Mulberry fruit as an antioxidant component in muesli

doi:10.4236/as.2013.45B024

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Vol.4, No.5B, 130-135 (2013) Agricultural Sciences

doi:10.4236/as.2013.45B024

Mulberry fruit as an antioxidant component in muesli

Joanna Kobus-Cisowska, Anna Gramza-Michalowska, Dominik Kmiecik, Ewa Flaczyk,

Józef Korczak

Faculty of Food Science and Nutrition, Poznan University of Life Sciences, 31 Wojska Polskiego Str., 60-624 Poznan, Poland;

*Corresponding Author: joannak@up.poznan.pl

Received 2013

ABSTRACT

The aim of the study was to develop the tech-

nology of muesli via analyzing the addition of

white mulberry fruit. The first step involved the

selection of ingredients from bioactive com-

pounds. Next, the technology of production was

developed. Then, the antioxidant activity of new

muesli with potentially pro-health model sys-

tems was estimated. Muesli was characterized in

terms of its basic nutritional value, and antioxi-

dant potential. The ability to inactivate DPPH

and ABTS radicals was estimated. In addition,

the amount of fat in muesli was calculated. As

part of this calculation, fatty acid composition

and content of primary and secondary oxidation

products were ascertained. The last stage of the

project included sensory analysis of the resul-

tant products. Our studies showed that the new

product-muesli had high nutritional value, high

antioxidant potential and a positive fatty acid

composition. And sensory analysis showed that

the product was attractive.

Keywords: Mulberry; Morus Alba; Muesli;

Antioxidant Properties; Bioactive Food

1. INTRODUCTION

There is an increasing awareness of diseases associ-

ated with diet. Numerous analyses have shown that foods

containing phytochemicals with antioxidant compounds

have a strong protective effect against major diseases; for

example cardiovascular diseases, diabetes, cancer, obesity

and many others [1-3]. Most antioxidant compounds in

food are derived from different plants and belong to vari-

ous classes of compounds with a wide variety of physical

and chemical properties – carotenoids, dietary glutathione

polyphenols, and vitamins. Moreover, these compounds

also play a significant role in the assurance of high quality

food, including mainly fat containing food, by the inhibi-

tion of oxidative changes to this fat. Lipid oxidation is a

highly deteriorative process in foods, which lead first of

all to unacceptable properties for the customer and most

importantly a loss in nutritional value [4-7].

The mechanism of polyphenol activity is highly differ-

entiated. They might act as reducing substances, com-

pounds blocking free radicals, chelating metal ions, cata-

lysts of oxidation reaction, inhibitors of the activity of

enzymes contributing to free radical creation, e.g. xan-

thine oxidase, lipoxygenases or protein kinase. In addi-

tion, they may reduce reactive oxygen species to more

stable forms [7-9].

The most commonly grown species in the Morus ge-

nus are white mulberry, black mulberry, and red mul-

berry [10,11]. Mulberries (Morus alba) have long been

used in traditional medicine to improve eyesight, lower

blood pressure, prevent diabetes, protect the liver,

strengthen joint and treat fever. Fruits, leaves, sprouts as

well as bark are currently used in production processes,

and various forms of final products, including syrups,

jams, ice-cream, vinegar or alcohol, are produced from

them [11,12]. As may be concluded from numerous re-

ports, the fruits of the white mulberry (Morus alba L.),

due to their characteristic composition and properties,

may additionally profitably affect the human organism.

Mulberry fruits are rich in organic acids such as malic

acid, citric acid, and tartaric acid [3,10,12]. This fruit

contains essential fatty acids, as well as vitamins and

polyphenols which are effective antioxidants [11].

The present study aimed to develop a technology for

producing muesli with the fruit of the white mulberry.

This object was achieved by:

• Selection of materials containing potentially bioac-

tive compounds,

• Development of production technology by detailing

the percentage of each fraction,

• Evaluation of the antioxidant activity of the resulting

muesli with potentially pro-health model systems.

2. MATERIALS AND METHODS

2.1. Material

Ingredients used for muesli preparation were purchased

J. Kobus-Cisowska et al. / Agricultural Sciences 4 (2013) 1 30-135 131

in local stores, with a minimum three months of shelf- life.

These included: oatmeal (Kupiec Sp. z o.o., Krzymów,

Poland), linseed (Sante, Warsaw, Poland), granulated oat

bran (Sante, Warsaw, Poland), dried appled (Paula,

Kalisz, Poland), dried cranberry (Bakalland, Warsaw,

Poland), dried mulberry fruits (Bio Planet, Leszno, Po-

land).

All solvents were of analytical grade unless otherwise

specified. 2,2-Diphenyl-1-picrylhydrazyl. (DPPH.), 6-

hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

(Trolox), and Folin-Ciocalteau reagent were purchased

from Sigma-Aldrich (St. Louis, MO, USA). The other

chemicals were obtained from Merck (Darmstadt, Ger-

many) and POCH (Gliwice, Poland).

2.2. Muesli Preparation

A base muesli formulation, taken from the literature [9]

and slightly modified was used as a reference and was

prepared in the laboratory (sample A). Muesli samples

(with apple and cranberry - sample B and with an addi-

tion of apple, cranberry and mulberry fruit-sample C)

were prepared according to Table 1.

2.3. Chemical Characteristics of Muesli:

Protein, Total Dry Matter, Lipids, Ash,

Fatty Acid Composition

All analyses were performed on these 3 samples in 3

replications according to method AOAC [13]. The mois-

ture of samples was determined by drying at 103 ± 2℃

until they reached a constant weight. Protein content was

determined by the Kjejdal method (N × 6.25), in Kjeltec

2200 (Foss Tecator, Sweden). Fat content was deter-

mined using the method of multiple constant extraction

with petroleum ether, which was next evaporated, and

the weighing method was used in the fat content exami-

nation. A Soxtec HT6 apparatus manufactured by Foss

Tecator (Sweden) was used for the extraction. Ash con-

tent was examined by incineration in a muffle furnace –

Naberterm S 27- at a temperature of 535℃, with air ac-

cess.

Table 1. Composition of muesli flakes.

Ingredients: sample A: sample B: sample C:

oatmeal 300 g 300 g 300 g

oat bran (granulated) 90 g 90 g 90 g

linseed 60 g 60 g 60 g

dried cranberry - 60 g 60 g

dried apple - 60 g 60 g

dried mulberry - - 110 g

The percentage composition of fatty acids was deter-

mined using a gas chromatograph with an FID detector

(Agilent Technologies 7820A GC) according to method

described in PN-EN ISO [14]. The method involved dis-

solution of a fat sample with hexane, and its further

trans-esterification using sodium methanol solution.

2.4. Antioxidant Activity

The of total phenolic content (TPC) in muesli was de-

termined by visible spectrophotometry based on a col-

orimetric oxidation/reduction reaction according to Cheung

et al. [15]. Ferulic acid was the standard employed in this

work. The absorbance of the sample was measured at λ =

725 nm using a SPECORD® 40 (Analytik Jena AG,

Germany). Based on the construction of a standard curve,

TPC data for the preparations from muesli were ex-

pressed as mg ferulic acid equivalents/g dry extract.

The free-radical-scavenging potentials of muesli were

tested in a methanolic (80%) and water solution of DPPH

as described by Amarowicz et al. [16]. The extent of

discoloration of the solution indicated the scavenging

efficacy of the added substance. Absorbance was meas-

ured at λ = 517 nm (SPECORD® 40, Analytik Jena).

The ability of ABTS radical cation deactivation was

examined according to the method described by Re et al.

[17]. The examination involved determination of the de-

gree of scavenging of ABTS + radical cations formed

from ABTS by oxidation with potassium persulfate. The

scavenging was measured spectrophotometrically at a

wave length of 734 nm (SPECORD® 40, Analytik Jena,

Germany). Antioxidant ability with ABTS radical cation

application was calculated per Trolox for a standard

curve, and the result was given as mg of Trolox/1 g d.m.

of an extract.

2.5. Oxidative Stability Determination

Lipid extraction

The process was run with the Folch mixture (chloro-

form: methanol 2:1 v/v). After the separation phase,

chloroform was evaporated in a vacuum evaporator and

the lipid fraction was obtained [18].

Pero xi d e val ue

The peroxide value (PV) was determined by titration

with 0.02 M sodium thiosulfate and was expressed in

meq O2/kg [19].

Anisidine value

The determination was performed based on PN-ISO

[20]. The method is based on the reaction of aldehydes

present in the sample with a p-anisidine solution in

ice-cold acetic acid and on the spectrophotometric ab-

sorbance measurement of the formed yellow complex at

a wave length of 350 nm (Metertek SP-830, Taiwan).

J. Kobus-Cisowska et al. / Agricultural Sciences 4 (2013) 1 30-135

132

2.6. Sensory Analysis

The descriptive panel comprised 20 assessors (14 fe-

male and 6 male, aged 22 to 54 y), who were students or

staff of the university.

Sensory assessment of the experimental samples of oat

muesli was conducted in the sensory laboratory fulfilling

the requirements provided in standard PN-ISO, [21].The

method of descriptive quantitative analysis, i.e. sensory

profiling, was used in the study. Qualitative determinants

of color, smell, taste and consistency were evaluated.

The intensity of each quality score was determined using

a 10cm structured linear scale with suitable margin deno-

tations. The color (beige, brown, red, orange, green, purple)

was evaluated on a scale from slightly intense to intense.

Marginal designations, i.e. imperceptible - intense, were

used for smell (fruit, cereal, foreign) and taste descriptors

(sweet, salty, bitter, cloying, fruity, vegetable, and for-

eign). Marginal designations: large, small, were used for

consistence descriptors (compactness, degree of steeping),

while crunchiness was assessed on a soft-hard scale. Also,

a general assessment of the product was performed. The

results obtained were replaced with numerical values

expressed in points.

2.7. Statistical Analysis

All analyses were made in at least three replications.

Average and standard deviation values were calculated

with the use of Microsoft Office Excel 2007 software.

Pearson’s correlation indexes were calculated with the

use of STATISTICATMPL 7.0 StatSoft. The significance

of differences between mean values was determined at p

≤ 0.05 applying the analysis of variance (ANOVA) fol-

lowed by Tukey’s multiple range test.

3. RESULTS

3.1. Chemical Characteristics of Muesli

The chemical characteristics of muesli are given in

Table 2.

The total protein content of muesli was between 12.05

(sample C with mulberry) and 16.43 (sample A - control),

respectively. Control muesli (sample A), which had the

lowest moisture content, had the highest total fat (9.81

%), followed by sample B with apple and cranberry (8.53)

and sample C with mulberry (6.93). The ash content of

the examined muesli was between 1.20% (sample A -

control) and 1.60 (sample C with mulberry), respectively.

A similar chemical composition for muesli was pre-

sented by Pernille at al. researches [4,5], where protein

and fat content was in a range up to 9% and 14%, re-

spectively. Also, the humidity of muesli in the study of

the above-mentioned authors was the same as in the pre-

sented experiment, i.e. at a level of 4% - 7%.

Fatty acid analysis showed that the muesli studied

contained six major fatty acids. Oleic acid, C18:1, was

the dominant fatty acid (34.68% - 36.31%) in all muesli

samples, followed by linoleic acid C18:2 (28.51% -

29.97%). Our results are in agreement with Pernille et al

[4], who reported that the fatty acids found at the highest

levels of muesli were oleic acid (around 48% - 49%) and

linoleic acid (20% - 31%). The examined muesli in the

study by Pernille et al. [5] also contained the highest

amount of oleic acid and linoleic acid, while stearic acid

and linolenic acid were noted at the level of 2% - 3% and

1% - 5%, respectively.

3.2. Antioxidant Activity

Antiradical activity towards ABTS and DPPH is pre-

sented in Figures 1 and 2.

Table 2. Chemical composition of muesli.

Index / Sample A B C

Protein 16.43 13.19 12.05

Fat 9.81 8.53 6.93

C 16:0 12.86 10.24 10.93

C 18:0 2.14 2.36 2.27

C18:1 34.68 36.26 36.31

C 18:2 29.94 28.51 29.97

C 18:3 20.29 22.45 20.28

C 20:1 0.11 0.17 0.24

Ash 1.20 1.50 1.60

Dry matter 93.65 92.89 92.67

Figure 1. Antiradical potential of muesli extract

measured with ABTS radicals.

Figure 2. Antiradical potential of muesli extract

measured with DPPH radicals.

J. Kobus-Cisowska et al. / Agricultural Sciences 4 (2013) 1 30-135 133

The muesli sample with added mulberry fruit was

characterized by the highest activity towards ABTS: 7.71

mg Trolox/ g d.m. for water extract and 12.73 mg Trolox/

g d.m. for methanolic extract. Lower activity was noted

in the control sample where the values were 2.55 mg

Trolox/g d.m. for water extract, and 5.71 mg Trolox/g

d.m. for methanolic extract. In turn, the lowest activity

towards ABTS radical cation was demonstrated for the

sample with added cranberry and apple: 1.64 mg Trolox/

g d.m. for water extract, and 2.62 mg Trolox/g d.m. for

methanolic extract, respectively.

Similar results were obtained in the test with DPPH.

The highest values for antiradical activity were noted for

muesli with white mulberry. Methanolic extract demon-

strated a scavenging ability value of - 180.14 mM

Trolox/g d.m. of an extract, a 6-fold lower value for this

sample was noted in the case of water extract (31.70 mM

Trolox/g d.m. of an extract). The other two samples were

characterized by considerably lower values. Antiradical

activity for the control sample was at a level of 9.71 mM

Trolox/g d.m. for water extract, and 31.57 mM Trolox/g

d.m. for methanolic extract. In turn, for the sample with

added apples and cranberry it was 5.71 mM Trolox/g d.m.

for water extract, and 13.72 mM Trolox/g d.m. for

methanolic extract. Fruit addition, mainly mulberry, af-

fected the antiradical activity of muesli. Numerous pa-

pers demonstrating the beneficial antioxidative effect of

mulberry fruits are available in the literature. Chon et al.

[11] demonstrated that ethanolic extracts of mulberry

fruits scavenge radicals to the highest degree.

The total content of polyphenols is presented in Fig-

ure 3. The highest level of total polyphenols was noted

in the sample of muesli with added mulberry fruit, in

water extract, and then in methanolic extract: the level of

polyphenols was 5.65 and 3.29 mg of ferulic acid/g d.m.

of an extract, respectively. In turn, the muesli sample

with added apple and cranberry contained 1.71 mg of

ferulic acid /g d.m. for water extract, and 0.55 mg of fer-

ulic acid/g d.m. for methanolic extract.

An increase in polyphenol levels in muesli is ex-

plained by the increase in the amount of fruits which are

a source of these components. It was demonstrated in the

Figure 3. Polyphenolic content in muesli extract.

study by Ercisli and Orhan [10] that white mulberry

fruits contain as much as 181 mg in fresh mass. Similar

results comparing polyphenol content in various ana-

tomical parts of white mulberry were presented by Chon

et al. [11], who noted total polyphenol content in mul-

berry fruits at a level of 213 mg/kg. As may be con-

cluded from the study by Bae et al. (2007) mulberry fruit

extracts contained as much as 2570 µg/g of polyphenols

expressed as gallic acid. The relationship between poly-

phenolic compound content and antiradical activity was

also confirmed in this study. As demonstrated by Gundogdu

et al. [12] and Pawlowska et al. [23] rutin, chlorogenic,

gallic and caffeic acids are the predominant polyphenols

in white mulberry fruits.

3.3. Oxidative Stability Determination

Muesli is susceptible to lipid oxidation due to the high

content of unsaturated fatty acids. The oxidative proc-

esses also depend on the content of compounds which

may theoretically inhibit these unprofitable changes.

Oxidative stability was determined in the study by ex-

amination of the content of primary and secondary oxi-

dation products, and the results are presented in Table 3.

The content of primary oxidation products was in the

range of 5.78 meq O2/kg for the control sample to 9.47

meq O2/kg for the sample containing mulberry fruits.

The content of secondary oxidation products expressed

as an anisidine number was in turn nearly two-fold lower

in sample C (18.01) when compared to the reference

sample A (33.43). Various contents of primary and sec-

ondary oxidation products result probably from the dif-

ferent compositions of the examined muesli. Moreover,

as may be concluded from the study by Bae et al. [22],

active compounds of mulberry fruits considerably mod-

erate fat oxidation. It was demonstrated in model tests

that formation of secondary oxidation products was as

much as 23% higher.

3.4. Sensory Analysis

The results of sensory assessment are presented in

Figure 4. Beige color was predominant characteristic for

all samples. Purple and red colors were also noted in the

sample with apple and cranberry, while orange color was

additionally noticed in the sample with mulberry.

Table 3. Oxidative stability of muesli recorded by peroxide and

anizidine value.

Peroxide value [meq O2/kg] Anizidine value

Muesli A5.78 ± 0.76 33.43 ± 1.44

Muesli B5.92 ± 0.28 34.85 ± 1.95

Muesli C9.47 ± 0.47 18.01 ± 0.98

J. Kobus-Cisowska et al. / Agricultural Sciences 4 (2013) 1 30-135

134

(a) colour of muesli

(b) taste of muesli

Figure 4. Sensory analysis of muesli (a) colour of

muesli;(b)taste of muesli;(c)odor of muesli and

crunchiness, density, softening).

Cereal smell was predominant in all samples. Further,

a fruity smell was also perceived, which was especially

distinct in the sample with added apple and cranberry,

and mainly in the sample with added white mulberry.

Sweet and fruity taste was especially highly evaluated

in the examined samples. Also, a subtly cloying taste was

noticed, and even bitter to a small degree in the control

sample without fruits. Comparable crunchiness evalua-

tion results were obtained in the examination, however

the highest crunchiness was noted for muesli with white

mulberry – 4.66 points, then for the control sample –

4.09 points, and muesli with apple and cranberry –

3.81points (on a 1 - 10 scale, where 1 means soft muesli

flakes, and 10 – hard ones). People tend to like contrasts

in foods, and products which undergo changes in texture

during mastication are well liked [6]. As may be con-

cluded from these authors’ research, the choice of muesli

is mainly affected by a sweet taste, which may be de-

rived from dried fruits. It can be noticed in the results of

this study that sweet taste was to the highest degree de-

tectable in the sample of muesli C with added mulberry

fruits.

4. CONCLUSIONS

Muesli including oat flakes, linseed, granulated bran,

cranberry, apple and white mulberry fruit was character-

ized by high nutritional value. The content of saturated

acids in muesli was at a level of about 13%, monoun-

saturated at a level of 37%, and polyunsaturated 50%.

Oleic acid was found in the highest amount, and linoleic

acid was predominant among polyunsaturated ones. The

study demonstrated less advanced oxidative changes of

the product with added white mulberry fruit. Moreover,

the examined samples exhibited antioxidative properties

measured in free radical tests with ABTS and DPPH,

which resulted from a higher content of polyphenolic

compounds also originating from dried fruits. The con-

ducted sensory analysis allowed us to notice that an ad-

dition of mulberry fruits profitably influenced the sen-

sory attractiveness of the product.

Thus, mulberry fruits may be an attractive component

of muesli due to an increase in antioxidative activity, as

well as profitable sensory properties and nutritional value.

Muesli based on oat flakes, granulated bran and linseed

with an addition of white mulberry fruits may constitute

an important element of a diet.

5. ACKNOWLEDGEMENTS

Financially supported by EU Project Nr POIG 01.01.02-00-061/0.

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