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STUDIES ON THE KINETIC CHANGES OF MIXED FRUIT MARMALADE

DEVELOPED FROM LEMON AND ORANGE

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 Journal of Advance Research in Food, Agriculture and Environmental Science ISSN: 2208-2417

STUDIES ON THE KINETIC CHANGES OF MIXED FRUIT MARMALADE

DEVELOPED FROM LEMON AND ORANGE
U.S. Poson1, M.W. Ahmed2 and M.A. Alim3
1
Instructor, Department of Food Technology, Moulvibazar Polytechnic Institute, Bangladesh
2
Assistant Professor, Department of Agricultural Engineering, Sher-e- Bangla Agricultural
University, Dhaka, Bangladesh
3
Professor, Department of Food Technology and Rural Industries, Bangladesh Agricultural
University, Mymensingh, Bangladesh
ABSTRACT
The aim of this study was to assess the quality of the developed mixed marmalade using
lemon and orange juice with peels in different ratios. The developed mixed fruit marmalades
were analyzed for proximate compositions, microbiological status and sensory attributes. The
kinetic degradation of vitamin-C and effect of storage temperature on marmalade samples
were also investigated. Storage study was conducted on the mixed fruit marmalades for six
months at room temperature (23-30ºC) and refrigeration temperature (4ºC) at relative
humidity 80 to 85%. The Sample S3 (lemon: orange: orange peel=6:2:1) was found as the
best of all samples with respect to chemical composition and sensory attributes. The sample
S3 has a composition of 27.15% moisture, 0.75% ash, 66.3% total sugar, 67.85% TSS and
15mg/100gm vitamin-C. Small amount of mold and bacterial attack was also observed in all
samples after 48hours of incubation period at room temperature (27 ºC). The result showed
that vitamin-C degradation was followed first order kinetic model in all developed
marmalade samples. The activation energy for the vitamin-C degradation of the sample S1,
S2, S3 and S4 was estimated to be 3.009 kcal/ mol, 2.082 kcal/mol, 2.022 kcal/mol and
2.090 kcal/ mol respectively.

Keywords: Marmalade. Lemon. Orange. Mixed fruit. Vitamin-C.

INTRODUCTION
Marmalade is a fruit preserve made from the fruit ingredients together with sweetener, food
acids and jellying agent which is prepared by boiling to minimum 65% of total soluble solid
(Dauthy, 1995). It is mainly prepared from citrus fruits with or without peel from same fruits
(James & Kuipers, 2004). Peel supplies extra nutrition to the marmalade and reduce the
wastage of citrus fruits, moreover requires no jellying agent though citrus peel is a rich source
of pectin (Bagde, Dhenge, & Bhivgade, 2017). Mixed fruit marmalade can be prepared using
different citrus fruits but the quantity of citrus fruit ingredients used in the manufacturing of
1000 g of finished product must not be less than 200 g of which at least 75 g must be
obtained from the endocarp (CODEX, 2009).Although consumption of marmalade is not
wide spread in Bangladesh but now a days this situation is going to be changed. Lemon
(citrus limon) and sweet orange (Citrus sinensis) are citrus fruits, very much popular for their
excellent flavour, colour and taste. In addition to their unique Phytochemical properties,
these fruits are excellent source of vitamin-C one of the most important antioxidants in
nature. Citrus fruits such as lemon contain antioxidant that neutralize free radicals and help to
reduce chronic metabolic diseases like gouty arthritis, kidney stones, hypertension, even
cardiovascular diseases in humans (Wang, Cheng, Lin, Ma, & Deng, 2017). During
processing and storage of citrus fruit juices considerable amount of vitamin-C is lost which
mostly follows first order kinetic model (Burdurlu, Koca, & Karadeniz, 2006). In fact,
ascorbic acid is readily destroyed by heating and oxidation (Mercali, Jaeschke, Tessaro,
Marczak, & Damasceno, 2012) and its protection is particularly difficult to achieve. Storage

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temperature, water activity, pH and metal ions especially iron and copper also affects the
quality of marmalade and other fruit juice concentrates during storage (Gordon &
Samaniego-Esguerra, 1990). During storage, marmalade or any other citrus fruit products
may get spoiled by non enzymatic browning, by fugal attack or by acid resistant bacteria
(Tiwari, 2018).
The aim of the study was to develop mixed fruit marmalades using four different ratios of
lemon and orange juice and peels and to observe the degradation rate of different quality
parameters (TSS, pH, vitamin C, moisture, ash and sugar content etc.) and microbial load of
developed mixed fruit marmalade with respect to storage conditions. Sensory evaluation of
different samples was performed by non technical panelists to find the most acceptable
product by statistical analysis.

MATERIALS AND METHODS

Raw materials
Fresh lemons and fully ripped, healthy oranges were purchased from the local supermarket of
Mymensingh, Bangladesh. The fruit leaves were removed out, followed by washing and
peeling prior to extraction of juice. Sugar, pectin and citric acid also used to have a highly
acceptable finish product.
Juice extraction
Clean and peeled lemon and orange were cut into smaller chunks, followed by blending in
separate blenders (Philips Liquidificador Daily Collection HR2105). The obtained pulp was
then strained to remove the solid portion of fruit from the juice. The extracted juices were
pasteurized at 83ºC for 5 minutes and followed by cooling and storage in closed glass
container.

Preparation of fruit peel

Inner whitish portion of lemon and orange peel was removed manually by using sharp knife.
After washing, both type of peel were cut into 3cm×2cm size and were soaked in 65% sugar
solution for 24 hours.

Preparation of mixed fruit marmalade

Lemon and orange juice, sugar, pectin, citric acid were weighed as per prescribed amount
(Table 1). Sugar and pectin were mixed thoroughly which is followed by addition of lemon
and orange juice and boiling. Soft and sugar soaked lemon and orange peels were added
separately as per stated proportion. Boiling of mixture was done by medium flame to stop
non enzymatic browning. Finally citric acid was added to the mixture before reaching 67º
brix of the finish product (TSS). Immediately after reaching desired brix, marmalade was
poured into sterile glass bottles and followed by room temperature cooling. After then
paraffining was done to stop microbial contamination of prepared marmalade from free air.
Then the bottles were capped properly and stored at room temperature (23-30ºC) and
refrigerated condition (4ºC) to observe the kinetics of different quality parameters.
Table1: Formulation of mixed fruit marmalade
Lemon Orange Lemon Orange Sugar Pectin Citric
Sample Juice Juice Peel peel acid
(g) (g) (g) (g) (g) (g) (g)
S1 300 100 50 - 550 5 5

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S2 100 300 - 50 550 5 5

S3 300 100 - 50 550 5 5
S4 100 300 50 - 550 5 5

Chemical analysis
The freshly formulated marmalades and stored at different temperature conditions were
analyzed for moisture, Total Soluble Solids (TSS), total sugar ash, pH and vitamin-C as per
the methods of Ranganna (1992) and AOAC (2016). All the trials were done in triplicate and
the results were taken as average value.
Kinetics of vitamin C degradation
Vitamin-C content in the marmalade was determined stored in both conditions after each 30
days interval for total 6 month. Sapeia & Hwa (2014) and Burdurlu, Koca, & Karadeniz
(2006) have shown that degradation of vitamin C follows first order reaction kinetics in citrus
juice and the following expressions can be used to predict vitamin C degradation. Using all
data of Equation 1, we calculated the reaction rate constant by using MS excel tool.
C
lnC t =−Kt
O
or, lnCt =ln Co −Kt …………….…………. (1)
Where,
𝐶𝑡 =Concentration at any time (t);
K=Reaction rate constant
𝐶𝑂 =Initial concentration;
A line was obtained when concentration of vit-C was plotted against time (t).
The temperature influence of vitamin-C degradation can be expressed in terms of the
activation energy (Ea) and adequately described by Arrhenius kinetics (Eq. 2) where K1 and
K2 are the rate constant at average storage temperatures of T1 (27 ºC) and T2 (4 ºC),
respectively. Ea is the activation energy for the ascorbic acid degradation (kcal/ mol), R is the
universal gas constant (1.987 kcal/ mol. K)
K1 = 𝐾2 . exp[−ERa (T11 − T12)]…………….…………. (2)
The activation energy was calculated from the slope of line obtained by plotting the logarithm
of the rate constant, k, versus the inverse temperature, 1/T.

Microbial Profile
Bacteria and mold count of the formulated marmalades were done after 48hours of incubation
period according to the method as described in the “Standard Methods for the
Microbiological Examination of Foods” (Bartram, Goresline, Cameron, James, Highlands, &
Etchells, 2011).

Sensory evaluation
The consumer’s acceptability of developed products was evaluated by a taste testing panel.
The hedonic rating test was used to determine this acceptability. The panelists were asked to
rate their acceptability for the product on a hedonic scale, 1 to 9, ranging from as “dislike
extremely” to as “like extremely”. The data were analyzed in Randomized Complex Block
Design using Computer Program MSTATC. Fisher’s LSD Multiple Range Test was done to
compute the storage condition variation.

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RESULT AND DISCUSSION

Chemical composition of processed marmalades
Mixed fruit marmalades were prepared from lemon and orange juice with peels in different
ratios. Composition, degradation of vitamin-C and acceptability of mixed fruit marmalades
were investigated for six month storage period. The obtained results of various experiments
conducted during the study period are shown in table 2.
Table 2: Chemical compositions of freshly formulated marmalades

Sample
Components (%)
S1 S2 S3 S4
Moisture content (wb) 27.5 28.35 27.15 28.5
TSS 67.2 67.65 67.85 67.5
Total sugar 66.74 72.18 66.3 72
Ash 0.7 0.85 0.75 0.96
pH 2.85 2.75 2.93 2.73
Vitamin C
12 20 15 18
(mg/100gm)

The moisture content of sample S1, S2, S3 and S4 was found to be 27.5%, 28.35%, 27.15
and 28.5% on wet weight basis (wb) while the corresponding total sugar content was found
67.2, 67.65%, 67.85 and 67.5% respectively. It was found that all the samples has high acid
content where highest pH value observed in S3 sample (2.93) which is followed by sample
S1 (2.85), S2 (2.75) and S4(2.73) . The vitamin-C content (mg/100gm) of the sample S2
(20), and S4 (18) was determined higher compare to sample, S1 (12) and sample S3 (15).
Here all the composition is quite similar to the mixed fruit marmalade developed from
Mango, Malta and pineapple reported by (Inam, Hossain, Siddiqui, & Easdani, 2012).
These variations of composition may be due the variation of juice ratio and other ingredients.
Higher amount of vitamin-C content in sample S2 and S4 was due to higher proportion of
orange juice because orange has higher amount of vitamin-C content compare to lemon
(Fatin & Azrina, 2017).

Storage changes of formulated marmalades

The quality parameters of mixed fruit marmalade samples were assessed in each 30 days
interval for a total period of 6 month storing in room temperature (23-30ºC) and refrigerated
condition (4°C). TSS, pH, vitamin C, moisture, ash and sugar content of different stored
samples from both conditions were analyzed and shown in table 3 and table 4.

Table3: Changes of chemical composition during storage at room temperature (23-30ºC)

Storage period (Days)
Parameter Sample 0 30 60 90 120 150 180
S1 27.5 27.58 27.62 27.65 27.71 27.74 27.80
Moisture S2 28.35 28.39 28.42 28.46 28.50 28.56 28.61
(%) S3 27.15 27.19 27.73 27.29 27.33 27.35 27.39
S4 28.35 28.54 28.59 28.62 28.66 28.69 28.73
S1 67.2 67.25 67.26 67.29 67.29 67.30 67.36
TSS (%) S2 67.65 67.58 67.5 67.47 67.45 67.41 67.4
S3 67.84 67.85 67.86 67.87 67.88 67.89 67.92

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S4 67.5 67.52 67.53 67.54 67.55 67.56 67.67

S1 66.74 66.74 66.73 66.71 66.70 66.69 66.89
Total sugar S2 72.18 72.15 72.13 72.14 72.12 72.11 72.10
(%) S3 66.3 66.25 66.23 66.21 66.18 66.17 66.15
S4 72.00 72.00 71.98 71.96 71.92 71.90 71.90
S1 0.7 0.7 0.69 0.69 0.68 0.68 0.68
S2 0.85 0.85 0.85 0.84 0.84 0.84 0.84
Ash (%) S3 0.75 0.75 0.75 0.73 0.73 0.73 0.73
S4 0.96 0.96 0.96 0.95 0.95 0.95 0.95
S1 2.85 2.84 2.82 2.80 2.78 2.73 2.67
pH S2 2.75 2.75 2.72 2.71 2.68 2.65 2.60
S3 2.93 2.90 2.90 2.88 2.87 2.84 2.82
S4 2.73 2.71 2.69 2.68 2.65 2.37 2.58
S1 12 10 8 6.5 5 4 3.86
Vitamin C S2 20 16.3 13.6 12.5 9.85 8.5 7.54
(mg/100g) S3 15 12 10 8 5.89 4.8 3.84
S4 18 14.5 12.2 10.3 8.1 6.4 5.57

Table 4: Changes of chemical composition during storage at refrigerated temperature (4°C)

Storage period (Days)
Parameter Sample 0 30 60 90 120 150 180
S1 27.5 27.5 27.52 27.54 27.55 27.55 27.60
Moisture S2 28.35 28.28 28.22 28.20 28.19 28.18 28.18
(%) S3 27.15 27.13 27.12 27.09 27.05 27.04 27.01
S4 28.5 28.44 28.42 28.40 28.37 28.35 28.32
S1 67.2 67.14 67.3 67.32 67.34 67.45 67.5
TSS (%) S2 67.66 67.68 67.72 67.75 67.78 67.90 68.23
S3 67.85 67.85 67.87 67.89 67.93 67.95 67.98
S4 67.5 67.54 67.55 67.56 67.58 67.59 67.68
S1 66.74 66.70 66.68 66.65 66.60 66.57 66.50
Total sugar S2 72.18 72.15 72.14 72.12 72.12 72.11 72.09
(%) S3 66.3 66.23 66.22 66.21 66.18 66.17 66.10
S4 72.00 71.98 71.97 71.96 71.91 71.90 71.86
S1 0.7 0.7 0.69 0.69 0.68 0.68 0.67
S2 0.85 0.85 0.84 0.84 0.84 0.84 0.83
Ash (%) S3 0.75 0.75 0.75 0.73 0.73 0.73 0.73
S4 0.96 0.96 0.96 0.95 0.95 0.95 0.95
S1 2.85 2.85 2.84 2.84 2.82 2.78 2.70
pH S2 2.75 2.75 2.73 2.73 2.72 2.71 2.69
S3 2.97 2.94 2.93 2.92 2.90 2.87 2.84
S4 2.73 2.70 2.70 2.66 2.63 2.62 2.62
S1 12 11 9 7.3 6.15 4.89 4.46
Vitamin C S2 20 18.5 15.6 14.2 11.87 10.98 10.5
(mg/100g) S3 15 13.2 11.2 8.96 7.6 5.43 4.89
S4 18 15.3 13.6 11.8 10.5 8.67 6.9

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Table 3 and table 4 shows that moisture content of all samples was nearly constant up to 6
month of storage period. Similar result was also found by Inam (2012) and reported as, slight
increase in moisture was occurred may be due to slight imperfection in sealing or mechanical
error in moisture determination by air oven.
It was observed that, TSS content of all samples was slightly increased with respect to time in
both storage conditions. Islam (2014) found, slight increase of total soluble solid in different
storage condition of mixed fruit juice developed from orange and pineapple. This slight
increase of TSS content might be due to acid hydrolysis of polysaccharides especially pectin
and gums of fruit peel.
Total sugar and Ash content of all samples was quite stable in both storage conditions.
Similar result was found by several researchers and it was reported that sucrose present in
samples get hydrolysis during prolong storage that leads conversion of reducing sugar to non
reducing sugar (Asare, Saalia, & Dedeh, 2014). Storage condition has no significant effect on
ash content of marmalade (Inam 2012; Islam 2014)
There was a significant decrease observed in pH content in both storage conditions for all
samples but higher decrease in room temperature compare to refrigerated storage. Inam
(2012) found a similar result for mixed fruit marmalade developed from malta, mango and
pineapple. The variation of pH of samples due to the variation of titrable acidity (Islam 2014)
and oxidation of reducing sugar (Ehsan, Naeem, Anzum, & Nazir, 2003).

Kinetics of vitamin C degradation

The degradation of Vitamin-C in formulated marmalades was studied in each 30 days storage
interval for 6 months at room temperature (27°C) and refrigerated condition (4°C). It was
observed that the degradation of vitamin-C was decreased gradually with time in both storage
conditions. The degradation of vitamin-C fitted by first order kinetics was shown in fig.1. Rate
constant of different marmalade samples and temperature influence (as activation energy, Ea)
on vitamin-C is given in table 5.

.
15 25
Vitamin-C( mg/100 gm)
Vitamin-C (mg/100 gm)

Room Temp. 20 Room Temp.

Refrigerated Temp. Refrigerated Temp.
10 15

10
5
5

0
0
0 100 200
0 100 200
Time (day)
Time (day) Sample S2
Sample S1

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20 20
Room Temp.

Vitamin-C(mg/100gm)
Vitamin-C( mg/100gm)
Refrigerated Temp.
15 Room Temp. 15
Refrigerated Temp.

10 10

5 5

0 0
0 100 200 0 100 200
Time (day) Time ( day)
Sample S3 Sample S4

Figure 1: Degradation kinetics of vitamin- C in mixed fruit marmalades

Table 5: First order kinetics of rate constant and temperature dependence of vitamin-C
concentration on different marmalade samples

Rate constant K ( R2 value)

Activation energy Ea
Sample
at average room at refrigerated temp. (Kcal/mol)
temp.(27°C) (4°C)
S1 0.046 (0.955) 0.043(0.973) 3.009
S2 0.064(0.943) 0.056(0.963) 2.082
S3 0.061(0.973) 0.058(0.987) 2.022
S4 0.068(0.973) 0.059(0.992) 2.090

Figure 1 and table 5 shows that, the first order rate constant is higher at room temperature
compare to refrigerated condition. Similar result was found by Sapei and Hwa (2013) and
also reported that Ascorbic acid concentration reduce more rapidly at the beginning of
storage due to the reaction with dissolved oxygen.
Activation energy of all marmalade samples was calculated by using equation 2 and found
highest in sample S1 (3.009 Kcal/mol) which is followed by sample S3 (2.090 Kcal/mol),
sample S2 (2.082 Kcal/mol) and sample S3 (2.022 Kcal/mol) respectively. Here higher
activation energy indicates higher temperature dependency of vitamin-C degradation. The
activation energy for the vitamin-C degradation in sweetened strawberry juice is 1.90 kcal/
mol reported by Sapei and Hwa (2013) that is quite similar to our result.

Microbial study of mixed fruit marmalade

The study of bacterial count was performed by standard plate count (SPC) method. The
viable bacterial load is not uniform. The viable bacteria was counted in‘’cfu/ml’’unit. The
total numbers of viable bacteria and mold in samples S1, S2, S3 and S4 in 48 hours incubation
at room temperate (27°C) has been shown in Fig. 2.

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5 5
Viable bacterial count(log

Total mold count (log cfu/ml)

no.of bacteria no.of mold
4 4
cfu/ml)

3 3

2 2

1 1

0 0
S1 S2 S3 S4 S1 S2 S3 S4
Sample no. Sample no.
Figure 2: Graphical presentation of viable bacterial and mold count (incubation period 48hr.
at 32°C)
It is shown in fig.2 that, maximum number of viable bacteria was found at S4 and minimum
bacteria found at S2. Growth of bacteria is closely related to the pH of the media. Since the pH
of different samples was not same thus the bacterial counts also were not same at same
incubation period. The number of total mold was found maximum in S4 and minimum in S2.
This variation of mold number may be related to the variation of pH content of mixed fruit
marmalade. Higher number of mold was found from higher pH sample where lower pH sample
gives lower number of mold as like bacterial count.
Sensory Evaluation of the samples
Table 6 shows the sensory analysis of the all formulated marmalade samples. In sensory
evaluation color, flavor, taste and overall acceptability were taken in consideration. After 6
month of evaluation period it was found that color, flavor, taste and overall acceptability was
not uniform for all samples. From table 6 it is clear that except sample S3 all characteristics
are quite similar and has no significant difference. The color change of the all samples may
be due to the fungal action and chemical reactions in the product. From the sensory score it is
found, the sample S3 has the highest value of color (8.273), flavor (8.182), texture (8.273)
and overall acceptability (8.091) which was finally selected as the best product. This
product had 300g lemon juice, 100g orange juice and 50g orange peel.
Table 6: Mean score for colour, flavour, texture and overall acceptability of mixed
marmalade
Sensory attributes
Sample Overall
Colour Flavour Texture
acceptable
S1 7.364b 7.00c 7.000c 7.182b
S2 7.818b 7.273bc 7.545b 7.545b
a a a
S3 8.273 8.182 8.273 8.091a
S4 7.545b 7.545c 7.455c 7.455b
Same letter suffix do not differ at 5% (P<0.05) level of significance.

Conclusion
This study indicates that lemon and orange juice with peel could be used to produce quality
value added product. Quality attributes of developed product were deteriorates over 6 months
of storage period where faster at room temperature compare refrigerated condition. The

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degradation of vitamin-C concentration of formulated marmalade samples ranged from 12 to

20 mg/100gm product and followed first order kinetic model upon storage. The reaction rate
constants of vitamin-C degradation were decreased when the marmalades were stored at the
refrigerated temperature. Microbial analysis indicates that, at room temperature the
formulated product is prone to bacterial and mostly fungal deterioration. So this formulated
marmalade is suggested to store at refrigerated condition for better quality and sensory
attributes.

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