Application of different methods for carbohydrates determination in raw materials
and confectionery products enriched with dietary fibres
Draženka Komes, Ana Belščak-Cvitanović, Dunja Horžić, Lana Babahmetović
Faculty Of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia (e-mail: [email protected])
ABSTRACT
Due to a great variety of positive health effects, dietary fibres are gaining growing interest in modern
nutrition, especially in the production of functional food products. In that respect, due to frequent
consumption of various confectionery products in the modern lifestyle, the application of dietary fibers in the
production of various confectionery products is desirable.
The purpose of this work was to examine and compare the use of different methods for the determination of
dietary fibers and sugars in confectionery products, in order to implement these methods in the regular
analytical practice of producing companies. Raw materials used in the production of biscuits (two types of
wheat flour T-550 and T-400), milk chocolate without sugar, plain biscuits as well as the final product (milk
chocolate with biscuit) were examined. Three different content of dietary fibres (0.5%, 1%, 3%) were added
to either wheat flours used in the production of biscuits, or to chocolate mass used for the production of milk
chocolates. The content of total dietary fibers was evaluated by the enzymatic-gravimetric methods.
Enzymatic assays were used to determine the contents of sucrose, glucose, lactose and fructose, while
standard analytical procedure was also applied to compare the contents of lactose and sucrose. Water activity
and the content of cellulose were determined in all samples. Effect of the addition of different content of
dietary fibres on the sensory properties of plain biscuits and chocolates was evaluated.
The obtained results indicate that it is possible to trace the addition of dietary fibers by applying these
analytical methods, as well as that the total dietary fiber content is varying according to the addition of
dietary fiber content. The enzymatic determination of sugars provides more precise and more reliable results
in comparison to the standard analytical procedure. The addition of dietary fibers causes an increase in
cellulose content of the tested samples, while the water activity is decreasing. The addition of lower content
of dietary fibers (0.5%, 1%) doe not affect the sensory properties of the tested products, while the addition of
3% of dietary fibers alternates their structure and appearance.
Keywords: Carbohydrates; confectionery products; dietary fibres; enzymatic assays
INTRODUCTION
Among the functional ingredients used by the food industry, dietary fibres are the most well known to the
consumers. The importance of food fibres has led to the development of a large and potential market for
fibre-rich products and ingredients, which initiated the interest for finding new sources of dietary fibre that
can be used as ingredients in the food industry [1].
Dietary fibres (DF) as a class of compounds include a mixture of plant carbohydrate polymers, both
oligosaccharides and polysaccharides, e.g., cellulose, hemicelluloses, pectic substances, gums, resistant
starch, inulin, that may be associated with lignin and other non-carbohydrate components (e.g., polyphenols,
waxes, saponins, cutin, phytates, resistant protein). Published reports indicate numerous health benefits
associated with an increased intake of dietary fibre, including reduced risk of coronary heart disease,
diabetes, obesity, and some forms of cancer. Not only does dietary fibre help evade hydrolysis, digestion and
absorption in the human small intestine, but it achieves at least one of these functions: increases the faecal
bulk, stimulates colonic fermentation, reduces postprandial blood glucose (reduces insulin responses) and
reduces pre-prandial cholesterol levels [2].
The intake of fibre and fibre containing foods is well below the recommended levels in Western countries
[3].
Supplementation with dietary fibre can result in fitness-promoting foods, low in calories, cholesterol and fat.
According to current recommendations [4], the average daily requirement of dietary fibre is 25 g per day for
women younger than 50, 21 g per day for women older than 50; 38 g per day for men younger than 50, and
30 g per day for men older than 50. Most nutritionists and diet experts suggest that ca 20–30% of our daily
fibre intake should come from soluble fibre.
Dietary fibre can also impart some functional properties to foods, e.g., increase water holding capacity, oil
holding capacity, emulsification and/or gel formation. Dietary fibre incorporated into food products (bakery
products, dairy, jams, meats, soups) can modify textural properties, avoid synaeresis (the separation of liquid
from a gel caused by contraction), stabilise high fat food and emulsions, and improve shelf-life. Among
foods enriched in fibre, the most known and consumed are breakfast cereal and bakery products such as
integral breads and cookies [5, 6], as well as milk and meat derived products. The addition of DF to bakery
products also improves their nutritional quality since it makes possible to decrease the fat content, by using
DF as a substitute of fat without loss of quality [7,8]. Incorporation of rich-fibre by-products, including wheat
bran in breakfast cereals, rice bran, sugarcane bagasse, wheat bran in bread and peach dietary fibres
concentrate in jam have been investigated [9-11]. Brewers’ spent grain was used as a source of fibre for
incorporation in cookie formulation [12]. Leelavathi and Rao [13] reported that raw wheat bran up to 30%
could be used to substitute flour in the preparation of high fibre biscuits. Both full fat rice bran and defatted
rice bran have been incorporated into many bakery products such as multigrain breads, doughnuts, pancakes,
muffins and waffle mixes, among others [14]. High fibre sugar – snap cookies were prepared using different
cellulose of different particle size and carboxymethylcellulose or pectin coated cellulose [15].
The chemical nature of fibres is complex; dietary fibres are constituted of a mixture of chemical entities. The
choice of analytical method to investigate fibres depends on the composition of each particular fibre. The
measurement of dietary fibres in foods is a complex issue, associated with the definition of fibre in the
analytical method chosen. Methods for the determination of dietary fibre may be divided into three
categories: non-enzymatic-gravimetric, enzymatic-gravimetric, and enzymatic-chemical methods. The later
includes enzymatic-colourimetric and enzymatic-chromatographic (GLC/ HPLC) methods. Nowadays, the
most commonly used methods for dietary fibre measurement are the enzymatic-gravimetric Association of
Official Analytical Chemists (AOAC) method [16] and enzymatic-chemical method [17].
Although a significant number of papers has been published on the addition of dietary fibers to food
products, there is no report regarding the influence of fiber addition on the content of individual sugars,
cellulose and sensory properties of milk chocolate. The aim of this study was therefore to develop a novel
fiber- enriched functional product, which is regularly consumed by people of all ages. Additionally, the
application of different analytical procedures for the determination of dietary fibers content and individual
sugars was compared. Since a standardized method for the determination has not yet been established, two
different enzymatic-gravimetric methods were employed in this study. Also, the differences between the
standard Luff-Schoorl method for the determination of disaccharides sucrose and lactose and enzymatic
methods were examined. In this way, faster and more reliable assays for a routine analysis of individual
sugars could be employed in the quality control aspect of confecionery industry.
MATERIALS & METHODS
Sample preparation
Three raw materials (two types of wheat flours T-550 and T-400 and milk chocolate without sugar) and two
final products (plain biscuits, milk chocolate with biscuits) produced in the local confectionery industry (Kraš
d.o.o.,Croatia) were analyzed. Three different amounts (0.5, 1, 3%) of dietary fibers (Vitacel, Rosenberg,
Germany) were added to wheat flours used in the production of biscuits, or to chocolate mass used for the
production of milk chocolate. The contents of dietary fibers and carbohydrates were determined in all the
enriched samples, as well as the samples without the addition of dietary fibers. For all the analytical
determinations, the samples were previously defatted using the Soxhlet extraction.
Determination of total dietary fibers (TDF) content
Total dietary fibers content was determined using two enzymatic-gravimetric methods: the official AOAC
2001.03 method [18] for the determination of total dietary fibers and the AOAC 991.43 method [19] for the
determination of total, soluble, and insoluble dietary fibers.
Determination of carbohydrates content
The content of sucrose, glucose and fructose was determined enzymatically using the K-SUFRG assay kit
(Megazyme, Ireland) based on the method developed by Outlaw and Mitchell [20], Beutler [21] and Kunst et
al. [22], while the content of lactose was determined as well enzymatically using the K-LACSU assay kit for
the determination of lactose, glucose and sucrose in flours. All reagents were prepared according to the
manufacturer`s instructions. The procedures suggested by the manufacturer were strictly followed, and the
content of individual sugars calculated and expressed as percentage (%). The contents of sucrose and lactose
were additionally determined using the standard procedures according to Luff-Schoorl [23]. The content of
cellulose was determined according to the standard procedure of Kürschner-Hanak [24].
Sensory analysis of final products
The samples were evaluated for their sensory properties using quantitative descriptive analysis procedures.
The experimental samples were subjected to sensory evaluation using a laboratory type panel, comprising 15
people with previous experience in taste panel procedures. Four basic attributes (appearance, odour, structure
and flavour) were evaluated. The sensory properties were presented on a 5-point scale, and the average point
number was calculated for each sample. Warm water (about 60°C) was provided for rinsing between
samples.
RESULTS AND DISCUSSION
In this study the potential of substituting carbohydrates with dietary fibers in the production of confectionery
products was evaluated, aimed for the development and production of functional products for a wide range of
consumers. In order to enable the regular analytical control of TDF and individual carbohydrates content in
different raw materials and final products of confectionery industry, in this study two different enzymaticgravimetric methods for the determination of dietary fibers content were applied, as well as enzymatic
methods for the determination of individual sugars, and a standard procedure for the comparison of sugar
content.
As can be seen on Figure 1. the content of TDF is increasing proportionally with the increase of added
dietary fibers in all samples. No significant differences were observed between the TDF content determined
using the two AOAC methods, which indicates that both applied methods can be readily employed for the
determination of dietary fibers in confectionery industry.
4.5
4
3.5
%
3
2.5
2
1.5
1
0.5
0
0
0.5
1
3
Plain milk chocolate
without sugar
0
0.5
1
3
Wheat flour 1
0
0.5
1
3
Wheat flour 2
AOAC 2001.03
0
0.5
1
Biscuit
3
0
0.5
1
3
Milk chocolate with biscuit
AOAC 991.43
Figure 1. Total dietary fibers content (%) in the analyzed samples using the two enzymatic-gravimetric methods
Among the samples with no addition of dietary fibers, the highest TDF content was determined in milk
chocolate with biscuit (1.15%), while the addition of 3% of dietary fibers resulted with 4.09% of TDF in the
same sample, which is in agreement with the amount of added dietary fibers. The obtained results indicate
that dietary fibers can be easily implemented in the production of confectionery products for the development
of novel functional products, and that these beneficial ingredients are not affected by the processing
conditions.
According to the results displayed in Table 1., it can be observed that the content of individual sugars is
decreasing in an almost proportional ratio to the increase of added dietary fibers content. The inverse relation
was confirmed for all sugars, which implies that the substition of carbohydrates with dietary fibers has been
achieved succesfully in the final products. The addition of biscuit to milk chocolate caused an increase in the
lactose content of about 0.6%, which is not surprising considering that raw materials used in the production
of biscuits (powdered milk, powdered whey and honey) are the source of this disaccharide. Plain biscuit was
characterized with the highest content of fructose (2.11-2.36%), due to the presence of this compound during
production of biscuits, which also resulted with an increase in the content of fructose in milk chocolate with
biscuit. Since the chocolate without sugar examined in this study was produced in the same technological
facility as the regular chocolates, usually a small content of sucrose and glucose can be contained in the
following produced product, as a consequence of the residual sugars left in the equipment after the
production of previous product. Therefore the chocolate without sugar contains a small amount of sucrose
and glucose, although the sum of sugars (sucrose-0.42%, glucose-0.42%, fructose-0.07%) amounts to below
<1%, which is the allowed limit of total sugar content in milk chocolates without sugar, according to the
companies practice.
The contents of sucrose and lactose determined using the standard method according to Luff-Schoorl,
revealed a higher content of these sugars in the investigated samples, compared to the contents determined by
the enzymatic method. An average 1.32-fold higher values of sucrose and 1.03-fold for lactose contents were
obtained by the Luff-Schoorl method, compared to the ones obtained using the enzymatic method. The
results indicate that the standard procedure according to Luff-Schoorl is not as appropriate for the
determination of low concentrations of sugar as the enzymatic methods, which provide faster analyses, better
accuracy and higher precision. The content of cellulose is increasing proportionally with the increase in the
added dietary fibers content. The addition of 0.5% of dietary fibers increases the average cellulose content for
0.3%, while the addition of 3% of dietary fibers increases that content in approx. 1.3%. The highest content
of cellulose in the samples without the addition was determined in milk chocolate with biscuit (1.2%), while
the lowest content was determined in milk chocolate without sugar (0.6%).
Table 1. The content of individual sugars (%) and cellulose (%) in the analyzed samples enriched with dietary fibers
Sucrose
Enzymatically
Luff Schoorl
Plain milk chocolate
0,42 ± 0,04
0,45 ± 0,04
0%
0,36 ± 0,02
0,38 ± 0,02
0.5 %
0,29 ± 0,01
0,38 ± 0,05
1%
0,08 ± 0,01
0,31 ± 0,02
3%
Wheat flour 1
0,81 ± 0,06
0,95 ± 0,04
0%
0,74
±
0,06
0,95
± 0,08
0.5 %
0,65 ± 0,05
0,76 ± 0,04
1%
0,45 ± 0,03
0,76 ± 0,06
3%
Wheat flour 2
0,79 ± 0,04
0,95 ± 0,08
0%
0,73 ± 0,05
0,95 ± 0,06
0.5 %
0,65 ± 0,05
0,76 ± 0,04
1%
0,45 ± 0,03
0,76 ± 0,03
3%
Biscuit
15,47 ± 0,59
16,42 ± 0,89
0%
15,40 ± 0,78
16,26 ± 0,45
0.5 %
15,35 ± 0,98
16,11 ± 0,57
1%
15,03 ± 0,68
15,88 ± 0,39
3%
Milk chocolate with biscuit
36,15 ± 2,45
38,38 ± 1,29
0%
36,08 ± 1,78
38,22 ± 1,56
0.5 %
36,00 ± 3,01
38,00 ± 0,74
1%
35,74
±
1,26
37,62 ± 0,93
3%
Lactose
Glucose
Fructose
Cellulose
Enzymatically
Luff Schoorl
Enzymatically
Enzymatically
KürschnerHanack
10,23 ± 0,45
10,20 ± 0,65
10,12 ± 0,74
10,00 ± 0,98
10,55 ± 1,01
10,47 ± 0,56
10,42 ± 0,78
10,35 ± 0,98
0,42 ± 0,03
0,38 ± 0,04
0,31 ± 0,02
0,10 ± 0,01
0,07 ± 0,00
0,07 ± 0,01
0,06 ± 0,00
0,06 ± 0,01
0,60 ± 0,03
0,95 ± 0,07
1,28 ± 0,08
1,86 ± 0,12
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0,26 ± 0,01
0,19 ± 0,01
0,13 ± 0,01
0,06 ± 0,00
0,16 ± 0,02
0,15 ± 0,01
0,15 ± 0,01
0,14 ± 0,02
0,90 ± 0,08
1,20 ± 0,07
1,54 ± 0,06
2,16 ± 0,02
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0,22 ± 0,01
0,17 ± 0,02
0,10 ± 0,03
0,05 ± 0,01
0,14 ± 0,01
0,13 ± 0,01
0,12 ± 0,01
0,12 ± 0,00
0,78 ± 0,06
1,10 ± 0,08
1,50 ± 0,04
2,03 ± 0,06
3,53 ± 0,12
3,47 ± 0,28
3,41 ± 0,21
3,30 ± 0,18
3,97 ± 0,46
3,64 ± 0,24
3,47 ± 0,17
2,95 ± 0,15
2,78 ± 0,02
2,72 ± 0,12
2,68 ± 0,16
2,48 ± 0,65
2,36 ± 0,05
2,28 ± 0,04
2,20 ± 0,02
2,11 ± 0,03
1,20 ± 0,02
1,45 ±0,10
1,80 ± 0,07
2,40 ±0,08
10,90 ± 1,01
10,78 ± 0,78
10,71 ± 0,45
10,61 ± 0,64
11,27 ± 0,56
11,15 ± 0,12
11,08 ± 0,29
10,85 ± 0,36
0,50 ± 0,01
0,44 ± 0,03
0,37 ± 0,02
0,16 ± 0,01
0,35 ± 0,03
0,35 ± 0,02
0,34 ± 0,02
0,33 ± 0,01
1,20 ± 0,03
1,46 ± 0,05
1,82 ± 0,06
2,40 ± 0,07
The addition of dietary fibers to foods modifies the texture and stability of the product (A report by the
Amercian Council on Science and Health). Dietary fibers combine the functional properties of product (low
fat content, low energy) as well as the technological and sensory properties (texture, moisture, breakage).
Significant differences (p<0.05) in the evaluated sensory attributes among the samples (biscuit, milk
chocolate without sugar and milk chocolate with biscuit) with and without the addition of dietary fibers were
observed. According to the results of sensory analysis, the products without the addition of dietary fibers or
with a minimum content of added fibers (0.5%) were preffered over the products containing the highest fiber
content.
Plain biscuit with no added fibers exhibited slightly lower score for appearance and structure, and exhibited
the final average score of 4.6. In all examined products, odour and flavour were not altered by the addition of
dietary fibers. Both attributes were characteristic for the product and were scored highest (odour- 5.0, flavour
4,8). When compared to the samples without the addition of dietary fibers, no significant differences were
observed in the sensory properties of samples with the lower content of added dietary fibers (0.5%). These
results can also be interperted by the fact that differences observed by the descriptive analysis panel were
insufficient to affect preferences of this consumer panel.
The addition of 1% of dietary fibers to samples, affected the structure and solubility of products, so a
decrease in the final score was observed (4.8). The increase in the content of added fibers to 3%, resulted
with a significant changes in the evaluated sensory properties. All evaluated samples exhibited a rough
surface, harder texture and less solublity. Especially chocolates containing 3% of dietary fibers exhibited
grainy surface, weaker breakage and altered consistency.
The obtained results indicate that further investigations are required in order to achieve the most efficient
addition of dietary fibers, and to optimise the functional, physical and sensory properties of confectionery
products. As can be seen on Figure 2., plain milk chocolate without sugar was significantly more preffered
over the milk chocolate with biscuit.
0%
5
4.75
4.5
4.25
3%
0.50%
4
1%
Biscuit
Milk chocolate without sugar
Milk chocolate with biscuit
Figure 2. Spider chart representing mean scores of the evaluated sensory attributes for biscuit, milk chocolate without
sugar and milk chocolate with biscuit affected by the addition of dietary fibers
CONCLUSIONS
The content of TDF is increasing proportionally with the increase of added dietary fibers in all samples, as
well as the content of cellulose, while the content of individual sugars is decreasing in an almost
proportional ratio to the increase of added dietary fibers content. No significant differences were observed
between the TDF content determined using the two AOAC methods, which indicates that both applied
methods can be readily employed for the determination of dietary fibers in confectionery industry. The
standard procedure according to Luff-Schoorl is not as appropriate for the determination of low
concentrations of sugar as the enzymatic methods, which provide faster analyses, better accuracy and higher
precision. According to the results of sensory analysis, the products without the addition of dietary fibers or
with a minimum content of added fibers (0.5%) were preffered over the products containing the highest fiber
content.
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