Impact of Cherries, Strawberries, Bilberries, and Cornelian Cherry Addition on the Antioxidant Activity of Yogurt

Abstract

Increasing awareness of the negative health effects associated with high sugar intake has led to a growing demand for reducing added sugar in food products. In this study, the antioxidant properties of commercial yogurts containing pasteurized fruits were evaluated and compared with natural yogurts freshly enriched with 3–20% thawed fruits (bilberries, cherries and strawberries). Additionally, yogurts enriched with cornelian cherry were analyzed. Antioxidant activity was assessed using the ABTS and DPPH methods, along with measurements of total polyphenol content and reducing power. The effect of fruit addition on the number of yogurt bacteria was also investigated. The results showed that the addition of fruits significantly increased the yogurts’ ability to neutralize free radicals, attributed to the presence of natural antioxidants and polyphenols. The addition of fruits helped maintain the vitality of lactic acid bacteria, with bacterial counts remaining well above the minimum threshold of 10⁷ cfu/g. The findings demonstrated that cornelian cherry has great potential as a source of polyphenols with antioxidant properties. These results confirm the high nutritional value of yogurts enriched with thawed fruit, which may serve as a valuable component of a healthy diet and a healthier alternative to sweetened yogurts commonly available in stores.

1. Introduction

In recent years, there has been a growing interest in the role of diet in the prevention and treatment of many diseases, including metabolic disorders, cardiovascular disease, type 2 diabetes, and various forms of cancer—conditions increasingly linked to excessive sugar consumption [1,2,3,4]. Rising awareness of the adverse health effects associated with high sugar intake is driving efforts to reduce the amount of added sugar in food products. Yogurt manufacturers frequently add substantial amounts of sugar to meet consumer taste preferences. Sucrose, fructose, and glucose are among the most commonly added sugars in processed foods [5]. One of the simplest strategies for reducing sugar content in yogurt is to eliminate added sugars altogether. However, such yogurts may be less appealing to consumers due to their natural acidity, the natural result of fermentation, as well as the widespread preference for sweet-tasting products [6]. Consequently, there is a growing need to identify alternative ingredients that can replace sugars while maintaining the desirable sweetness and texture of food products [7]. Given the increasing prevalence of obesity and its numerous complications, it is both timely and necessary to implement sugar-reduction strategies aimed at offering consumers healthier and nutrient-rich dairy products [8].

In the context of healthy eating, particular attention is given to fermented dairy products, especially natural yogurts, which not only provide valuable nutrients, but also show a number of health benefits, such as reducing LDL cholesterol levels and improving nutrient absorption and digestion [9,10]. Yogurt stands out among dairy products not only as an excellent source of high-quality protein that supports the feeling of satiety but also for its numerous health-promoting properties. Yogurt is a rich source of calcium, magnesium, vitamin B12, and conjugated linoleic acid, which is known for its antioxidant, anti-diabetic, anti-cancer and anti-atherosclerosis effects [11,12,13,14]. In addition, yogurt contains beneficial bacterial species, such as Lactobacillus casei, Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium bifidum, which often makes it a source of probiotics [15]. Probiotic yogurts support the immune system and positively influence the gut microbiota by inhibiting the growth of pathogenic microorganisms. The main—and often only—disadvantage of many commercially available yogurts is their high sugar content [16]. Nevertheless, it is generally better to consume yogurt than not, as numerous epidemiological and clinical studies have shown an association between yogurt consumption and a reduced risk of chronic diseases, including metabolic syndrome, type 2 diabetes, cardiovascular disease, and obesity [17].

Yogurt parfait can provide probiotics, prebiotics, high-quality protein, and a variety of vitamins and minerals that may work synergistically to promote health. Antioxidant potential is considered an important nutritional property of foods. Consuming foods rich in antioxidants, such as fruits, can help protect the body from oxidative stress, which is implicated in most diet-related chronic diseases [18]. Both fruits and dairy products, including yogurt, are recognized as healthy components of the diet, as reflected in national and global diet guidelines and recommendations [11].

Fruits, due to their rich contents of antioxidants, polyphenols, and prebiotic fiber and their relatively low energy density, have the potential to reduce oxidative stress, which may contribute to the prevention of the development of certain diseases [19]. In particular, cornelian cherry (Cornus mas L.), used in this study for its high antioxidant potential and functional properties, may play an important role in the development of new food products. Cornus mas L., commonly known as cornelian cherry or edible dogwood fruit, belongs to the dogwood family and is native to the southern and eastern regions of Europe and the Middle East. Cornelian cherry fruits are oval to pear-shaped, and their color ranges from red to purple [20]. Cornelian cherry is a good source of vitamin C, ranging from 29 to 300 mg per 100 g of fresh weight—significantly higher than that of strawberry and kiwi fruit, which contain between 29 and 80 mg per 100 g [21,22]. It is also particularly rich in potassium (approximately 1490 mg/100 g), as well as other minerals such as zinc, manganese and iron [23]. Additionally, this fruit is a source of various phytocompounds, including anthocyanins, phenolic acids, flavonoids, tannins, catechins (elagoic acid, epicatechnia, catechin and chlorogenic acid), as well as caffeic acid and iridoids —many of which have documented anti-inflammatory and antioxidant properties [24]. Numerous studies have demonstrated that, due to its high phenolic content, cornelian cherry exhibits strong radical scavenging activity [25,26] and has antioxidant, anti-inflammatory, anti-parasitic, anti-cancer and protective effects on the cardiovascular system, liver, and kidneys [27,28,29,30,31].

Previous studies on the addition of fresh or processed fruits to yogurts have shown that this is a commonly used strategy to increase the content of phenolic compounds in the product [32]. Phenolic compounds are natural substances with strong antioxidant properties that can contribute to enhancing the health-promoting qualities of yogurts [33]. Enriching yogurt with fruits not only increases its nutritional value, but also positively affects its antioxidant properties, which is particularly important in protecting the body against oxidative stress. Raikos et al. [34] showed that the addition of fruit extracts rich in polyphenols to yogurt beverages improved their antioxidant activity. Research into the functional properties of yogurt, including its antioxidant activity, is an important topic in the scientific literature. However, studies focusing specifically on the impact of fruit addition on the antioxidant activity and health-related properties of ready-to-eat natural yogurt remain relatively scarce. Therefore, further research in this area is warranted. To our knowledge, no published study has evaluated the antioxidant activity of yogurts containing a wide variety of fruit additions in a single study. Such investigations can provide valuable insights useful for the development of functional dairy products and offer a comprehensive understanding of their antioxidant potential, which is crucial not only for product quality but also for consumer health benefits.

There are many fruit-containing yogurts on the market; however, the fruits in commercial yogurts are usually processed, e.g., pasteurized. Fruit yogurts can easily be prepared at home by mixing natural yogurt with fresh or thawed fruits. When consumed immediately after preparation, such yogurts offer a pronounced fruit flavor without added sugars. Nevertheless, questions arise as to whether the addition of fruit to natural yogurt truly enhances its antioxidant properties and whether it negatively affects the bacteria present in the yogurt. The aim of this study was to evaluate the antioxidant properties of natural yogurts enriched with thawed fruits (bilberries, cherries and strawberries) and to compare their antioxidant activity with that of commercial yogurts containing the same processed fruits. These particular fruits were selected due to their popularity and availability. Additionally, the impact of fruit addition on the number of yogurt bacteria was assessed. Furthermore, we analyzed the antioxidant properties of yogurts with the added cornelian cherry. Given its high antioxidant potential, we aimed to compare its effects with those of more commonly used fruit additives in yogurt. To the best of our knowledge, this is the first study to analyze the antioxidant properties of yogurt enriched with cornelian cherry.

2. Materials and Methods

2.1. Material

The materials used in the study included commercial stirred yogurts (natural and fruity) made from cow’s milk, as well as frozen fruits including cornelian cherry, strawberry, cherry and bilberry. Both the yogurts and most of the fruits were purchased from a local supermarket in Lublin, Poland, in August 2024. All fruits and yogurts were produced by Polish manufacturers. Fresh cornelian cherry fruit was purchased from a local orchard, then washed, pitted and frozen for later use. A detailed list of the basic compositions of the yogurts used in the analysis are presented in

Table 1. Fruit content and nutritional composition of the commercial yogurts *.

Yogurt	Fruits [g/100 g]	Protein [g/100 g]	Fat [g/100 g]	Carbohydrate [g/100 g]	Sugars [g/100 g]
N1	0.0	4.1	2.0	6.0	5.6
N2	0.0	4.5	2.0	6.0	6.0
N3	0.0	4.8	3.1	4.0	4.0
N4	0.0	4.5	2.0	6.8	6.8
N5	0.0	5.0	2.0	5.9	5.6
N6	0.0	4.5	3.4	5.1	5.1
N7	0.0	4.9	6.0	6.5	6.5
S1 (3)	3.0	3.1	2.8	14.8	13.9
S2 (7.5)	7.5	2.5	2.5	12.5	11.0
S3 (10)	10.0	2.7	2.5	12.6	11.3
S4 (10)	10.0	3.5	2.7	12.0	11.4
S5 (13)	13.0	3.5	2.5	12.9	12.0
C1 (3)	3.0	3.1	2.8	14.9	13.8
C2 (6.7)	6.7	2.5	2.5	12.5	11.0
C3 (9)	9.0	3.5	2.6	14.8	13.8
C4 (10)	10.0	2.7	2.5	13.4	11.4
C5 (13)	13.0	3.5	2.5	12.9	12.0
B1 (3)	3.0	3.1	2.5	11.0	11.0
B2 (6)	6.0	2.5	2.5	12.5	11.0
B3 (6.3)	6.3	3.5	2.7	11.3	10.7
B4 (8)	8.0	2.7	2.5	12.6	11.5
B5 (13)	13.0	3.5	2.5	12.9	12.0

* according to the manufacturer’s declaration; N1–N7—commercial natural yogurts from different manufacturers; S—commercial strawberry yogurt; C—commercial cherry yogurt; B—commercial bilberry yogurt; values in parentheses indicate fruit content (%).

. All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Method of Preparing Fruit Yogurts

Preparing fruit yogurts with varying amounts of frozen fruit (3%, 7%, 10%, 13%, or 20%) involved initial thawing of the selected fruits (cornelian cherry, bilberry, strawberry, and cherry) at room temperature under sterile conditions. After weighing the natural yogurt, the appropriate proportion of fruit was added, and the ingredients were blended using a blender. The entire procedure was repeated three times for each type of yogurt. All analyses were carried out immediately after yogurt preparation.

2.3. The pH Value of Yogurts

The pH measurement was carried out using a pH meter (Elmetron, Zabrze, Poland) equipped with a pH electrode (ERH-111, Hydromet, Syców, Poland).

2.4. Preparation of Extracts

To obtain the extracts for analysis, 10 g of yogurt was weighed and mixed with 30 mL of water. The pH of the mixture was adjusted to 4.6 using 1 mol/L HCl and then homogenized with a homogenizer (T 25 basic Ultra-Turrax, IKA) for 1 min. The mixture was then incubated in a water bath at 40 °C for 1 h, after which the homogenate was centrifuged at 5000× g at 4 °C for 20 min. After centrifuging, the top layer of fat was removed and the supernatant was filtered through a Whatman No. 42 filter. The resulting samples were used to determine the antioxidant activity using the ABTS, DPPH, and reduction power assays, as well as to evaluate the polyphenols content [35].

2.5. Analysis of Antioxidant Activity

2.5.1. Determination of the Anti-Radical Properties by the ABTS•⁺ Method

The antioxidant activity in the sample extracts was determined using the method described by Re at al. [36], based on the reaction with the ABTS•⁺ cation radical. The ABTS•⁺ radical was generated by reacting 2,2′-azinobis-3-ethylbenzothiazolin-6-sulfonic acid with potassium peroxide carrier and allowing the mixture to stand for 16 h. The prepared ABTS solution was then diluted with a sodium phosphate buffer (0.02 mM at pH 6.6) to obtain an absorbance of A = 0.7 ± 0.02 at wavelength 734 nm. A sample of 10 μL and 240 μL of ABTS solution was mixed and incubated for 10 min at room temperature. The absorbance was then measured at 734 nm using a 96-well microplate reader. The results were expressed as a percentage of radical inhibition (%). Compounds with antioxidant properties present in the extracts reduced the concentration of cationic ABTS*, as evidenced by a decrease in absorbance at 734 nm. The percentage inhibition of the ABTS•⁺ radical was calculated using the following formula:

$$\%{\mathrm{RSA}}^{\mathrm{ABTS}}=\left[\left(\mathrm{Abscontrol}-\mathrm{Abssample}\right)/\mathrm{Abscontrol}\right]\times 100$$

where Abssample is absorption of the sample and Abscontrol is absorption of the control sample (ABTS solution).

2.5.2. Determination of DPPH Radical Scavenging Activity

The method of determining the scavenging activity of 2,2-diphenyl-1-picrylohydrazyl hydrate (DPPH) was performed as described by Pavithra and Vadivukkarasi [37] with modifications. First, 100 μL of DPPH solution (0.2 mol/L prepared in methanol) was added to 100 μL of the sample and the mixture was incubated at room temperature for 30 min in the dark. The absorbance was measured at a wavelength of 517 nm using a 96-well microtiter plate reader (Thermo Scientific, Multiskan Sky, Waltham, MA, USA). For the control sample, 100 μL of methanol was used instead of the test sample. The results were expressed as a percentage of DPPH radical scavenging activity (RSA) according to the following formula:

$$\%{\mathrm{RSA}}^{\mathrm{DPPH}}=\left[\left(\mathrm{Abscontrol}-\mathrm{Abssample}\right)/\mathrm{Abscontrol}\right]\times 100$$

where Abssample is absorption of the sample and Abscontrol is absorption of the control sample (DPPH solution).

2.5.3. Determination of Reducing Power (RP)

The reduction power of the extracts was determined by colorimetric measurement of the degree of discoloration of iron (III) solutions (i.e., from the chemical point of view of reducing Fe³⁺ ions to Fe²⁺) according to the method described by Oyaizu [38]. For this purpose, a sample volume of 2 mL of solution was mixed with 2 mL of sodium phosphate buffer (0.2 M; pH 6.6) and 2 mL of 1% hexacyanoferric (III) potassium solution (K₃[Fe(CN)₆]). The prepared mixture was incubated in a water bath at 50 °C for 20 min, then 2 mL of trichloroacetic acid (10%) solution was added. After 10 min, the absorbance was determined at 700 nm with a Nicolet Evolution 300 spectrophotometer (Thermo Electron Corp., Waltham, MA, USA)). The increased absorbance indicated an increased reducing power of the test solution.

2.5.4. Determination of Total Reducing Capacity (TRC) by the Follin–Ciocalteu Assay

The method developed by Singleton [39] was used. The Folin–Ciocalteu reagent was first diluted with distilled water (1:5). Then, 0.4 mL of this diluted reagent and 0.1 mL of distilled water were added to the extract (0.1 mL). After 3 min, Na₂CO₃ solution (10%; 2.5 mL) was added. The mixture was thoroughly mixed and then incubated in the dark at room temperature for 30 min. After incubation, the absorbance was measured at a wavelength of 725 nm. The final results were expressed as the concentration of total phenolic compounds, which was determined using a standard curve prepared for gallic acid. The content of polyphenol compounds was expressed in milligrams per milliliter of extract as gallic acid equivalent (GAE) [mg GAE/mL].

2.6. Microbiological Analysis

According to the method of Gustaw et al. [40], serial decimal dilutions in sterile peptone water (0.1%) were prepared from every type of yogurt (1 g sample). Then, 1 mL aliquots were plated over selected culture media as follows: M-17 agar for Streptococcus thermophilus and M-MRS agar for Lactobacillus. The method of plate counting was used in three repetitions. Streptococcus thermophilus was incubated in 37 °C aerobically for 48 h and Lactobacillus was incubated in 37 °C anaerobically for 72 h (GasPak System—Oxoid). The results were given in colony-forming units per gram (CFU/g).

2.7. Statistical Analysis

Data were analyzed using bidirectional variance analysis (ANOVA). The significance of differences between mean values was calculated at p < 0.05, using the t-test for the Tukey range. Results were expressed as mean ± standard deviation. Two-factor ANOVA analysis was used for statistical comparisons among fruit type, addition, and selected parameters (ABTS, DPPH, RP, TRC). All calculations and comparisons were analyzed using Statistica software version 13.3 (Dell, Inc., Round Rock, TX, USA). All analyses were performed three times.

3. Results

Table 2. pH values (mean ± standard deviation) of the tested yogurts.

Yogurt	pH	Yogurt	pH
N1	4.76 ± 0.02 z	DD (3)	4.38 ± 0.01 jk
N2	4.68 ± 0.02 y	DD (7)	4.31 ± 0.01 fgh
N3	4.58 ± 0.02 tuw	DD (10)	4.25 ± 0.01 c
N4	4.61 ± 0.02 uw	DD (13)	4.21 ± 0.01 b
N5	4.68 ± 0.02 y	DD (20)	4.14 ± 0.01 a
N6	4.59 ± 0.01 tuw
N7	4.66 ± 0.02 y
S1 (3)	4.41 ± 0.02 klm	DS (3)	4.55 ± 0.01 r
S2 (7,5)	4.54 ± 0.02 prs	DS (7)	4.52 ± 0.01 ps
S3 (10)	4.35 ± 0.01 hij	DS (10)	4.49 ± 0.01 no
S4 (10)	4.55 ± 0.02 rst	DS (13)	4.48 ± 0.01 n
S5 (13)	4.39 ± 0.01 jkl	DS (20)	4.44 ± 0.01 m
C1 (3)	4.37 ± 0.01 ijk	DC (3)	4.58 ± 0.01 tw
C2 (6,7)	4.56 ± 0.02 rt	DC (7)	4.49 ± 0.01 no
C3 (9)	4.43 ± 0.01 lm	DC (10)	4.43 ± 0.01 m
C4 (10)	4.34 ± 0.02 ghi	DC (13)	4.31 ± 0.01 efg
C5 (13)	4.29 ± 0.01 def	DC (20)	4.26 ± 0.01 cd
B1 (3)	4.32 ± 0.01 efgh	DB (3)	4.61 ± 0.01 u
B2 (6)	4.58 ± 0.01 tuw	DB (7)	4.51 ± 0.01 op
B3 (6,3)	4.55 ±0.02 rst	DB (10)	4.48 ± 0.01 n
B4 (8)	4.44 ± 0.02 m	DB (13)	4.39 ± 0.01 k
B5 (13)	4.31 ± 0.01 efgh	DB (20)	4.29 ± 0.01 e

a–z Values are means; means with different letters are significantly different (p ≤ 0.05); N1–N7—natural yogurts from different manufacturers; S—commercial strawberry yogurt; C—commercial cherry yogurt; B—commercial bilberry yogurt, DS—natural yogurt (N6) with added strawberry; DC—natural yogurt (N6) with added cherry; DB—natural yogurt (N6) with added bilberry; DD—natural yogurts (N6) with added cornelian cherry; values in parentheses indicate fruit content (%).

presents the results of the pH analysis of natural yogurts; natural yogurts with the addition of cornelian cherry, bilberries, cherries, and strawberries; as well as commercial fruit yogurts. In the following sections, natural yogurts with added fruit will be referred to as diet yogurts to distinguish them from commercial plain natural yogurts (without fruit) and commercial fruit yogurts. The pH analysis of the diet yogurts showed that their pH values tended to decrease with increasing amounts of added fruit.

The results of the evaluation of the antioxidant contents (ABTS, DPPH, reduction power, phenolic compounds) of natural yogurt extracts are presented in

Table 3. Antioxidant activity (ABTS, DPPH, RP) of extracts from commercial natural yogurts.

Sample	ABTS [% RSA]	DPPH [% RSA]	RP
N1	67.36 ± 1.89 abc	91.30 ± 1.08 c	0.24 ± 0.02 a
N2	67.85 ± 1.08 abc	87.82 ± 1.36 ab	0.35 ± 0.01 b
N3	65.47 ± 2.42 a	90.31 ± 2.27 bc	0.63 ± 0.04 cd
N4	65.28 ± 4.25 a	91.97 ± 1.44 c	0.67 ± 0.02 d
N5	66.29 ± 1.15 ab	86.28 ± 3.72 a	0.37 ± 0.02 b
N6	70.85 ± 2.36 c	90.94 ± 2.01 bc	0.65 ± 0.05 d
N7	69.56 ± 1.38 bc	90.49 ± 2.13 bc	0.58 ± 0.02 c

a–d Values are means; means with different letters for the same column are significantly different (p ≤ 0.05); N1–N7—natural yogurts from different manufacturers.

. The analyzed yogurts did not differ substantially in their ABTS and DPPH values, although statistically significant differences (p ≤ 0.05) were found between individual manufacturers. The highest values of the antioxidant parameters measured by the DPPH method and the reduction power (RP) were recorded for N, N4 and N6, with N6 also showing the highest ABTS value among them. Based on these results, yogurt N6 was selected for the preparation of fruit-enriched yogurts. Fruit contents ranging from 3% to 13% were selected to approximate the fruit levels found in commercial yogurts, while a 20% addition was included to create a strongly fruity yogurt and to assess how such a high fruit level influenced the antioxidant properties of the diet yogurts.

As shown in

Table 4. Antioxidant activity (ABTS, DPPH, RP, TRC) of extracts from diet yogurts (natural yogurts with varying additions of different fruits) and commercial fruit yogurts.

Yogurt (Percent Fruit Content)	ABTS [% RSA]	DPPH [% RSA]	RP	TRC [mg GAE/mL]
DS (3)	63.89 ± 1.20 abc	86.97 ± 0.52 f	0.32 ± 0.03 ab	0.63 ± 0.01 a
DS (7)	66.27 ± 1.70 bcd	88.59 ± 1.06 f	0.37 ± 0.03 abce	0.64 ± 0.02 a
DS (10)	64.70 ± 1.26 abc	87.15 ± 1.13 f	0.37 ± 0.04 abce	0.66 ± 0.01 ab
DS (13)	65.91 ± 0.69 abcd	87.63 ± 0.88 f	0.41 ± 0.03 abce	0.68 ± 0.03 ab
DS (20)	70.06 ± 2.18 cde	87.95 ± 1.11 f	0.45 ± 0.05 abcdeg	0.69 ± 0.02 ab
S1 (3)	59.77 ± 3.18 ab	74.83 ± 5.33 e	0.49 ± 0.05 abcdefg	1.33 ± 0.06 gh
S2 (7,5)	59.40 ± 13.81 a	27.97 ± 8.96 b	0.75 ± 0.06 defghij	1.56 ± 0.14 ij
S3 (10)	61.03 ± 3.69 ab	43.31 ± 5.91 c	1.14 ± 0.25 ijk	1.46 ± 0.14 hi
S4 (10)	65.75 ± 3.96 abcd	71.33 ± 1.45 e	0.41 ± 0.05 abcdeg	1.48 ± 0.02 hi
S5 (13)	79.62 ± 3.15 hijk	76.06 ± 2.95 e	0.62 ± 0.11 bcdefgh	1.61 ± 0.02 ijk
DB (3)	85.56 ± 1.27 kl	76.20 ± 1.43 e	0.63 ± 0.09 cdefg	0.76 ± 0.03 bc
DB (7)	93.99 ± 0.41 m	83.44 ± 0.61 f	1.07 ± 0.17 ik	0.90 ± 0.06 de
DB (10)	93.95 ± 0.33 m	84.80 ± 0.64 f	1.67 ± 0.43 l	0.98 ± 0.02 ef
DB (13)	93.96 ± 0.21 m	87.73 ± 0.32 f	1.83 ± 0.27 l	1.06 ± 0.03 f
DB (20)	94.04 ± 0.21 m	89.50 ± 0.27 f	3.02 ± 0.32 m	1.20 ± 0.02 g
B1 (3)	71.56 ± 3.09 def	73.35 ± 2.83 e	0.68 ± 0.04 bcdefghj	1.99 ± 0.22 l
B2 (6)	80.79 ± 7.36 ijk	24.19 ± 10.73 b	0.69 ± 0.14 bcdefghj	1.91 ± 0.09 l
B3 (6,3)	73.72 ± 1.61 efgh	76.62 ± 1.40 e	0.18 ± 0.08 a	1.62 ± 0.06 ijk
B4 (8)	74.72 ± 3.09 efghi	74.49 ± 3.03 e	0.72 ± 0.12 bcdefghij	1.71 ± 0.23 jk
B5 (13)	93.63 ± 0.83 m	64.47 ± 2.71 e	0.65 ± 0.10 bcdefgh	3.23 ± 0.09 n
DC (3)	68.25 ± 1.07 cde	89.49 ± 0.97 f	0.44 ± 0.07 abceg	0.63 ± 0.02 a
DC (7)	71.30 ± 1.39 def	89.53 ± 0.80 f	0.48 ± 0.06 abcdeg	0.66 ± 0.02 ab
DC (10)	79.08 ± 1.52 ghijk	89.29 ± 0.75 f	0.63 ± 0.07 cdefg	0.70 ± 0.02 ab
DC (13)	77.02 ± 0.82 fghij	89.34 ± 0.77 f	0.63 ± 0.15 cdefg	0.73 ± 0.05 ab
DC (20)	83.68 ± 0.74 jkl	90.06 ± 0.77 f	0.73 ± 0.08 dfhj	0.76 ± 0.03 bc
C1 (3)	60.85 ± 2.59 ab	70.62 ± 9.62 e	0.26 ± 0.04 abc	1.49 ± 0.10 hi
C2 (6,7)	73.88 ± 2.92 efgh	14.67 ± 9.00 a	0.79 ± 0.09 dfghij	1.92 ± 0.21 l
C3 (9)	72.85 ± 6.78 efg	71.95 ± 3.35 e	0.48 ± 0.02 abcdefg	1.80 ± 0.06 kl
C4 (10)	71.53 ± 2.77 def	58.85 ± 4.70 d	1.01 ± 0.17 hijk	1.63 ± 0.10 ijk
C5 (13)	81.96 ± 4.51 jk	68.1 ± 1.72 e	0.63 ± 0.05 bcdefgh	2.42 ± 0.18 m
DD (3)	80.50 ± 1.99 hijk	88.64 ± 0.64 f	0.56 ± 0.05 bcdefg	0.73 ± 0.05 ab
DD (7)	89.88 ± 3.14 lm	88.55 ± 1.61 f	0.78 ± 0.12 fhij	0.84 ± 0.04 cd
DD (10)	92.86 ± 2.36 m	88.41 ± 0.60 f	1.07 ± 0.23 ik	0.93 ± 0.05 de
DD (13)	93.50 ± 0.96 m	87.92 ± 0.89 f	1.17 ± 0.17 k	0.95 ± 0.04 de
DD (20)	94.33 ± 0.11 m	86.30 ± 0.60 f	1.67 ± 0.25 l	1.21 ± 0.05 g

a–n Values are means; means with different letters for the same column are significantly different (p ≤ 0.05); DS—natural yogurt (N6) with added strawberry; DC—natural yogurt (N6) with added cherry; DB—natural yogurt (N6) with added bilberry; DD—natural yogurt (N6) with added cornelian cherry; S—commercial strawberry yogurt; C—commercial cherry yogurt; B—commercial bilberry yogurt.

, the values of DPPH, ABTS reduction power and phenolic compounds varied depending on the fruit content in the yogurts. The highest antiradical activities measured by the ABTS method were observed in diet yogurts containing 20% bilberries and cornelian cherry, whereas the lowest values were recorded for most commercial strawberry yogurts (Table 4). The lowest ability to neutralize free radicals and the lowest reduction power were observed in yogurts with lower fruit content. On the other hand, the content of phenolic compounds was higher in commercial fruit yogurts (strawberry, bilberry, cherry) than in the corresponding diet yogurts. Analyzing the antioxidant activity of diet fruit yogurts, we note that TRC ranged from 0.66 mg GAE/mL (commercial strawberry yogurt) to 0.98 mg GAE/mL (commercial bilberry yogurt). Among the diet yogurts, the samples with 20% additions of cornelian cherry and bilberries, DD (20) and DB (20), respectively, had the highest phenolic content. The antioxidant activity of yogurts with cornelian cherry, cherry, and strawberry, as measured by the DPPH method, did not differ significantly between samples. However, an increase in the addition of cornelian cherry was associated with a consistent rise in antioxidant activity (as measured by ABTS), reduction power and phenolic content.

Since the detailed analysis presented in Table 4 may be difficult to interpret unambiguously, several simplifications have been made for statistical purposes. Specifically, values for each level of fruit addition (

Table 5. Effect of fruit addition on the antioxidant activity of diet yogurts.

Addition [%]	ABTS [% RSA]	DPPH [% RSA]	RP	TRC [mg GAE/mL]
3	73.61 ± 9.23 a	85.25 ± 5.57 a	0.49 ± 0.13 a	0.69 ± 0.07 a
7	80.62 ± 12.18 ab	87.59 ± 2.63 b	0.67 ± 0.29 ab	0.76 ± 0.12 ab
10	82.35 ± 12.21 b	87.51 ± 1.88 b	0.94 ± 0.55 b	0.82 ± 0.14 b
13	82.44 ± 12.16 b	88.22 ± 1.01 b	1.01 ± 0.58 b	0.85 ± 0.16 b
20	86.03 ± 10.03 b	88.59 ± 1.64 b	1.47 ± 1.04 c	0.96 ± 0.25 c

a–c Values are means; means with different letters for the same column are significantly different (p ≤ 0.05).

) were calculated. The results showed that increasing the amount of fruit in diet yogurts significantly enhanced the antioxidant activity, as measured by both the DPPH and ABTS methods, as well as the reduction power and total phenolic content (p < 0.05). However, it is worth noting that even a 7% fruit addition was sufficient to significantly improve the antioxidant activity. Further increases in fruit content generally did not lead to substantial additional improvements in these parameters. Notably, significantly higher RP and TRC values were observed only at the 20% fruit addition level.

In the case of diet fruit yogurts, a similar trend was observed: bilberry-enriched yogurts had the highest total phenolic content (0.98 ± 0.15 mg GAE/mL), while strawberry yogurts had the lowest (0.66 ± 0.03 mg GAE/mL). Comparable differences were seen in reduction power. As with the commercial yogurts, DPPH values did not significantly differ among fruit types.

As in Table 5, to facilitate the interpretation of the results, mean values for phenolic compounds concentration, reduction power and free radicals scavenging capacity were calculated based on the type of fruit added to the yogurt and then compared with those of commercially available fruit yogurts (

Table 6. The effect of the type of fruit additive on the antioxidant activity of commercial and diet yogurts.

Flavor Variant	ABTS [% RSA]	DPPH [% RSA]	RP	TRC [mg GAE/mL]
S	65.12 ± 10.12 a	59.49 ± 20.95 a	0.65 ± 0.27 a	1.48 ± 0.13 c
C	72.22 ± 7.92 b	60.95 ± 20.04 a	0.63 ± 0.28 a	1.85 ± 0.35 d
B	78.89 ± 8.90 c	63.75 ± 20.62 a	0.58 ± 0.23 a	2.09 ± 0.62 e
DS	66.08 ± 2.51 a	87.68 ± 1.10 b	0.39 ± 0.06 a	0.66 ± 0.03 a
DC	76.14 ± 5.57 bc	89.55 ± 0.82 b	0.58 ± 0.14 a	0.70 ± 0.06 a
DB	92.61 ± 3.19 d	84.33 ± 4.71 b	1.64 ± 0.86 c	0.98 ± 0.15 b
DD	90.79 ± 5.05 d	88.03 ± 1.22 b	1.05 ± 0.42 b	0.93 ± 0.17 b

a–e Values are means; means with different letters for the same column are significantly different (p ≤ 0.05); S—commercial strawberry yogurt; C—commercial cherry yogurt; B—commercial bilberry yogurt; DS—natural yogurt (N6) with strawberry addition; DC—natural yogurt (N6) with added cherry; DB—natural yogurt (N6) with bilberry addition; DD—natural yogurt (N6) with added cornelian cherry.

). The antioxidant activity of both commercial and diet fruit yogurts is presented as means ± standard deviation, with statistically significant differences indicated at p ≤ 0.05. Among the commercial yogurts, the highest antioxidant activity measured by the ABTS method was observed in bilberry yogurts, while the lowest was in strawberry yogurts. These differences were confirmed in the case of TRC analysis. However, no statistically significant differences were found in DPPH radical scavenging activity or reduction power between the different flavors of commercial yogurts (Table 6). Analyzing the antioxidant activity of diet fruit yogurts, as in the case of commercial yogurts, we can see that the highest total content of phenolic compounds was present in bilberry yogurts (0.98 ± 0.15 mg GAE/mL) and the lowest in strawberry yogurts (0.66 ± 0.03 mg GAE/mL). Comparable differences were seen in reduction power. As with the commercial yogurts, DPPH values did not significantly differ among fruit types. Notably, the highest values for reduction power and ABTS radical scavenging capacity were recorded in natural yogurts enriched with added cornelian cherry (DD) and bilberry (DB). Overall, this simplified analysis suggests that natural yogurts with the addition of thawed fruit generally exhibit higher antioxidant activity than their commercial counterparts containing the same type of fruit.

The simplifications used in Table 5 and Table 6 are confirmed by the results presented in

Table 7. Basic technological parameters characterizing the tested yoghurts.

Effect	p
Type of fruit	***
Addition	***
Type of fruit × addition	***

***—p < 0.001.

, which shows the results of two-way ANOVA analysis for factorial designs. Fruit type, addition, and fruit type x addition were shown to result in statistically significant (p < 0.001) differences in all four determinants of biological activity (i.e., ABTS, DPPH, RP, and TRC).

The addition of fruit to natural yogurts did not significantly affect the number of bacteria of the genus Streptococcus (

Table 8. The number of Lactobacillus and Streptococcus thermophilus cells in yogurts with the addition of 10% of fruits.

	Number of Bacteria [log CFU/g]
Yogurt	Lactobacillus	Streptococcus
Cornelian cherry	8.1 a	10.5 a
Strawberry	8.4 a	10.3 a
Bilberry	8.5 ab	10.6 a
Cherry	8.6 ab	10.7 a
Natural	9.0 b	10.3 a

a–b Values are means; means with different letters for the same bacteria strain are significantly different (p ≤ 0.05).

). Similar results were observed for the genus Lactobacillus but this only applied to yogurts with the addition of bilberries and cherries. In the case of the addition of cornelian cherry and strawberries, significantly smaller numbers of Lactobacillus bacteria were found when compared to natural yogurt.

4. Discussion

Epidemiological studies clearly indicate that the diet plays a significant role in the prevention of many chronic diseases [41]. Fruit yogurts have become a popular product in the diet of many people, but their high content of added sugars is controversial. Excessive consumption of products with a high content of sugars not only increases the calorific value of the product, but may also contribute to the development of obesity and its complications [42]. An alternative could be the consumption of natural yogurt (without added sugars), although its flavor profile may make it less appealing to some consumers. Natural yogurt with the addition of fresh or thawed fruit may be a better alternative to commercial fruit yogurts. We decided to investigate whether the amount and type of relatively unprocessed fruit (frozen) has an impact on the antioxidant activity of yogurts. Natural yogurt with fruit without added sugar can be a much healthier option. These products contain only naturally occurring sugars from the fruit, which help preserve the desirable flavor without adding excessive calories [43]. Differences in antioxidant compounds, such as solubility, ability to form hydrogen bonds, and ionic conditions, can affect the results obtained from methods used to assess antioxidant activity [35]. Various methods, including ABTS and DPPH assays, can be used to determine the antioxidant activity of yogurts. It should be noted that the methods used differ in their conditions, such as sample incubation time and solution concentration [44]. When analyzing DPPH radical scavenging activity, all natural yogurts with the addition of different fruits showed higher antioxidant capacity compared to commercial yogurts, which was not always confirmed by the ABTS method (Table 4). Each method has its limitations; for example, the Folin–Ciocalteu assay, often applied to measure total phenolic content, can be influenced by the presence of some amino acids, vitamins and their derivatives (e.g., ascorbic acid), and inorganic reducing ions [45]. Although the majority of the recorded results are likely affected by the phenolic content, for clarity of message, in this study, we used the term “total reducing capacity”. To avoid bias, we employed different methods. While none of these methods is perfect, we assumed that statistically significant differences between samples would allow us to identify, among fruit yogurts, those with higher nutritional value.

An interesting phenomenon can be observed when comparing Table 3 with Table 4: yogurts with added strawberry (DS) generally showed lower values for ABTS, DPPH, and RP compared to the selected commercial natural yogurt. There are several possible explanations for this observation.

First, plant-based antioxidants (e.g., polyphenols, flavonoids) can form complexes with milk proteins (e.g., casein), which reduces their availability and reactivity in antioxidant tests such as DPPH and ABTS. For example, Arts et al. [46] observed that the antioxidant capacity of several green and black tea components with α-, β- and κ-casein or albumin is not additive; that is, part of the total antioxidant capacity is masked due to these interactions. This masking effect depends on both the protein and the flavonoid involved. Similarly, the study by Bourassa et al. [47] supports this finding, showing that the presence of α-casein reduces the antioxidant activity of tea polyphenols in the ABTS test by about 11–27%. Another possible reason for the decreased antioxidant activity in fruit-added yogurts compared to natural yogurt is the lowering of pH caused by the fruit additives. Low pH can affect the stability and redox reactions of some antioxidants [48], making them less effective in ABTS and DPPH tests [49].

Numerous scientific studies have shown that antioxidant activity is directly proportional to the overall polyphenol content. TRC was found to be higher in all commercial fruit yogurts compared to natural yogurts with the addition of selected fruits, except for yogurts with 20% bilberry addition and cornelian cherry, where the values were similar (Table 4). A similar relationship was noted by Blassy et al. [50], who reported that enriching yogurt with mango and guava significantly increased (p < 0.05) the total phenolic content as the fruit pulp content increased. Bioactive polyphenols—such as phenolic acids, flavonoids and anthocyanins—present in bilberry and cornelian cherry play a key role in total antioxidant activity, which largely depends on the presence of free polyphenols [51,52]. Since interactions between phenolic compounds and proteins affect both their content and antioxidant capacity [53], this may explain the discrepancies between the polyphenol content and free radical neutralization ability in this study. Furthermore, the tested commercial yogurts contained carrot juice concentrate, aronia juice concentrate, beet juice concentrate and grape juice concentrate, which could also influence TRC. It is worth noting that a high content of phenolic compounds was reported for yogurts with the addition of bilberry and cornelian cherry. These results particularly highlight the need for more advanced scientific research, as, to the best of our knowledge, no previous studies on yogurt enriched with cornelian cherry have been reported in the literature.

The increase in the antioxidant activity of yogurts observed in Table 5, corresponding with the increasing percentage of fruit, is most likely due to the presence of bioactive compounds such as polyphenols, vitamins (e.g., vitamin C and E), carotenoids and flavonoids, which are known for their ability to neutralize free radicals [19]. Fruits are recognized as rich sources of these compounds, which explains their positive influence on the antioxidant properties of yogurt. A significant increase in antioxidant activity is observed when fruit is added at a 7% level. At this point, the concentration of bioactive compounds likely reaches a threshold sufficient to produce a measurable effect. Many phenolic compounds attain optimal concentrations in foods when a certain fruit content is achieved. For example, research conducted by Mena et al. [54] suggests that even small amounts of fruits can significantly increase polyphenols levels in dairy products, enhancing their antioxidant activity. However, beyond this threshold (in this case, 7%), further increases in fruit content do not result in significant changes in antioxidant activity (Table 4). Rui et al. [55] showed that excessive fruit content can lead to a decrease in the product’s pH, which can affect the stability of certain antioxidant compounds. It is well-documented that pH can influence the antioxidant activity of polyphenols, which may explain the plateau in activity observed at higher fruit concentrations. In our study, we also recorded a significant decrease in pH value with increasing fruit content in the yogurts, which may have influenced the antioxidant results due to the impact of pH on compound stability. Nevertheless, the pH values of the tested yogurts remained within the ranges reported in the literature [54].

When analyzing the antioxidant activity of different flavor variants, particularly bilberry yogurts, interesting results were observed for both commercial and diet versions (Table 6). This study showed that the diet bilberry yogurt exhibited significantly higher radical scavenging activity, as demonstrated by the ABTS method. Furthermore, the commercial bilberry yogurt showed a much higher content of phenolic compounds compared to other commercial flavor variants. Similar trends were observed for yogurts with cornelian cherry. These findings are likely strongly correlated with the presence of bioactive phenolic compounds found in bilberry and cornelian cherry, including anthocyanins, chlorogenic acid and quercetin [56].

In the studies of yogurts with added fruit, particular attention was paid to the vitality of yogurt bacteria, which is a key factor influencing the health-promoting properties of these products. Notably, the results showed that in all yogurt samples containing selected fruits (strawberries, bilberries, cherries and cornelian cherry) the content of yogurt bacteria remained well above the minimum threshold of 10⁷ cfu/g at the time of consumption, as recommended by the Codex Alimentarius (CXS 243-2003) and the World Health Organization (Table 8) [57]. In contrast to findings by Boycheva et al. [58], where the addition of aronia and blueberry juices to goat’s milk yogurt increased the lactic acid bacteria counts compared to control samples, and Blassy et al. [50], where guava pulp addition enhanced bacterial viability, in our study we observed a decrease in the number of Lactobacillus bacteria with fruit addition compared to native yogurt. This decrease is somewhat unexpected, especially considering that fruits contain bioactive compounds that may serve as substrates for the growth of lactic acid bacteria [59].

5. Conclusions

The effect of adding cherries, strawberries, bilberries and cornelian cherry to natural yogurts on their antioxidant activity was investigated, highlighting the potential health benefits of such a combination. A practical implication of this study is that plain yogurts enriched with fruit can serve as a health-promoting alternative without added sugars. The inclusion of fruits in natural yogurts increased their ability to neutralize free radicals, primarily due to the presence of natural antioxidants and polyphenols. Simplifying the complexity of the results, it can be concluded that natural yoghurts with the addition of thawed fruit generally demonstrated higher antioxidant activity than commercial yoghurts containing the same fruit. In most cases, the addition of strawberries, cherries and cornelian cherry to natural yogurts also supported the vitality of lactic acid bacteria. Our study indicates that cornelian cherry has strong potential as a source of polyphenols with antioxidant properties, and yogurt may serve as an effective carrier for this relatively sour fruit. As this is the first known use of cornelian cherry in yogurt, further research is warranted. Future studies should examine the impact of cornelian cherry on yogurt microbiota during extended storage, analyze the bioaccessibility and profile of bioactive compounds in this carrier, and explore its effects on consumer health parameters. In addition, more specific analytical techniques such as HPLC should be applied to identify and quantify individual phenolic compounds. It is also essential to optimize the sensory profile of such yogurts through sensory analysis and to further investigate the interactions between phenolic compounds and proteins. These studies may lead to a better understanding of antioxidant mechanisms and improve the bioavailability of these components in the human diet.