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JOURNAL OF TROPICAL BIOLOGY AND CONSERVATION 10: 31-41, 2013

Research Article

Biochemical properties of rice wine produced from three different startercultures

*Kishneth Palaniveloo and Charles Santhanaraju Vairappan

1Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation, UniversitiMalaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia.*e-mail csv@ums.edu.my

ABSTRACT. Starter cultures influence aromaand flavour in rice wine production. The

biochemical characterization of rice wineproduced from common and glutinous rice

5using three starter cultures; 1) bitter (1.51x104CFU/g), 2) bitter-sweet (5.5x10 CFU/g), and 3)

5sweet (4.47x10 CFU/g), were investigated.The volume of wine, with pH ranging from 4.3to 4.7, obtained from glutinous rice was twicethe volume of common rice. Glucose levels ofwine from glutinous rice ranged between300.270.28 ~ 440.1429.97 mg/ml, twice ofthat in common rice wine. The wine from

common rice contained higher alcohol(9.960.08 ~ 12.531.35%) as compared towine from glutinous rice. Volatile hydrocarbonsin both rice wines were analysed and reported.Rice wine and fermented rice cakes were testedfor their antioxidant and fibrinolytic activities.However, only fermented rice cakes fromcommon rice displayed positive antioxidant andfibrinolytic activities. Best fibrinolytic activitywas exhibited by a bitter-sweet starter with IC504.670.51 mg/ml and 0.320.01 uni tplasmin/mg.

Keywords: Rice wine, starter cultures,fermentation, antioxidant, fibrinolytic activity.

INTRODUCTION

Fermentation is the oldest transformationmethod used to preserve and enhance flavour,aroma and nutritive values of food (Steinkraus,2002). In this regard, rice fermentation for theproduction of alcoholic beverage is an ancient

process practiced in most Asian countries. Ricewine is widely consumed during social and

cultural events, and is part of offerings for agood harvest, traditional medicine and post-natal recovery (Chiang et al., 2006). TheChineseHuangjiu, Indian Sonti, Japanesesake,and Korean Yakju are a few examples offermented rice wines (Steinkraus, 2002). In ricewine production, starch (sugar) from rice isconverted into alcohol via fermentation byyeast, fungus and lactic acid bacteria (LAB)(Chuenchomrat et al., 2008). The choice of rawmaterial plays a vital role in producing wine of

good quality and volume. Aroma determines thequality of wine produced through the volatilecompounds produced during fermentation(Gobetti et al., 1995). The use of different startercultures with varying microbial content and ricevariety has been associated with the productionof wine with different tastes and flavours.Variety ofSaccharomyces cereviseae has beenreported to produce wine with a diverse flavourprofile (Chen & Xu, 2010). In addition, the typeof rice fermented can influence the quantity andquality of wine. Glutinous rice for instance is arich source of starch, protein and variousmicroelements that are used by microbes duringthe fermentation process to produce more wine(Que et al., 2006). The taste and quality of wineproduced can al so be affected by loca lproducers' recipe depending on availability ofraw materials and starter cultures (Dung et al.,2005).

In Borneo, rice wine is produced and usedduring cultural functions. It is produced using

Received 28 February 2013. Revision accepted 27 March 2013

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BIOCHEMICAL PROPERTIES OF RICE WINE

common and glutinous rice with a variety ofstarters based on preference. The brewers use

three different starter cultures; 1) bitter, 2)bitter-sweet and 3) sweet. The microbial strainsused in each of the cultures are similar but varyin density. The addition of spices such ascinnamon, pepper, dried chili and local herbsresults in the unique flavour of these starters(Gandjar, 2003). However, no scientificinformation is available on the chemicalqualities and nutritional properties of this localbeverage. Hence, this report providescomparative data on the biochemical propertiesof rice wine and cake produced using three

different starters currently unavailable locallyso as to complement the available microbial data(Chiang et al., 2006).

MATERIALS AND METHODS

Fermentation

A total of 3.3 kg glutinous and common riceobtained from Tenghilan, Sabah was used ineach set of fermentation (4 L/jar, 3 jars/set). Rice

was cooked (rice grain : water = 2:1) and cooledat room temperature (28~30C). A total of 80 gof starter culture was mixed with the cooledwhite and glutinous rice. This mixture was putinto jars without additional water and left toferment for four weeks. Three starter cultureswere used; 1) bitter, 2) bitter-sweet, and 3)sweet. After fermentation, rice wine and cakewere separated through filtration. The wine wasstored under refrigeration (< 4C) prior toanalysis and rice cake (1 kg) was extracted inethanol for bioassay and chemical analysis.

Sugar Analysis - High Performance LiquidChromatography (HPLC)

Rice wine and cake extracts were diluted (10x)in distilled water and analysed under theseconditions: Shimadzu low pressure gradient,NH Luna Phenomenex 5 m 100A column2(250 x 4.6 mm ID), RI detector, isocratic mode,mobile phase (80% acetonitrile) at flow rate of 3ml/min, 10 l injection volume, and column

oven at 35C.

32

Gas Chromatography Mass Spectrometer(GCMS) analysis

Alcohol content analysis of rice wine sampleswere done by direct injection of diluted ricewine (5x dilutions) into GCMS using a Q-Plot(30 m x 0.25 mm) column. Instrumentconditions during analysis were as follows: GCtemperature: 100-180C at 5C/min, heliumcarrier gas: 2.88 ml/min, the injectortemperature: 250C, interphase temperature:280C and mass range: 450 a.m.u. Analysis ofvolatile hydrocarbon was done by injection ofconcentrated hexane (solvent-solvent partition

with rice wine and cake extract) into GCMSequipped with BPX 5 (30 m x 0.25 mm).Instrument conditions during analysis were asfollows: GC temperature programme: 50-280C at 3C/min, helium carrier gas: 0.8ml/min, injector temperature: 250C, interphasetemperature: 280C and mass range: 450 a.m.u.Resulting peaks were analysed and compared tothe National Institute of Standards andTechnology (NIST) and Flavour, Fragrance,Natural and Synthetic Compounds (FFNSC)

database.

Microbial enumeration for starter cultureand fermentation

A total 1 g of finely pounded starter culture; 1)bit ter, 2) bitter-sweet, and 3) sweet, wassubjected to 5x dilutions and enumerated fortotal microbial count using spread plate methodas described by Chiang et al. (2006). Twospecific agars were used; 1) de Man, Rogosa,and Sharpe (MRS) agar, 2) Dichloran RoseBengal Choramphenicol (DRBC) agar. Similarenumerations were also conducted on

s t thspecimens of the 1 and 28 day of fermentation.

Antioxidant assay

DPPH (1,1-diphenyl-2-picryhydrazyl) assay(Kumaret al., 2008) was used. Absorbance wasmeasured at 517 nm using a micro-plate reader(Infinite M200, TECAN, Switzerland) and

MAGELLAN software. The Free RadicalScavenging ability was calculated as IC and50

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KISHNETH PALANIVELOO & CHARLES SANTHANARAJU VAIRAPPAN

expressed as ascorbic acid equivalentantioxidant capacity (AEAC) in mg AA/g

amount as follows:

AEAC (mg AA/g) = [IC (ascorbate) / IC50 505(extract)] x 10

Fibrinolytic enzyme assay

Fibrinolytic enzyme assay was carried out basedon a method described by Mine et al. (2005).Fibrinogen solution consisting 0.8% (w/v) ofbovine fibrinogen in 0.8% (w/v) of agarosesolution at 37C was prepared. The solution was

mixed with 0.2% (v/v) of 1 unit/L of thrombinprotease (Amersham Pharmacia Biotech,Netherlands) and 10 ml was poured into a 6 cmglass Petri dish. The Petri dish was left at roomtemperature for two hours to form a fibrin clotlayer. The sample was inserted into pre-punchedhole (5 mm) prior to incubation for 17 hours atroom temperature. The diameter of the clear zonewas measured and expressed in plasmin units.

Statistical analysis

Experimental values were means SD of threereplicates. A multivariate analysis of variancewas performed using SPSS v11.5 for Windows.

Pvalues < 0.05 were regarded as significant.

RESULTS AND DISCUSSION

Fermentation dynamics

Total microbial load of starters

Total microbial load in starters were enumeratedusing DRBC and MRS agars due to theirspecificity to assist the growth of yeast and lacticacid bacteria, respectively (Chiang et al., 2006).Total microbial load of yeast and lactic acidbacteria (LAB) in all three starters showed aconsistent trend as shown in Table 1;sweet>bitter-sweet>bitter for both common andglutinous rice. Total microbial load in sweetstarters were 196.1% and 712.7% more than bitterand bitter-sweet for yeast. Similarly, microbial

load in sweet starters were 285.4% and 757.5%more than bitter and bitter-sweet for LAB.

The total microbial load in starters playsan important role in fermentation dynamics.

Varying bacterial load has proven to affect themicrobial growth during fermentation and itsrelation to the reduction in rice cake weightduring fermentation, volume and pH of wineproduced (Dung et al., 2005). The microbialcontent of each starter culture varied by densitybut the diversity of strains was the same andsimilar to those as reported by Chiang et al.(2006). The differences in the density ofmicrobes of the starters could be due to theinfluence of herbs as promoters or inhibitors ofmicrobial dynamics (Gandjar, 2003). Spices and

herbs are also known to improve aroma, tasteand t o p r even t g rowth o f unwan tedmicroorganisms (Dung et al., 2005). In thisstudy, Saccharomyces cereviseae was theprimary yeast in the starters and was the mostdominant due to its ability to survive in lowacidity levels (Gandjar, 2003). Among the otheryeast found in the starter culture were Candidakrusei, C. pelliculosa, C. utilis, C. sphaerica, C.magno l ia and Rhodo turu la g lu t in i s .

Lactobacillus plantarum andL. brevis were the

major lactic acid bacteria detected in the startercultures as reported by Chiang et al. (2006).

Microbial increment during fermentation

Comparative microbial increment during the 28day fermentation for common and glutinous ricewith the three starters is shown in Table 1. Ricefermented with sweet starter showed the highestincrement of microbial growth, between579.311.2% and 581.727.9% in common ricefor yeast and LAB, respectively. A similar trendwas also observed for glutinous rice at543.534.2% and 559.619.7% for yeast andLAB, respectively. Fermentation using a bitter-sweet starter showed higher microbial growth inglutinous rice as compared to common rice,contrary to the trend showed by sweet and bitterstarters. The overall percentage of microbialincrement for bitter-sweet was almost 50% ofthat shown by the microbes in sweet starterfermentations. On the other hand, microbialgrowth profile in fermentation using bitter

starter was the lowest for both types of rice.

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BIOCHEMICAL PROPERTIES OF RICE WINE34

Table

1.Microbialcountsofstarterc

ulturesandtheirincrementafterfermentationusingcommonandglutinousrice.

DRBC(CFU/g)

B

BS

S

5

(1.510.4

2)x10

5

(2.630.5

4)x10 5

(9.421.9)x10

208.722

.5

5

(2.371.2

1)x10

5

(5.820.7

0)x10

157.813

.6

4

(5.50.85)x10

4

(6.141.76)x10

5

(1.910.70)x10

224.118.7

4

(7.350.39)x10

5

(2.330.51)x10

278.213.7

5

(4.473.27)x10

5

(3.151.65)x10

6

(2.151.10)x10

579.311.2

5

(4.180.53)x10

6

(2.690.33)x10

543.534.2

B

BS

S

5

(1.030.26)x10

5

(2.30.12)x10

5

(8.180.90)x10

229.116.8

5

(2.151.13)x10

5

(6.133.46)x10

185.113.9

4

(4.630.36)x10 4

6.151.30)x10

5

(2.580.14)x10

251.210.4

4

(5.430.72)x10

5

(2.530.27)x10

367.820.9

5

(3.972.98)x10

5

(2.891.48)x10

6

(1.860.94)x10

581.727.9

5

(3.322.39)x10

6

(2.221.64)x10

559.619.7

MicrobialLoad

Day0

Day28

%Increment

Day0

Day28

%Increment

Starter

s

Comm

on

Rice

Glutinous

Rice

MRS(CFU/g)

(Note:

Valuesaremeansstandarddeviatio

ns(n=3).

(B-Bitter;BS-Bitter-Sweet;S-Sweet;DRBC-DichloranRoseBengalChoramphenicol;MRS-Man,RogosaandSharpe)

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KISHNETH PALANIVELOO & CHARLES SANTHANARAJU VAIRAPPAN

This variability can be related to the originalmicrobial load of the starter. The microbial

growth during the fermentation processundergoes the classic dynamics of incrementduring the early stages and eventually decreasestowards the end of fermentation. The microbialgrowths were calculated to reflect the highestincrement of the microbes in the fermentationprocess. The differences in microbial growthprofiles could be associated to the additions ofherbs and spices during the starter preparation(Gandjar, 2003). The sweet starter exhibitedrelatively higher microbial growth due to theaddition of sugar during the starter preparation.

Similarly, bitter and bitter-sweet starters wereprepared using a combination of herbs thatwould have resulted in their interaction with themicrobes incorporated in the starters. Thedifferences in microbial dynamics between ricetypes could be attributed to the total starchcontent and rice type (Dung et al., 2005).

Weight reduction in rice during fermentation

The efficiency of this fermentation could be

predicted based on bacterial dynamics (Table 1)and the reduction rice weight (Table 2). Thehighest reduction was recorded for common andglutinous rice fermented with sweet starterculture. The average percentage of reductionwas calculated to be 57.104.20% and55.104.70%, respectively. This was followed

by bitter-sweet starter culture with a reductionof 52.802.40% and 50.701.80% for commonand glutinous rice, respectively. The lowestreduction in utilisation of rice was recorded forbitter starter cultures with a total reduction of48.102.30% and 46.103.10%. Reduction inthe weight of the rice cake showed a trendrelated to the microbial load of the starters.Starter culture with higher microbial load gavehigher increment of microbes during thefermentation process and this caused significantreduction in rice weight (Wu et al. 2010). Thereduction in weight of common rice was greaterthan of glutinous rice due to the higher contentof starch in the latter. Starch is comprised ofamylose and amylopectin, which degrades into

simple sugar that can be utilised by microbes(Raimbault , 1998). In glutinous rice,

amylopectin is much higher than amylose,compared to common rice. Since amylopectin is

more soluble, glutinous rice is more capable ofabsorbing more water, thus adding to its weight(Raimbaul t , 1998) . Af te r 28 days of fermentation, common rice, being of lowers tarch level would have exhausted i tamylopectin content resulting in greater weightloss (Dung et al., 2007). However, total weightreduction in glutinous rice was more since therewas plenty of starch for utilisation by microbes.

Rice wine volume, pH, alcohol and sugar profile

The rice wine volume, pH and sugar profiles areshown in Table 2. Fermentation with sweetstarter culture produced the highest amount ofwine, followed by bitter-sweet then bitter startercultures. There was also a consistent trend thatglutinous rice produced twice the wine volumeof common rice. In bitter starter culture,glutinous rice wine was 209% more thancommon rice and 228% more when bitter-sweetstarter culture was used. Fermentation usingsweet starter culture produced 205% more wine.

Relatively higher wine volume could beassociated to the much higher starch content inglutinous rice comparatively (Que et al., 2006).

The pH of the produced wines were in therange of 4.3 to 4.7 and in accordance topublished reports (Chiang et al., 2006). Wineproduced using sweet starter cultures recordedpH of 4.5 and 4.7 for common and glutinous ricerespectively. However, wine produced usingbitter-sweet starter cultures had a slightly lowerpH level of 4.3 and 4.4 for common andglutinous rice. The low pH level in the wine iscontributed by the presence of alcohol, variousorganic acids and by products during theanaerobic process (Chiang et al., 2006).

Sugar profiling in the produced rice winewas performed using High Performance LiquidChromatography (HPLC). Resulting peak wascompared to sugar standards; maltose, lactose,glucose, galactose and fructose. The sugardetected in the wine sample was glucose. Rice

wine from common rice fermented with sweetstarter culture contained the highest amount of

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Table

2.Biochemicalpropertiesofricewinebystartercultureandricetype.

pH

VOLUME

(L)

4.270.06

4.330.20

4.530.08

4.700.23

4.370.11

4.720.27

1.400.10

0.730.25

2.000.00

3.000.00

1.670.58

4.000.00

12.531.35

11.481.40

9.960.08

8.380.00

7.920.12

7.090.10

Ic

(mg/ml)

50

120.640.21

270.420.32

310.335.83

300.270.28

320.250.00

440.1429.97

21.001.73

20.030.12

19.620.58

68.000.01

61.210.58

70.000.01

5.260.42

5.490.03

5.610.23

1.620.01

1.790.04

1.570.01

C-Bitter

C-Bitter-Sweet

C-Sw

eet

G-Bitter

G-Bitter-Sweet

G-Sw

eet

DPPH

DPPH

IC

wascalculatedusing10mlofricewine.

50

(C-co

mmon,G-glutinous;Valuesaremeansstandarddeviations(n=3).Valuessignificantlydifferent(p 18%/mg) in both rice cakes. Thedetection of acetic acid (ca. 4%/mg) was evidentin both the analysed rice cake samples. Aceticacid explains the sour feeling in the rice cake.All rice cake samples contained ethyl oleate,while glutinous rice cakes had an additionalaldehyde (14-hexadecenal) and 3-eicosene.Hydrocarbons were much lower in compositioncompared to rice wine samples, mainly rangingfrom 1-3%/ml. Differences in the volatileorganics were significant between rice typesrather than starter cultures.

Hydrocarbons produced in thesefermentations are mainly influenced by the type

of rice. The hydrocarbons formed duringfermentation could have originated from the

outer membranes of rice grains or intermediateproduc ts of fe rmen ta tion. However, the

presence of yeast and LAB which is introducedthrough the starter culture plays an importantrole since volatiles are byproducts offermentation by these microbes (Chen & Xu,2010). Detection of lactic acid is a goodindicator of the presence and importance ofLAB in these fermentations (Gobetti et al.,1995). This is supported by the 14%/ml averagecomposition of lactic acid detected in the ricewine samples.

In addition, butanediol in both rice wine and

cake samples is a byproduct during thebreakdown of pyruva te during ethanolproduction by yeast. This alcohol is a commonproduct from the fermentation of sugar and can bedetected in most alcoholic beverages undergoinganaerobic microbial fermentation. Mateo et al.(2001) also detected this compound in winesamples they tested. It is a common byproduct inanaerobic microbial fermentation. The otheralcohols detected in the wine samples wereisoamyl alcohol, 1-butanol and phenylethyl

alcohol. Isoamyl alcohol is a byproduct producedby almost all strains of S. cerevisiae(Chuenchomrat et al., 2008), while the presenceof phenylethyl alcohol could explain the aroma inthe wine produced (Chen & Xu, 2010).Phenylethyl alcohol was reported to be acompound produced by strains of commercial S.cerevisiae (Chen & Xu, 2010) and it gives outfloral odour. On the other hand, 1,3-dioxolanedetected in the rice wine samples is a productfrom the acetalisation of acetaldehydes andglycerol. This compound could be an indicator ofthe wine age (Camaraet al., 2003).

Antioxidant (DPPH) and fibrinolytic enzymeassays

DPPH radical scavenging activity of rice wineand cake extracts was expressed by thepercentage reduction (IC ) of the radical ion by50samples. In rice wine, the best radicalscavenging activity was observed by sweetstarter (IC , 19.620.58 mg/ml), followed by50bitter-sweet (IC , 20.030.12 mg/ml) and50bi tter (IC , 21.001.73 mg/ml) . As for50

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BIOCHEMICAL PROPERTIES OF RICE WINE

glutinous rice wine, the activity was displayedusing bitter-sweet starter (IC , 61.210.5850

mg/ml), followed by bitter (IC , 68.000.0150mg/ml) and sweet (IC , 70.000.01 mg/ml).50The radical scavenging potential of rice cakeswere also tested and common rice cakefermented with bitter-sweet starter exhibited thebest activity (IC , 1.870.20 mg/ml), followed50by bitter (IC , 4.000.80 mg/ml) and sweet50(IC , 4.480.20 mg/ml) starter. Meanwhile,50glutinous rice cake fermented with bitter-sweetstarter displayed the best activity (IC ,504.600.32 mg/ml) followed by sweet (IC ,5010.801.15 mg/ml) and bitter (IC , 11.200.3550mg/ml). Radical scavenging activity of ricewine and cakes were compared to ascorbic acid(IC , 0.0110.01 mg/ml) and is expressed in50Ascorbic Acid Equivalent Antioxidant Capacity(AEAC). Data is displayed in Tables 2 and 3.

Based on these findings, both commonrice wine and cake displayed better antioxidantproperties compared to glutinous rice. Theantioxidant activity can be related to the type ofrice. Rice contains phytochemicals such as

tocopherols, oryzanol and polyphenols (Rohrer& Siebenmorgen, 2004). Polyphenols, such asvanillic acid, quercetin and other phenolics areknown to be potent antioxidants which have thepotential to be protective against cardiovasculardiseases (Que et al., 2006). In addition, lowmolecular weight polysaccharides, proteins orpeptides are also known to influence theantioxidant properties (Kumaret al., 2007).

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Fibrinolytic enzyme assay

Fibrinolytic enzyme is a major component infermented food (Ashipala & He, 2008). Only ricecakes that showed activities and results are shownin Table 3. Fibrinolytic activity was expressed inplasmin units. Common rice cake extractsfermented with bitter-sweet starter displayed thebest fibrinolytic activity (0.320.01 unit/mg),followed by sweet (0.280.01 unit/mg) and bitter(0.230.00 unit/mg). In glutinous rice cake, thestrongest inhibition was observed using bitter-sweet starter (0.170.00 unit/mg) followed bysweet (0.160.00 unit/mg) and bitter (0.140.01

unit/mg). The use of a different starter did notinfluence the fibrinolytic enzyme activity.Instead, the choice of rice showed a cleardifference with common rice displaying anactivity twice, as shown by glutinous rice.

Fibrinolytic enzymes have been closelyassoc ia ted wi th fe rmenta t ion re la tedmicroorganisms from the genusBacillus (Wong &Mine, 2004). The presence of fibrinolytic enzymedisrupts the formation of fibrin clots in the blood

circulatory system, thus aiding in the restoration ofblood clots. Since commercial enzymes areexpensive, fermented food could be an alternativeoption. Oral administration of fermented food (ricecake) containing fibrinolytic enzyme couldpromote the introduction of plasminogen activatorin humans as a means to disrupt the formation offibrin clots (Wong & Mine, 2004), directly helpingto reduce cardiovascular problems.

Table 3. Antioxidant and fibrinolytic enzyme activities of rice cake by starter culture and rice type.

C- Bitter

C - Bitter-Sweet

C - Sweet

G - Bitter

G - Bitter-Sweet

G - Sweet

4.00 0.80

1.87 0.20

4.48 0.20

11.20 0.35

4.60 0.32

10.80 1.15

3(5.67 1.16) x 10

3(11.87 1.37) x 10

3(4.93 0.23) x 10

3(1.96 0.06) x 10

3(4.81 0.34) x 10

3(2.03 0.23) x 10

0.23 0.00

0.32 0.01

0.28 0.01

0.14 0.01

0.17 0.00

0.16 0.00

Ic50 (mg/ml) AEAC (mgAA/g)FIBRIN (unit/mg)DPPH

DPPH IC was calculated using 2.5 g of rice cake.50(C- common, G- glutinous; Values are means standard deviations (n=3). Values significantly different(p

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KISHNETH PALANIVELOO & CHARLES SANTHANARAJU VAIRAPPAN

CONCLUSION

Biochemical properties of rice wine wereinfluenced significantly by the type of rice ratherthan the starter culture. Glutinous rice producedtwice more wine compared to common rice andthe wines were much sweeter due the presence offree sugar, glucose. The alcohol level is at least 40~ 50% higher in common rice compared toglutinous rice. It was also shown that bitterstarters produced highest alcohol level but lowestvolume of wine regardless of rice type. Thereduced wine volume could have resulted in thehigher alcohol level of wine produced in batches

fermented with this starter. On the other hand, gaschromatographic analysis revealed that thevolatile hydrocarbons were almost similar incomposition. In addition, rice cake samplesdisplayed positive antioxidant and fibrinolyticproperties making it healthy for consumption.Therefore, through this study, it could beconcluded that using different rice types anddifferent starters produce significant differencesin the quality of wine. It also produces rice cakeswith high bioactive potentials that could be

utilised as a functional food.

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