Introduction
Cardiovascular disease (CVD) is the leading cause of
death worldwide, with 80% of cases occurring in low- and
middle-income countries (1).
Indonesia was the 6th highest country in the world for CVD deaths
in 2019, with an estimated 375,479 deaths (2). One of the factors causing coronary
heart disease is the pattern of fiber consumption, which is still
low. Dietary factors play a crucial role in the development and
progression of CVD. Individuals consuming the highest amounts of
dietary fiber intake can significantly reduce their incidence and
mortality from CVD. Dietary fiber can be divided into the soluble
and insoluble types. Soluble dietary fiber is derived from grains,
legumes, nuts, fruits and vegetables (3-5).
In addition to a low fiber intake, the low
consumption of vitamin C also causes coronary heart disease.
Vitamin C does not exert its antioxidative function only by direct
interaction with reactive oxygen species, but also via the
maintenance of the redox regulation by increasing levels of other
cellular radical scavengers (6).
Vitamin C inhibits the oxidation of low-density lipoprotein,
thereby reducing atherosclerosis. Vitamin C deficiency is
associated with a higher risk of mortality from CVD; vitamin C may
slightly improve endothelial function and lipid profiles,
particularly in those with low plasma levels of vitamin C (7). Vitamin C, also known as ascorbic
acid, is an essential nutrient that humans cannot synthesize,
rendering its intake crucial for health (8). One of the foods deemed to have a high
vitamin C content is seaweed (9).
The habitual consumption of seaweed may provide a
nutritionally rich addition to the diet. Seaweed is high in fiber
and contains numerous other potentially bioactive compounds. The
rich mineral and trace element content of seaweed compared to
terrestrial foods can negatively affect its organoleptic
characteristics. However, it has been shown to be acceptable to
consumers when baked into breads (9).
Seaweed can be dried and processed into seaweed
flour to be used for longer period of time. Once it becomes seaweed
flour, it can be used in other food ingredients. Cookies are a food
that are much loved by the public, particularly by children and the
elderly due to their soft texture, and various flavors and shapes.
Seaweed powder has been substituted for wheat flour in varying
amounts to fortify the nutritional content of cookies (10). The present study aimed to determine
the effects of seaweed (Eucheuma cottonii) powder
substitution on the dietary fiber, vitamin C and polyphenol content
in cookies. These cookies may prove to be beneficial for patients
with coronary heart disease.
Materials and methods
Study design and materials
The present experimental study used three treatment
groups with one control group design. It was conducted at the
Dietetic and Culinary Laboratory, Universitas Respati Yogyakarta,
Yogyakarta, Indonesia. Seaweed drying was carried out traditionally
in Waingapu, East Sumba, Indonesia using sunlight for 6 days, with
a drying time of 6 h each day. The seaweed flouring process was
carried out at the Gadjah Mada University Food and Agricultural
Technology Laboratory. Analysis for dietary fiber, polyphenols and
vitamin C levels were conducted at the Chem-Mix Pratama Laboratory
in Yogyakarta.
The present study used seaweed (Eucheuma
cottonii) flour to make cookies with various substitutions. The
present experimental study prepared three different variations of
cookies and one cookie formulation used as the control, as follows:
i) Cookies A (the control): The cookies were prepared from 100%
wheat flour (without seaweed flour substitution); ii) cookies B
were prepared from the combination of 80% wheat flour and 20%
seaweed flour; iii) cookies C were prepared from the combination of
70% wheat flour and 30% seaweed flour; iv) and cookies D were
prepared from the combination of 60% wheat flour and 40% seaweed
flour. The ingredients used in making cookies included seaweed
flour, wheat flour, butter, eggs, skim milk, salt, powdered sugar
and baking powder. Cookies for each variation of the mixing
composition were made twice in repetition, with two experimental
units each. The cookie-making process began with mixing flour, skim
milk, eggs, powdered sugar, butter, baking powder and salt in a
bowl. Once all the ingredients were combined, the following step
was to stir using a mixer for ~5 min until all the dough was
combined. Seaweed flour was added to the dough and the dough was
ready to be molded. The molded dough was then baked in an oven at a
temperature of 130-150˚C for 30 min. Once cooked, the cookies were
removed from the oven.
The dependent variables analyzed include the content
of dietary fiber, vitamin C and polyphenols. Researchers analyzed
these contents at the Chem-mix Pratama Laboratory in Bantul,
Indonesia. They measured dietary fiber using the multi-enzyme
method, determined vitamin C through titration, and assessed
polyphenols using spectrophotometry. The analysis followed the
procedures in force at the Chem-Mix Pratama Laboratory in
Yogyakarta. The procedure for analyzing the dietary fiber content
was as follows: A total of 0.5 g of sample was placed into an
Erlenmeyer flask, followed by the addition of 50 ml pH 7 phosphate
buffer. Subsequently, 0.1 ml alpha amylase enzyme was added to the
Erlenmeyer flask containing the sample and heated in a water bath
at 100˚C for 30 min, stirring occasionally. The sample was then
removed and cooled. A total of 20 ml of distilled water and 5 ml of
1 N HCl were then added. Following this, 1 ml of 1% pepsin enzyme
was added to the Erlenmeyer flask containing the sample and heated
in a water bath for 30 min. The Erlenmeyer flask was then removed,
and 5 ml of 1 N NaOH were then added, followed by the addition of
0.1 ml β-amylase enzyme. The Erlenmeyer flask was then covered and
incubated in a water bath for 1 h. The solution was filtered using
a constant-weight filter paper of known weight. The sample was then
washed with 2x10 ml ethanol and 2x10 ml acetone. The sample was
oven-dried at 105˚C overnight, cooled in a desiccator, and then
weighed to determine the insoluble dietary fiber content. The
filtrate was adjusted to a volume of 100 and 400 ml warm 95%
ethanol were added. The filtrate was allowed to settle for 1 h. The
filtrate was then filtered through ash-free filter paper and washed
with 2x10 ml ethanol and 2x10 ml acetone. The filtrate was
oven-dried overnight at 105˚C, then placed in a desiccator, and
weighed to determine the soluble dietary fiber content. The total
dietary fiber content=insoluble dietary fiber content + soluble
dietary fiber content.
The procedure for analyzing the polyphenol content
was as follows: A total of 5 g of the ground sample was weighed and
transferred to a 100 ml Erlenmeyer flask. The sample was diluted
with distilled water to a volume of 100 ml. The solution was then
filtered/centrifuged until a clear solution/filtrate was obtained.
A total of 1 ml of the clear solution/filtrate was taken,
transferred to a test tube, and 0.5 ml of Denis' Follin (1:1
Follin) was added; 1 ml saturated Na2CO3
solution was then added to the filtrate and allowed to stand for 10
min. Subsequently, 10 ml distilled water were added to the solution
and vortexed until homogeneous. The sample absorbance was read
using a spectrophotometer at a wavelength of 730 nm. The data
obtained were recorded and then calculated using a phenol standard
curve.
The procedure for the analysis of the vitamin C
content was as follows: A total of 200-300 g sample was weighed and
ground in a blender until a slurry was obtained. Subsequently,
10-30 g of slurry were placed in a 100-ml volumetric flask,
distilled water was added to the mark, and the solution was then
filtered using a Gooch crucible or centrifuged to separate the
filtrate. This was followed by pipetting 5-25 ml of the filtrate
into a 125-ml Erlenmeyer flask; 2 ml of 1% starch solution (soluble
starch) were then added, and 20 ml distilled water were added if
deemed necessary. The filtrate was titrated with 0.01 N standard
iodine. The vitamin C content was calculated as follows: 1 ml of
0.01 N iodine=0.88 mg of ascorbic acid.
Statistical analysis
Data were analyzed using SPSS version 16 software.
The dietary fiber data were analyzed using the Kruskal-Wallis and
followed by the Bonferroni test as a post hoc test. The
Kruskal-Wallis test was used as the polyphenol data was not
normally distributed, then followed by the Bonferroni test as a
post hoc test as the result of the Kruskal-Wallis Test was
significant. The vitamin C and polyphenol data were analyzed using
one-way ANOVA followed by the Games-Howell Test. One-way ANOVA was
used as the vitamin C and polyphenol data were normally
distributed, followed by the Games-Howell as post hoc test as the
result of one-way ANOVA was significant and the test of homogeneity
revealed that the variances of the four groups were not the same. A
value of P<0.05 was considered to indicate a statistically
significant difference.
Results
Production of seaweed flour
Seaweed flour is brown and has black spots. This is
due to the fact that the seaweed used is seaweed which is dried in
the sun; thus, it has a brownish color. Seaweed flour was prepared
by soaking seaweed (Eucheuma cottonii) in fresh water for 10
h to remove impurities, then rinsing with running water and
draining. It was then soaked in 5% lime solution which was made by
the authors for 5 h and drained again, and dried for 24 h (11). It was then milled and sieved using
an 80-mesh sieve.
The present study analyzed the differences in
dietary fiber, vitamin C and polyphenol content in cookies A as the
control, and cookies B, cookies C and cookies D with different
compositions of wheat flour and seaweed flour.
Effect of the variations in seaweed
flour substitution on the dietary fiber content in cookies
As demonstrated in Table I, cookies D had the highest food
fiber content, namely 11.466%, while cookies A had the lowest food
fiber content with a value of 5.759%. Analysis using the
Kruskall-Wallis yielded a P-value of 0.003 (P<0.05), indicating
that there were differences in the dietary fiber content of the
cookies prepared with variations in seaweed flour substitution.
Analysis was continued using the Bonferroni test to determine the
differences between each group. The results revealed significant
differences (P<0.05) between cookies A and B, cookies A and C,
cookies A and D, cookies B and C, cookies B and D, while there were
no significant differences (P>0.05) between cookies C and D.
These results indicated that the seaweed (Eucheuma cottonii)
flour substitution significantly affected the dietary fiber content
in the cookies.
 | Table IEffects of seaweed (Eucheuma
cottonii) flour substitutions on the dietary fiber content in
the cookies. |
Table I
Effects of seaweed (Eucheuma
cottonii) flour substitutions on the dietary fiber content in
the cookies.
| Cookies | Unit | Mean ± SD | P-value |
|---|
| A | % |
5.759±0.710a | 0.003 |
| B | % |
8.923±0.585b | |
| C | % |
10.687±0.183c | |
| D | % |
11.466±0.354c | |
Effect of the variations in seaweed
flour substitution on the vitamin C content in cookies
The results of the analysis of the effects of
seaweed (Eucheuma cottonii) flour substitution on the
vitamin C content in the cookies are demonstrated in Table II. It was found that cookies D had
the highest vitamin C content (44.176%), while cookies A had the
lowest vitamin C content (19.115%) (Table II).
| Table IIEffects of seaweed (Eucheuma
cottonii) flour substitutions on the vitamin C content in the
cookies. |
Table II
Effects of seaweed (Eucheuma
cottonii) flour substitutions on the vitamin C content in the
cookies.
| Cookies | Unit | Mean ± SD | P-value |
|---|
| A | mg/100 g |
19.115±1.549a | <0.001 |
| B | mg/100 g |
26.363±1.442b | |
| C | mg/100 g |
31.791±4.677b | |
| D | mg/100 g |
44.176±6.200c | |
The results of one-way ANOVA revealed a P<0.001,
indicating that there were differences in the vitamin C content of
the cookies with variations in seaweed flour substitution. The
analysis was continued using the Games-Howell as a post hoc test to
determine the differences between each group. The results revealed
that there was a significant difference (P<0.05) between cookies
A and B, cookies A and C, cookies A and D, and cookies B and D,
while no significant differences were found between cookies B and
C, and cookies C and D (P>0.05).
Effect of the variations in seaweed
flour substitution on the polyphenol content in cookies
As demonstrated in Table III, cookies D had the highest
polyphenol content, namely 0.356% or 35.6 mg per 100 g of cookies,
while cookies A had the lowest polyphenol content with a value of
0.175% or 17.5 mg per 100 g of cookies. The results of one-way
ANOVA revealed a P<0.001, indicating that there were differences
in the polyphenol content of cookies with variations in seaweed
flour substitution. The analysis was continued using the
Games-Howell post hoc test to determine differences between the
groups. The results revealed that there were significant
differences (P<0.05) between cookies A and B, cookies A and C,
cookies A and D, cookies B and C, cookies B and D, and cookies C
and D.
| Table IIIEffects of seaweed (Eucheuma
cottonii) flour substitutions on the polyphenol content in the
cookies. |
Table III
Effects of seaweed (Eucheuma
cottonii) flour substitutions on the polyphenol content in the
cookies.
| Cookies | Unit | Mean ± SD | P-value |
|---|
| A | % |
0.175±0.006a | <0.001 |
| B | % |
0.218±0.011b | |
| C | % |
0.274±0.004c | |
| D | % |
0.356±0.0116d | |
Discussion
Effect of the variations in seaweed flour
substitution on the dietary fiber content in cookies. Based on the
results of the present study, cookies D had the highest dietary
fiber content among all variations. They contained 11.5% dietary
fiber, relevant to 11.5 g of dietary fiber per 100 g of cookies.
There were differences in the dietary fiber content of the cookies
with variations in seaweed flour substitution (P<0.05): The
greater the seaweed flour substitution, the greater the dietary
fiber content in the cookies. Wet seaweed (Eucheuma
cottonii) contains 11.6 g of fiber per 100 g, while in the form
of flour, it contains 57.2 g of fiber per 100 g of seaweed flour
(12). The results of the present
study are in line with those of previous research related to mixing
seaweed powder in the preparation of cookies; the higher the
percentage of seaweed substitution, the higher the fiber content of
the cookies (10).
Dietary fiber is the edible part of plants that is
undigested and cannot be absorbed in the human small intestine.
Dietary fiber can be fully or partially fermented in the large
intestine. Dietary fiber can be divided into soluble and insoluble
fiber based on its solubility in hot water (13).
Dietary fibers provide health benefits through three
primary mechanisms in the body: Bulking, viscosity and
fermentation. Soluble fibers slow down digestion and control
nutrient absorption; thus, they can reduce the effects of
postprandial blood glucose and lipid increases, which are risk
factors for CVD. Both soluble and insoluble fiber can increase
gastric distension and have an effect on gut hormones that elevate
satiety, leading to reduced food intake, weight reduction and
improved glucose metabolism. Dietary fiber increases the rate of
bile acid excretion, which reduces total and low-density
lipoprotein cholesterol. Once fermented in the colon, dietary fiber
produces short-chain fatty acids that inhibit the synthesis of
cholesterol. Dietary fiber has been shown to have an impact on
plaque stability by decreasing pro-inflammatory cytokines known to
affect plaque stability (14).
There is evidence to indicate an inverse association between
dietary fiber intake and the risk of coronary heart disease, where
greater consumption corresponds to a reduced risk (15). However, the physicochemical
properties of various dietary fibers (such as solubility,
viscosity, and fermentability) vary significantly depending on
their origin and processing, and are important determinants of
their functional characteristics and clinical utility (16). Therefore, it is important to
consume dietary fiber from a variety of fiber sources in the daily
diet.).
Effect of the variations in seaweed
flour substitution on the vitamin C content in cookies
Based on the results obtained in the present study,
cookies D (combination of 60% wheat flour and 40% seaweed flour)
had the highest vitamin C content, 44.176 mg/100 g of cookies.
There were differences in the vitamin C content of cookies with
variations in seaweed flour substitution (P<0.05): the higher
seaweed flour substitution, the higher the vitamin C content in the
cookies.
The various forms of Eucheuma cottonii
seaweed (fresh, dried, and flour) have different nutritional
contents. Fresh Eucheuma cottonii contains 3.35% vitamin C,
dried Eucheuma cottonii 5.76%, and floured Eucheuma
cottonii contains 6.34% (17).
Vitamin C is a water-soluble vitamin. Seaweed is a
source of vitamin C and E. Seaweed also contains antioxidants. The
seaweed that was tested herein underwent a process of soaking and
mixing with demineralized water, reducing its vitamin C content
(18). Moreover, in the present
study, cookies D had high levels of vitamin C. The higher the
seaweed flour substitution, the higher the vitamin C content for
the four cookie preparations.
Vitamin C plays a pivotal role in several processes
involved in the pathogenesis of cardiovascular disease. Vitamin C
and antioxidants play a crucial role in endothelial function.
Patients with coronary heart disease can obtain adequate vitamin C
from foods or dietary supplements (6,19).
Modified foods, such as cookies D (combination of 60% wheat flour
and 40% seaweed flour) prepared in the present study may be an
alternative food for patients with coronary heart disease, as they
have a high dietary fiber and vitamin C content.
Effect of variations in seaweed flour
substitution on the polyphenol content in cookies
Cookies D in the present study had the highest
polyphenol content, with 0.356% or 35.6 mg per 100 g of cookies.
Variations in seaweed flour substitution varied the polyphenol
content of cookies. The higher the seaweed flour substitution, the
higher the polyphenol content in cookies.
As previously demonstrated, the total polyphenol
content increases with the increasing temperature and extraction
time in the extraction of seaweed (Eucheuma cottonii). The
total polyphenol content was shown to be highest at 6 min of
extraction time at 60˚C and then decreased when the extraction
temperature was 60˚C (20).
The content of polyphenolic compounds decreases at
an extraction temperature of 80˚C. The total phenolic content is
highest at an extraction temperature of 60-80˚C for
traditional extractions. An increase in temperature will increase
the content of extracted polyphenolic compounds, which can damage
or increase the hydrolysis bonds of some polyphenolic compounds and
cause them to be easily extracted (21).
In the present study, the polyphenol content went
through three stages of heating, which could reduce the polyphenol
content in seaweed flour. The first heating was performed by drying
at 26˚C during the traditional drying process in Waingapu, East
Sumba. The second heating is done using a cabinet dryer at 60˚C for
~17 h in Yogyakarta, and the third heating involved heating the
seaweed cookies for 30 min at a temperature of 130-150˚C.
The longer the extraction process, the longer the
contact between the solvent and the solute; thus, the process of
dissolving polyphenolic compounds will continue. The longer
extraction time can cause more exposure to oxygen. This occurs when
the extraction process lasts >8 min at 60˚C. This can increase
the chances of oxidation of polyphenolic compounds so that the
total content of extracted polyphenols decreases. The polyphenol
content of seaweed cookies baked at 130-150˚C for 30 min
demonstrates a reduction in polyphenol levels. The results of the
analysis of polyphenol levels for the four cookies with various
flour substitutions indicated that there were significant
differences between cookies A and cookies D. The polyphenol content
of the cookies was 0.175-0.356; this proved that the cooking
temperature used during the cookie-making process affected
polyphenol levels.
Polyphenols are bioactive compounds found mostly in
cereals, fruits, vegetables, dry legumes, chocolate and beverages,
such as coffee and tea. They have beneficial effects on the
vascular system by lowering blood pressure, improving endothelial
function, increasing antioxidant defenses, inhibiting platelet
aggregation and low-density lipoprotein oxidation and reducing
inflammatory responses (22).
The present study had certain limitations which
should be mentioned. One limitation was in the drying of the
seaweed. Sun-drying alone does not sufficiently dehydrate seaweed;
therefore, the drying process was conducted twice to achieve the
desired level. The procedure combined sunlight exposure with
cabinet drying to ensure optimal results. Another limitation
pertains to the number of indicators tested. A very small number of
indicators were tested. Three indicators were analyzed herein,
namely dietary fiber, vitamin C and polyphenol content. Due to
financial constraints related to the laboratory analysis, the
authors did not analyze other indicators.
In conclusion, the present study demonstrates that
variations in the substitution of seaweed (Eucheuma
cottonii) flour in the manufacture of cookies affect the
dietary fiber, vitamin C and the polyphenols content. Cookies D,
which had the highest proportion of seaweed flour substitution
among the other variations, also had the highest dietary fiber,
vitamin C and polyphenol content, among all the cookie variations.
Cookies D contained 11.5 g of dietary fiber, 44.176 g of vitamin C,
and 35.6 mg of polyphenols per 100 g of cookies. The higher
proportion of seaweed flour substitution, the higher content of
dietary fiber, vitamin C, and polyphenols in cookies.
Further research related to seaweed flour is
warranted. Further studies are required to investigate all the
contents of seaweed flour apart from the content of dietary fiber,
vitamin C and polyphenols. Further studies are also required to
examine the water content of seaweed as when dried, seaweed is
still tough and moist. Experimental research for providing cookies
for patients with coronary heart disease needs to be carried out to
determine its effect on certain variables, including nutritional
status, immune status or inflammation status.
Acknowledgements
The authors would like to thank Universitas Respati
Yogyakarta, Indonesia, for granting permission to conduct this
research.
Funding
Funding: No funding was received.
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
YDAB, as the principal investigator, coordinated the
research. FLW and SW were involved in the conception and design of
the study, as well as in data analysis. All authors have read and
approved the final manuscript. FLW and SW confirm the authenticity
of all the raw data.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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