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Materials and Methods

Juice extractionFresh M. citrifolia were blended at a ratio of 1:1

(water: fruit). The blend was ltered using a cottoncloth and centrifuged at 4000 rpm for 20 min. Then

the extract was sterilized by autoclaving at 121o

C for15 min and was stored at 4 oC.

Yeast strainsS. cerevisiae ATCC 62418 was purchased from

the American Type Culture Collection (ATCC62418). Yeast cultures were routinely grown at 30Cin Yeast Peptone Dextrose (YPD) medium (Suutari etal ., 1996).The yeast was stored in 10% (v/v) glycerolat -18C as a culture stock (Sherman et al. , 1991).

Fermentation processThe yeast inoculum size used for fermenting the

extract were 0, 1.5, 3, 4.5 and 6% v/v. Fermentationwas carried out, in capped sterile asks containing 100ml of autoclaved M. citrifolia extract. The inoculatedextracts were incubated at different temperatures: 30,33.5, 37, 40.5 and 44 oC; substrate concentration: 40,50, 60, 70 and 80% w/v; and fermentation time: 0, 1.5,3, 4.5 and 6 days. The treatment combinations were

based on the Central Composite Rotatable Design asshown in Table 1. The fermented M. citrifolia extractwas subsequently analyzed for physico-chemical

characteristics.Total sugar content

Total sugar content was determined by phenol -

sulphuric methods (Dubois et al. , 1956). A standardcurve was prepared to quantify the total sugar content

by glucose.

pH Measurement of pH value was done in room

temperature using a pH meter (Model PHM 210,Radiometer Analytical). For each measurement, 10ml of samples were used (AOAC 1990).

Titratable acidityAcidity was expressed as g of citric acid per 100

g of extract. Tests were done using 5 ml of sampleand titrated with 0.1N NaOH with phenolphthalein asindicator (Cheng et al. , 2007).

TurbidityTurbidity was determined using

Spectrophotometer (Model Spectronic 20 PRIM,Secomam) by measuring the absorbance at 580 nm.Distilled water was used as a reference (Sin et al .,2006).Total soluble solid

Total soluble solid was measured using a hand-held refractometer (Model ATAGO, Japan) with arange of 0-50% Brix.

Total polyphenol content Total polyphenol content was determined using

the Folin-Ciocalteu reagent (Shahidi and Naczk,1995), which contains sodium phosphomolibdateand sodium tungstate. The epicatechin (EAE) (g /

Table 1. Treatment combination and responses

Run Actual and coded ( ) v ariable level pH (y1 ) Titrata blea cidity (y2 ) Turbidity (y3 ) Total Soluble Solid (y4 ) Total Phenolic Content (y5 )

1 0 (60) 0 (3) 0 (37) -2 (0) 3.85 2.24 2.37 5 1092

2 1 (70) -1 (1.5) -1 (33.5) -1 (1.5) 3.87 3.2 4.47 6 1112

3 1 (70) 1 (4.5) 1 (40.5) -1 (1.5) 3.89 3.2 3.85 5.5 10684 -1 (50) -1 (1.5) -1 (33.5) 1 (4.5) 3.82 2.56 3.9 4.5 4805 0 (60) 2 (6) 0 (37) 0 (3) 3.91 2.88 4.96 6 680

6 -1 (50) 1 (4.5) -1 (33.5) 1 (4.5) 3.9 2.56 4.06 4 3087 1 (70) -1 (1.5) 1 (40.5) 1 (4.5) 3.87 3.52 5.4 6 7328 0 (60) 0 (3) 2 (44) 0 (3) 3.88 2.88 4.91 5 4369 -2 (40) 0 (3) 0 (37) 0 (3) 3.91 1.92 3.03 4 206

10 -1 (50) -1 (1.5) 1 (40.5) -1 (1.5) 3.86 2.56 4.47 4 92211 -1 (50) -1 (1.5) 1 (40.5) 1 (4.5) 3.88 2.56 4.03 5 43612 0 (60) 0 (3) 0 (37) 0 (3) 3.88 2.88 4.08 5 682

13 -1 (50) 1 (4.5) 1 (40.5) -1 (1.5) 3.91 2.24 4.15 4 78414 2 (80) 0 (3) 0 (37) 0 (3) 3.88 3.52 7.49 7 954

15 -1 (50) -1 (1.5) -1 (33.5) -1 (1.5) 3.85 2.56 3.72 4.5 46416 0 (60) 0 (3) 0 (37) 0 (3) 3.85 2.56 4.77 5 49817 0 (60) 0 (3) -2 (30) 0 (3) 3.86 2.88 4.95 5.5 516

18 1 (70) -1 (1.5) 1 (40.5) -1 (1.5) 3.84 3.2 5.87 5.5 84419 0 (60) 0 (3) 0 (37) 0 (3) 3.87 2.88 4.41 5.5 54620 0 (60) 0 (3) 0 (37) 2 (6) 3.85 2.88 0.5 6.5 91821 1 (70) 1 (4.5) -1 (33.5) 1 (4.5) 3.87 3.2 3.68 6 95422 1 (70) -1 (1.5) -1 (33.5) 1 (4.5) 3.81 3.2 4.9 6 88423 0 (60) 0 (3) 0 (37) 0 (3) 3.86 2.88 4.47 5 716

24 1 (70) 1 (4.5) -1 (33.5) -1 (1.5) 3.91 3.2 3.34 6 826

25 -1 (50) 1 (4.5) 1 (40.5) 1 (4.5) 3.92 2.56 3.66 4 15826 1 (70) 1 (4.5) 1 (40.5) 1 (4.5) 3.89 3.2 6.15 6 61027 0 (60) 0 (3) 0 (37) 0 (3) 3.89 2.88 4.45 5.5 65628 0 (60) -2 (0) 0 (37) 0 (3) 3.85 2.88 5.64 5 742

29 -1 (50) 1 (4.5) -1 (33.5) -1 (1.5) 3.93 2.24 3.62 4 43630 0 (60) 0 (3) 0 (37) 0 (3) 3.86 2.88 5.6 5 838a: X 1 = substrate concentration (%) w/v; X 2 = inoculum size (%) v/v; X 3 = temperature (

oC); and X 4 = fermentation time (days)

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ml) stock solution was prepared prior to use.

Experimental designA central composite rotatable design (CCRD)

was used to allocate treatment combinations in thisexperiment (see Table 2) using Design Expert Version

6.0.10 (Stat-ease, Inc) software. The independentvariables were substrate concentration, X 1 (40, 50, 60,70 and 80%) (w/v), inoculum size, X 2 (0, 1.5, 3, 4.5and 6%) (v/v), temperature, X 3 (30, 33.5, 37, 40.5 and44 oC) and fermentation time, X 4 (0, 1.5, 3, 4.5 and6 days). Each independent variable had ve levels:-2, -1, 0, +1 and +2. A total of thirty combinations(including ve replicates at the center point with eachvalue coded as 0) were chosen in random order. Theactual and coded values (x) of the four independentvariables together with the responses are shownin Table 1. The responses functions (y) were pH,titratable acidity, turbidity, total soluble solid andtotal polyphenol content.

The analysis of variance (ANOVA) tables weregenerated and the effect and regression coef cientsof individual linear, quadratic and interaction termswere determined. The signi cance of all terms in the

polynomial was judged statistically by computing theF-value at a probability (p) of 0.05. The regressioncoef cients were then used to make statisticalcalculation to generate contour maps from theregression models.

Results and Discussion

The models and analysis of variance for veresponses, namely pH, titratable acidity, turbidity,total soluble solids and total polyphenol content are

presented in Table 2. The high R 2 values (> 0.80)for all responses indicate a good agreement betweenthe experimental results and the theoretical values

predicted by the model (Weisberg, 1985). The lack-of- t was not signi cant (p > 0.05) for all the ten

responses. Based on the results in Table 2, models ofthe parameters showed that they t the model well andthus represented the experimental data adequately.The coded and actual models for all responses are

presented in Table 3.

Total sugar content Total sugar content was determined in the

M. citrifolia extract where the value is 49.9 g/L.According to the Chunghieng (2003), the glucoseand fructose content of the fruit were 11.9 0.2g/L and 8.2 0.2 g/L respectively. While EuropeanCommission (2002), the glucose and fructose contentof the fruit were 3 4 g/100 g and 3 4 g/100 g.

pH From Table 2, b 1, b 2, b 3, b 11, and b 34 were

signi cant (p < 0.05) for pH value. Substrate

Table 2. Statistical analysis of models representingthe response surface of pH, titratable acidity, turbidity,total soluble solids and total polyphenol content duringthe fermentation process of M. citrifolia extract by S.

cerevisiaeResponse Model P value R 2 Lack-of-fit Test P value

pH

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concentration showed a negative linear effect on pH where pH decreased with increasing substrateconcentration. Higher substrate concentration

produced lower pH due to the higher acid contentin the extract. Farine et al. (1996) reported that 83%of the volatile components in M. citrifolia consist ofcarboxylic acid. At the same time, the signi cant (p< 0.05) quadratic coef cient (b 11) suggests that thereis a maximum point for this model. Figure 1 showsthat pH value of fermented M. citrifolia extract as afunction of temperature and fermentation time for50% substrate using 3% inoculum size. At xedsubstrate concentration, pH of fermented M. citrifoliaextract increased with increasing temperature. Within

the experimental region, the effect of temperatureshowed a clear relationship with pH (signi cant pHand temperature interaction) (Arroyo-Lopez et al.,2009). The interaction effect between fermentationtime and temperature showed a signi cant positive(p < 0.05) suggesting that the effect of temperatureduring fermentation S. cerevisiae depended onrelationship fermentation time during the speci ctemperature.

Titratable acidityFrom Table 2, it may be observed that only

substrate concentration (b 1) and fermentation time(b4) have a signi cant effect on titratable acidity werea positive linear effect was observed. This shows thattitratable acidity increased with increasing substrateconcentration. Increase substrate concentrationmeans increased concentration of fruit acids, henceof higher pH. The positive linear effect of substrateconcentration on titratable acidity was in agreementwith values of pH. Laopaiboon et al. (2009) reportedthat pH of the sweet sorghum juice at all condition wasslightly decreased to 4.5 after 12 hours fermentation

and relatively constant afterward. It showed that thetitratable acidity increased as the pH decreased.

TurbidityTurbidity in fruit juices can be a positive or a

negative attribute depending on the expectationof the consumers (Hutchings, 1999). In the case of

M. citrifolia juices as well as apple juices, the clear juices are more desirable and acceptable by theconsumers. Inoculum size (b 2) and temperature (b 3)have signi cant (p < 0.05) linear effect on turbidity.Fermentation time (b 4) showed a signi cantlyaffected the turbidity in quadratic manner. Inoculumssize showed a negative linear effect on turbidity.This shows that turbidity decreased with increasinginoculums size. S. cerevisiae has been reported ascontaining chitin and chitosan in their cell wall andsepta (Gooday 1993), named fungal chitosan where

proved highly effective in reducing the apple juiceturbidity and gave lighter juices than the sample treated

with shrimp chitosan (Rungsardthong et al ., 2006).Fermentation time had a negative effect on turbidityat quadratic terms, showing a highly signi cant levelof p < 0.05. Absorbance has been reported to decreasefrom initial in the fermentation of lingonberry juice

by S. cerevisiae (Visti et al., 2003). From Table 2,temperature showed a signi cant (p < 0.05) positivelinear effect on turbidity which indicates that turbidityof fermented M. citrifolia extract increased withincreasing the temperature during the fermentation.This observation maybe due to the formation of haze

particles as the time of incubation increased (Visti etal ., 2003).

Total soluble solid From Table 2, only substrate concentration (b 1)

and inoculum size (b 2) signi cantly affected totalsoluble solid. Substrate concentration had a positivelinear effect on total soluble solid at a signi cant levelof p < 0.05. Thus, total soluble solid of fermented M.citrifolia extract increased with increasing substrateconcentration. As stated previously in titratableacidity, increasing the substrate concentrationcaused increased amount of total soluble solid in theextract. Total soluble solid was observed to decreasesigni cantly with increasing inoculum size duringfermentation. Visti et al. (2003) reported that citricacid and total sugar concentration in lingonberry

juice decreased during fermentation due to increaseof required total amount of yeast.

Total polyphenol content From Table 2, it was observed that total

polyphenol content was signi cantly (p < 0.01)

affected by the linear effect of substrate concentration(b1) and fermentation time (b 4). Increasing the

DESIGN-EXPERT Plot

pHX = C: TemperatureY = D: Inc Time

Actual Factors A: Subs Conc = 50.00B: Inoculum = 3.00

3 . 8 5 5 7 1

3 . 8 6 5 5 1

3 . 8 7 5 3

3 . 8 8 5 0 9

3 . 8 9 4 8 8

p H

3 3 . 5 0

3 5 . 2 5

3 7 . 0 0

3 8 . 7 5

4 0 . 5 0

1 . 5 0

2 . 2 5

3 . 0 0

3 . 7 5

4 . 5 0

C : T e m p e r a tu r eD : I n c T i m e

Figure 1. pH value of fermented Morinda citrifolia as afunction of temperature and fermentation time for 50%

substrate using 3% inoculum concentration

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substrate concentration would produce highertotal polyphenol content. However, increasingthe fermentation time could result in reduction oftotal polyphenol content. Table 2 also indicated asigni cant (p < 0.05) interaction between substrateconcentration and temperature. Figure 2 shows thattotal polyphenol content of fermented M. citrifoliaextract as a function of substrate concentration andtemperature using 3% of inoculum size for 3 days(72 hours) of fermentation. At xed temperature,

total polyphenol content of fermented M. citrifoliaextract increased with increasing amount of substrateconcentration of M. citrifolia. Increased substrateconcentration corresponds with an increase in theamount of M. citrifolia fruits that were used in thesamples. It has been described that M. citrifolia fruitscontain some bioactive components such as phenoliccompounds in particular coumarins and avonoidsand iridoids (Potterat et al., 2007). Dang et al .(2010) also reported that a few other compoundssuch as scopoletin, quercetin and rutin occurring insigni cant, although much less quantities. Taviriniet al. (2008) told that fruits with high antioxidantactivity generally contain large quantity ofantioxidant substrate especially phenolic compounds

and speci cally avonoids. Figure 3 shows that total polyphenol content of fermented M. citrifolia extractas a function of temperature and fermentation timefor 60% substrate using 3% of inoculum size. Athigher fermentation time approximately more than3 days, total polyphenol content decreased withincreasing the temperature. Despite the popularity ofcommercially available fermented M. citrifolia juice,fermentation process greatly decreased the radicalscavenging activity (RSA) of the product within2 weeks. The total phenolics of M. citrifolia juiceduring fermentation are stable at 1.952.41 mg ml -1

for 10 weeks then decreases by 30 40% from 10 to12 weeks (Yang et al. , 2007). However, in this study,total polyphenol content of M. citrifolia graduallydecreased during 6 days of the fermentation process

by S. cerevisiae .

Conclusion

The methodology of the experimental designwas shown to be very useful for the evaluation offour responses for fermentation of M. citrifoliaextracts by S. cerevisiae. The different conditions(substrate concentration, inoculum size, temperatureand fermentation time) for the fermentation showedthat all these variables markedly affect the pH,titratable acidity, turbidity, total soluble solid andtotal polyphenol content. From the results, pH,titratable acidity, turbidity, total soluble solids andtotal polyphenol content were successfully tted withmathematical models.

Acknowledgements

The authors would like to thank UniversitiMalaysia Terengganu for the nancial assistance andscholarship and Universiti Kebangsaan Malaysia(UKM) for their nancial support under theINDUSTRI-2011-004 Research grant.

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DESIGN-EXPERT Plot

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3 4 7 . 3 5 9

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6 0 8 . 0 1 9

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