fermentation

Article
Studies on Proximate Composition, Mineral and Total Phenolic
Content of Yogurt Bites Enriched with Different Plant
Raw Material
Jurgita Kulaitienė, Nijolė Vaitkevičienė * and Dovilė Levickienė

Department of Plant Biology and Food Sciences, Vytautas Magnus University Agriculture Academy,
Donelaičio Str. 58, 44248 Kaunas, Lithuania; jurgita.kulaitiene@vdu.lt (J.K.); dovile.levickiene@vdu.lt (D.L.)
* Correspondence: nijole.vaitkeviciene@vdu.lt; Tel.: +370-(37)-752-326

Abstract: Yogurt products are consumed by millions of people every day. Consumers’ priority for
ready-to-eat yogurt snacks enriched with various plant raw materials have increased each year.
Therefore, the aim of this study was to prepare freeze-dried yoghurt bites with the addition of
powders of beetroot, mulberry leaves, nettle leaves and rosehip fruit and to investigate these raw
materials’ influence on the proximate composition, mineral and total phenolic content. The moisture,
protein, fat, carbohydrate and sugar content of the yogurt bites were established using standard
methods: mineral composition—using an inductively coupled plasma mass spectrometer (ICP-
MS); total phenolic content—by the spectrophotometric method. The results demonstrated that the
addition of different raw material powders to the formulation of yogurt bites had no significant
influence on carbohydrate, protein and total fat amounts. However, the incorporation of powders





 of beetroot, mulberry leaves, nettle leaves and rosehip fruit in yogurt bites allowed a significant
increase of the amounts of all investigated minerals and total phenolic content of the manufactured
Citation: Kulaitienė, J.; Vaitkevičienė,
bites. Among all investigated yogurt bites, the highest amounts of K, P, Mg, Fe and Zn were
N.; Levickienė, D. Studies on
Proximate Composition, Mineral and
determined for yogurt bites enriched with nettle leaves. In conclusion, the enrichment of yogurt bites
Total Phenolic Content of Yogurt Bites with freeze-dried plant raw material powders can increase amounts of selected minerals and total
Enriched with Different Plant Raw phenolic content.
Material. Fermentation 2021, 7, 301.
https://doi.org/10.3390/fermentation Keywords: yogurt bites; nettle leaves; mulberry leaves; rosehip fruit; beetroot; calcium; potassium;
7040301 phosphorus; iron

Academic Editor: Senaka Ranadheera

Received: 16 November 2021 1. Introduction

Accepted: 9 December 2021
With the intense development of the food industry, a lot of products of the ready-to-eat
Published: 9 December 2021
type are being produced for the customer and the amount of such produced food products
is increasing rapidly. People that are interested in healthy and functional food enriched
Publisher’s Note: MDPI stays neutral
with additional vitamins, minerals, bioactive ingredients and probiotic bacteria grows
with regard to jurisdictional claims in
published maps and institutional affil-
each year [1]. Such products are gaining more widespread popularity and acceptance
iations.
throughout the developed world. Therefore, functional products need to satisfy advanced
product properties: extended shelf life, attractive market appearance, appropriate and
attractive taste, smell and colour, and maintain these properties during transportation and
storage time.
Greek yoghurt is one of the most popular fermented milk products. It is a great source
Copyright: © 2021 by the authors.
of high-quality protein, calcium, phosphorus and potassium, and various vitamins [2–4],
Licensee MDPI, Basel, Switzerland.
but it is considered a poor source of phenolic compounds. Raw materials such as berries,
This article is an open access article
distributed under the terms and
vegetables or leaves are a promising source of phenolic components [5]. However, yoghurt
conditions of the Creative Commons
shelf life is short compared to other dairy products such as cheese. In addition, the yoghurt
Attribution (CC BY) license (https://
requires appropriate temperature conditions and refrigeration equipment [6]. Currently,
creativecommons.org/licenses/by/ the world market of freeze-dried yoghurt snacks/bites is fragmented. Yoghurt snacks are
4.0/). mostly enriched with berries and fruit concentrates (strawberries, raspberries, blueberries,

Fermentation 2021, 7, 301. https://doi.org/10.3390/fermentation7040301 https://www.mdpi.com/journal/fermentation

Fermentation 2021, 7, 301 2 of 11

blackberries, peaches, etc.). There is no such yoghurt snack or bite which is enriched
with nettle and mulberry leaves, or beetroot and rosehip powder. Nettle (Urtica dioica L.)
has a long history of usage and is currently receiving attention as a source of fibre and
alternative medicine. In many cultures, nettle is also eaten as a leafy vegetable. The
entire plant of nettle can be utilised for a variety of applications, including food, fodder,
medicinal, cosmetics, biodynamic agriculture and textile manufacture [7,8]. Chlorophyll,
carotenoids, vitamins, proteins, lipids, carbohydrates, organic acids, minerals and trace
elements are abundant in the leaves of nettles [9–12]. In many regions of the world,
beetroot (Beta vulgaris L.) is a traditional and popular vegetable. Beetroot includes a high
concentration of biologically active components such as phenolic compounds, betalains,
carotenoids, B-vitamins (B1 , B2 , B3 , B6 and B12 ), fibre and low-energy carbohydrates [13].
Furthermore, manganese, potassium, phosphorus, magnesium, sodium, iron, copper, zinc,
boron, silica and selenium are all minerals found in beetroots [14–16]. Beetroot is produced
for food rather than for the manufacturing of sugar. Sucrose is the primary sugar in beets,
with small levels of glucose and fructose [17]. Rose plants (Rosa spp.) produce fruits that
are high in polyphenols, vitamins A, C and E, essential fatty acids, galactolipid, minerals
(Ca, Mg, K, S, Si, Se, Mn and Fe) and other bioactive components [18,19]. Due to its
chemical content and pharmacological properties, the white mulberry plant (Morus alba L.)
is one of the traditional herbs that has been use in medicine since ancient times. Mulberry
leaves have been established as a high-protein, high-carbohydrate, high-vitamin and high-
dietary fibre food source [20–22]. Additionally, the mulberry leaves contain a variety of
macroelements. Mineral elements play important roles in biological reactions and have
structural functions [20,23–25].
As the demand for healthier food products with health-promoting effects has been
increasing significantly worldwide, consequently, the incorporation of lyophilised raw
material powder in yogurt can be a popular approach to increase the phenolic and mineral
element content of the product. Therefore, the purpose of this research is to prepare
freeze-dried yoghurt bites with the addition of powders of beetroot, mulberry leaves, nettle
leaves and rosehip fruit, and to investigate the influence of these plant raw materials on
the proximate composition, mineral and total phenolic content of the final product.

2. Materials and Methods

2.1. Materials
The plant raw materials were collected in 2019 from spring to autumn. White mulberry
(Morus alba) leaves were collected in June, and nettle (Urtica dioica) leaves in March, on an
organic farm in Kaunas district, Lithuania (54◦ 530 5000 N 23◦ 530 1000 E). Rosehip (Rosa rugosa)
fruits were harvested in August, and red beetroot (Beta vulgaris) in the end of September at
an organic farm in Pakruojis district, Lithuania (56◦ 100 29.000 N 23◦ 490 02.600 E). Collected raw
materials were frozen at −35 ◦ C and lyophilised using a Freeze-Drying Plant Sublimator
(ZIRBUS technology GmbH, Bd Grund, Germany). The samples were lyophilised for 48 h,
then were milled (Grindomix GM 200, Retsch GmbH, Haan, Germany), their chemical
composition determined in dry matter (DM) (Table 1) and stored in sealed containers at
5 ◦ C in the dark until yogurt bites preparation.
Greek-style yogurt was purchased at local supermarkets in the city of Kaunas (Lithua-
nia). Nutrition facts of Greek-style yogurt are shown in Table 2.

2.2. Yogurt Bites Preparation

In this research, the experiment consisted of 5 treatments as follows (Figure 1):
1. Control (yogurt bites without plant raw materials);
2. Yogurt bites with 1% rosehip fruit powder;
3. Yogurt bites with 1% nettle leaves powder;
4. Yogurt bites with 1% mulberry leaves powder;
5. Yogurt bites with 1% beetroot powder.
 32.92 ± 0.20 11.89 ± 0.17 8.46 ± 0.14 38.08 ± 0.24
g−1)
2500.01 ±
K (mg 100 g−1) 1630.14 ± 29.3 2950.25 ± 41.3 2630.46 ± 40.0
38.2
Ca (mg 100 g−1) 220.03 ± 9.20 2870.08 ± 36.7 2680.16 ± 35.6 590.33 ± 11.15
Fermentation 2021, 7, 301 P (mg 100 g ) 410.22 ± 13.2 430.88 ± 10.2
−1 770.47 ± 16.1 690.81 ± 14.31
3 of 11
170.06 ±
Mg (mg 100 g−1) 270.68 ± 9.21 430.58 ± 14.5 340.66 ± 10.0
12.00
Fe (mgcomposition
Table 1. Chemical 100 g−1) 3.81
of ± 0.07 plant
freeze-dried 7.93raw
± 0.11 13.25 ±DM.
material powders, 0.41 7.52 ± 0.13
Zn (mg 100 g−1) 2.43 ± 0.04 2.73 ± 0.09 3.34 ± 0.19 2.55 ± 0.17
Nutrition Facts B (mgBeetroot
100 g−1) 1.99Mulberry
± 0.01 Leaves2.71 ± 0.03 Nettle Leaves
3.28 ± 0.04 Rosehip
3.19 Fruit
± 0.04
Moisture (%) (mg±100
Mn 3.07 0.03g )
−1 3.26 ± 0.041.82 ± 0.03
1.49 ± 0.02 3.51 ±1.93
0.08± 0.03 ± 0.05
1.73
3.24 ± 0.02
Protein (g·100 g−1 ) ± 0.11
4.31phenolic
Total 3.42 ± 0.09 5.28 ± 0.13 3.93 ± 0.07
Fat (g·100 g−1 ) 1.42 ± 0.02 224.69 ±1.15
7.13± 0.02
301.94 ± 8.99 1.39313.46
± 0.03 ± 12.58 461.94
0.98 ± 14.31
± 0.02
−1 (mg 100 g−1)
Sucrose (g·100 g ) 31.66 ± 0.29 9.96 ± 0.13 5.57 ± 0.09 18.57 ± 0.14
Glucose (g·100 g−1 ) 1.27 ± 0.02 1.41 ± 0.05 1.39 ± 0.03 9.13 ± 0.12
Fructose (g·100 g−1 ) Greek-style
<0.02 yogurt was0.53 purchased
± 0.02 at local supermarkets
1.50 ± 0.03 in the city11.08
of Kaunas
± 0.10 (Lith-
Total sugar (g·100 g−1 ) uania). Nutrition
32.92 ± 0.20 facts of Greek-style
11.89 ± 0.17yogurt are shown in Table
8.46 ± 0.14 2. 38.08 ± 0.24
K (mg·100 g−1 ) 2500.01 ± 38.2 1630.14 ± 29.3 2950.25 ± 41.3 2630.46 ± 40.0
Ca (mg·100 g−1 ) Table220.03 ± 9.20 facts of Greek-style
2. Nutrition 2870.08 ±yogurt.
36.7 2680.16 ± 35.6 590.33 ± 11.15
P (mg·100 g−1 ) 410.22 ± 13.2 430.88 ± 10.2 770.47 ± 16.1 690.81 ± 14.31
Mg (mg·100 g−1 ) 170.06Nutrition
± 12.00 Facts 270.68 ± 9.21 As±Sold
430.58 14.5 for 100 g 340.66 ± 10.0
Fe (mg·100 g−1 ) Energy
3.81 ± 0.07 (kcal) 7.93 ± 0.11 13.25 ± 0.41121 7.52 ± 0.13
Zn (mg·100 g−1 ) 2.43 ± 0.04
Fat (g) 2.73 ± 0.09 3.34 ± 0.19 10 2.55 ± 0.17
B (mg·100 g−1 ) ± 0.01
1.99Saturated fat (g) 2.71 ± 0.03 3.28 ± 0.04 6.5 3.19 ± 0.04
Mn (mg·100 g−1 ) 1.49 ± 0.02 1.82 ± 0.03 1.93 ± 0.03 1.73 ± 0.02
Carbohydrates (g) 4.0
Total phenolic (mg·100 g−1 ) 224.69 ± 7.13 301.94 ± 8.99 313.46 ± 12.58 461.94 ± 14.31
Sugars (g) 4.0
Proteins (g) 3.7
Table 2. Nutrition facts of Greek-style yogurt.
Salt (g) 0.1
Nutrition Facts As Sold for 100 g
2.2. Yogurt Bites Preparation
Energy (kcal) 121
In this research,
Fatthe
(g) experiment consisted of 5 treatments as 10
follows (Figure 1):
1. Saturated
Control (yogurt fat (g)
bites without plant raw materials); 6.5
Carbohydrates (g) 4.0
2. Yogurt bites with 1% rosehip fruit powder;
Sugars (g) 4.0
3. Yogurt bitesProteins
with 1% (g)nettle leaves powder; 3.7
4. Yogurt bites with
Salt (g) mulberry leaves powder;
1% 0.1
5. Yogurt bites with 1% beetroot powder.

Figure 1. Yogurt bites: yogurt bites without plant raw materials (control) (1); yogurt bites with 1%
rosehip fruit powder (2); yogurt bites with 1% nettle leaves powder (3); yogurt bites with 1% beetroot
powder (4); yogurt bites with 1% mulberry leaves powder (5).

To prepare yogurt bites, Greek yogurt, plant raw material powder, fruit pectin, corn
starch and maltodextrin were mixed in a mixer until a smooth mixture was obtained. The
obtained yogurt samples of 7 mL of yoghurt were poured into silicone moulds and frozen
to −36 ◦ C overnight before being transferred to the freeze dryer. After 24 h, the samples
were lyophilised using a Freeze-Drying Plant Sublimator 30 40 5 (ZIRBUS Technology GmbH,
Fermentation 2021, 7, 301 4 of 11

Bd Grund, Harz, Germany). The samples were transferred to the freeze dryer operating at
−40 ◦ C in a condensation chamber under vacuum at a minimum pressure of 0.011 kPa and
maintained during freeze-drying. The freeze-drying was completed in 72 h. The resultant
bites were kept in zip-lock plastic bags until chemical analyses were established. For yogurt
bites’ quality, laboratory analyses of 500 g (165 bites) of final lyophilised product were
prepared from each treatment. All the chemical analyses were replicated tree times.

2.3. Proximate Composition Analysis

The amounts of total sugar in plant raw materials and yogurt bites were identified
using a standard enzymatic assay (second method) [26]. The total fat in yogurt bites and
plant raw materials was established by the Soxhlet extraction method using SOXTHERM
(Gerhardt, GmbH & Co., Königswinter, Germany) according to AOAC932.06/2012 [27],
and the total protein by the Kjeldahl method according to EN ISO 8968-3:2007 using KJEL-
DATHERM (Gerhardt, Königswinter, Germany) [28]. Total carbohydrates were determined
by calculating the percentage remaining after all the other components were measured.

2.4. Mineral Elements Analysis

Mineral contents (K, Ca, P, Mg, Fe, Zn, B, Mn) in plant raw materials and yogurt
bite samples were carried out using a CEM MARS 6® (CEM Corporation, Matthews, NC,
USA) digestion system equipped with 100 mL Teflon vessel. Approximately 0.3 g of the
homogenised sample was accurately weighed into a Teflon vessel and digested using a
nitric (HNO3) and hydrochloric (HCl) acid mixture (5:1). Digestion was performed under
the following conditions: temperature—180 ◦ C; pressure—800 psi; ramp time—20 min;
hold time—20 min; microwave power—800 W. The digested sample was cooled down and
thoroughly transferred into a 100 mL volumetric flask and diluted using bidistilled water
till the mark. Each sample was prepared in triplicate and the blank sample was included in
each digestion run. Qualitative and quantitative evaluation of elements was performed
using an inductively coupled plasma mass spectrometer (ICP-MS). Inductively coupled
plasma mass spectrometry was performed under a helium collision cell (He-cell) with
kinetic energy discrimination mode to remove polyatomic interferences. Samples were
introduced using an autosampler with ASXpress. A rapid uptake module (CETAC ASX-
520, Teledyne Technologies Inc., Omaha, NE, USA) through a PEEK nebuliser (Burgener
Mira Mist, Burgener Research Inc., Mississauga, Canada) was used. The standard mixture
solution of multiple mineral elements in 2% nitric acid was obtained from CPAchem
(Bulgaria). Bidistilled water was prepared by means of distillation apparatus from Thermo
Scientific (Fremont, CA, USA). The limits of detection (LOD) and quantification (LOQ) are
shown in Table 3.

Table 3. LOD and LOQ (mg·kg−1 ).

Elements LOD LOQ

K 0.0027 0.009
Ca 0.0056 0.0187
P 0.01 0.033
Mg 0.01 0.033
Fe 0.0155 0.0511
Zn 0.008 0.264
B 0.0231 0.106
Mn 0.0042 0.0138

2.5. Determination of Total Phenolic Content

Total phenolic compound content was measured by the spectrophotometric method
using Folin–Ciocalteu reagent [29]. In this study, 0.25 g freeze-dried plant raw materials
or yogurt bite samples were mixed with 10 mL ethanol (70% v/v) and extracted in an
ultrasonic bath for 30 min. Then, the extract was centrifuged at 3000 rpm for 30 min.
Fermentation 2021, 7, 301 5 of 11

Next, 0.2 mL prepared extract was mixed with 1 mL Folin–Ciocalteu reagent, 0.8 mL
sodium carbonate (7%) and 5 mL pure water. After 60 min of incubation at 20 ◦ C in the
dark, the absorbance was measured at 760 nm using a spectrophotometer (Labomed Inc.,
Los Angeles, CA, USA). Total phenolic content was measured with the calibration curve
using gallic acid equivalent standards. The results were expressed in milligrams per 100 g
of dry matter (mg·100 g−1 DM).

2.6. Statistical Analysis

Data on the proximate and mineral composition of yogurt bites were processed by
Microsoft® Excel® 2016 MSO software and the Statistica 10 (StatSoft, Inc., Tulsa, OK, USA)
package. The reliability of the results was evaluated by one-way analysis of variance, using
the ANOVA software package. The statistical significance of differences between the means
was estimated by Tukey’s test (p < 0.05).

3. Results and Discussion

3.1. Proximate Composition
The proximate composition such as moisture, protein, carbohydrates and fat of all
yogurt bite samples is summarised in Table 4. In this study, the amount of moisture varied
from 4.13 to 4.76% (Table 4). The significantly highest moisture amount was found in
yogurt bites enriched with rosehip fruit powder, in comparison with all yogurt samples.
Among all investigated yogurt bites, the highest amount of total protein was determined
for yogurt bites with nettle leaves, followed by the yogurt bites with beetroot, control bites
and yogurt bites with mulberry leaves (14.13, 13.84, 13.72 and 13.57 g·100 g−1 , respectively).
Our data showed that total protein amount was established 4.88% lower in the yogurt
bites enriched with rosehip powder compared with the control. In addition, all different
raw material powder additives used in the yogurt bites had no significant influence on
carbohydrates and total fat amounts. Our investigated plant raw material powders (rosehip
fruit, beetroot, mulberry leaves and nettle leaves) are not rich in proximate compounds
(protein, carbohydrates and fat) (Table 1). Agustini and other researchers [30] established
that the additive of spirulina powder to yogurt samples had significant differences in
protein, but no significant differences in moisture or carbohydrate compared to control.
Addition of freeze-dried raw material powder did not significantly affect the energy amount
of yogurt bites, which ranged from 527 to 534 kcal. Similar results were determined by
Barakat et al. [31], confirming that proximate chemical composition of pumpkin yoghurt by
adding different pumpkin pulps had no significant impact on caloric values. Additionally,
similar findings were reported by several other researchers [32–34].
Table 5 shows that the amount of sucrose was found to range from 19.25 to 21.29 g·100 g−1
in all investigated yogurt bites, and the maximum amount of sucrose was established in
yogurt bites with beetroot powder. According to the literature, fresh beet/beet powder or
extracted pigments are used to improve the red colour of sauces, desserts, sweets, jams,
jellies, soups and ice cream [35,36]. As can be seen in Table 1, the highest amount of sucrose
was found in beetroot powder (31.66 g·100 g−1 ). Bavec and other authors [17] have also
established that the main sugar in beetroot was sucrose, followed by small amounts of glucose
and fructose. Data showed that beetroot additive significantly increased sucrose amount in
yogurt bites by as much as 7.24%, compared with the control.
Among all our investigated yogurt bites was established the significantly highest
glucose and fructose amounts in yogurt bites enriched with rosehip fruit powder (2.34
and 1.46%, respectively). Additionally, by 1.18 times, the significantly highest total sugar
amount was established in bites enriched with rosehip fruit, compared with the control
product. It could be due to rosehip fruit powder having the highest amount of total
sugar (38.08 g·100 g−1 ) compared with other investigated raw materials (Table 1). A
lesser amount of total sugar in rosehips (8.68–22.44 g·100 g−1 ) has been reported by
Kadakal et al. [37]. Therefore, there were no significant differences of total sugar between
our other investigated samples (Table 5). According to the literature, sugars are the most
Fermentation 2021, 7, 301 6 of 11

common soluble constituents of berries and fruit. These sugars have an important effect
on taste, nutritive properties and shelf life, and they are acceptable by consumers [38].
Moreover, sugars participate in polyphenol biosynthesis [39]; thus, higher sugar content
in fruit implies higher polyphenol concentration [40], and the same is confirmed by the
data of our study, where rosehip fruit powder was used as an additive in the production of
yogurt bites (Table 5).

Table 4. Proximate composition of yogurt bites.

Control YBB YBM YBN YBR p-Values

Moisture (%) 4.31 ± 0.11 b1 4.13 ± 0.11 b 4.31 ± 0.12 b 4.23 ± 0.13 b 4.76 ± 0.23 a 0.0035
Protein (g·100 g−1 ) 13.72 ± 0.22 ab 13.84 ± 0.27 a 13.57 ± 0.32 ab 14.13 ± 0.26 a 13.05 ± 0.15 b 0.5400
Carbohydrates (g·100 g−1 ) 46.66 ± 0.61 a 45.94 ± 0.83 a 46.66 ± 1.04 a 46.18 ± 0.71 a 46.77 ± 1.03 a 0.0044
Total fat (g·100 g−1 ) 32.00 ± 0.67 a 32.80 ± 0.85 a 32.00 ± 0.73 a 32.00 ± 0.95 a 32.00 ± 1.01 a 0.7152
Energy (kcal) 530 ± 5.31 a 534 ± 6.32 a 529 ± 4.99 a 529 ± 4.16 a 527 ± 5.11 a 0.5752
Energy (kJ) 2210 ± 15.02 a 2230 ± 14.21 a 2208 ± 8.30 a 2209 ± 10.23 a 2201 ± 6.30 a 0.0855
1Different letters in the same line represent significant differences between samples (p < 0.05). Control—yogurt bites without plant raw
materials, YBB—yogurt bites with 1% beetroot powder, YBM—yogurt bites with 1% mulberry leaves powder, YBN—yogurt bites with 1%
nettle leaves powder, YBR—yogurt bites with 1% rosehip fruit powder.

Table 5. The sugar content of yogurt bites, g·100 g−1 .

Control YBB YBM YBN YBR p-Values

Sucrose 19.75 ± 0.70 b 1 21.29 ± 0.79 a 20.09 ± 0.81 b 19.21 ± 0.62 b 20.49 ± 0.65 b 0.0463
Glucose 0.63 ± 0.05 c 0.74 ± 0.05 c 0.52 ± 0.06 c 1.20 ± 0.08 b 2.34 ± 0.23 a <0.0001
Fructose 0.21 ± 0.03 b 0.08 ± 0.01 c 0.25 ± 0.04 b 0.23 ± 0.03 b 1.46 ± 0.22 a <0.0001
Total sugars 20.59 ± 0.99 b 21.37 ± 0.96 b 20.86 ± 0.85 b 20.64 ± 0.88 b 24.29 ± 1.03 a 0.0031
1Different letters in the same line represent significant differences between samples (p < 0.05). Control—yogurt bites without plant raw
materials, YBB—yogurt bites with 1% beetroot powder, YBM—yogurt bites with 1% mulberry leaves powder, YBN—yogurt bites with 1%
nettle leaves powder, YBR—yogurt bites with 1% rosehip fruit powder.

3.2. Mineral Composition

A macro- and micro-element amount of yogurt is directly related to the original
milk used for the production of these products. The mineral composition of milk and
yogurt does not change during fermentation [41]. Yogurt is a nutrient-dense food that is
an excellent source of some minerals such as calcium, magnesium, phosphorus and so
on. These essential micronutrients have a positive influence on human health, especially
decreasing the risk of diseases. Their deficiency causes various health disorders such
as mental retardation, learning disabilities, blindness, low work efficiency as well as
early death [42,43]. Increasing essential micronutrient bioavailability results in improving
the society’s healthcare [43,44]. Various strategies such as yogurt product enrichment
with plant raw materials and freeze-drying of final product may be employed to reach this
purpose. Therefore, one of the aims of this study was to manufacture novel yogurt products
with a higher amount of minerals by combining different freeze-dried raw material powder.
Furthermore, there is little information available in the literature regarding the macro- and
micro-element amounts of freeze-dried yogurt bites.
The mineral element amounts of yogurt bites are depicted in Table 6. The average
amount of mineral elements can be represented as K > Ca > P > Mg > Fe > Zn >
B > Mn. Results showed that of the studied minerals, K has the highest amount in all
samples, ranging from 315.70 mg·100 g−1 in yogurt bites without plant raw materials to
603.30 mg·100 g−1 in yogurt bites with 1% nettle leaves powder. Yogurt bites enriched
with different plant raw material powders had significantly higher amounts of this element
by 38.09–91.10% compared with the control. The highest K amount was determined for
yogurt bites enriched with nettle leaves powder (603.30 mg·kg−1 ), and the yogurt bites with
rosehip fruit powder showed the second highest amounts of this element (546.22 mg·kg−1 ).
As can be seen in Table 2, freeze-dried nettle leaves powder contained the highest amount
of K, followed by the rosehip fruit, beetroot powder and mulberry leaves. According
Fermentation 2021, 7, 301 7 of 11

to the literature, nettle leaves [11] and rosehip fruit [45] are excellent additives for the
development of enriched foods with an increased amount of potassium. The amount
of K can range from 2590 to 3600 mg·100 g−1 for nettle leaves [46] and from 1110 to
4540 mg·100 g−1 for rosehip fruit [47]. The recommended daily allowance (RDA) for
K is set at 2000 mg/day for adults [48]. Assuming a yogurt bites intake of 100 g/day,
the nettle powder enrichment bites supply the 30.17%, the rosehip powder enrichment
bites—27.31% of K RDA. The yogurt bites with beetroot (435.94 mg·100 g−1 ) and mulberry
leaves (445.20 mg·100 g−1 ) powders exhibited similar amounts of this mineral, where no
difference was found (approx. 22% of recommended daily intake, respectively).

Table 6. Mineral element composition of yogurt bites, mg·100 g−1 .

Mineral
Control YBB YBM YBN YBR p-Values
Elements
K 315.70 ± 6.41 d 1 435.94 ± 9.04 c 445.20 ± 5.00 c 603.30 ± 35.03 a 546.22 ± 5.74 b <0.0001
Ca 198.78 ± 4.74 d 213.83 ± 5.20 c 240.81 ± 3.32 a 226.24 ± 2.00 b 186.21 ± 3.40 d <0.0001
P 146.70 ± 2.39 e 160.73 ± 4.21 d 202.57 ± 2.61 c 255.50 ± 3.60 a 241.52 ± 5.53 b <0.0001
Mg 14.52 ± 1.51 e 24.80 ± 2.50 d 34.80 ± 1.09 c 51.93 ± 1.12 a 40.28 ± 1.39 b <0.0001
Fe 1.98 ± 0.04 d 2.35 ± 0.16 c 3.30 ± 0.14 b 5.29 ± 0.26 a 3.53 ± 0.19 b <0.0001
Zn 1.82 ± 0.02 d 2.27 ± 0.12 c 2.54 ± 0.09 b 3.08 ± 0.17 a 2.73 ± 0.13 b <0.0001
B 0.09 ± 0.008 c 0.23 ± 0.02 b 0.23 ± 0.03 b 0.32 ± 0.02 a 0.29 ± 0.02 a <0.0001
Mn 0.03 ± 0.036 c 0.14 ± 0.006 b 0.11 ± 0.03 b 0.20 ± 0.01 a 0.23 ± 0.03 a <0.0001
1Different letters in the same line represent significant differences between samples (p < 0.05). Control—yogurt bites without plant raw
materials, YBB—yogurt bites with 1% beetroot powder, YBM—yogurt bites with 1% mulberry leaves powder, YBN—yogurt bites with 1%
nettle leaves powder, YBR—yogurt bites with 1% rosehip fruit powder.

Calcium and phosphorus are other important minerals found in yogurt bites. Signif-
icantly, the highest amount of Ca was found for the sample containing mulberry leaves
at 240.81 mg·100 g−1 , while the control sample (198.78 mg·100 g−1 ) and yogurt bites with
rosehip fruit (186.21 mg·100 g−1 ) exhibited the lowest amount. As is obvious in Table 1,
mulberry leaves powder had the highest amount of Ca (2870.08 mg·100 g−1 ), followed
by the nettle leaves (2680.16 mg·100 g−1 ), rosehip fruit (590.33 mg·100 g−1 ) and beetroot
(220.03 mg·100 g−1 ) powders. From these results, it follows that mulberry leaves as well
as nettle leaves are excellent food resources with high amounts of macronutrients such as
calcium. Previous research showed that the amount of Ca can range widely from 1121 to
2924 mg·kg−1 in mulberry leaves [49] and from 2210 to 3050 mg·kg−1 in nettle leaves [46].
The RDA is 800 mg/day for Ca in adults [48]. Therefore, the consumption of 100 g/day of
yogurt bites enriched with mulberry leaves supplies 30.10% of the RDA for Ca. The Ca
in 100 g of yogurt bites with nettle leaves supplies 28.28% of the RDA. Maietti et al. [50]
also stated that the addition of nettle to bread can significantly increase the content of
calcium. Statistical analysis demonstrated that the differences in the amount of P among
all yogurt samples were statistically significant. The maximum amount of P was found
for yogurt bites with nettle leaves, followed by the yogurt bites with rosehip fruit, yo-
gurt bites with mulberry leaves and yogurt bites with beetroot at 255.50, 241.52, 202.57
and 160.73 mg·100 g−1 , respectively. The control sample showed the lowest P amount
(146.70 mg·100 g−1 ). The researchers reported that high amounts in P allow the classifica-
tion of nettle leaves being the food able to bring supplements with this mineral [10]. The
amount of P in nettle leaves can range between 719 to 1002 mg·100 g−1 [46]. Rosehip fruits
are also considered an important source of P and its amount can vary widely depending
on the genotype [47]. The RDA for P is set at 700 mg/day for adults [48]. Therefore, 100 g
of yogurt bites enriched with nettle powder and yogurt bites enriched with rosehip fruit
powder can be particularly considerable sources of P, providing 36.50% and 34.43% of
the RDA.
The results of this study indicated that the incorporation of different plant raw material
powder in yogurt bites allowed the increase of the Mg amount of the manufactured product
by 1.71 to 3.58 times. The Mg amount was minimum in yogurt bites without plant raw
Fermentation 2021, 7, 301 8 of 11

materials (14.52 mg·100 g−1 ), whereas the maximal amount was determined for yogurt
bites with 1% nettle leaves powder (51.93 mg·100 g−1 ), followed by the yogurt bites with
rosehip fruit (40.28 mg·100 g−1 ). According to the literature, both rosehip fruits and nettle
leaves accumulate quite similar amounts of this mineral. The Mg can vary from 420 to
810 mg·100 g−1 for nettle leaves [46] and from 430 to 690 mg·100 g−1 for rosehip fruit [45].
The RDA for magnesium is 375 mg/day for adults [48]. The consumption of 100 g/day of
yogurt bites enriched with nettle leaves supplies the 13.85% of the Mg RDA, while 100 g of
control sample—only 3.87% of the Mg RDA.
The addition of freeze-dried raw material powders to the formulation of yogurt bites
allowed the significant increase in the tested amount of microelements (Fe, Zn, B, Mn) of
the final products. The sample enriched with freeze-dried raw material powders contained
18.79–167.17% higher values of Fe and 24.67–69.23% more Zn than the yogurt bites without
additives. The yogurt bites enriched with nettle leaves powder had the significantly highest
amounts of Fe and Zn (5.29 and 3.08 mg·100 g−1 , respectively), and the yogurt bites with
rosehip fruit powder showed the second highest amounts of both these elements (Fe—
3.53 mg·kg−1 , Zn—2.73 mg·100 g−1 ). In yogurt bites, the B amount varied from 0.09 to
0.32 mg·100 g−1 , and the Mn amount ranged from 0.03 to 0.23 mg·100 g−1 . The yogurt
bites with nettle leaves and rosehip fruit powders showed the significantly highest amounts
of B and Mn. These values were not significantly different at p < 0.05. The samples with
beetroot and mulberry leaves powders exhibited similar amounts of both these minerals,
where no difference was found. The lowest amounts of Fe, Zn, B and Mn were determined
for the control yogurt sample. As can be seen in Table 1, freeze-dried nettle leaves powder
is rich in Fe, Zn, B and Mn. Due to this reason, nettle leaves can be used as an ingredient in
foods and beverages. The RDAs are 14 mg/day for Fe, 10 mg/day for Zn and 2 mg Mn
in adults [48]. Therefore, the consumption of 100 g of the nettle powder enrichment bites
would supply 37.79% of the RDA for Fe, 30.80% of the RDA for Zn and 10% of the RDA
for Mn, while 100 g of the control sample would account for 14.14% of RDA for Fe, 18.20%
of the RDA for Zn and 1.5% of the RDA for Mn. Literature data also confirm that nettle
leaves are rich in mineral elements. The amount of these substances is about 20% of the dry
mass. The major microelements in nettle are iron and zinc [51]. The rosehip fruit powder
also is rich in minerals such as Fe, Mn and Zn (Table 1). Assuming a yogurt bites intake of
100 g/day, the rosehip fruit enrichment bites supply 25.21% of the RDA for Fe, 27.30% of
the RDA for Zn and 14.50% of the RDA for Mn.

3.3. Total Phenolic Amount

Regardless that dairy products (including yoghurt and yogurt products) have a high
nutritive value, they are poor sources for phenolic compounds. Plants are the major sources of
phenolic compounds in the human diet [5]. Therefore, the combination of plant raw materials
with various yogurt products could be nutritionally beneficial. Figure 2 shows that yogurt
bites enriched with different plant raw material powders had a significantly higher amount
of total phenolic content by 1.86–3.54 times compared with the control sample without
additives. The highest total phenolic amount was established for yogurt bites enriched with
rosehip fruit powder (49.05 mg·100 g−1 ), and the yogurt bites with nettle and mulberry leaves
powders showed the second highest amounts of this element (40.51 and 38.81 mg 100 g−1 ,
respectively). As illustrated in Table 1, rosehip fruit powder had the highest amount of
total phenolic content (461.94 mg·100 g−1 ), followed by the nettle leaves (313.46 mg·100 g−1 ),
mulberry leaves (301.94 mg·100 g−1 ) and beetroot (224.69 mg·100 g−1 ) powders.
As our study has shown, these plant raw materials are excellent sources of phenolic
compounds for enrichment of yogurt bites. However, there is no information about the
effect of plant raw materials used in our study on the amount of total phenolic content of
yogurt bites. Rosehip fruits are known as rich in phenolic compounds and their content
can range from 130.83 to 766.0 mg·100 g−1 DW, depending on species, harvesting time
and environmental conditions [52,53]. Previous research has shown that incorporation of
rosehip powder in cookies has definitely increased the total phenolic content [54]. A signif-
 raw materials with various yogurt products could be nutritionally beneficial. Figure 2
shows that yogurt bites enriched with different plant raw material powders had a signif-
icantly higher amount of total phenolic content by 1.86–3.54 times compared with the con-
trol sample without additives. The highest total phenolic amount was established for yo-
gurt
Fermentation 2021, 7, 301 bites enriched with rosehip fruit powder (49.05 mg 100 g ), and the yogurt bites with
−1
9 of 11
nettle and mulberry leaves powders showed the second highest amounts of this element
(40.51 and 38.81 mg 100 g−1, respectively). As illustrated in Table 1, rosehip fruit powder
had the highest amount of total phenolic content (461.94 mg 100 g−1), followed by the nettle
leaves (313.46 mg icant100
increment of totalleaves
g−1), mulberry phenolic content
(301.94 mg was
100 galso observed
−1) and in nettle-enriched
beetroot (224.69 mg bread [50],
100 g ) powders.
−1 beetroot juice-enriched yogurt [55] and mulberry leaf extract-enriched yogurt [56].

60.00
Total phenolic amount, mg 100 g-1

49.05 a
50.00
40.51 b
38.81 b
40.00

30.00 25.70 c

20.00 13.84 d

10.00

0.00
Control YBB YBM YBN YBR

Yogurt bites samples

Figure 2. Total phenolic content of yogurt bites, mg 100 g−1. Different letters represent significant
Figure 2. Total phenolic content of yogurt bites, mg·100 g−1 . Different letters represent significant differences between
differences between samples (p < 0.05). Control—yogurt bites without plant raw materials, YBB—
samples (p < 0.05). Control—yogurt bites without plant raw materials, YBB—yogurt bites with 1% beetroot powder,
yogurt bites with 1% beetroot powder, YBM—yogurt bites with 1% mulberry leaves powder, YBN—
YBM—yogurt bitesbites
yogurt withwith
1% mulberry
1% nettleleaves
leavespowder,
powder,YBN—yogurt
YBR—yogurtbitesbiteswith
with1%1%nettle leaves
rosehip powder,
fruit YBR—yogurt bites
powder.
with 1% rosehip fruit powder.
As our study has shown, these plant raw materials are excellent sources of phenolic
4. Conclusions
compounds for enrichment of yogurt bites. However, there is no information about the
In conclusion, the 1% powder of raw material additive added to yogurt bites showed
effect of plant raw materials used in our study on the amount of total phenolic content of
no changes on proximate composition, except for the significantly highest moisture and to-
yogurt bites. Rosehip fruits are known as rich in phenolic compounds and their content
tal sugar amounts that were established in yogurt bites enriched with rosehip fruit powder,
can range from 130.83 to 766.0 mg 100 g−1 DW, depending on species, harvesting time and
compared with the control. There were differences in mineral and total phenolic content
environmental conditions [52,53]. Previous research has shown that incorporation of rose-
of yogurt bites enriched with plant raw materials compared to the control. Importantly,
hip powder in cookies has definitely increased the total phenolic content [54]. A signifi-
1% nettle leaves powder additive had significantly increased amounts of K, P, Mg, Fe
cant increment of total phenolic content was also observed in nettle-enriched bread [50],
and Zn in yogurt bites followed by the rosehip fruit powder. The study clearly showed
beetroot juice-enriched yogurt [55] and mulberry leaf extract-enriched yogurt [56].
that the additive of freeze-dried mulberry leaves powder had a significantly increased Ca
amount of the yogurt bites. The highest amount of total phenolic content was established
for yogurt bites enriched with rosehip fruit powder. Therefore, this study revealed that
nettle leaves, rosehip fruit, mulberry leaves and beetroot powder additives improve yogurt
bites’ nutritional value, as well as being a beneficial additive for food production.

Author Contributions: Conceptualisation, J.K., N.V., D.L.; methodology, J.K., N.V., D.L.; software
J.K., N.V., D.L.; formal analysis, J.K., N.V., D.L.; investigation, J.K., N.V., D.L.; data curation, J.K., N.V.,
D.L.; writing—original draft preparation, J.K., N.V., D.L.; writing—review and editing, J.K., N.V.,
D.L.; visualisation, J.K., N.V., D.L.; supervision J.K; project administration J.K. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the European Structural Fund, within the international
project Eureka E!13405 LYBEF “Lyophilized yoghurt bites enriched with non-traditional functional
raw materials”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Fermentation 2021, 7, 301 10 of 11

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