The Extracts of Thermal Processed Ginger (Zingiber officinale Rosc.) Rhizome Combined with Honey as

Natural Antioxidant to Produce Functional Drink

Hari Purnomo1, Firman Jaya2* and Simon Bambang Widjanarko3 1 Dept.Animal Food Technology, Faculty of Animal Husbandry, Brawijaya University, Malang, East Java,Indonesia.2 Alumni Post Graduate Program,Faculty of Agricultural Technology, Brawijaya University, Malang, East Java, Indonesia. 3 Dept Food Technology, Faculty of Agricultural Technology, Brawijaya University, Malang, East Java, Indonesia.

ABSTRACT

The thermal processing of 6 minutes and boiling at 100oC gave the best effect of its antioxidant activity of ginger (Zingiber officinale Rosc.) rhizome extracts. Whilst the best functional drink was obtained from the combination of 15 mL honey in 25 mL of ginger water extracts. It had the following properties: free radical scavenger activity of 35.51±0.02%; phenolic total of 1.31±0.018 mg/g; and gingerol content of 0.74±0.010 mg/g; taste score of 4.95; colour score of 7.65; and aroma score of 7.4 by the hedonic scale scoring of 1-9. The analysis with GC-MS identified 1-hexadecene (47.81%); hexadecanoic acid (14.25%); and 9-octadecenoic (15.33%) compounds as antioxidant agents. The FTIR analysis showed that CH2 kel. bid. def. vibration at 816.80 cm-1 wave was found for the hexadecene compound, and OH stretch H-bonded at 2944.13 cm-1 wave were found for the hexadecanoic acid and 9-otadecenoic compounds. It was proven that the functional drink produced by the combination of 15 mL honey in 25 mL of ginger water extracts had been organoleptically accepted by the panelists.

Keywords: ginger rhizome, antioxidant, thermal processing, honey concentration

* Corresponding author address: lone_manutd@yahoo.com

INTRODUCTION

Ginger (Zingiber officinale) rhizome has become a very popular spice and used widely in Indonesian cuisine as well as in other countries. It is a common food additive in a number of foods and beverages and it is valued due to the volatile components especially the aromatic compounds which give a spicy, pungent and pleasant smell. Bartley and Jacobs (2000) noted that these aroma compounds only partially contribute to the flavor of fresh ginger rhizome and the oleoresin content plays an important role for its pungency.

However, there is very little information on the effect of thermal processing on the antioxidant quality of Indonesian local elephant ginger rhizome extract. Jaya (2008) reported that in Indonesian traditional beverages the ginger rhizome was boiled at temperature of around 100ºC for about 20–30 minutes and it is aimed to get a better ginger taste and less bitter taste. He also reported the effect of type and time of themal processing in ginger (Zingiber officinale Rosc.) rhizome antioxidant quality and found that 6 minutes of boiling at 100oC gave the best effect of its antioxidant activity where methyl ester (0.17%), 9-octadecenoic (0.32%), nortrachelogenin (0.30%) were the compounds of antioxidant agents, while zingerone compound total peak area was increased from 5.68% to 6.32%.

Currently, ginger is a common interest of society with high potentially developed as health drink. Combining honey as other antioxidant is one of the ways to maintain ginger drink product in consequence of thermal processing. Antony, Rieck and Dawson (2000) reported the addition of up to 15% honey inhibited the development of oxidative compounds in cooked turkey meat, with little further inhibition observed compared to 20% honey. While the study of Brown, Henderson and Hunt (2006) reported that pinocembrin has been credited with affording the high radical scavenging abilities of such substances as Thai ginger and honey/ propolis. The flavonoids in these products, as well as in a number of pine trees, prove to be antimicrobial as well as effective in vitro antioxidants. It is used as one of the anti-HIV cocktail of drugs.

Honey is known to be rich in both enzymatic and non-enzymatic antioxidants, including glucose oxidase, catalase, ascorbic acid, flavonoids, phenolic acids, carotenoid derivatives, organic acids, maillard reaction products, amino acids and proteins. It was reported that the composition and antioxidant capacity of honey depend on the floral source used to collect nectar, seasonal and environmental factors, as well as processing which also possibly have an effect on honey composition and antioxidant activity. Honey colour depends on the potential alkalinity and ash content, as well as on the antioxidatively active pigments, such as carotenoids and flavonoids (Baltrušaityte, Venskutonis dan Čeksterytė, 2005). It is also noted that colour accounted for over 60% of the variance in antioxidant capacity for the honeys examined (r2 = 0.634), with darker colour having greater antioxidant capacity (D’Arcy, 2005).

Ginger and honey are antioxidant resources that synergistically able to be combined each other. Weiner (1994) reported that some plants or specific combinations of herbs in formulations may act as antioxidants by exerting superoxide scavenging activity or by increasing superoxide dismutase (SOD) activity in various tissue sites. Each of these groups of compounds are

2

substances that may exert that cell-protective action by more than one biochemical mechanism.

However, there is limited information available on the combination of ginger rhizome extracts and honey based on antioxidant activity in Indonesia. The objectives of current study were to find out the potential of honey as natural antioxidant if combined with extracts of thermal processed ginger rhizome to produce functional drink.

1. MATERIALS AND METHODS

1.1 Sample preparation

The thermal processing of 6 minutes and boiling at 100oC was carried out as described by Jaya (2008). Honey as one of the ingredients were obtained from one nectar source, such as kapok tree which is taken in second harvest approximately on May-June. Honey samples were stored at room temperature for several weeks to produce a darker colour, with expectation of a high antioxidant properties will be achieved. The amount of honey used were 15, 30 and 45 mL based on the report of FAO (1996) where 236.58 mL potable water were needed for 47.316 mL honey.

1.2 Determination of antioxidant activity

The mixture of ginger water extract and honey were analyzed for its antioxidant activity using DPPH (1,1 diphenyl-2-picrylhydrazyl) radical scavenging assay as described by Khalaf et al. (2007), total phenol assessed by the method of Miliauskas (2000) and the gingerol content following the method as desribed by Puengphian and Sirichote(2007).

1.3 GC-MS and FTIR

The antioxidant compounds were analyzed using GC-MS (QP2010S-Shimadzu) under the following condition : column used were Rtx-5MS,30 m length and inner diameter of 0.25mm and the initial column temperature was 70oC and final temperature was 280oC (5oC/minute), while the injector temperature was 300oC with split mode injector and split ratio of 72.6 and pressure of 14.0 kPa. The flow rate was 40 mL/minute and the flow within the column was 0.50 mL/minute. The detector temperature was 300oC and using Helium as the gas carrier with EI (Electron Impact); and the samples volume injected was 1µl. Compounds were identified by comparing retention indices/comparing mass spectra of each compound with those of authentic samples and library. While for functional unit determination the Shimadzu Fourier Transform Infrared Spectrophotometer - FTIR-8400S were used.

The IR spectra were recorded on FTIR-8400S (Shimadzu Deutchland GmbH) spectrophotometer in KBr and polyethylene pellets. Samples were weigh-in at 0.01 g and homogenized with 0.01 g KBr anhydrous by mortar agate. The mixture of sample and KBr were pressed by vacuum hydrolic (Graseby Specac) at 1.2 psi to obtain transparancy pellet. Scanned sample

3

passed through infrared, where its continuing wave by detector that connected to computer and gave a description of tested sample spectrum. Samples were usually scanned in the absorption area of 600-4000 cm-1. The results of analysis were consisted of chemical structure, molecular binding form and certain functional group of tested sample as basic of spectrum type.

1.4 Statistical analysis

All statistical analyses were carried out using Microsoft Excel 2003. Analysis of variance (ANOVA) followed by Duncan Multiple Range Test at a level of P<0.01 if there was significant differences between samples. The best treatment was determined by effectivity index method as described by Susrini (2005). Organoleptic test analyzed following non-parametric statistical method by Friedman test (Steel and Torrie, 1989). Identification and in order to elucidate its structure of antioxidant compounds in functional drink was accomplished by using gas chromatography-mass spectrometry (GC-MS) and fourier transform infrared spectrophotometer (FTIR), respectively, with computerized integrated data processing and descriptively disscussed based on literature.

2. RESULTS AND DISCUSSIONS

3.1 Determination of ginger water extracts volume

Ginger water extracts volume of ginger rhizome boiled for 6 minutes at 100oC determined by organoleptical procedure including taste, colour and aroma using hedonic grade test by 30 panelists.

The results showed that the avarage of panelist acceptance to ginger water extracts taste were ranging from 3.2-5.6 (Figure 1). The amount of ginger extract of more than 25 mL had a more pungent sense whilst if less it lost the ginger taste, therefore the panelists prefered as 25 mL sample.

Zingerone were non-volatile compound that contribute to give the pungent sense of ginger. Jaya (2008) reported that analysis of total zingerone compound area of ginger rhizome boiled for 6 minutes at 100oC using GC-MS increased from 5.68% to 6.32%. Friedi (2000) suggested that pungent sense of ginger corresponding with non-volatile compound was the alcohol group of oleoresin.

4

4.43.83.9

4.95.6

43.63.43.2

0

1

2

3

4

5

6

5 10 15 20 25 30 35 40 45

Volume (mL)

Score

4.2 4.2

5.7

3.9

5.1 54.5 4.7

4.2

0

1

2

3

4

5

6

5 10 15 20 25 30 35 40 45

Volume (mL)

Score

Figure 1. Average score of ginger water extracts taste

The score of colour in the organoleptic test showed that the average of panelists acceptance were ranging from 3.9-5.7 (Figure 2),and the best combination treatment was 15 mL volume.

Volatile compound contribute to the colour of ginger, extract boiled at 100oC as described by Jaya(2008) was edulan II compound. This compound was reported as a compound which gave a red colour on tomato, and its form was dark brown concentrated liquid with characteristic odour and taste and contained 15-30% ginger oil (Anonymous, 1999; Hayati, 2005).

The score of aroma showed that the average of panelists acceptance were ranging from 3.3-5.8 (Figure 3) and the best combination treatment was 25 mL volume of ginger water extracts.

Volatile component of ginger rhizome was atsiri oil, which is the compound that gave a typical smell. The main compound that responsible to the aroma was sesquiterpene hydrocarbon volatile oil (Hayati, 2005). Jaya (2008) reported that ginger rhizome boiled for 6 minutes using GC-MS produced geranyl acetate and 3,6-dimethyl-2,3,3a,4,7a-hexahydrobenzofuran, which are volatile components contribute to herbal plants aroma.

Figure 2. Average score of ginger water extracts colour

5

4.34.14.3

55.8

3.83.3

4.24.1

0

1

2

3

4

5

6

7

5 10 15 20 25 30 35 40 45

Volume (mL)

Score

Figure 3. Average score of ginger water extracts aroma

The best treatment of combination between ginger rhizome boiled for 6 minutes and solvent (potable water), using effectivity index method as described by Susrini (2005) was 25 mL volume, where the taste score was 5.6; colour score 5.1; and aroma score 5.8 by the hedonic scale scoring of 1-9.

3.2 Chemical characteristic of honey as raw material

The determination results of honey which will be used for functional drink for its moisture content, HMF value and diastase activity were showed on Table 1.

Table 1. Average of moisture content (%), HMF value (mg/kg) and diastase enzym activity (DN) of honey

Parameters AmountMoisture (%)HMF (mg/kg)Diastase enzym activity (DN)

18.73 ± 0.1230.28 ± 1.11 12.51 ± 1.41

HMF and diastase enzym activity of this honey were 30.28±1.11 mg/kg and 12.51±1.41 DN, respectively. While Bogdanov et al. (2004) reported that before testing these parameters, it should be checked that the HMF content is below 15 mg/kg. Meanwhile, the Indonesian National Standard (2004) noted that HMF value generated was lower than 50 mg/kg. Whereas, Jaya (2008) reported that diastase enzym acivity of several honey in Indonesia was 15.52 DN. Furthermore,Bogdanov et al. (2004) reported that enzym activity of honey decreases after storage.

The moisture content of honey used in this research was 18.73 ± 0.12 %, and Bogdanov et al. (2004) noted that the moisture content is a quality parameter, which affected the physical properties of honey (viscosity, crystallisation) and also influenced the value of the glucose/water ratio. While the Indonesian National Standard (2004) noted that the moisture content should be of 22%. Therefore, the honey sample could be used for functional drink combined with ginger water extracts and potable water.

6

0

1

2

3

4

5

6

7

8

15 30 45

Honey concentration (mL)

Score

4.95 5.05

7.55

3.3 Organoleptic evaluation of functional drink

The result of organoleptic evaluation on taste score combining 25 mL water ginger extract and concentration of honey at various volume (15, 30 and 45 mL) using hedonic scale scoring test by 25 panelists were ranging from 4.95-7.55 (Figure 4). The best concentration of honey was 30 mL, as most of the panelists could accept the pungent of ginger and sweetness of honey. Jaya (2008) reported that the non-volatile compound produced by ginger rhizome boiled for 6 minutes was zingerone, which gave the effect of pungent, and the total compound areas of ginger rhizome boiled for 6 minutes using GC-MS increased from 5.68% (fresh ginger rhizome) to 6.32%.

Sweet sensation of functional drink was due to fructose content in honey. National Honey Board (2005) reported that in most honey, fructose predominates and tends to make honey taste slightly sweeter than sugar. On the average, honey is 1 to 1.5 times sweeter (on a dry weight basis) than sugar. HMF is a compound that results from the breakdown of simple sugars (such as glucose or fructose) at pH 5 or lower. HMF occurs naturally in honey, especially in warm climates.

Figure 4. Average of taste score of ginger-honey functional drink

The colour showed that the average of panelists preference were in the range of 4.8-7.65 (Figure 5). The best concentration of honey was 15 mL, where most panelists preferred yellowish honey colour. The GC-MS analysis of the ginger rhizome water extracts sample showed that it contained edulan II compound which classified as carotenoid group and gave a yellowish-red colour on tomato and carot as reported by Jaya (2008). Zeb and Mehmood (2004) also reported that common carotenoids degradation product as Edulan was found in purple passion fruit, osmanthus, burley tobacco, virginia tobacco which play an

7

01

234

567

89

15 30 45

Honey concentration (mL)

Score

7.65

4.85.1

important potential role in human health by acting as biological antioxidants, protecting cells and tissues from the damaging effects of free radicals and singlet oxygen.

The aroma scores showed that most panelists preference were ranging from 5.05-7.4 (Figure 6). The best concentration of honey was 15 mL, where most panelist accepted the beverage with a slightly pungent aroma. These pungency ginger originated from volatile compounds of ginger rhizome as sesquiterpene hydrocarbon group, such as: α-pinene, camphene, β-phellandrene, ar-curcumene and zingiberene. These evidence were corresponding with the production of other aroma compounds, such as geranyl actetate and ethylgluaiacol from ginger rhizome boiled for 6 minutes using GC-MS as described by Jaya (2008). Bartley and Jacobs (2000) noted that it is also possible that the heat involved in the drying process is sufficient to promote esterification of these terpene alcohols with natural acetic acid (or acetic acid produced by the thermal decomposition during drying). It is significant that citronellyl and geranyl acetates have been implicated by previous workers in imparting important characteristics of ginger flavor.

Figure 5. Average of colour score of ginger-honey functional drink

The carbohydrates found in honey have the ability to improve the intensity of desirable flavors and reduce the intensity of others, which transform starch to other carbohydrates (dextrins, oligo-, di- and monosaccharides) by diastase enzyme (National Honey Board, 2005). The results of diastase enzyme activity in this study were 12.51±1.41 DN larger than noted by the Indonesian National Standard (2004) of 3 DN. These results indicated that starch was transformed as carbohydrate in honey and have the ability to improve the intensity of desirable flavors. However, honey aroma was less promenently than ginger aroma, which is noted by Cuevas-Glory, Pino, Santiago and Sauri-Duch (2007), who reported that aroma compounds were present in honey at very low concentrations as complex mixtures of volatile components of different functionality and relatively low molecular weight.

8

0

1

2

3

4

5

6

7

8

15 30 45

Honey concentration (mL)

Score

7.4

5.05 5.05

The best treatment according to organoleptic test results were the combination of 25 mL water ginger extract and 15 mL honey concentration. This sample had a taste score of 4.95; colour score of 7.65; and aroma score of 7.4 by the hedonic scale scoring of 1-9. Whereas, antioxidant activity as free radical scavenging activity was 35.51±0.02%, total phenolic content was 1.31±0.018 mg/g and gingerol content was 0.74±0.010 mg/g.

Figure 6. Average of aroma scores of ginger-honey functional drink

According to antioxidant activity parameter, the sampel with 15 mL concentration of honey was smaller than the others (30 and 45 mL concentration of honey). However, the selected honey concentration with its low antioxidant activity had been preferred from its sensory properties. It is because honey can be used as sweetener in beverage if used in advisable amount. Terna and Ayo (2002) reported that Kunun Zaki of traditional drink in Nigeria, as the functional food had an acceptable sensory characteristics such as appearance, colour, texture and taste by consumer and gave less contradiction and side effects to other nutrient metabolism if used in advisable amount.

3.4 Determination of antioxidant activity on functional drink

Data in Table 2 showed that the average of free radical scavenging activity using DPPH assay of ginger boiled for 6 minutes (25 mL) combined with honey at various volume (15, 30, 45 mL) were ranging from 35.51 to 42.73%. The percentage of functional drink free radical scavenging activity was smaller than ginger rhizome with/without thermal processing (> 60%) as reported by Jaya (2008). It is possibly due to the combination of ginger water extracts, honey and potable water. Ramadan (2008) noted that the antioxidative properties are correlated not only with the total amount of antioxidants, but also with the presence of selected compounds. The significantly stronger antiradical action of some beverages may be due to the differences in content and composition of bioactives, the differences in kinetic behaviors of potential antioxidants, a synergism of bioactives with other components present in each beverage and the diversity in structural characteristics of potential phenolic antioxidants present.

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In regards of the average of total phenolic in this study it was found that the combination of 25 mL water ginger extract and honey concentration were ranging from 1.30 -2.07 mg/g (Table 2). Schramm and Keen (2005) reported that consuming 150 grams of corn syrup or buckwheat honey could increase the availability of phenolic content. This investigation support the conclusion that phenolic antioxidants from honey are bioavailable and convey antioxidant protection to healthy human subjects.

While, the average of gingerol content was ranging from 0.74-0.96 mg/g (Table 2). The addition of potable water as total solution to dissolve combination of ginger water extract and honey could decrease the gingerol content. Shibuya, Moriwaki and Tsuji (2008) suggested that a water-soluble ginger root extract substantially is low gingerol by subjecting a water extract or a hydrous alcohol extract of a ginger root to isolation and purification such as adsorption treatment.

Table 2. Average of free radical scavenging activity (%), total phenolic (mg/g) and gingerol content (mg/g) combined between ginger water extract (25mL) and concentration of honey at various volume Concentration of honey

(mL)

Average of free radical scavenging activity

(%)

Average of total phenolic(mg/g)

Average of gingerol content(mg/g)

15 35.51 ± 0.02 a 1.31 ± 0.018 a 0.74 ± 0.010 a30 37.65 ± 0.03 b 1.71 ± 0.013 b 0.84 ± 0.017 b45 42.73 ± 0.03 c 2.07 ± 0.018 c 0.96 ± 0.018 c

Means standard deviation in the same column with different letters are significantly different (P 0.01)

3.5 Identification antioxidant compounds of ginger-honey functional drink

A typical gas chromatogram of ginger-honey functional drink is shown in Figure 7 and a list of the compounds identified appears in Table 3. According to %RA, three compounds were found that dominate ginger-honey functional drink, namely as 1-hexadecene, 9-octadecenoic and hexadecanoic acid.

Murphy, Taylor and McCormick (2004) reported that 1-hexadecene has an aromatic rings and is highly resistant to oxidation. Whereas Woollett, Daumerie and Dietschy (1994) suggested that 9-octadecenoic is unsaturated fatty acid groups commonly used which corresponding with drugs, and also biologically neutral and does not regulate the low density lipoprotein (LDL) receptor as the cis isomer does in the hamster. Guillou, Rioux, Catheline, Thibault, Bouriel, Jan, D’Andrea and Legrand (2003) noted that hexadecanoic acid contribute as antioxidant compound and vitamin A where added in low fatty milk to subtitute vitamin that decreased cause by the milk fat removal.

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Retention time (minute)

Respond detector

1-hexadecene

Zingiberene

Zingerone9-Octadecenoic

Hexadecenoic Acid

Figure 7. GC-MS chromatogram of ginger-honey functional drinkVerzera, Campisi, Zappala and Bonnacorsi (2001) reported that

hexadecanoic acid is one of predominating compounds in orange honey and chestnut honey which is having peak areas between 20-40% using GC-MS.

Table 3. Main compound in ginger-honey functional drinkPeak Molecular

Form Name Compound group Amount (%)

5

8

13

17

22

C15H24

C11H14O3

C19H38

C17H34O2

C9H36O2

Zingiberene

Zingerone

1-Hexadecene

Hexadecanoic Acid

9-Octadecenoic

Monocyclic sesquiterpene

Hydroxil phenol

Alkene

Carboxylic acid

Carboxylic acid

0.67

5.15

47.81

14.25

15.33

The infrared spectrum of ginger-honey functional drink and its functional group analyzed using Fourier Transform Infrared (FTIR) was shown in Figure 8. Data in Table 4 showed that wave length range from 615.25 cm-1 to 3339.51 cm-1 were found, and although 13 functional compounds were found but only 11 compounds were identified, as peak no 10 and 11 were not identified (Figure 8). These unidentified peaks were probably due to the relatively low energy vibration which could not be identified by the infrared spectrophotometer.

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Figure 8. Infrared spectrum of ginger-honey funcional drink

The IR spectrum of the compound has been studied in order to elucidate its structure. Data in Table 4 showed that the presence of substituted alkene (816.80 cm-1), alcohol secondary (1146.60 cm-1), aldehyde (1342.36 and 1416.62 cm-1) and keton (1635.52 cm-1) were not exist neither in fresh ginger rhizome or ginger boiled for 6 minutes as reported by Jaya (2008). These functional compounds, excluding alkene, were implied in oxigenated groups, which were identified using GC-MS as 9-octadecenoic (15.33%), hexadecanoic acid (14.25%) and zingerone (5.15%).

1-hexadecene was the predominating compound in ginger-honey functional drink using GC-MS, which is implied in substituted alkene group at 816.60 cm-1. Ruoff (2006) suggested that the most characteristic differrences among honey were observed between 800 and 1500 cm-1. While the largest variation in the spectra of honey types were found in the C-O and C-C stretching regions of the saccharides between 950 and 1050 cm-1.

Table 4. Functional compounds of ginger-honey funcional drink analyzed using FTIR.

No. Wave length (cm-1)

Vibration type Functional compound

12345678910111213

615.25816.80105.81

1146.601258.471342.361416.621455.191635.522078.162360.712944.133339.51

O-H bendCH2 kel. bid. def C-OH stretch C-OH stretch C-O-C stretch vinyl ether H-C=O bend. aliphatic aldehyde H-C=O bend. aliphatic aldehyde Ring aromatic stretch (4p) C=C stretch konj.--OH stretch H-bondedOH stretch; Phenol

PhenolSubtituted alkene Alcohol primer (-CH2OH)Alcohol secondary (-CHROH)Ether (R-O-R); Aldehyde (R-CHO)Aldehyde (R-CHO)C=C AromaticKeton (R-CO-R)--Carboxylic acid (RCOOH)OH

12

50075010001250150017502000250030003500400045001/cm

-10

0

10

20

30

40

50

60

70

80

%T

3339

.51

2944

.13

2360

.71

2078

.16

1635

.52

1455

.19

1416

.62

1342

.36

1258

.47

1146

.60

1059

.81

816.

80

615.

25

Sampel :

3. Conclusion

Honey as natural antioxidant is synergistically able to combine with thermal processed ginger rhizome extracts to produce functional drink. It is concluded that ginger-honey functional drink can be produced which could maintain the antioxidant properties under such conditions, moreover, the products also organoleptically acceptable by the panelists.

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