang River in theYunggui area (Yunnan-Guizhou provinces) of China in 4000 years ago.Together with the distribution of rice, the culture of making rice winespread from Yunggui area to SEA, as concentric cycle. Because of thereligion, some area of Southeast Asia, like Indonesia and Malaysia, didnot develop the rice wine. Leastways, we suggested that rice wine isoriginated from the Yunggui area (China).

Unfortunately, from our several times survey, currently, there is no moreproduction of brewing alcohol beverage from cereal or rice in Yungguiarea. Alcohol beverages that available in the market of this area areshochu (distilled spirit).

2. Rice and Cereals wine in SEA

As mentioned in the previous section, the methods and custom of drinkingalcohol in SEA is said, were come from Yunggui area of China as anorigin. Yunggui area is divided into two provinces, namely Yunnan andGuizhou area. In Yunnan province, there are about 30 minorities and inGuizhou area, there are about 15 minorities. From our survey severaltimes in Yunnan, we found that Bai, Dai, Jinpo, and Nazi ethnic minoritiesof China make alcohol beverages from rice. In the farm householder, whomakes rice wine, simple ceramic distiller for making distiller spirit orunrefined rice wine is equipped in their house. From our visit, every 1 or 2farmer is equipped with a unit of making alcohol beverages.

In the village which making cereals wine, the materials used for makingcereals wine is usually rice. However, Miao Minority in Kummin (capitalcity of Yunnan) used corn, red-corn, or barley in making cereals wine,instead of rice. Furthermore, the ethnic minorities of Red Zao, Zai,Kumong in North Vietnam, in every farmer house, which make rice wine,were equipped with distiller.

On the other hand, the people of the tribe of E de, Mnon, and Coho inSouth or central west side of Vietnam, usually make rice wine, and drinkin traditional way when festivals.

In other SEA area, for example Lao tribe in Laos, Lao-tai tribe in NorthThailand, Ifugao tribe in Philippine, and Bali people in Indonesia, ricewine are also be widely produced. Because of rice wine is the product ofthe certain tribes or people and not full-scale production, the distributionof rice wine is local and spot.

3. Method for making cereals wine

Generally, the method for producing cereals wine in Southeast Asia is asfollowed. Wash the rice, immerse the washing rice in water for 1 hour,and steam it. Spread out on cloth for cooling down the steamed rice toroom temperature. Disperse the amount of starter. Starter containsRhizopus which have the ability to decompose amylase andSacharomycopsis which have the ability to promote alcohol fermentation.Transfer the steamed rice spread with starter in wide-mouthed Chinese jarand place it at room temperature for 1 or 2 days. In the continental of Asia,they mix steamed rice with chaff. By the way, rice wine from Bali island,brem, and from Luzon island, tapuy, did not be added with chaffs.

From our survey, traditional rice wine from the continental and the islandsof Asia have differences on the adding of rice chaff. There are also someplaces, after first stage of fermentation, they added chaff again, andtransfer this moromi to other narrow-mouthed jar. Then, fill up the mouthof jar with chaffs, place banana leaves, clothe with plastics or papers, tieup tightly with lace, and ferment it at room temperature for 1 month.When drink, get out of the chaff, add with water, leave for 1 h, and suck

with long fine bamboo straw. To filtrate the rice grains or chaff, small holeor slit is incised at the edge of long fine bamboo straw. If one jar of winewas opened, in this day, it should be finished. Figure 1. present the generalmethod of making rice wine in SE Asia.

Rice powder adding with herb or ginger forming and shaping to ballor square fermentation starter

Rice or Cereals washing immersing on water splashing the water steaming for 1 hour cooling down spreading the starter first

stage fermentation (saccharifying) transferring to a new jar secondstage fermentation aging rice wine

Fig. 1 Basic procedure for making starter and rice or cereals wine

4. Characteristics of traditional rice wine

The rice wine in SEA has the characteristics as follows:

1. The raw material for making rice wine is paddy, red or white glutinousrice, or ordinary rice.

2. The starter used for making rice wine is the small rice powder ballmixed with molds, yeast and bacteria.

3. Chinese ceramic jar is usually used as container for making rice wine.

4. During fermentation, rice chaff has been applied or not.

5. The fermentation process of rice wine is conducted in one or two jars.

6. The manner of drinking is traditionally used fine bamboo strawwithout filtration.

From the above characteristics, we found the application of rice chaff infermentation process is unique and differed between the places and

peoples. There are four way of making rice wine in accordance toapplication of rice chaff (Fig. 2).

1. Upper layer type (Black Thai tribe, Laos)

Rice chaff added 2. Mixed type (Mon-khmer, South Laos)

Rice wine 3. Upper and bottom type (M’nong, South Vietnam)

4. Without rice chaff (Khmer, Northeast Thailand; Ifugao, Luzon)

Fig. 2 Classification of rice chaff application.

5. Other traditional brewing alcohol in SEA

SEA countries located in the belt of the palm culture, and the ratio ofapplication of palm is extremely high than other plants. Palm wine wasmade broadly in SEA region. Main varieties of palm, which used for palmwine making, are sugar palm (Arenga pinnata), palmyra palm (Borassusflabellifer), coconut palm (Cocos nucifera), and nipa palm (Nypafruticans). Coconut palm grows near river or sea side, nipa palm wasfound frequently in brackish water, and palmyra palm grows in warmclimate inlands. The process of making palm wine is simple and wellknown. Oozed sap from palm are collected in bamboo tube, the volume ofoozed sap from palm can be reached until 50-60 ml/h. Bamboo tube usingfor collecting is adhered by many yeasts and lactic acid bacteria. Soalcohol fermentation already started while bamboo tube was hung in thecutting inflorescence. Palm wine include about 2% ethanol. Collecting ofpalm wine is mostly distilled by simple method in the farmer houses. Thedistilled spirit from palm wine is the main alcohol beverage in SEAregion. And from palm wine, they make palm brown sugar. Same as ricewine, palm wine was distilled to be distilled spirit, contained high

concentration of alcohol. In the palm wine making, if they did not docarefully, insects will come in, and the palm wine will become sour.

In Luzon Island of the Philippine and in Annamese Cordillera of CentralVietnam, some farmer makes sugar cane wine for own consumption. Inthe Philippine, it is called “basi” and in Vietnam it is called as “abietatau”. The making place, the product, and the consumption of sugar canewine are very limited in SEA.

From our survey, we found that generally, cereals wine are made by theminority people, but palm wine are made by general people.

6. Distilled spirit from rice wine in SEA region

Rice wine was made by ethnic minority houses of South Yunnan provincein China until 15 years ago. However, nowadays, rice wine is all almostdistilled. By way of distillation, distilled spirit from rice wine can bestorage longer, easier for transportation, and has higher alcoholconcentration content than brewing rice wine. Because of these reasons,the rice wine is shifted gradually by the distilled spirit.

Almost the minorities live in the border area between Yunnan andcountries of SEA, Vietnam, Laos, and Myanmar, made distilled spiritfrom rice wine. Red Zao tribe in the north Vietnam near Yunnan use thewhole paddy for making wine. They called their paddy wine as “bongruou”. According to the explanation of farmer, the method for making“bong ruou” is as follows. Steam the dried paddy for 1 hour and coolthem. Spread 1.5 kg “men” (starter) to 30 kg steamed paddy. In order tosaccharification, pile the paddy and cover them by cotton cloth, and leavefor 2 days in the room. Transfer the sweetening paddy to drum andferment for 15-20 days. After fermentation, rice wine is distilled by theEast Asian type distiller. The procedure of making “bong ruou” is shownin Fig. 3.

Paddy sun dry steaming (1 hr) cooling spreading “men” piling leaving (2 days) transfer to drum fermentation distillation “bong ruou”

Fig. 3 “Bong ruou” making

Inder tribe live around Inde Lake in Myanmar, are well known of makingdistilled rice wine and it is called as “aie pyu” (aie=alcohol, pyu=white).Generaly, local people used crunch rice as raw materials for making thisrice wine. After quick wash, the crunch rice were steamed for 60 min,spread on bamboo mat for cooling, and spread 55 pieces of starter ball (1pieces=15 g). After that, put while push the rice into jar and ferment themfor 6 days. After 6 days, mix with a bucket of palm brown sugar solutionand continue 4 days more fermentation. The product of rice wine wasdistilled by western type distiller. The taste of this alcohol beverage issweet, a little bitter, and contained about 50% alcohol. If the farmer salethis distiller spirit to the market, they diluted the product into 40%.Procedure of “aieu pyu” making are presented in Fig. 4. The starter usedfor making “aieu pyu” was called “garse”. The making process of “garse”is shown in figure 5.

Crunch rice steaming (1 hr) cooling spreading of starter ball (55pieces/60 kg rice) transferring to four jars first fermentation (6 days)

adding palm sugar solution second fermentation (4 days) distillation product (±50% alcohol) dilution (35-40% alcohol)

Fig. 4 “Aiu pyu” making

Glutinous rice washing steaming for 1 h pounding shaping place on rice chaff spread old starter powder inoculation with molds

sun drying product

Fig. 5 “Garse” starting making

Technology Development of Sake Fermentation in Japan

Toshiomi YOSHIDA

JSPS (Japan Society for the Promotion of Science) Bangkok LiaisonOffice

113 TWY Office Center, 10th Fl., Serm-mit Tower, 159 SukhumvitSoi 21, Bangkok 10110, THAILAND

Brewing of sake is one of Japanese traditional technologies developed in along experience of peoples spending a long time over several thousands ofyears in their history. Their continuous efforts for technology developmenthas now resulted in a large scale industry producing various kinds of sakein a big scale with the most modern technologies including facilities offull automation and operation of the processes with computer controlsystems. The products are now well appreciated by the people with theworld wide distribution of them. This article will review recentdevelopment of technologies in sake brewing including computerizationof brewing processes for adaptive control of so sophisticated processes ofmashing sake, and knowledge based control of the process utilizing non-conventional methods of fuzzy theory and expert systems for brewingsake and ginjo sake, which is one the most complicated processes withmany unknown factors and phenomena involved, and the product shouldbe evaluated in non-conventional ways with delicate sensory tests byhighly experienced persons.

Dissolution of Steamed Rice in Saké Mashing Process

The dissolution of substances from steamed rice on the addition of waterwas studied to formulate an operational equation to describe thedissolution phenomena. The following phenomena were taken intoconsideration in order to construct kinetic equations. (1) A significantincrease of total sugar concentration was observed at an early stage of

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dissolution. (2) The high concentration of produced sugar decreased α-amylase activity and consequently dissolution was greatly suppressed.During the fermentation, as the glucose concentration was decreased byconversion to ethanol, the steamed rice was significantly dissolvedwithout being influenced by the glucose inhibition. (3) When water wasadded to steamed rice, the volume of the liquid phase remarkablydecreased during the 2 h after mixing due to adsorption of water into thegrains and afterwards the liquid volume increased due to the release of thewater on disintegration of rice grains. The operational equations forsteamed rice dissolution formulated based on the experimental resultsmentioned above could well simulate the experimental observations undervarious operational conditions.

Dissolution of Koji in Saké Mashing

The dissolution of koji was investigated in order to formulate a set ofempirical equations for the process control of saké mashing. Experimentalanalysis resulted in the following conclusions, which provided importantrationales for formation of process equations. 1) Koji contained two kindsof soluble substrates, one readily soluble without α-amylase, and the otherslowly soluble by the action of α-amylase. 2) The dissolution of the lattercomponent varied greatly with the variety of the rice. Koji dissolution wassuccessfully simulated by use of the process equations. Combination of

VS

SrC

SSrJVVS

SVV

EV

S

STE

dt

SSd

t

t

no

n

/)1(

)())(1(

20.0

)/9260exp(105.19)/(

0

000

0

0

00

110

−=

−⋅+−−+=

=

−⋅×−=

the equations of dissolution of koji and steamed rice allowed a closeestimation of the dissolution of the materials during saké mashing.

Fig. 1 A model of dissolution of steamed rice, hydroxylation of starch,and ethanol fermentation

InsolubleStarch

SolubleStarch

Glucose Ethanol

YeastGluco-amylase

α-Amylase

Steamedrice + water

Wateradsorbed

Ricedissolution &hydroxylation

Fermentation

Fig. 2 Simulation of dissolution of various kinds of rice in sake mash.Broken line: partially digested rice in koji; dotted line: steamedrice; solid line: whole mash including both steamed rice andkoji; one dot line: koji; two dots line: residual starch in koji.

Yeast Growth and Ethanol Production in Saké Mashing

Growth and fermentation of Saccharomyces cerevisiae kyokai 601(awanashi) were examined aiming the set-up of equations for theestimation of yeast growth and ethanol production in saké mashing, andthe following were concluded.

Specific growth rate µ (1/h) and specific ethanol production rate (g-ethanol/g-cell/h) in a liquid medium were expressed as functions ofglucose concentration G (g/dl) and ethanol concentration Et (g/dl) takinginto account the effect of temperature:

0

20

40

60

80

100

120

0 5 10 15 20

Time (d)

Dissolution rate (

where all letters except µ, ν, G, Et and T are experimental constants.

2. Equations of specific growth rate and specific production rate weremodified for saké mashing, taking into account the effect of cellconcentration on specific growth rate and correlation between specificproduction and specific growth rate:

where Xm (108 cells/ml) is the maximum X and �is constant.

Adaptive Control of the Mashing Process in Saké Brewing

A simulation model of the saké mashing (moromi) process, the mainprocess of saké brewing, was constructed on the basis of mass transfer andbiochemical reactions in a multi-phase system of solid and liquid. Thetemperature in the mashing process was controlled using this simulationmodel and a microcomputer, and fine saké was brewed on an experimentalscale. It appeared that the mashing process was divided practically intotwo phases. In the first phase, the reactions of rice dissolution, glucoseproduction, and ethanol fermentation did not interact with each other,while in the second phase they were limited by the rate of rice dissolution.

+⋅⋅⋅⋅

++⋅=

+⋅⋅⋅⋅

++⋅=

T

kGkEk

k

GkG

Ga

T

KGKEK

K

GKG

GA

t

sis

t

sis

32

212

32

212

)exp(exp

)exp(exp

π

µ

ss

ms X

X

µαππ

µµ

⋅+=

−= )1(

SteamedRice

Aqueous Phase

Koji

InsolubleStarch

PartiallyDigestedStarch

HardStarch

Enzymes

α-Amylase

S o l u b l eStarch

Glucose

Ethanol

α-AmylaseGluco-

amylase

Yeast

Water

Water

Water

Absorption

into rice

SteamedRice

V

Sm-Smo

So-Sk

Ct

1-S/So

G X

dX/dt

d(Sh/Sho)/dt

d(Sd/Sdo)/dt

dG/dt

dE/dt

d(Sm/Smo)/dt

CO2

Koji

πµ

α-

Gluco-

T

T

Ekig

kie

Inhi-

bitionT

Fig. 3 Element analysis and signal flow diagram of the sake mashing

Fig. 4 Simulation of sake mashing process with 3 times feeding ofrice.

Computer Control of Sake Mashing in Pilot Scale

This study deals with pilot-scale fermentation of sake mash (Moromimash) by on-line basis computer control. A test batch of the mashingusing 1,000 kg of polished rice was conducted, following an ordinarymethod of three stages mashing in a closed fermentor of 3 kl capacity. Asthe dead time and the time lag have been observed in response of mashtemperature, a cascade system was adopted for effective control of mashtemperature. An adaptive control system with the aid of a microcomputerwas constructed of continuous monitoring of the temperature and ethanolconcentration in the mash. The ethanol concentration was estimated from

0

20

40

60

80

100

120

140

160

180

0 100 200 300 400 500Tim e (h)

EtOHnol (ml/l); G (g/l); S (g/l);

Diss. rate (%); Yeast (x50/l)

Ethanol Glucose Dissolution Total Sugar Yeast

Temperatureprofile

evolution rate of carbon dioxide gas measured with a dry-gas-flow-meter.The control of the mashing was started after the final addition of materials.In a test run, temperature of the mash was controlled in a range of ±1°Cdeviation from the optimized set value of temperature, and the processwas completed on the schedule. Observed values of ethanol concentration,one of the most important state variables, coincided well with a referenceprocess, a little deviation was observed on sake-meter. The quality ofproduced sake was evaluated similar to that of the reference processcharacteristically.

Fig. 5 A cascade control of sake mashing process for adaptive control.

Controller1

DPC

Wall TempTw

Temp @Center

Tc

Mash TempTav

Minor Loop

Major Loop

Controller2

Computer

Coolingunit

Switches unit

DPC

Computer

Fermentor

Pulse generator

Gasmeter

TcTw

Fuzzy control of sake mashing

Tsuchiya Y., et al constructed fuzzy rules and a fuzzy simulator based onthe control techniques of Hiroshima tohji (experts). The linguistic rule -base for control of moromi mashing process in a sake brewery inHiroshima is as follows. Their results of controlling sake mashing weresuccessful, and the technologies have been extensively developed by Oishiet al to be industrially utilized in the biggest sake brewing company inKyoto.

Table 1. The fuzzy rules for the moromi mashing process in a sake brewery

1. IF MN < 7 THEN FO = 0

2. IF MN > 7 AND BE is B(H) THEN BFO(H) =k1 B(H)

3. IF MN > 7 AND BE is B(M) THEN BFO(M) =k2 B(M)

4. IF MN > 7 AND BE is B(L) THEN BFO(L) =k3 B(L)

5. IF MN > 7 AND AL is A(H) THEN AFO(H) =k4 A(H)

6. IF MN > 7 AND AL is A(M) THEN AFO(M) =k5 A(M)

7. IF MN > 7 AND AL is A(L) THEN AFO(L) =k6 A(L)

8. IF MN > 7 AND MN < 15 THEN FO = (BFO+AFO)/2

9 IF MN > 15 AND A(M) = 1 THEN FO = BFO

10 IF MN > 15 AND A(M) ≠ 1 THEN FO = (BFO+AFO)/2

11 IF MN = 17 AND B(L) = 1 THEN END

12 IF MN = 18 AND B(H) ≠ 1 THEN END

13 IF MN = 19 THEN END

MN, brewing time; BE, baume; AL, alcohol; FO, fuzzy output variable

B(H),B(H),B(H),A(H),A(H),A(H), values membership function

Framework rules for fuzzy control of ginjo sake making

Hanai, T., et al have developed a fuzzy control system for ginjo sakemaking. Their linguistic rules for the control of ginjo sake brewing areshown in the following Table 2. Ginjo sake has been successfullyproduced in a commercial scale in Nagoya since their works. They haveextended their research works to develop a method to control the qualityof products, ginjo sake, with use of a data mining technique to extractimportant information of a long-term accumulation of the data of actualproduction.

Table 2. Control strategies and parameters of membership functions

Parameter ofmembership

functionControlregion

Period Temperaturecontrol Reference

s b

1 Days 1 - 9Regularincrease

(0.4�/day)-

Ratio oflength 0.5 1.5

2Day 10 -

bouze

Increase byfuzzy

control �Degree -15 15

Ratio oflength 0.5 1.5

3Bouze -baume>

2.0

Decrease byfuzzy

control

Straightline A - B

plot

�Degree -15 15

�BMD -2 2

4Baume <

2.0

Increase ordecrease by

fuzzycontrol

Straightline BMD

curve Temperature 5.0 8.0

Bouze means the disappearance of the fermentation foam from the moromi.A - B plot: Alcohol vs. Baume; BMD curve, Cumulative baume vs. time.

Yeast Diversity in Thai Traditional Alcoholic Starter

Savitree LIMTONG, Somporn SINTARA, Poonpilai SUWANNARITand Napha LOTONG

Department of Microbiology, Faculty of Science, KasetsartUniversity, Bangkok 10900, THAILAND

Forty-three isolates of yeast were obtained from 38 samples loog-pang-kao-mag (starter for alcoholic sweetened rice) and 49 were obtained fromloog-pang-lao (starter for rice wine) 19 samples. Among them, 31 isolatesfrom loog-pang-kao-mag and 20 isolates from loog-pang-lao wereidentified as Saccharomycopsis fibuligera. The other yeast speciespresented in both types of loog-pang were Pichia anomala (8),Issatchenkia orientalis (6), P. burtonii (6), P. fabianii (4), Candidarhagii (4), C. glabrata (3), Torulaspora globosa (3), P. mexicana (2) and1 isolate each of P. heimii, Rhodotorula philyla, Saccharomycescerevisiae, T. delbrueckii and Trichosporon asahii. S. fibuligera was thesingle yeast species found in 23 samples (60.53%) of loog-pang-kao-magand 7 samples (36.84%) of loog-pang-lao. In addition, 7 samples(18.42%) of loog-pang-kao-mag consisted of S. fibuligera together with 1-2 species of other yeast, while 10 samples (52.63%) of loog-pang-laoconsisted of S. fibuligera and 1-4 other yeast species. Forty-six from 47isolates of S. fibuligera revealed strong amylolytic activity, express asratio of clear zone diameter and colony diameter on soluble starch agar, inthe range of 1.93-3.25. However, most of them produced low ethylalcohol, i.e., less than 2 %v/v from 18 % glucose at 48 hours. On thecontrary other yeast species showed low amylolytic activity but high ormoderately high in alcohol fermenting ability. Among isolates thatfermented high alcohol contents were T. globosa YKM032 (6.03%v/v), I.orientalis YMK036 (6.01%v/v), P. burtonii YKM034 (6.00%v/v) and P.burtonii YL048 (5.99%v/v). The only one isolate of S. cerevisiaeproduced relatively low ethyl alcohol at 4.68%v/v.

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Introduction

“Loog-pang”, commonly is known as "Chinese yeast cake" to the Westernpeople, is a Thai term for dry form of “fermentation starter” for productionof traditional fermented products from starchy raw materials, i.e., kao-mag(alcoholic sweetened rice), lao (rice wine) and num som sai chu (vinegar)(Lotong, 1992). Use of this type of fermentation starter is believed to beoriginated in China and have been transferred to many countries in Asia.Various local names are used differently in Asian countries, such as banhmen in Vietnam, chu in Chinese, koji in Japanese, nuruk in Korean,murcha in Indian, ragi in Indonesia, ragi tapai in Malaysia and bubod inPhilippines. These starters are apparently mixed cultures of molds, yeastsand bacteria grown on rice or other cereals. In certain localities nativeherbs are added (Batra and Millner, 1974; Saono, 1982; Lotong, 1998 andThanh et al., 1999). Saccharomycopsis fibuligera is a common yeastspecies reported in many starter cakes including loog-pang. While Pichiaanomala is presented in bubod, loog-pang and murcha, Candida spp. arethe common yeast in ragi. Saccharomyces spp. have been found only insome samples of banh men, bubod and loog-pang, ragi (Chatisantien,1977; Chaowsungket, 1978; Dijen, 1972; Saono, 1982; Lotong, 1998 andThanh et al., 1999). Among microorganisms resented in these startercakes only some species play important roles in product formation(Lotong, 1992; Haard et al. 1999). Though there are some reportsconcerning with microorganisms in loog-pang and their possible roles inproduct formation but only few isolates of microorganism were collectedand maintained properly. From past to present, loog-pang is difficult toget because its preparation process is known only in some families. Dueto the limited knowledge some key microorganisms tend to loss resulted indecrease in quality of loog-pang. Therefore, to control the quality offermentation products, isolation, selection and conservation of the keymicroorganisms in loog-pang which could be used as pure cultures forproduction of the fermented products is gaining attention. This workreports the isolation and identification of yeasts from loog-pang, includingtheir amylolytic activity and ethyl alcohol fermenting abilities.

Materials and Methods

Sources of loog-pang

Thirty-eight samples of loog-pang-kao-mag and 19 samples of loog-pang-lao were collected from many provinces in various parts of Thailand.They were stored in refrigerator until use.

Isolation of yeast

To obtain as many yeast species as possible, isolation was carried out by 3protocols namely, enrichment in acidified (pH 3.7-3.8) YM broth (0.3%yeast extract, 0.3% malt extract, 0.5% peptone and 1.0% glucose),enrichment in YM broth containing 300 g/l glucose and preparation offermented products from each type of loog-pang followed by isolationfrom fermented mash. Pure cultures obtained were preserved on YM agarand stored in a refrigerator.

Identification of yeast

Morphological including ascospore formation, physiological andbiochemical characteristics of yeast isolates were examined according tothe standard methods as described by Yarrow (1998). Assimilation ofcarbon compounds were tested by API 20 C AuxTM (Bimerieux, France).

Amylolytic activity

Active yeast culture grown on YM agar was point inoculated on thesurface of soluble starch agar (4 % soluble starch, 5 % yeast extract and1.5 % agar) at 4 positions. Two replications were performed for 1 isolate.After incubation for 72 h at room temperature the culture plate wasflooded with Lugol’s iodine solution (2 g iodine, 2 g ammonium sulfateand 300 ml deionized water) for 1 min and diameters of clear zone andcolony were measured. The amylolytic activity was expressed as ratio ofclear zone diameter to colony diameter.

Ethyl alcohol fermentation

Active yeast cultures grown on yeast extract peptone dextrose (YPD) agar(1 % yeast extract, 2% peptone, 2% glucose and 1.5% agar) for 24 h wastransferred to 50 ml of YPD broth in 250 ml Erlenmeyer flasks and

incubated on rotary shaker (170 rpm) at room temperature for 16-20 h.They were then used as inoculum by transferring appropriate volume to100 ml of YPD broth containing 18 % glucose to obtain assigned initialcell concentration. The fermentation was carried out on a rotary shaker at70-80 rpm, room temperature for 48 h. Growth was measured as opticaldensity at 660 nm by spectrophotometer (Shimadzu UV-190, Japan).Ethyl alcohol concentration was measured by gas chromatography(Shimadzu GC-9A, Japan) using propanol as internal standard.

Results and Discussion

Isolation and identification of yeasts from loog-pangIsolation of yeasts were carried out by 3 protocols, to obtain as many kindof yeast as possible, resulted in 43 and 49 isolates from loog-pang-kao-mag and loog-pang-lao, respectively. Identification following taxonomickeys from “The Yeasts: a Taxonomic Study, 4th edition” (Kurtzman andFeel, 1998) revealed that most isolates obtained from loog-pang-kao-magwere Saccharomycopsis fibuligera (31 isolates) (Table 1). The remainingisolates were Torulaspora globosa (2), Issatchenkia orientalis (3), P.burtonii (2) and one isolate each was C. rhagii, P. fabianii, P. anomala,Rhodotorula philyla and T. delbrueckii. Many samples of loog-pang-kao-mag (23) contained only S. fibuligera while the other 7 samples consistedof S. fibuligera together with other 1-2 species of yeast. Only 2 samplesdid not have S. fibuligera but I. orientalis or T. delbrueckii was presented(Table 3). In loog-pang-lao 20 from 49 isolates of yeast were S. fibuligera(Table 2). The other isolates were P. anomala (7), P. burtonii (4), C.glabrata (3), C. rhagii (3), I. orientalis (3), P. fabianii (3), P. mexicana(2), P. heimii (1), Saccharomyces cerevisiae (1), T. globosa (1) andTrichosporon asahii (1). It should be noted that only S. fibuligera wasfound in 7 samples. While in other 10 samples contained S. fibuligeraand other 1-4 yeast species. There were 2 samples that were absent of S.fibuligera whereas P. anomala was found in 6 samples (Table 4).Surprisingly, S. cerevisiae was found only in 1 sample of loog-pang-lao,which may resulted from long term storage of loog-pang. However, thisfinding is agreed with Chatisantien (1977) and Chaowsungket (1978).

Many of the species reported in this work were also commonly found inother fermentation starters, for example, S. fibuligera and I. orientalis instarter cakes, P. anomala in bubod, murcha and ragi, P. fabianii in ricekoji and S. cerevisiae in banh men and ragi (Dijen, 1972; Saono, 1982;Lotong, 1998 and Thanh et al., 1999).

Amylolytic activity of yeast isolates obtained from loog-pangInvestigation on all isolates of S. fibuligera revealed strong amylolyticactivity on starch agar, 1.93-3.25, expressed as ratio of clear zone diameterand colony diameter, except YKM025 that the ratio was 1.18. While theother yeast species showed relatively low activities (ratio as 1) and manyspecies including C. rhagii, C. glabrata, I. orientalis, P. heimii P.mexicana, R. philaya, S. cerevisiae, T. globosa and T. delbrueckii did nothave activity (Table 1 and 2). The strong amylolytic activity of S.fibuligera agree with the roles of this yeast in loog-pang on hydrolysis ofstarch to sugar that has been indicated by many authors (Sukhumavasi etal., 1975; Chatisantien, 1977 and Chaowsungket, 1978).

Ethyl alcohol fermentation of yeast isolates obtained from loog-pangStudy on ethyl alcohol fermentation in 18% glucose medium usingshaking cultivation at room temperature after 48 h of fermentationrevealed that most isolates of S. fibuligera produced relatively low ethylalcohol (less than 2%v/v). Eleven isolates of this species produced 2.01-3.00 %v/v whereas 3 isolates produced more than 3.00%v/v, i.e.,YMK001 (3.66%v/v), YMK019 (3.43%v/v) and YMK026 (3.04%v/v).Most of the remaining yeast species produced ethyl alcohol less than4%v/v and 2 isolates, i.e., R . philyla and T. asahii could not ferment.High ethyl alcohol fermenting yeast were found in seven isolates i.e. P.anomala YL024, , I. orientalis YL047, P. anomala YKM006, T. globosaYKM009, P. anomala YL046, P. anomala YL002 and I. orientalis YL040at 5.28, 5.11, 5.00, 4.67, 4.52, 4.44 and 4.05%v/v, respectively. Theisolates that produced exceptionally high ethyl alcohol were T. globosaYKM032 (6.03%v/v), I. orientalis YMK036 (6.01%v/v), P. burtoniiYKM034 (6.00%v/v) and P. burtonii YL048 (5.99%v/v). The only oneisolate of S. cerevisiae produced ethyl alcohol at 4.68%v/v. Yeast species

that produce low concentration of ethyl alcohol might have another role incontribution of desirable flavor of the products.

In order to maintain diversity of these yeasts, all isolates were preservedby lyophilization and cryopreservation for further studies.

Conclusions

The results of this work showed that most samples of loog-pang-kao-magand loog-pang-lao comprised of S. fibuligera, which showed strongamylolytic activity. Its character related to the role of this yeast in starchhydrolysis during product formation. About half of the samples containedonly S. fibuligera. This may due to low quality of these loog-pangs and/orlong term storage in undesirable conditions. Though most of S. fibuligeraisolates could produce only low concentration of ethyl alcohol they mightcontribute to certain extent on ethyl alcohol production. Moreover, theresults of this work indicated that S. cerevisiae might not be the main ethylalcohol producer in loog-pang but other yeast species could be the majorplayers, for example T. globosa, I. orientalis, P. burtonii and P. anomala.

Table 1 Number, isolation and identification of yeasts in loog-pang-kao-mag including amylolytic activity and ethyl alcohol fermentation.

Isolation

Sampleno. No. of

isolate Method

Yeastisolatecode

Identification

Amylase activity(diameter ofclear zone

/ diameter ofcolony)*

Ethanol(%v/v)

1 1 YM30YKM001 S. fibuligera 2.84 3.66

2 1 KM YKM002 T. delbrueckii 0 2.24

5 2 YM (pH 3.5) YKM003 P. fabianii 1.00 2.27

YM (pH 3.5) YKM004 S. fibuligera 2.44 2.00

6 2 YM (pH 3.5) YKM005 S. fibuligera 2.61 1.96

YM (pH 3.5) YKM006 P. anomala 1.00 5.00


Table 1 (continued)

Isolation

Sampleno. No. of

isolate Method

Yeastisolatecode

Identification



Ethanol(%v/v)

YM30 YKM008 S. fibuligera 2.38 1.67

YM30 YKM009 T. globosa 0 4.67


18 0 - - - - 0.00

19 1 KM YKM011 S. fibuligera 2.20 1.87

20 0 - - - - -

21 1 YM30 YKM012 S. fibuligera 2.42 2.76

23 0 - - - - -

24 1 KM YKM014 S. fibuligera 2.39 2.11


YM30 YKM016 R. philyla 0 0.05



KM YKM019 S. fibuligera 2.38 3.43




60 0 - - - - -

64 1 YM (pH 3.5) YKM023 I. orientalis 0 3.4865 2 YM (pH 3.5) YKM024 C. rhagii 0 2.73

YM (pH 3.5) YKM025 S. fibuligera 1.18 1.28

66 1 Y M(pH3.5)

YKM026 S. fibuligera 2.53 3.04

67 0 - - - - -

Table 1 (continued)

Isolation

Sampleno. No. of

isolate Method

Yeastisolatecode

Identification



Ethanol(%v/v)

68 1 YKM027 YKM027 S. fibuligera 2.54 2.32

69 0 - - - - -

70 0 - - - - -





YKM032 YKM032 T. globosa 0 6.03


84 5 YKM034 YKM034 P. burtonii 1.00 6.00

YKM035 YKM035 S. fibuligera 2.25 2.29

YKM036 YKM036 I. orientalis 0 6.01

YKM037 YKM037 P. burtonii 1.00 3.11

YKM038 YKM038 I. orientalis 0 3.43







Note: YM (pH 3.5) = Enrichment in acidified YM broth (pH 3.7-3.8).YM30 = Enrichment in acidified YM broth containing 30% glucose.KM = Preparing alcoholic sweeten rice fermentation (kao-mag)

using loog-pang-kao-mag and isolated from kao-mag.

Table 2 Number, isolation and identification of yeasts in loog-pang-laoincluding amylolytic activity and ethyl alcohol fermentation.

Isolation

Sampleno. No. of

isolate MethodYeast isolate

codeIdentification



Ethanolconc.

(%w/v)

3 4 YM (pH 3.5) YL001 P. anomala 1.00 3.95

YM (pH 3.5) YL002 P. anomala 1.00 4.44

YM (pH 3.5) YL003 S. fibuligera 2.71 0.49

L YL004 S. cerevisiae 0 4.68

4 2 YM30 YL005 S. fibuligera 2.33 0.71


7 1 YM (pH 3.5) YL007 S. fibuligera 2.08 1.15


14 2 L YL009 S. fibuligera 2.64 1.04

L YL010 P. anomala 1.00 3.16

17 1 L YL011 S. fibuligera 2.30 0.66

26 3 L YL012 C. glabrata 0 2.42


L YL014 S. fibuligera 2.24 1.66


YM (pH 3.5) YL016 C. glabrata 0 3.61

30 3 YM30 YL017 T. globosa 0 3.87

YM (pH 3.5) YL018 T. asahii 1.00 0.00


33 2 YM30 YL020 P. anomala 1.00 2.85

L YL021 P. heimii 0 3.28

48 1 L YL022 S. fibuligera 2.35 2.34

Table 2 (continued)

Isolation

Sampleno. No. of


codeIdentification



Ethanolconc.

(%w/v)


58 2 YM30 YL024 P. anomala 1.00 5.28



YM (pH 3.5) YL027 C. rhagii 1.00 2.96


L YL029 C. rhagii 0 2.14

L YL030 P. burtonii 1.00 3.20


YM (pH 3.5) YL032 P. fabianii 1.00 2.32

L YL033 P. anomala 1.00 2.91


YM (pH 3.5) YL035 C. rhagii 0 3.34

L YL036 P. mexicana 0 3.61

L YL037 I. orientalis 0 3.76


YM30 YL039 P. fabianii 1.00 3.80L YL040 I. orientalis 1.00 4.05

L YL041 P. fabianii 1.00 3.68

L YL042 C. glabrata 0 3.01

87 6 YM30 YL043 P. burtonii 1.00 3.24

YM (pH 3.5) YL044 P. mexicana 0 3.59


L YL046 P. anomala 1.00 4.52

Table 2 (continued)

Isolation

Sampleno. No. of


codeIdentification



Ethanolconc.

(%w/v)

L YL047 I. orientalis 0 5.11



Remark: YM (pH 3.5) = Enrichment in acidified YM broth (pH 3.7-3.8).YM30 = Enrichment in acidified YM broth containing 30% glucose.L = Preparing rice wine (lao) using loog-pang-lao and

isolation from fermenting mash.

Table 3 Summary of yeast species in loog-pang-kao-mag.

Yeast No. of samples

S. fibuligera 22

S. fibuligera + T. globosa 2

S. fibuligera + C. rhagii 1

S. fibuligera + P. anomala 1S. fibuligera + P. fabianii 1

S. fibuligera + R. philyla 1

S. fibuligera + P. burtonii + I. orientalis 1

I. orientalis 1

T. delbrueckii 1

Table 4 Summary of yeast species in loog-pang-lao.

Yeast No. ofsamples

S. fibuligera 7

S. fibuligera + C. glabrata 2

S. fibuligera + P. anomala 2

S. fibuligera + P. anomala + S. cerevisiae 1

S. fibuligera + T. globosa + T. asahii 1

S. fibuligera + C. rhagii + P. burtonii 1

S. fibuligera + P. anomala + P. fabianii 1

S. fibuligera + C. rhagii + P. mexicana + I. orientalis 1

S. fibuligera + C. glabrata + P. fabianii + I. orientalis 1

P. anomala + P. heimii 1

P. anomala + P. burtonii + P. mexicana + I. orientalis 1

Literature cited

Batra, L.P., and P.D. Millner. 1974. Some Asian fermented foods andbeverages and associated fungi. Mycology 66: 942-950.

Chaowsungket, M. 1978. Selection of yeast and mould strains for ricewine production. M.S. Thesis, Kasetsart University, Bangkok.

Chatisantien, C. 1977. Selection of mould and yeast strains in loog-pangkao-mag fermenattion. M.S. Thesis, Kasetsart University, Bangkok.

Djien, K. S. 1972. Tape fermentation. Appl. Microbio. 23: 976-978.

Haard, N.F., S.A. Odunfa, C.H. Lee, R.Q. Ramorez, A.L Quinones, andC.W. Radarte. 1999. Fermented Cereals. A Global Perspective. FAO

Agricultural Services Bulletin. No.138. Food and AgricultureOrganization of the United Nations. Rome.

Kurtzman, C. P. and J. W. Fell. 1998. The Yeasts: A Taxonomic Study, 4th

edition. Elsevier Science, Amsterdam.

Kulapreecha, S. 1978. Effects of culture media of sporulation of Rhizopusspp. Isolated from loog-pang. M.S. Thesis, Chulalongkorn University,Bangkok, Thailand.

Lotong, N. 1992. Seed Inoculum and Their Production Technology (inThai). Funny Plublishing, Bangkok.

Lotong, N. 1998. Koji. pp. 658-695. In Microbiology of Fermented Food.p. 658-695. Vol. 2. 2nd edition (J.B Wood, ed.) Blackie Academic andProfessional. London.

Saono, JK.D. 1982. Microflora of Ragi, its compositions and as a sourceof industrial yeasts, pp. 241-250. In S. Saono, F.G. Winarmo and D.Karjadi (eds.). Traditional Food Frementation as Industrail Resources inASCA Countries. The Indonesian Institute of Sciences (LIPI), Jakarta.

Sukhumavasi, J., K. Kato, and T. Harada. 1975. Glucoamylase of strain ofEndomycopsis fibuligera isolated from mold-bran (loog-pang) ofThailand. J. Ferment. Technol. 53: 559-565.

Thanh, H.P., T.L. Thuoc, H. Ino, and M. Kosaki. 1999. Ruou can (tubewine) in Vietnam. Proceeding of International Conference Asian Networkon Microbial Research. Chiang Mai, Thailand. 520-528.

Yarrow, D. 1998. Method for isolation, maintenance and identification ofyeasts. pp.77-100. In C. P. Kurtzman and J. W. Fell (eds.), The Yeasts: ATaxonomic Study, 4th edition. Elsevier Science, Amsterdam.

How to Screen Favorable Lactic Acid Bacteria Strain, from

the Case of Bacteriocin Producing Strain and Silage

Fermentation Starter Strain

Sadahiro OHMOMO1), Sunee NITISINPRASART2) and SupanitHIRANPRADIT3)

1 )Japan International Research Center for Agricultural Sciences(JIRCAS) Thailand Office (c/o Soil Science Division, Department ofAgriculture, Chatuchak, Bangkok 10900, THAILAND).2) Department of Biotechnology, Faculty of Agro-Industry, KasetsartUniversity Chatuchak, Bangkok 10900, THAILAND3) Plant Pathology and Microbiology, Division, Department ofAgriculture, Ministry of Agriculture and Cooperatives Chatuchak,Bangkok 10900, THAILAND

Summary

Through out our research topics on the development of new bacteriocinsand silage fermentation starter using lactic acid bacteria (LAB), how toisolate excellent LAB strains is introduced. The isolation and selection ofan excellent strain will be a promising choice to get the successful results.

Introduction

Trends of researches on the application of lactic acid bacteria (LAB) areworldwide topics and are actively studied in various research fields. Infood industry, the application of LAB strains and/or their metabolites tothe quality maintenance as additives that are generally recognized as safe(GRAS) are expected (Muriana and Luchansky, 1993). We also have agreat interest in LAB. The following research topics from the point of

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view of the effective application of LAB for agriculture and agro-products. “Development of new bacteriocin (BAC) produced by LAB”and ”Development of silage fermentation starter adapted to tropicalconditions” were studied.

BACs produced by LAB are small molecular weight proteins showingbactericidal activity and are expected as a safe additive supported byGRAS status instead of the use of antibiotics and bactericides of whichthey are regulated strictly. Nisin is a typical BAC produced byLactococcus lactis and is only one BAC used as an additive for food inmore than 50 countries of the world.

Silage, a kind of stored forage, is prepared through out the process ofnatural lactic acid fermentation by LAB and is just like a fermentedvegetable. The quality of silage is influenced by the performance of LAB.To make good quality silage, LAB additives are widely used as a starter.However in tropical regions, good quality silage preparation is ratherdifficult due to the tropical environments and the shortage of LAB formaking good quality silage.

In either case, how to isolate excellent strains using easy and certaindetection methods and suitable isolation sources are very important.Getting an excellent strain will be promised the success in future. Namely,it is not too much to say that success in isolating a strain having strong andstable activity means the finish of the half part of research. In thisopportunity, our hot results on BAC producing strains and silagefermentation starter strains are introduced, especially in focusing on howto screen a suitable LAB strain.

Outline of LAB and Fermented Foods Associated with LAB

LAB are heterotroph, Gram positive and facultative anaerobic bacteriawhich convert more than 50% of sugar into lactic acid, and consist of 12genera such as Lactobacillus, Streptococcus, Lactococcus Enterococcus,Pediococcus, Leuconostoc, etc (Suzuki, 1996). In addition, it is generallyrecognized that the genus Bifidobacterium is also a member of LAB. Ineither case, LAB have a long history of effective use for making popular

foods such as fermented milk products (cheese, yogurt), fermentedsausage, bread, soy sauce “Shoyu”, Miso, fermented vegetables, etc.Consequently, LAB strains are considered to be safe and useful microbes.For this reason, selected LAB strains are used as starter cultures forvarious fermented food industries to make products, certainly and safety.It is, however even nowadays, known that many products are naturallyfermented while the quality of products sometimes becomes poor. Andactually, many scientists have great interests in these natural fermentedfoods.

Development of New BAC Produced by LAB

BAC

A BAC produced by certain bacteria is a small molecular weight proteinand shows various biological activities against animals andmicroorganisms. Among them, BAC produced by LAB is highly valuatedbecause of their bactericidal activity. Namely, it is possible to repress thegrowth of certain food spoilage and/or pathogenic bacteria and to extendthe preservation time of food products. Nisin produced by Lactococcuslactis is already used in the food industry as an antagonistic additive inmore than 50 countries. The use of nisin is, however, limited because itshows bactericidal activity only in acidic conditions and dissolves in waterat low levels. Information on other BAC produced by LAB is notsufficient, and the application of BAC other than nisin to the food industryis not recognized (Daeschel, 1993). Therefore, many research groups havebeen screened LAB strain to produce a new BAC.

We also study BAC produced by thermophilic LAB strains with greatinterests and have ideas for applying them to the process control andquality maintenance of fermented foods. In addition, the BAC productionby a thermophilic strain has advantages to be easily controlled the cultureand protected from the microbial contamination. Actually, we havescreened LAB to produce new BAC and found Enterocin ON-157 fromEnterococcus faecium (Ohmomo et al., 2000), Enterocin SE-K4 fromEnterococcus faecalis (Eguchi et al., 2001) and Mundticin KS fromEnterococcus mundtii (Kawamoto et al., 2002).

Screening method

On the screening of LAB strains to produce, it is very important how toscreen a strain easily, rapidly and surely. Various screening methods havebeen tried by many researchers as follows: spot-on-lawn assay (Hooverand Harlander, 1993), microtiter plate assay (Geis et al., 1983), agar welldiffusion assay (Barefoot and Klaenhammer, 1983), multi-well plate assay(Toba et al., 1991), etc. In here, the agar well diffusion assay is consideredbecause this is a simple and low-cost method. Usually, this method is usedin combination with direct agar plate assay (spot-on-lawn assay).

First step: Test strains are spotted on the surface of agar plate (dried for 2hr), incubated overnight and overlayed with soft agar containing a targetstrain. After overnight culture of the target strain, a clear zone (growthinhibition zone) formed around the test strains is observed. If bacterialstrains except LAB are used as target strains, the clear zone involves theformation due to the effect of lactic acid produced by LAB. However, thestrains forming clear zone are selected to the next step.

Second step: The culture filtrate of each selected strain is spotted on agarplates containing a target strain and then agar plate is cultured. Afterovernight culture, the clear zone formed around the spot is observed. Onthe spotting, a well directly formed on the agar plate is used. A paper disk(for the determination of antibiotics activity) is also used instead of thewell. An aliquot volume of culture filtrate (usually 30-50 �l) is pouredinto the well or adsorbed onto the paper disk.

Cautions: On the second step, following cautions should be paid attentionto carry out the screening.

(1) If the pH of the culture filtrate is lower than 4.0, it should be adjustedto 5.0-5.5. Especially in using bacterial strains except LAB as a targetstrain, the pH adjustment is necessary because the growth of target strainusually inhibited at less than pH 5.

(2) The cell concentration of target strain should be about 106 cfu/mlbecause the high cell concentration weakens the clear zone formation.

(3) The agar plate should keep at about 4° C for 2 hr before culturing atarget strain to make a stable appearance of the clear zone formation.Because the active compounds in the culture filtrate diffuse sufficientlyinto the agar medium in this meantime.

(4) The medium for screening should be considered. The MRS broth iswidely used by many research groups. But recently we are using amodified medium instead of MRS broth to clear the problem of droppingpH to less than 4. Our medium is consisted of low concentration ofglucose (0.2%) and high concentration of potassium phosphate (2 timeshigher than that of MRS broth). By using this modified medium, theculture filtrate was kept at more than pH 5.5 in the presence of 0.25%lactic acid with the enough growth of LAB strain.

Isolation source

On the screening of LAB to produce BAC, it is very important whatmaterials are used for the isolation sources. The efficiency of isolatingLAB strains is easily influenced by the isolation sources. Of course, thematerials associated with lactic acid fermentation such as natural cheeses,natural fermented milk products, fermented vegetables, silages, etc, shouldbe used. Until now, many European groups have used natural cheeses,fermented milk products, fermented sausage, etc, originated from animalproducts as the isolation sources (Ennahar et al., 1996). On the other hand,there are various kinds of fermented vegetables associated with lactic acidfermentation in Asian countries. These materials should use for theisolation sources of LAB strains to find new BAC because there are fewreports using these materials. Namely, the use of these materials willpromise us to find new BAC with high frequency. Further, it is proposedthat BAC and/or culture products originated from BAC producing strainsisolated from common fermented foods have the advantage of obtainingpermission for the use as food additives.

The detecting frequency of BAC (BAC-like activity) producing LABstrains is about 1-10% when these materials were used as isolationsources. For example obtained from our laboratory, only 8 strains (0.57%)among 1,404 strains showed the inhibition activity against some LAB and

pathogenic bacterial strains. They were isolated from only fermentedfoods purchased at the market in Provinces of Chonburi, Nakhon Pathom,Khon Kaen, etc. However, interestingly, no LAB strain was isolated fromthe fermented vegetable (Pak Dong) purchased at the urban area market inBangkok. The reason is not know yet. It is assumed that this vegetableshould be soaked in lactic acid solution instead of fermentation. Namelythis is a fermented-like product, not real fermented product. If thesesources are used for the isolation, it will be very hard to isolate LAB. So,the use of hand-made fermented vegetables purchased at the rural marketsis better way for the isolation of LAB. Additionally, 446 strains of LABhave been isolated from silages. Total of 1,850 strains were investigatedand only 14 LAB strains exhibited BAC like activity. Among them, themicrobial properties and BAC activities of typical two strains, IMC 21-1and CC 1-12, are found and shown in Table 1.

Silages are also very nice sources to isolate not only LAB strains but alsoyeast strains because silage is considered to be a kind of naturalenrichment culture of LAB. The use of silage is recommended as a sourcefor isolating LAB. In fact, on the said screening, we detected the inhibitionactivity in 14 strains (8.0%) among 175 strains that were isolated from 14silages prepared in Provinces of Chonburi, Saraburi, Nakhon Pathom andKhon Kaen. The preparation of silage is not a common technique inThailand and the use of silage for the isolation source might not bepopular. The microbial flora of silage prepared in Thailand is generallyconsisted of abundant LAB, moderate yeasts and bacteria except LAB andpoor clostridia (BAB) in good quality silage. The poor quality silage hasthe microbial flora of abundant yeasts, bacteria except LAB, poor LABand moderate BAB. In either case, the use of silage for the isolation ofLAB is worth trying.

Table 1. Properties of typical LAB strains and their BAC activities

Strain RGT1)

(C)

Shape andFP2)

Isolationsource

pH of

CF3)

Target strains4)

1 2 3 4 5 6

IMC21-1

40-45 Short rod,

Chain,

Hetero

Fermented

vegetable

4.22 - ++ - - ++ -

(-) (+)

<-> <+>

CC

1-21

40-45 Short rod,

Chain,

Hetero

Fermented

Corncob

4.40 ++ - +++ ++ ++ ++

(-) (+) (+) (+) (+)

<-> <-> <-> <-> <->

1) Range of growth temperature; 2) Pattern of the lactic acid fermentation;3) Culture filtrate; 4) BAC activity of culture filtrate (pH adjusted to 5.0)against target strains. Target strain �: Bacillus subtilis TISTR 025; �:Staphylococcus aureus TISTR 029; �: Salmonella typhimurium TISTR292; �: Escherichia coli TISTR 527; �: Lactobacillus plantarum TISTR541; �: Leuconostoc mesenteroides TISTR 473. Inside of () shows theactivity after digested with proteinase K. Inside of <> shows the activityafter digested with pepsin.

Development of Silage Fermentation Starter Adapted toTropical Environments

Silage

The silage is a kind of stored forage for the winter season in temperate andcold regions. The method of preparing silage (ensiling) developed in themiddle of the 19th century originated from a technique depicted in anancient Egyptian mural. In the 1950s, Thai government introduced thetechnology of dairy farming with ensiling method through the support ofDenmark and Germany (Ranong, 1999). However, dairy farming (mainly

for milk production) was not popular in Thailand until recent years. Rawmilk production (RMP) in Thailand did not achieve a remarkable develop-ment and silage usage did not also become a common feeding method. Inthe 1980s, the consumption of fresh milk and fermented milk rapidlyincreased due to the changing of the life style associated with the growthof the economy and the production of fresh milk was unable to satisfythe demand. Dairy farming as well as the livestock industry is recognizedas an important industry under the future strategy for agricultureencouraged by the Thai government. At the same time, the use of silage isgradually recognized as an important technique to increase the RMP. Forexample, about 4,000-4,500 Kg/head/lactation was recorded as the RMPby feeding of good quality silage (Napiergrass silage, corn silage) throughout the year. This record is about 1.4-1.5 times higher than that of theaverage production in Thailand. Namely, ensiling and feeding of silage isan important technique to make good use of plant materials with thehighest nutrient value through out a year not for the dry season in thetropical zone (Ohmomo et al., 2002a).

Silage preparation method

Ensiling is based on natural lactic acid fermentation whereby LABferment sugars to mainly lactic acid under anaerobic environments. Thefermentation is mainly performed under anaerobic conditions in acontainer that is called silo. In such an environment, natural lactic acidfermentation generates an acidic environment. Name- ly, with themaintenance of an anaerobic and acidic (about pH 4) environment, silagescan be preserved over long periods of time without spoilage (McDonald,1991). Homo-fermentative lactobacilli play an active part in good qualitysilage. Particularly, Lactobacillus plantarum is recognized as apredominant homo-fermentative microorganism in silage fermentationand a typical strain originating from plant materials. Some kinds oflactococci also contribute to the creation of an acidic environment at theearly stage of silage fermentation and then lactobacilli becomepredominant microorganisms for the generation of an acidic environment.Following points attach great importance to make good quality silage(Ohmomo et al., 2002b): dry matter content in materials (35-40%),sufficient sugar content in materials (more than 2% in fresh matter base),

high packing density and rapid sealing (air-tight), storage temperature,presence of home-fermentative LAB, etc. However, the preparation ofgood quality silage is not so easy. It depends on the performance ofnatural LAB even though ensiling is carried out under the conditionsmentioned above. Therefore, a starter strain of LAB (silage additive) isused as a sure and safe making method. Silage additives are sold in coldand temperate zones, but nothing for the use in tropical zone.

Microbial flora of Thai silage

The microbial flora of silage prepared in Thailand was considerablydifferent from that prepared in Japan (Ohmomo et al., 2002a). Needless tosay, the main and important microorganism in good quality silageprepared in both of Japan and Thai- land were LAB. However, moderateor poor quality silages prepared in Thailand contained a relatively highnumber of coli-form bacteria (CFB) and yeast, but low number of BABcomparing with that prepared in Japan. Sometimes, good quality silagesprepared in Thailand contain a large number of yeasts. The inhibition ofthe growth of yeasts and CFB in silage in Thailand is important while theinhibition of BAB growth is essential for making good quality silage inJapan. Because yeasts utilize lactic acid as a carbon source for the growthunder aerobic conditions (after opening silo) and then the pH value ofsilage is changed to neutral. Consequently, it promotes the growth ofaerobic bacteria (referred to as aerobic deterioration) resulting in the lossof nutrients in silage.

Model of fermentation system

Culture conditions such as medium composition, size of inoculation,culture temperature and aeration as well as types of microorganisms areessential parameters to be considered for microbial growth. Silagefermentation is a kind of microbial culture and the culture conditionsshould be considered. Therefore, it is necessary to construct a modelsystem for silage fermentation with a constant medium composition andmicrobial flora. The system should reflect the properties of silagefermentation consisting of a solid, mixed and non-sterilized culture. Basedon the idea mentioned above, we constructed a model system of silagefermentation, named Pouch method (Tanaka and Ohmomo, 1995). This

method uses a bag made from air-tight plastic film (oxygen permeability:about 1 ml/m2/day) and a powder of the same lot of alfalfa hey cube as aplant material which is pasteurized and adjusted both the contents of sugarand moisture. Further, CFB and BAB together with LAB are inoculated astypical silage microorganisms. The bag is sealed by using a vacuum sealermachine after inoculation of silage microorganisms and cultured undercontrolling temperature. An LAB strain is selected as a silage fermentationstarter if the growth of CFB and BAB are inhibited or repressed by theLAB strain tested. The pouch method was developed to screen LABstrains used as inoculants for silage making in Japan (Ohmomo et al.,2002b). This system can easily apply to screen LAB strains using inThailand by the modification of microorganisms reflecting the propertiesof microbial flora in silage prepared in Thailand.

It is thus obvious that the silages prepared in Thailand are not always agood quality due to natural silage fermentation under adverse conditions.On the other hand, the use of LAB, that is adapted to the tropical climateand the natural environment of Thailand and inhibit the growth of yeastand CFB, could be more suitable. However, LAB strains as silageadditives for preparing good quality silage in tropical regions are notavailable. Therefore, we hereby screened LAB strains by using amodification of pouch method (Ohmomo et al., 2003) and selected somestrains. Among them, strain SP 1-3 isolated from corn silage prepared inNakhon Pathom showed very good fermentation properties in a pouch asshown in Table 2. The inoculum size of LAB of 103 higher than that ofCFB and yeast enhanced the amount of lactic acid (2.03 times increased at24 hr culture and 1.43 times increased at 5.4 hr culture) and repressed thegrowth of CFB and yeast. The strain SP 1-3 should be a favorable LABstrain for making good quality silage in Thailand.

Table 2 Effect of inoculum size on lactic acid fermentation by strain SP1-3. Medium was consisted of about 2 cm length cut Napiergrass(moisture content 75%) and 1.5% of glucose (w/w). The mediuminoculated with LAB (Lactobacillus plantarum SP 1-3), CFB (Coli- formbacteria: Enterobacter SG 1-1T) and yeast (Saccharomyces serevisiaeSG 2-1Y) was cultured at 45*C, anaerobically. Inoculum size (cfu/g) of A:LAB 102, CFB 102 and Yeast 102; B: LAB 105, CFB 102 and Yeast 102.

Inoculum

size(cfu/g)

Culturetime (h)

Medim

pH

Lactic

acid (%)

Count of microorganisms (cfu/g)

LAB CFB Yeast

A 6

24

504

6.18

4.39

4.05

<0.01

0.393

1.021

3.1x105 1.6x105 3.8x104

5.4x106 1.4x107 4.7x106

1.1x105 3.9x105 9.3x103

B 6

24

504

4.60

3.88

3.84

0.402

0.797

1.461

4.0x106 9.7x104 9.3x103

1.7x108 5.0x104 7.9x105

8.9x106 5.4x105 2.2x103

References

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