Food Technology Research Journal, Vol.

2, special issue 2, 73-85, 2023

Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves

on Shelf Life of the Salty Biscuits
1*
Neveen, A.M. Arfa & 2Shaimaa, M. Hassan
1*
Experimental Kitchen Research Unit ,Food Technology Research Institute, Agriculture Research
center, Giza, Egypt
2
Department of Special Foods and Nutrition, Food Technology Research Institute, Agriculture
Research center, Giza, Egypt
Original Article ABSTRACT
Stinging nettle (Urtica dioica) is an edible plant, well-known for its nutraceutical proper-
ties. Stinging nettle leaves are typically rich in fibers, minerals and vitamins, as well study
was to evaluate the sensory properties and the rheological properties of wheat salted bis-
cuits after adding of dried nettle leaves to flour wheat at different concentrations of 5,10
Article information and 15 % antioxidant compounds, i.e., phenols and flavonoid. along with the control sam-
Received 27/12/2023 ple. Also, changes in the microbial load of biscuits added to nettle leaves during storage
Revised 9/12/2023 periods (1,3,6 and zero time) were investigated. Showed that total bacterial counts were
Accepted 27/12/2023 not detected at all directly after processing with all used concentration increased gradually
Published 30/12/2023 with time. At 5%, and 10% concentration, total bacterial count appeared a month later zero
Available online
30/12/2023
time. During storage period, total bacterial count appeared and a month later with (3.08
and 2.30 log cfu/g) and increased gradually to reach (3.34 and 2.77 log cfu/g) respectively,
while 6 months later of storage, total bacterial counts increased gradually reaching (5.50,
4.91 and 4.56 log cfu/g) with concentrations 5%,10% and 15% respectively compared to
control. It means that, 15% can be enough as preservation treatment for biscuits. concen-
tration with 15% had higher effect, where total bacterial counts appeared after 3 months
(2.30 log cfu/g) and increased to (4.65 log cfu/g) after 6 months of storage. fungi was not
Keywords: detected (ND) through increasing time of storage due to the low moisture in biscuits sam-
Nettle leaf powder; sensory
evaluation; minerals; farino- ples and reducing the numbers of fungies in the various concentrations of nettle leaf. The
graph and extensograph antimicrobial activity of ethanolic and methanolic extracts of nettle they demonstrated no
parameters; antioxidant con- effects against yeast-like fungi (Candida albicans). Although all extracts exhibited an anti-
tent; microbiological.
microbial activity against all tested bacteria with inhibition zones ranging from 10.0±0.58
to 12.7±0.67 mm., they were more effective against gram-positive bacteria (Bacillus cere-
us and Staphylococcus aureus) than against gram gram-negative bacteria (Salmonella
typhimurium and Escherichia coli).
1. Introduction hairs. The species name dioica means ‘two houses’ be-
Nettles (Urtica diocia L.), or stinging nettle, is a per- cause the plant usually contains either male or female
ennial plant growing in temperate and tropical wasteland flowers. Nettle has a well-given character for giving a
areas around the world. It grows 2 to 4 meters high and savage sting when the skin touches the hairs and
produces pointed leaves and white to yellowish flowers, bristles on the leaves and stems. They have also been
and it belongs to the family Urticaceaea (Kregiel et al., used for centuries in traditional medicine and have been
2018). The rubric name Urtica comes from the Latin verb accepted as a healing plant because of their
urere, meaning ‘to burn,’ because of these surcharging considerable effects on human health in

Journal website: https://ftrj.journals.ekb.eg/ *Corresponding Author

Published by Food Technology Research Institute, ARC Email: neveenmaher2810@gmail.com
 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits 74

many countries all over the world (Adhikari et al., 2. Materials and Methods
2016). They are used as a diuretic, antihypertensive, Materials
anti-diabetic, hemostatic, anti-asthenia, antianemic,
Plant materials
antispasmodic, antirheumatic, and as a remedy for
The nettle samples (Urtica diocia L.) were ob-
headaches and chills (Bnouham et al., 2002). Nettle
tained from the Faculty of Agriculture, Cairo Uni-
is also used to treat spleen, renal, and dermal disor-
versity, Giza, Governorate. The biscuit ingredients
ders (Borges Botelho et al., 2014). The whole plant
(wheat flour, butter, eggs, salt, and baking powder)
is used as a diuretic, antihypertensive, anti-diabetic,
were purchased from one of the markets near the
hemostatic, anti-asthenia, antianemic, antispasmod-
Food Technology Research Institute in Giza, Gov-
ic, antirheumatic, and as a remedy for headaches
ernment. 2.1.2. Bacterial and fungal strains aggre-
and chills (Belščak-Cvitanović et al., 2015). The
gated from five strains from gram-positive bacteria,
presence of precious biologically important compo-
gram-negative bacteria, and incentive-such fungi
sites such as proteins, vitamins, phenolic factors,
(Salmonella typhimurium NCTC 12023/ATCC
macro- and microelements, tannins, flavonoids,
14028, Escherichia coli ATCC 25922, Bacillus ce-
sterols, adipose acids, carotenoids, and chlorophylls
reus ATCC 33018, Staphylococcus aureus ATCC
contributes to the application of stinging nettle in
25923, and Candida albicans CAIM-22) were pro-
different ways (Nica et al., 2012; Kopyt'ko et al.,
vided by the Microbiological Resources Center
2012). Therefore, nettle is used as a dietary supple-
(MIRCEN), Faculty of Agriculture, Ain Shams
ment as well as for the production of functional
University, Cairo, Egypt.The fungal strain was
foods. However, it is still an underrated plan (Man
maintained on potato dextrose agar, whereas bacte-
et al., 2019). Stinging nettle is one such raw materi-
rial strains were cultivated on nutrient agar.
al. Thus far, it has been successfully used as a func-
Methods
tional ingredient in bread (Ðurovi´c et al., 2020) or
as a component of a herbal mixture added to wheat Preparation of raw materials
bread baked without salt (Wójcik et al., 2021). It Nettle leaves were thoroughly washed with run-
has also been used to coagulate milk proteins in the ning water under the tap. When done, the nettle was
cheese-making process (Fiol et al., 2016), and con- dried in an air oven (Type: VENTICELL55- Artikel
centrated and lyophilized stinging nettle extract was Nr. 000721/10000 - Temperaturberech: 250°C –
used to manufacture chocolates (Belšˇcak- Anschlub:230V AC 50/60Hz–Leistungaufn.:1250W
Cvitanovi´c et al., 2015). In order to enhance the Germany) at 40°C and passed through a sieve of
activity of bioactive compounds contained in nettle, 250 μm for 24 hours. (Akubor and Owuse 2020).
attempts have been made to nanocapsulate its ex- Dried dry nettle leaves were crushed using an elec-
tracts, and the research findings indicate the possi- tric grinder (Type: Moulinex MFP626, 220V, 50-
bility of. In this study, nettle leaf powder was added 60Hz, 1000W, 250 ml, France), passed through 250
to salted biscuit dough to improve the nutritional μm to obtain a uniform size, packaged in polyeth-
intake of minerals, fiber, protein, and antioxidant ylene bags, and stored in a desiccator until used for
compounds, as well as increase the shelf life of the further analysis (Salama et al., 2022).
biscuit. the obtained nanocomposites in various Production of biscuits
food matrices (Rutakhli et al., 2019 and Delfanian The biscuits were prepared using the straight
and Sahari, 2020). dough method as described by (Akubor 2016). The
Nettle is a raw material not only of high health- ingredients used were 20% buter, 10% homoge-
promoting potential. It is rich in chlorophyll and nized whole egg, 2% salt, and 0.5% baking powder.
metallic elements and can be used alongside The ingredients were weighed and thoroughly
vegetable powders and concentrates as a natural mixed manually. The wet ingredients were first
coloring component. mixed in a mixing bowl at an evenly low speed for

Food Technology Research Journal, Vol. 2, special issue 2, 73-85, 2023

75 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits

5 minutes. The dry ingredients were added, and the lic acid fellow (GAE) per 100 g sample.
mixing was continued at a high speed until a uni- Determination of Total Flavonoid Percentage
form texture was obtained. Using an electric perine- Total flavonoid content (TF) was measured us-
um (RBSFOODMIXERPRO, Cuizimate, Thailand) ing AlCl3, a colorimetric method A.O.A.C. (2006).
at low (80 rpm, for 1 min) and moderate speeds The absorbance was measured at 510 nm using the
(100 rpm, for 10 min), use wheat flour to make bis- spectrophotometer. The TF of the excerpts was ex-
cuits, adding dry nettle leaves in proportions of 5, pressed as milligrams of catechin fellow (GAE) per
10, and 15% to the wheat flour. The dough samples 100 g sample.
were then manually kneaded, molded, shaped, and Rheological properties
placed on the baking tray. and baked in a thermo- Farinograph test
statically controlled baking oven at 170°C for 20 Farinograph tests were carried out on wheat
minutes. The biscuits produced were cooled to am- flour and its mixtures of nettle leaves were added to
bient temperature (32 oC) and packaged in polyeth- it in different proportions (5, 10, and 15) in the fol-
ylene bags prior to analysis. The 100% wheat flour lowing constant flour weight procedure according
biscuit served as the control (Thliza et al., 2021). to method A.A.C.C. (2002), in the rheology labora-
Formula of crackers tory of the Food Technology Research Institute in
Control = 100% wheat flour Cairo, Egypt.
A = 5% addition of nettle leaves + wheat flour Extensograph test
B = 10% addition of nettle leaves + wheat flour Extensograph parameters were measured with a
C = 15% addition of nettle leaves + wheat flour Brabender Extensograph (Brabender OHG) accord-
Methods of analysis ing to A.A.C.C. (2002) in the rheology laboratory
All analyses of crude protein, fat, fibers, and ash of the Food Technology Research Institute in Cairo,
content were done according to the Association of Egypt. Extensograph measurements included elas-
Analytical Chemists Methods (A.O.A.C. 2000). ticity (B.U.), extensibility (mm), proportional num-
carbohydrates = 100 (% crude protein + % crude fat ber, and energy (cn2).
+ % ash + % crude fibers) were estimated by differ- Preparation of powder
ence. humidity and titratable acidity were deter- The upstanding corridor of U. Dioica was shade
mined according to (A.O.A.C. 2012). -dried at room temperature for ten days and also
Determination of minerals ground to a fine greasepaint using an electronic
The mineral content of iron (Fe), zinc (Zn), cal- blender. Extraction was done from one gram of
cium (Ca), potassium (K), and manganese (Mn) was obtained powder using 10 mL of alcohol (ethanol or
determined using an Agilent Technologies (model methanol, Merck, Germany) and distilled water
4210 MPAES) atomic absorption spectrophotome- solution (8:2 v/v), centrifugation at 3000 rpm for
ter according to the method of A.O.A.C. (2011). 15 minutes, and harvesting the supernatant. This
Determination of Total Antioxidant Activity process was repeated three times, and solvents were
Total antioxidant content (TAC) was estimated evaporated by placing the yielded materials at room
by using a 2,2-diphenyl-1-picrylhydrazil (DPPH) temperature for seven days (Riehemann. et al.,
assay to measure the free radical scavenging capaci- 1999; Seyyednejad. et al., 2001).
ty (Sidaoui et al., 2015). Antimicrobial assay
Determination of Total Phenolic Percentage Antimicrobial activities were assessed with the
Total Phenolic Content (TP) of Extracts was de- agar well-diffusion method (Perez and Paul, 1990).
termined using the Folin-Ciocalteu reagent as de- In brief, 1ml of fresh bacterial or fungi culture was
scribed previously by (Ghaima et al., 2013). The TP pipetted in the center of a sterile Petri dish. Also,
of the excerpts was expressed as milligrams of gal- 15 ml of nutrient agar medium for bacterial strains

Food Technology Research Journal, Vol. 2, special issue 2, 73-85, 2023

 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits 76

or potato dextrose agar medium for fungal strains 3. Results and Discussions
was poured into the Petri dish containing the inocu- Proximate composition of dried nettle
lum and mixed well. A sterile cylinder (8 mm in
leaves
periphery) was used to make wells into the solidify-
The chemical analysis of nettle leaves powder
ing agar plates containing inoculums. After solidifi-
was carried out on dry weight basis and the results
cation, 100 µl of the diluted stock solutions
are presented in Table 1. stinging nettle powder
(extracts) were added to the wells. Wells containing
showed higher amount of carbohydrates content
reference antibiotics (tetracycline for bacteria and
40.86% while recording the protein of 30.59%. Al-
Nystatin for fungi) served as positive controls (50
so found that total ash content of nettle. Dietary fi-
µg/ml). The incubation period was either 24 hours
ber not estimated record 16.25% this may be
for bacteria or 48 hours for incentives. Antimicrobi-
caused nettle is rich in minerals. The result in the
al exertion was determined by measuring the zone
same table showed that the crude fiber contents in
of inhibition (including the well periphery) after the
nettle powder record 8.80%, it may note in this ta-
incubation period.
ble that the moisture and fat content are less in the
Microbiological analysis chemical composition of dry nettle leaves, as per-
To determine microbial counts, three replicas centages were recorded 3.81% and 3.50% respec-
from each treatment and control were aseptically tively. From the table of the chemical composition
opened. Ten grams from each sample were trans- of the nettle plant, it is clear that it has a high nutri-
ferred to a sterilized glass bottle containing 90 ml tional value that makes it a candidate plant for use
of sterile physiological saline (0.85% NaCl). The in the food industry, the results are in harmony with
bottle was shaken to homogenize the sample, and (Shonte et al., 2020)
then serial 10-fold dilutions were prepared. Appro- Table 1. Chemical composition of dry nettle
priate serial dilutions were duplicate plated (pour leaves powder
plate method) with Plate Count agar medium
Parameters (%) Nettle
(APHA, 1985) for the total bacterial count. Then
Moisture 3.81±0.36
the plates were incubated at 37 oC for 24-48 hours. protein 30.59±2.48
Yeast mold agar (Jong and Edwars, 1991) was used Fat 3.50±0.05
for counting yeast and molds at 25 oC for 5 days. Ash 16.25±0.04
Sensory evaluation Crude fiber 8.80±0.35
The sensory characteristics of the control bis- Carbohydrates 40.86±0.01
cuit samples and the different addition ratios 5, 10, *Chemical composition was calculated based on the dry weight
and 15 were evaluated in terms of color, taste, basis. Data are presented as means ± SDM (n=3)
smell, texture, crispness, appearance, and general Content of mineral elements of dry nettle
acceptance. The evaluation was carried out by 10 leaves
food tech judges. Rec. Using a 10-point pleasure
Stinging nettle is rich in minerals, as shown in
scale. Samples receiving an overall quality score of
Table 2. Current studies showed the nettle powder
7 or above were considered palatable. according to
contained ash content, which is much higher than
the method of (Al-Marazeeq and Angor 2017).
conventional cereals. It contains nettle powder at a
Statistical analysis high rate of Mg, Ca, and K (858.98, 673.56, and
one-way analysis of variance (ANOVA), with a 641.93 ppm), respectively, while the leaf powder
multiple range significant difference (LSD) test, recorded a good percentage of Fe and Na (67.71
and standard error (SE) (p < 0.05) were carried out and 30.44 ppm). It was the lowest content of the
using SPSS according to (Sarmento and Costa elements Mn and Zn (12.47 and 7.50 ppm) nettle
2019). powder probably is one of the richest sources of

Food Technology Research Journal, Vol. 2, special issue 2, 73-85, 2023

77 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits

minerals among the plant foods this is evident from Rheological properties
the study of the mineral content of nettle leaf pow-
der these results are in symmetry with (Adhikari et
Farinograph test
al. 2016) All measured values of the rheological properties
Table 2. Mineral content of dry nettle leaves (Farinograph and Extensograph) of wheat flour to
powder which dry nettle leaves are added are shown in
Table 4. Water absorption gradually increased with
Nettle Mineral elements (ppm)
the addition of dry nettle leaves; it increased from
Ca 673.56±3.16 61.5% for the control wheat flour sample to 62.5%,
K 641.93±3.80 63.0%,63.0 64.5%, respectively, for wheat flour
Mg 858.98±12.72 supplemented with 5%, 10%, and 15% dry nettle
Na 30.44±1.82 leaves. It is well known that an increase in fiber
content in a dough causes an increase in water ab-
Fe 67.71±2.36
sorption (Abdel-rahman 2014). The hydroxyl
Mn 12.47±0.70
groups of the fiber structure cause an interaction
Zn 7.50±0.61 between water and hydrogen bonds, and this illus-
trated the increase in the tendency of flour to hold
Antioxidant content of dry nettle leaves
water (Amir et al., 2015). Likewise, dough develop-
The results of content and antioxidant content
ment time increased and reached 3.0 min for adding
determined in the leaves of nettle are presented in
15% dry nettle leaves as compared to the corre-
Table 3. It was noticed from the table that the high-
sponding control (1.5 min). On the other hand,
est percentage was recorded for the total phenolic
dough stability time (min) was shortened in the
content of nettle powder, TP (206.00 mg GAE/100
dough in the percentage of addition 15% because
g), followed by the total antioxidant content, TA
the existence of fiber particles resulted in a disrup-
(64.27%), and the lowest total flavonoids, TF
tion of the starch-gluten network and thus a de-
(25.54 mg CAT/100 g). These results were recorded
crease in dough stability time, while this value in-
by Otles and Yalcin (2012), and it has been reported
creased and reached its maximum in the dough with
that the natural phenolic compounds play an im-
5% and 10% addition compared with the corre-
portant role in cancer prevention and treatment.
sponding control (Abdel-Ghafor et al. 2013). The
Phenolic compounds from medicinal herbs and die-
dough stability is better when the time is longer
tary plants include phenolic acids, flavonoids, tan-
(Mehfooz et al., 2018). They demonstrated that the
nins, and others. The colorful bioactivities of phe-
high concentration of dry nettle leaves mixed with
nolic composites are responsible for their chemo-
white flour led to a progressive elevation in water
preventive properties, e.g., antioxidant, anticarcino-
immersion, dough stability time, and appearance
genic, antimutagenic, and anti-inflammatory.
time. The degree of softening (B.U.) increased with
Table 3. Determination of total antioxidant con-
the addition of dry nettle leaves and reached its
tent (TA), total phenolic, (TP) and total flavo-
maximum in the dough blending at 15%. (El-Taib et
noids (TF) of dry nettle leaves powder
al., 2018) support our findings when they found that
Parameters Nettle the degree of weakening (BU) was raised progres-
sively by the increasing ratios of dry nettle leaf ad-
TA (%) 64.27±2.05
dition, and it can be attributed to lower wheat gluten
TP (mg GAE /100g) 206.00±2.02
content in the dough (El-Basyouny et al., 2019)
TF (mg CAT /100g) 25.54±5.10
Extensograph test
The Extensograph provides information about
the viscoelastic behavior of dough (Sharma et al.,

Food Technology Research Journal, Vol. 2, special issue 2, 73-85, 2023

 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits 78

et al., 2016). This equipment measures dough exten- ture the dough (high maximum resistance to elastici-
sibility and resistance to elasticity. A combination ty) (Sullivan et al., 2010) also stated that the elastic-
of good resistance and good extensibility results is ity of the dough lowered with the addition of dry
needed for dough properties (Rizk et al., 2016). The nettle leaves. It can be noticed that the proportional
data is shown in Table 4. illustrated that the elastici- number (P.N.) increased from 2.40 for the control
ty of the dough was reduced with the increase in dry sample to 3.16, 3.86, and 3.66, respectively, at lev-
nettle leaves, and the same phenomenon was ob- els 5, 10, and 15% of dry nettle leaves. These data
served in the extensibility. These data are on the were in agreement with (Aly et al., 2021), who
same line with (El-Taib et al., 2018), who men- demonstrated that the proportional number in-
tioned that the dough extensibility is the ability of creased as the levels of dry nettle leaves increased.
the dough to extend or stretch; it depends on the gli- Concerning the dough energy (cm2), the values were
adin protein in the dough. showed a pronounced re- reduced by increasing the proportion of dry nettle
tard as the percentage of dry nettle leaves increased leaves, suggesting that the incorporation of dry net-
the extensibility of the dough. Mixtures that have an tle leaves makes the dough more suitable for mak-
increased distance before rupture (high extensibil- ing biscuits (Wang et al., 2015).
ity) whereas the increasing force demanded to rup-
Table 4. Farinograph and Extensograph parameters of biscuit dough containing dry nettle leaves
Farinograph Extensograph

Sample Water Arrival Dough Degree of

Stability Elasticity Extensibility Energy
absorption time Development softening P. N
(min) (B.U) (mm) (cn2)
(%) (min) (min) (B.U)

Control 61.5 1.0 1.5 2.5 50 320 95 2.40 64

5% 62.5 1.5 2.0 10.0 60 300 125 3.16 46
10% 63.0 1.5 2.0 10.0 60 270 85 3.86 43
15% 64.5 1.5 3.0 9.5 80 250 70 3.77 32
P.N: Proportional number

Sensory evaluation of biscuits during leaves contain a pungent taste, as this taste appears
storage at room temperature in the high percentage of 15% added, but this per-
Table 5. illustrates the sensory characteristics of centage was not rejected by the arbitrators, as there
biscuits containing dry nettle leaves at zero time are consumers who desire this taste. (Mitrevski et
and after storage for six months at room tempera- al., 2023). Also, with regard to color, there was an
ture. It is noted that the highest scores were for the evaluation of the lowest scores between the propor-
control group, followed by the percentage of addi- tions added and the control group, where increasing
tion of 5%, which is close to the control scores the addition of dried nettle leaf led to a darkening of
(Rakshit and Srivastav 2022). While the percentage the color. However, it was not rejected by the arbi-
of addition of 10% recorded higher scores than the trators. Where the color was not dark to the point of
percentage of 15% in all sensory characteristics rejection (Ivanisˇova et al., 2019)
compared to the control during the storage periods.
The taste in the percentage of addition (15%) rec-
orded the lowest scores in evaluating the sensory
characteristics of the percentages added during the
storage periods (Godase et al., 2020). Dry nettle

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79 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits

Table 5. Sensory evaluation of dry nettle leaves biscuits with different addition ratios
Treatments Overall
Color Odor Texture Taste Crispness Appearance
acceptability
Zero time
control 9.36±0.04 9.22±0.03 9.27±0.02 9.48±0.03 9.24±0.15 9.18±0.10 9.15±0.01
5% 9.08±0.6 9.12±0.10 9.37±0.46 9.19±0.02 9.50±0.44 9.35±0.39 9.16±0.10
10% 8.93±0.15 8.96±0.02 8.89±0.10 9.13±0.32 8.94±0.02 8.98±0.03 8.97±0.03
15% 8.88±0.15 8.94±0.18 8.87±0.20 8.90±0.03 8.90±0.10 8.87±0.20 8.88.0.15
1 month
control 9.25±0.05 9.17±0.02 9.22±0.03 9.43±0.01 9.19±0.16 9.06±0.44 9.12±0.14
5% 8.92±0.03 8.91±0.20 8.87±0.02 8.89±0.10 8.89±0.03 8.93±0.02 8.84±0.03
10% 8.90±0.10 8.87±0.11 8.86±0.01 8.86±0.11 8.86±0.21 8.88±0.11 8.85±0.02
15% 8.83±0.04 8.86±0.10 8.80±0.10 8.83±0.13 8.83±0.03 8.84±0.15 8.86±0.02
3 months
control 9.13±0.15 9.6±0.43 8.97±0.21 8.92±0.10 8.87±0.01 8.88±0.03 8.79±0.30
5% 8.88±0.15 8.92±0.02 8.84±0.04 8.83±0.15 8.86±0.20 8.85±0.15 8.75.0.16
10% 8.84±0.10 8.87±0.14 8.82±0.20 8.78±0.13 8.85±0. 02 8.83±0.16 8.74±0.30
15% 8.83±0.06 8.84±0.15 8.81±0.15 8.75±0.25 8.81±0.01 8.80±0.03 8.60±0.34
6 months
control 8.95±0.10 8.89±0.25 8.78±0.15 8.79±0.03 8.80±0.01 8.75±0.02 8.76±0.10
5% 8.78±0.02 8.79±0.02 8.77±0.10 8.75±0.10 8.75±0.02 8.72±0.15 8.73±0.01
10% 8.75±021 8.76±0.15 8.75±0.21 8.73±0.25 8.74 ±0.10 8.71±0.20 8.71±0.15
15% 8.69±0.01 8.75±0.10 8.70±0.01 8.70±0.02 8.73±0.15 8.65±0.01 8.69±0.10

Effect of dry nettle leaves on the total bac- biscuits. concentration of 15% had a higher effect,
terial counting during storage periods where total bacterial counts appeared after 3 months
Microbial groups were counted in nettle leaves (2.30 log cfu/g) and increased to 4.65 log cfu/g after
with concentrations (5%, 10%, and 15%) and con- 6 months of storage. It can be concluded that there
trol biscuit samples. The counts of microbial groups are possibilities for these foods to become contami-
were determined directly after biscuit processing. nated during production and processing. Microbial
Samples were stored at room temperature with the spoilage in biscuits often depends on some factors
following microbial counts during the storage like storage temperature, humidity, storage hygiene,
period. Data in Table 6. showed that total bacterial packaging material, moisture content in the storage
counts were. Gradually during the storage period. area, etc. (Ahmed et al., 2020) and (Nerín, et al.,
detected at all directly after processing with all con- 2016). As a result of the previous reasons, the bacte-
centrations used at zero time. During the storage ria that are found in dried food may come during the
period, the total bacterial count appeared and in- production process and also after storage if the
creased gradually. The total bacterial count ap- packaging material is not intact or the storage condi-
peared a month later with 3.08 and 2.30 log cfu/g tion is in poor circumstances. During the production
and increased gradually to reach 3.34 and 2.77 log process, there are possible chances of contamination
cfu/g, respectively, after 3 months of storage. 6 occurring in mechanical areas of the processing line,
months later, after storage, total bacterial counts in- like the hopper with feeder, grader, peeler, feed con-
creased gradually, reaching 5.50, 4.91, and 4.56 log veyor, slicer, and flavor applicator. these areas
cfu/g, with concentrations of 5%, 10%, and 15%, where attention should be maximized to prevent
respectively, compared to the control. It means that such unwanted contamination (Ostadrahimi et al.,
15% can be enough as a preservation treatment for 2020).

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 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits 80

Table 6. Effect of dry nettle leaves on the total tested bacteria with inhibition zones ranging from
bacterial counting (log 10 Cfu/g) 10.0±0.58 to 12.7±0.67 mm., they were more effec-
Zero 1 3 6 tive against gram-positive bacteria (Bacillus cereus
Concentration
time month months months and Staphylococcus aureus) than against gram-
Control ND 3.81 4.15 6.49 negative bacteria (Salmonella typhimurium and
5% ND 3.08 3.34 5.50 Escherichia coli). This behavior could be due to the
10% ND 2.30 2.77 4.91 differences in their cell wall structures. Gram-
15% ND ND 2.30 4.65 negative bacteria have an extra-hydrophilic outer
Fungal changes in nettle biscuits membrane that functions as a preventive barrier.
It is evident from the Table 7. that the concen- This renders the Gram-negative bacteria generally
trations of nettle leaf on biscuits contributed to ob- more resistant to plant extracts than the Gram-
taining good results. fungies was not detected (ND) positive bacteria (Elswaby et al., 2022 and El-Rady
through increasing time of storage due to the low et al., 2023). The results are consistent with numer-
moisture in biscuits samples and reducing the num- ous studies (Harrison et al., 2022; Motomedi et al.,
bers of fungies in the various concentrations of 2014 and Mzid et al., 2017) that have documented
nettle leaf according to the level of addition com- the antibacterial potentiality of Urtica dioica against
pared with control treatment. Control samples data Gram-positive and Gram-negative bacteria, yeast,
showed that, the total fungi counts were not detect- and filamentous fungus. Solvent types can also af-
ed at zero time but increased in 1, 3 and 6 months as fect the antimicrobial properties of the extracts. Sol-
(2.9, 3.30 and 3.60 log cfu/g), respectively (Amir et vents differ in their extraction capabilities depend-
al., 2012). ing on their polarity and the solute’s chemical struc-
Table 7. Effect of dry nettle leaves dioica on the ture. Different solvent extracts have different solu-
total fungi (log 10 Cfu/g) ble phytoconstituents in different amounts, and
Zero 1 3 6
hence, they have different levels of antimicrobial
concentration properties. In the current report, methanol extracts
time month months months
Control ND 2.9 3.30 3.60 showed higher activity compared with the corre-
5% ND ND ND ND sponding ethanol extracts (Table 8.). This result
10% ND ND ND ND
suggests that most of the bioactive molecules in Ur-
tica dioica are soluble in methanol. Conflicting data
15% ND ND ND ND
have been reported regarding the superiority of
Antimicrobial activity methanol extracts over ethanol or other extracts in
The antimicrobial activity of ethanolic and nettle. Some studies have shown that methanol ex-
methanolic extracts of nettle appeared as a zone of tracts are superior to ethanol extracts (Motamedi et
inhibition for tested microorganisms. The results of al., 2014), but other studies have shown that ethyl
the antimicrobial activity are given in Table 8. acetate and hexane extracts have the strongest anti-
These results showed that while these extracts were bacterial activity against particular microorganisms
effective against both kinds of bacteria (gram- (Modarresi et al., 2012). Noteworthy to remark is
positive bacteria and gram-negative bacteria), they the fact that numerous factors contribute to varia-
demonstrated no effects against yeast-like fungi tions in the antibacterial characteristics of the same
(Candida albicans). The resistance of fungal spe- solvent, making it difficult to compare the results of
cies extracted from nettles might be attributed to different experiments. For instance, environmental
their morphological structure; fungi have thicker factors such as season, local climate and soil type,
cell walls and contain a higher percentage of chitin the status of plant raw material, and extraction con-
(Amer and Aly, 2019; Amer, 2018). Although all ditions (extraction time, extraction temperature, pH,
extracts exhibited antimicrobial activity against all etc.) can remarkably impact the solvent property

Food Technology Research Journal, Vol. 2, special issue 2, 73-85, 2023

81 Effect of Bioactive Compounds of Sting Nettle (Urtica Diocia L.) Leaves on Shelf Life of the Salty Biscuits

factors such as season, local climate and soil type, Staphylococcus aureus (MRSA) and vancomycin-
the status of plant raw material, and extraction con- intermediate Staphylococcus aureus (VISA). Excel-
ditions (extraction time, extraction temperature, pH, lent inhibition against Staphylococcus species
etc.) can remarkably impact the solvent property can be a hope for the discovery of new natural anti-
(Amer, 2018). In this work, Staphylococcus aureus bacterial agents against coagulase-positive and
was found to be the most susceptible microorgan- coagulase-negative staphylococci (Motamedi et al.,
ism to nettle extracts (in addition to Bacillus 2014).
cereus). Especially against methicillin-resistant
Table 8. Antimicrobial activity of ethanolic and methanolic extracts of dry nettle leaves
Zone of inhibition (mm.)
Staphylococcus Bacillus Salmonella Escherichia Candida albi-
Solvent extract aureus cereus typhimurium coli cans
Ethanol 10.0±0.58 10.7±0.33 ND ND ND
Methanol 12.3±0.88 12.7±0.67 6.0±1.00 4.7±1.20 ND
Tetracycline 16.0±1.00 16.3±0.88 10.3±0.88 8.0±0.58 -
Nystatin - - - - 13.3±0.8
Values are means of triplicate determination (n=3) ± standard errors. ND, no zone of inhibition was detected. Tetracycline and
nystatin served as positive controls for bacteria and fungi, respectively .
4.Conclusion with inhibition zones ranging from 10.0±0.58 to
From the study of dry nettle leaves, which have 12.7±0.67 mm., they were more effective
a high nutritional value, they can be added to salted against gram-positive bacteria (Bacillus cereus
biscuits to improve their properties and increase and Staphylococcus aureus) than against gram-
their nutritional value, as the nettle contains miner- negative bacteria (Salmonella typhimurium and
als, antioxidants, proteins, and fibers. Escherichia coli). This behavior could be due to
• Adding nettle leaves to wheat flour led to a the differences in their cell wall structures.
higher rate of water absorption, which made the • The nettle plant is important as it is considered a
dough soft, smooth, and easier to shape. preservative in foods intended for human con-
• bacterial load of biscuits added to nettle leaves sumption. Therefore, it can be used as an alter-
during storage periods. The addition rate of 15% native to industrial preservatives and also as an
can be enough as a preservation treatment for antibacterial and antifungal agent in the food
biscuits. concentration of 15% had a higher ef- industry, giving a high nutritional value to the
fect, where total bacterial counts appeared after product.
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