A Study of Chemical Compositional Characteristics of Citrus Fruits for  D- Limonene, Organic acids, Minerals and Sugars.

 

Sugandha D. Garway,  Dattatray  G. Garway,  Yogendra T. Gaikwad, Ramiz M. R. Azad, Yogesh B. Dhokey, Sandeep N. Patel, Girish H. Pandya*

Research and Development Division, Anacon Laboratories Pvt Ltd, FP-34, 35, MIDC, Butibori, Nagpur 441122, India

*Corresponding Author E-mail: pandyagh@rediffmail.com

ABSTRACT:

In this study, the chemical compositional characteristic of citrus fruits sampled from the central part of India has been carried out. Sugars, organic acids and minerals have been estimated in citrus fruits. D-limonene was also studied in orange and sweet lime fruit rinds and juice using GC/MS. D-limonene content in rinds of orange fruits ranged between 13.72 to 34.55 g kg-1 and in lime fruits between 17.33 to 38.71 g kg-1. The ratio of citric acid to isocitric acid ranged between 78.9 to 129 confirming the authenticity of fruit juices. Minerals such as Ca and P were also analyzed using ICP-AES. Ca/P ratio is useful for studying the nutritional requirements of the fruit juices. A probable mechanism of D-Limonene formation in fruits is also discussed.

 

KEYWORDS: orange, rinds, fruit juice, food analysis, organic acids, GC/MS, LC/MS, HPLC limonene, sugars, and minerals.

 


 

INTRODUCTION:

Citrus fruits are the most popular ones for consumer throughout the world due to their pleasant flavors and nutritional value. The fruits are both consumed fresh as well as industrially processed. The pulps, which are rich in soluble sugars, significant amounts of vitamin C, fiber and different organic acids, are mainly used to process into juice. Orange peel containing abundant fragrant substances are extensively applied for processing into essential oils which are used commercially for flavoring foods, beverages, perfumes, cosmetics, etc.

 

Analytical research on the various chemical compounds in orange fruit has been carried out for many years. Many researchers have studied the volatile compounds of this fruit using different analytical methods. The presences of terpenes with traces of oxygenated components in the orange essential oil and in its rinds have recently gained importance due its natural pesticide and insect repellent properties. One of the terpene i.e. Limonene has also been studied for its anti-carcinogenic properties1

 

Orange oil also contains a considerable amount of D-limonene, and has numerous applications including a combustant in engines 2, a powerful degreaser in cleaning applications, and a natural pesticide.

 

Nagpur city and its surroundings being central part of India, is a centre for cultivation of citrus fruits such as orange and sweet lime. Both orange (citrus sinensis) and the and sweet lime (citrus limetta) fruits contain significant amount of D- limonene in rinds and in their core fruit juice. The fruit juice under investigation is also likely to contain significant amount of organic acids such as citric and isocitric acids. Analysis of these acids helps in determining the authenticity of citrus juices. In the present investigation samples of orange and sweet lime were collected from orchards around Nagpur throughout the year and analyzed for D-limonene in rinds and the juice extract was subjected to analysis of organic acids as well as sugar (glucose, fructose, and sucrose) levels. Changes in organic acid levels were related to the citric /isocitric acid ratio for determining the adulteration of juices, if any. The orange and lime fruits also contain essential minerals such as calcium and phosphorus (as phosphate) and they are in many cases dissolved in cellular juice. Ca/P ratio thus helps in selecting the fruits. Besides the fruit samples, readily available orange and lime juice in tetra pack were also analyzed for acids, sugars and minerals. The results are discussed in terms of the citric/isocitric ratio, Ca/P ratio and the limonene contents in the samples. Over 30 samples were analyzed for D-limonene using advanced GC/MS technique. A probable pathway for formation of D- limonene in the orange fruits is also discussed.

 

MATERIALS AND METHODS:

Chemicals and standards:

D-limonene stock standard (Sigma Aldrich) of 1000 mgL-1 was prepared in n-Hexane. Working standard of 5, 50, 100, 150, 200, 250, 300 and 350 mgL-1 was prepared from the stock standard in n-Hexane. These were used for preparing calibration curve for D-limonene.

 

Glucose, fructose and sucrose standard stock solutions (MERCK, India) of 1000 mgL-1 each were prepared in aqueous acetonitrile. Working standard of 50,100, 250,500 mgL-1 of each was prepared separately from the stock standard in aqueous acetonitrile. These were used for preparing calibration curve for glucose, fructose and sucrose.

 

Citric acid stock solution (MERCK, India)  and Isocitric acid  stock solution ( Sigma-Aldrich, USA) of 1000 mgL-1 was prepared in aqueous acetonitrile .Working standard of 10, 50, 100, 200 mgL-1 was prepared from the stock standard in aqueous acetonitrile.These were used for preparing calibration curve for citric acid and isocitric acid.

Calcium standard stock solution (CertiPURE, MERCK, Germany) with a concentration of 1000 mgL-1 was diluted to prepare a working standard of 100 mgL-1  by accurately diluting the stock standard solution with double distilled water with a conductivity of 0.5μS cm-1. Calibration standards were prepared in the range 5 to 40 mg L-1.

 

Sample preparation:

Samples of orange and lime fruit rinds were carefully cut using a razor blade. The samples were checked to ensure that none of the white flesh under the rind was included in the sample. The white flesh contributes to the mass of the sample but contains little D- limonene, this makes the rinds appear to have a lower limonene concentration. Each sample was cut down to a mass of approximately 0.1 g. The rind samples were each placed in vials with 10 mL of n-Hexane. The vials were shaken in a sonicator for 5 minutes and then allowed to stand for additional 5 minutes. After the 10-minute extraction was complete.

 

The juice was prepared in the laboratory by squeezing the fruit on a special instrument called a "juicer" or a "squeezer." The juice was collected in a small tray underneath. Second set of samples of orange and lime juice were obtained from market. These were frozen orange juice concentrates made from freshly squeezed and filtered orange juice. The samples of orange and lime juice were filtered through Whatman Filter 42 and diluted (1:1 v/v) with water. Similarly, samples were prepared for determining the concentrations of glucose, fructose, sucrose, citric acid and isocitric acid in samples. For calcium and phosphorus, the samples of the fruits and juice were ashed at 450 0C to remove the organic content and the residue containg the minerals were dissolved in HCl (1%) and HNO3 (0.2%).

 

Analysis:

The analysis of the standards and samples for D-Limonene in fruits rind and  juice was performed with Thermo Scientific GC/MS. GC was fitted with a capillary injector port using a 4-mm standard glass liner packed with quartz wool configured for split operation.

 

Isocitric acid and the sugars (glucose, fructose, sucrose) were determined by LC/MS/MS.

 

Citric acid in the samples was determined using HPLC with Diode Array Detector.

 

Mineral elements such as Ca and P were analyzed simultaneously by ICP-AES.The method becomes advantageous as the other colorimetric and AAS method for these elements consume time and are analyzed one by one. ICP provides useful information on the relationship of elements in the samples. In this study ICP has been extensively used to estimate Ca and P (as phosphate) concentration in fruit juices.

 

Apparatus:

A Thermo Trace GC Ultra gas chromatograph (Thermo Fisher Scientific Instruments, San Jose, CA95134,USA) with electronic flow control (EFC) was used. A Thermo Fisher Scientific TSQ Quantum GC triple quadrupole mass spectrometer was coupled to the gas chromatograph. Samples were injected with a 1 ul syringe, into a split/splitless septum-equipped injector. A fused-silica analytical capillary column TR 5 – MS, 30m x 0.25mm x0.25 μm from Thermo Scientific was used .The mass spectrometer was operated in electron ionization (EI) mode at 70 eV. NIST library was applied for identification of D-limonene. The mass spectrometer mass scale was calibrated with perfluorotributylamine every 3 days. Helium (99.999%) at a flow rate of 1 mL min-1 was used as carrier gas; argon (99.999%) at a pressure of 2 mTorr was used as collision gas. Thermo Workstation software XCaliber was used for instrument control and data analysis. Rotary vacuum evaporator Model   Evator,   from Medica Instruments, India was used for concentration of the samples.

 

The elution of glucose, fructose and sucrose was performed on a Symmetry C18 column (Hypersil Gold C18, 100x 4.6mm), Thermo Scientific, USA) using TSQ Quantum Access Max: LC/MS/MS attached with a Finnigan Surveyor autosampler (Thermo scientific, USA). A gradient system with 60:40 acetonitrile-water was used as the mobile phase at a flow rate of 0.3 ml min-1. The injection volume of the sample was 20 μl. The total run time was 20 min. The mass spectrometer and the data system used included a TSQ Quantum Access Max Electrospray HESI Probe. The instrument was operated in MSMS-full scan positive ion mode. An electrospray voltage of 4.5 kV and a capillary temperature of 250 °C were used. Vaporizer Temperature: 250 0C, Sheath gas pressure was 50 psi, Auxiliary gas pressure: 10 psi. Tube lens Offset: 128, HPLC Thermo Scientific, USA, Model Surveyor with PDA Detector was used for analysis of citric acid. The mobile Phase A contain  50 mM KH2PO4 (99%), and mobile Phase B contained acetonitrile (1 %),The flow rate was maintained at  1ml/min, injection volume was 10 μl, and the run time 10 min, The analysis was carried out at 210 nm with Diode Array Detector.

 

Calcium and phosphorus were analyzed using ICP-AES, Thermo scientific, UK, Model 6300 Radial.  Argon gas with 99.999% purity was used. In put pressure was six bar. The Flush pump and Analysis pump rates were 100 and 30 rpm respectively. The flush stabilizing time was 5 minutes. The instrument was set for analysis at wavelength of 392 nm for calcium and177 nm for phosphorus. The applied RF power was 1150 W with an Auxilliary gas flow of 5 litre min-1. The radial/viewing height was 15mm. The temperatures of the Camera, Generator and the Optics were -47 to -48 0C, -23 to -27 0C, and -37 to -38 0C respectively.

 

RESULTS AND DISCUSSION:

D-Limonene:

Chromatogram and mass spectra for the standard D- limonene analyzed by GC/MS are illustrated in Figure 1.  It is observed that D-Limonene has a retention time of 3.52 min and the m/z of 67.78 other masses include 66.78, 92.71 and parent peak at 135.73.The limonene spectrum and retention time in the standard matched those of the fruit extract, and a NIST library search also supports the identification of D-limonene. The chromatogram for D-limonene at RT 3.52 min was chosen for quantification because it is a unique, high m/z peak that is relatively abundant; higher m/z peaks generally experience a better signal-to-noise ratio. The amount of limonene in each sample was quantified by plotting a calibration curve using the instrument response at m/z 67.78, shown in Figure 2.The linear regression analysis of the calibration curve in Figure 2 indicate coefficient of correlation as

0.9995.

 

Fig. 1: Chromatogram and Mass spectra of D-Limonene

 

Fig. 2: Calibration Curve for D-Limonene using GC/MS

 

The concentrations of D-limonene in various orange fruit and lime fruits were determined and the results are summarized in Table 1. It is observed that D-limonene content in rinds of orange fruits varied between 13.72 to 34.55 g kg-1.  In case of sweet lime, D-limonene concentration varied between 17.33 to 38.71 g kg-1. It is thus obvious that lime fruits are having greater amount of limonene content.  Table 2 summarizes the D-limonene content in juices of orange and lime fruits. It is observed that the concentration varies between 0.01 to 0.02 g kg-1 for oranges and 0.04 to 0.06 g kg-1for lime juice. The results thus indicate that D-limonene concentration decreases as one investigates from the surface of rinds to the inner parts of the fruits.

 

 


TABLE 1. Characterization of orange and lime fruits for D-limonene, organic acids, minerals and sugars.

Sample

In Rinds

In Juice

D-Limonene

(g Kg-1)

Ca

 

 (mg L-1)

P as Phosphate

(mg L-1)

Ca/P

Ratio

Glucose

(g L-1)

Sucrose

(g L-1)

Fructose

(g L-1)

Citric acid

(g L-1)

Isocitric acid

(mg L-1)

Citric acid/

Isocitric acid Ratio

Orange

34.55

200

330

0.61

27.92

31.72

23.44

7.3

60.20

121.2

Orange

26.57

210

390

0.54

19.44

35.42

16.12

7.9

63.0

125.4

Orange

13.72

170

270

0..63

22.70

27.80

13.50

10.1

79.5

127.0

Orange

25.38

100

150

0.67

14.90

23.39

11.60

9.3

71.6

129.8

Orange

25.70

165

240

0.69

24.32

32.0

9.00

8.5

84.2

100.9

Orange

21.38

245

360

0.67

24.19

33.20

20.90

8.9

80.2

110.9

Orange

29.54

220

360

0.61

13.65

26.30

11.67

9.8

84.3

116.2

Orange

28.40

220

360

0.61

20.12

36.5

12.54

8.4

77.2

108.8

Orange

33.28

210

300

0.70

15.61

35.21

12.50

9.4

91.0

103.3

Orange

19.77

180

300

0.60

14.80

29.60

10.90

8.6

85.5

100.6

Sweet lime

17.10

190

300

0.63

17.32

5.10

6.10

9.1

70.4

129.2

Sweet lime

13.94

170

240

0.71

10.20

8.19

9.80

9.3

85.3

109.0

Sweet lime

28.31

200

360

0.56

12.40

4.26

15.60

6.3

56.9

110.72

Sweet lime

38.71

198

321

0.62

15.28

6.39

13.53

8.3

76.2

108.9

Sweet lime

22.95

200

360

0.56

10.34

2.41

8.68

8.9

74.0

120.3

Sweet lime

17.33

220

390

0.56

15.42

2.02

12.65

7.3

65.0

112.3

Sweet lime

22.81

90

150

0.60

11.20

3.40

10.60

9.4

76.2

123.4

Sweet lime

24.04

180

300

0.60

18.96

5.89

15.02

9.0

79.0

113.9

Sweet lime

23.96

200

300

0.67

18.19

5.81

9.27

8.2

74.00

110.8

Sweet lime

20.01

170

270

0.63

16.28

3.35

10.57

10.0

81.3

123.0

Sweet lime

21.58

120

180

0.67

9.77

4.19

8.82

7.6

65.1

116.7

Sweet lime

28.89

220

300

0.73

13.10

6.90

9.10

7.5

95.0

78.9

Sweet lime

17.85

150

240

0.62

13.21

4.25

9.89

8.5

96.00

88.5

 

TABLE 2. Characterization of commercially available orange and lime juice for D-limonene, organic acids, minerals and sugars.

Sample

D-Limonene

(g Kg-1)

Ca

(mg L-1)

P

As Phosphate

(mg L-1)

Ca/P

ratio

Glucose

(g L-1)

Sucrose

(g L-1)

Fructose

(g L-1)

Citric acid

(g L-1)

Isocit-ric acid

(mg L-1)

 

Citric acid/Isocitric acid Ratio

Orange

0.02

220

300

0.73

22.2

29.3

17.8

9.4

85.0

110.6

Orange

0.01

200

330

0.61

21.6

31.9

22.1

9.1

98.0

92.8

Orange

0.02

200

375

0.53

11.9

3.3

6.4

8.2

91.2

89.9

Sweet lime

0.06

180

315

0.57

14.5

4.5

8.6

6.8

71.0

95.7

Sweet lime

0.05

173

360

0.48

11.8

2.1

8.8

7.8

95.0

82.1

Sweet lime

0.04

200

420

0.47

9.5

4.6

6.9

91.7

99.2

9.1

 

 


Synthesis of D-limonene in plants starts with 3-methyl-3butenyl pyrophosphate. This is then isomerizes by an enzyme to give 3-methyl-2 butenyl pyrophosphate.This process establishes an equilibrium where both the isomers are present. With the aid of another enzyme the two isomers can be joined to give geranyl pyrophosphate.  D-limonene is formed from geranyl pyrophosphate, via cyclization of a neryl carbocation or its equivalent as shown. The final step involves a loss of a proton from the cation to form the alkene.


 

 


Organic acids:

Adulteration of fruit juice has received attention due to its impact on health. Because of this fruit juices are subject for determination of organic acids in them 3. Isocitric acid is commonly used as a marker to detect the authenticity and quality of fruit products, most often citrus juices. In authentic orange juice, for example, the ratio of citric acid to isocitric acid is usually less than 130. An isocitric acid value higher than this is indicative of fruit juice adulteration. The concentrations of citric acid and isocitric acid were determined in the orange and lime juice samples under study. The results are summarized in Tables 1 and 2 respectively. It is observed that the citric acid content in orange and lime fruit juice extract ranged between 6.3 to 10.1 g L-1. Similarly the isocitric acid concentration ranged between 56.9 to 96.0 mg L-1. The ratio of citric acid to isocitric acid ranged between 78.9 to 129.8. This clearly signifies that the juice samples are not adulterated as the ratio is less than 130 4.

 

Minerals:

Many different minerals and other nutrients have interactions between them that affect their availability or absorption in the body. One of the most important, and often overlooked is the of these interaction between calcium and phosphorus. It has been reported that for every gram of phosphorus ingested in the diet, the body must match that with another gram of calcium before the phosphorus can be absorbed through the intestinal wall into the bloodstream. If the required calcium is not available from the diet, the body obtains it from wherever it can, such as from the storage depots in the bones. This idea leads to the concept of calcium-phosphorus ratio. The purpose of calculating such a ratio is to make sure that for every gram of phosphorus we are feeding, at least an equal amount of calcium (a 1:1 ratio or better) is fed, so that calcium isn't being continually mobilized from bones. The mineral element concentrations of calcium and phosphorus (as phosphate) in fruit juice of orange and lime in different samples are summarized in Tables 1 and 2. Ca concentration varied considerably, in a range of about 100 to 245 mg L-1 in the orange juice and 90 to 220 mgL-1 in the lime juice respectively. In the former species, Ca accumulated much more than in lime juice.  In orange juice phosphate was one of the ions for which the concentration varied between 150 to 390 mg L-1 as compared to lime which has phosphate concentration in the range 150 to 360 mg  L-1It is observed that Ca/P ratio for orange juice and lime juice is in the range of 0.54 to 0.70 and 0.56 to 0.73 respectively. The ratio confirms the nutritional requirements for juice. Most nutritionists recommend that the ideal levels are somewhere between 0.60 and 0.70. In case of juice obtained from commercial packs the ratio varies in the range of 0.48 to 0.73 (Table 2).

 

Sugars:

Sugars in fruit juices were estimated using advanced LC/MS/MS technique. The chromatogram and the mass spectra of sucrose are illustrated in Figure 3. The retention time for sucrose was 3.55 min with m/z of 203.08. The calibration curves for sucrose are summarized in Figure 4. It is observed that the coefficient of correlation for sucrose was 0.9992.

 

Fig.  3:  Chromatogram and Mass spectra of Sucrose

 

mg L-1

                  

Fig. 4: Calibration curve for  Sucrose using LC/MS/MS

 

The concentrations of glucose, fructose and sucrose in extracted and commercially available orange and lime juices are summarized in Tables 1 and 2.The concentration in the extracted orange juice for glucose range between 13.65 to 27.92, for fructose between 9.0 to 23.44 g L-1, and for sucrose between 23.39 to 36.50 g L-1 respectively. In sweet lime juice glucose varied between 9.77 to 17.32 g L-1, fructose between 6.10 to 15.60 g L-1, and sucrose 2.02 to 8.19 g L-1 respectively. Starch and sugars are the main source of the body’s energy. However, sugars are not essential foods; they provide energy but not nutrients. The fruit sugars mainly analyzed consist of glucose, fructose, and sucrose.

 

Validation of data for D- limonene:

The suitability of the D-limonene method was properly validated prior to its application in real samples of orange and sweet lime juice. The recovery data were obtained by spiking seven blank samples of orange juice at minimum concentration level of 5 mg kg -1 yielding recoveries in the range of 70 to110%. Precision values expressed as relative standard deviation (RSD) was 9.68 %. Linearity was studied in the range 5 to 350 mg L -1 and the coefficient of correlation was 0.9995 for D- limonene. Method Detection Limits (MDLs) and limits of quantification (LOQs) were also established. For MDL the standard deviation is multiplied by the correct Student's t-value obtained from the statistical Tables 5 . In the present study seven replicates were taken, hence six degrees of freedom with t as 3.143 was considered. The MDL was calculated for D-limonene.

MDL= (s) (t-value) = 0.0055 x 3.143= 0.001575 mg kg-1. Rounding to the correct number of significant figures, the calculated MDL becomes 0.0015 mg kg-1.Similarly; LOQs were subsequently established as 10 times the standard deviation of the recovered limonene. The limit of quantitation was   LOQ= 10 x (s) = 10 x 0.0055 = 0.0050 mg kg-1

 

CONCLUSION:

Fruits are selected largely for their agreeable taste. Most fruits are juicy, with high water and sugar content, and they become important mainly for vitamins, minerals, and fibers they contain. Fruits add variety and flavor to the diet. Fruits are living complex systems, and it is obvious that after the liberation of these chemical reactive components during size reduction, mashing, trimming, and any other destructive process, different deteriorative reactions takes place. Therefore, studies on the composition and changes occurring during ripening and storage are equally helpful to the nutritionist and the process analyst so as to optimize the processing parameters for avoiding undesirable reactions affecting organoleptic properties.

 

The study has demonstrated a simple quantification method for D-limonene in rinds and extracts of the fruit juice using GC/MS. The results also indicate the importance of determining organic acid contents, sugars and mineral content in fruit juices. These are useful in interpretation of the data for judging the suitability of juice for potability and adulteration. Students conducting this analysis will gain valuable experience in sample preparation, solid-liquid extractions, and  the most sensitive analytical techniques for the analysis of fruits and juices.

 

REFERENCES:

1.       Crowell, P.L., Gould, M.N. Chemoprevention and therapy of cancer by d-limonen, Crit Rev Oncog,   5; 1994: 1-22.

2.       Cyclone Power Technologies Inc. (2009) Why It’s Better. http://www.cyclonepower.com/better.html

3.       Wang, T., Gonzalez, A.R., Gbur, E.E. and Aselage, J.M. Organic acid changes during ripening of processing peaches. J. Food Sci. 58 ;  1993: 631–632.

4.       Isocitric acid : http:// Wikipedia.org/wiki/Isocitric acid

5.       Kelly, William D., Ratcliff, Thomas A. Jr., and Nenadic, Charles. Basic Statistics for Laboratories, A Primer for Laboratory Workers. Van Nostrand Reinhold, 1992.

 

 

 

 

Received on 23.11.2011         Modified on 26.12.2011

Accepted on 30.12.2011         © AJRC All right reserved

Asian J. Research Chem. 5(2):  February 2012; Page 299-304