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Article

Acidification Kinetics, Culture Viability, Physicochemical and Antioxidant Characteristics of Yogurt Fortified with Apple Pulp

by
Dimitra Dimitrellou
* and
Panagiotis Kandylis
Department of Food Science and Technology, Ionian University, 28100 Argostoli, Greece
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(8), 466; https://doi.org/10.3390/fermentation11080466
Submission received: 8 July 2025 / Revised: 30 July 2025 / Accepted: 9 August 2025 / Published: 13 August 2025
(This article belongs to the Section Fermentation for Food and Beverages)
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Abstract

Nowadays, there is interest in yogurts and fermented milks with incorporated fruits to fulfill the growing demand for healthier, nutritional, and functional foods. In the present study, the potential of apple pulp incorporation into yogurts was evaluated. Apple pulps from five cultivars (Gala, Starking, Jonagold, Golden, and Granny Smith) were characterized for their antioxidant activity and total phenolic content, with Granny Smith pulp selected for further application due to its superior functional properties. Apple pulp (0–15% w/w) significantly influenced the acidification kinetics by lowering the initial pH and reducing fermentation time. The viability of yogurt starters remained above 108 CFU/g throughout 28 days, fulfilling FAO/WHO criteria, although in yogurts with apple pulp, it was found to significantly decrease in a concentration-dependent manner compared to the control. Quality analyses revealed that apple pulp improved water-holding capacity and reduced syneresis. The antioxidant activity and total phenolic content of yogurts increased proportionally with apple pulp concentration, showing strong positive correlations. Yogurts containing 10–15% w/w apple pulp exhibited the most pronounced functional enhancements. These findings suggest that apple pulp serves as a promising sustainable natural ingredient for producing functional yogurts with improved health-promoting properties due to the antioxidant potential of apple-derived phenolics.

1. Introduction

Yogurt, a fermented dairy product, is consumed worldwide due to its sensory appeal and numerous health benefits. Several variations of yogurts and fermented milks are available, using traditional yogurt starters, combinations with probiotics, different types of milk, and the incorporation of prebiotics and postbiotics [1,2,3]. The popularity of yogurt, combined with the growing demand for healthier, functional foods, has spurred interest in enhancing its nutritional and functional properties through the incorporation of various additives, particularly fruits. Among the fruits explored for yogurt enhancement, apples stand out due to their rich content of bioactive compounds.
Apples are among the most widely consumed fruits globally because they are available year-round, either as fresh or processed products, versatile, and inexpensive. Furthermore, apples are very nutritious, containing bioactive compounds, and are an excellent source of carbohydrates, minerals, dietary fiber, and phenolics [4]. Moreover, the consumption of apples and their products has been associated with numerous health benefits and increased life expectancy [5,6,7]. It is well known that variations occur in antioxidant activity and total phenolic content among apple cultivars and fruit tissues [4,5,6,7,8].
There are numerous studies available in the literature regarding the addition of plant derivatives in yogurts, like fruits, vegetables, cereals, nuts, seeds, oils, plant or herbal extracts, and by-products from plant processing, aiming to mainly exploit their high content of health-promoting compounds such as fiber, phenolic compounds, vitamins, fatty acids and minerals [9]. In general, the addition of fruits and vegetables is a common trend in dairy products [10,11], and numerous studies evaluating the effect of adding fruit pulps to yogurts are available in the literature. These studies highlight improvements in flavor, antioxidant activity, sensory characteristics, and overall acceptability [12,13].
Apple pulp is high in polyphenols, pectins, and other compounds that have been linked to various health benefits, like improved gut health and reduced risk of chronic diseases, making it a very interesting additive for yogurts [14,15]. However, the addition of fruit pulps may alter the yogurt’s consistency and stability and subsequently negatively affect consumer acceptance [12,16]. Although many studies are available regarding the addition to yogurt of apple by-products, such as apple pomace (flour, syrup, etc.) [17,18,19,20] and apple fiber [21], only a limited number of preliminary studies have evaluated the addition of apple pulp [16,22,23]. When apple pomace (flour or powder) was incorporated into the milk base prior to yogurt production, it resulted in yogurts with increased probiotic and total phenolic content, antioxidant activity, and inhibited colon cancer cells’ viability [17], while reducing fermentation time, and may act as a natural stabilizer and texturizer [24]. In the case of apple pomace juice, an increase in fermentation time was reported (probably affecting the starter culture) in combination with increased phenolic content, antioxidant activity, and dietary fiber content [19,20]. Finally, apple fiber has been characterized as a promising potential prebiotic component in yogurts to improve the viability of probiotics during gastrointestinal conditions [21].
One of the existing studies with apple pulp focuses on a completely different product, examining the effect of incorporating apple pulp into already prepared yogurt rather than during the production process [16]. Moreover, previous research has not assessed the effect of apple pulp addition on the functional properties of yogurt, since total phenolic content and antioxidant activity were not measured. Additionally, the functional characteristics of apple pulps from different cultivars were not investigated prior to their incorporation into the milk base. These gaps highlight the need for more comprehensive studies to fully understand the impact of apple pulp addition on yogurt quality and functionality. In our recent study, we evaluated the effect of apple pulp addition on the textural characteristics, microstructure, volatile profile, and sensory acceptance of yogurts, revealing improvements in consistency and aroma with more fruity esters [25]. However, the effect on physicochemical and functional characteristics is still missing.
Considering these aspects, the present study aimed to evaluate the pulp of five well-known apple cultivars for their functional characteristics, such as total phenolic content and antioxidant activity. The apple pulp with the highest levels was selected and incorporated into yogurts at different concentrations. The resulting yogurts were then assessed for their physicochemical, antioxidant, and microbiological characteristics during 28 days of refrigerated storage.

2. Materials and Methods

2.1. Materials

Pasteurized and homogenized cow’s milk (Olympos, Greece; composition per 100 mL: fat 3.7 g; sugar 4.7 g; protein 3.4 g) and skim milk powder (Regilait, France; composition per 100 g: fat 0.8 g; sugar 52 g; protein 36 g) were employed in the present study. Apple samples from the following cultivars, Gala, Starking, Jonagold, Golden, and Granny Smith, cultivated in Greece, were purchased from a local store. The starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) used for yogurt production was the commercial CH-1 (YoFlex) (Chr. Hansen, Hørsholm, Denmark).

2.2. Apple Pulp Production and Extraction

The apples, fully ripened and ready for consumption, were rinsed with warm water (45 °C), followed by de-seeding and peeling. A hand blender (Severin Elektrogeräte GmbH, Sundern, Germany) was used to produce the apple pulp, which was then subjected to pasteurization at 79 °C for 69 s [26]. The pasteurized apple pulp (total solids 14.80 ± 0.30%) was either used for yogurt production (Section 2.3) or for the extraction described in the next paragraph.
The produced apple pulp was extracted according to the method of Senadeera et al. [26], with some modifications. Specifically, 5 g of apple pulp and 50 mL of 80% v/v methanol (99.9%; Sigma Aldrich, Burlington, MA, USA) solution were mixed for 3 min and left undisturbed overnight (18 h) at room temperature (25 ± 1 °C). The apple pulp extract was collected after filtration (Whatman No. 1) and stored at –20 °C until used for further analyses.

2.3. Yogurt Production and Extraction

Yogurt was produced mainly following the procedure by Dimitrellou et al. [27] as presented in Figure 1. More specifically, the apple pulp was added at 5, 10, and 15% w/w, 10 min before the addition of starter culture (Y5AP, Y10AP, and Y15AP, respectively). The mixture was incubated at 42 °C until the pH reached 4.6 (210–230 min) and then transferred to a refrigerator at 4 °C. A control sample without the addition of apple pulp was also manufactured (Y0AP). Yogurt water extract was prepared according to the method of Dimitrellou et al. [27] and stored at −20 °C until further use. In brief, the yogurt samples were diluted in water, acidified to pH 4.0 to precipitate casein, centrifuged, and the supernatant was neutralized to pH 7.0 and centrifuged again. The final supernatant was used for subsequent assays.

2.4. Analyses

2.4.1. Acidification Kinetics

The change in pH value during yogurt fermentation was determined every 25 min until the pH reached 4.6. Maximum acidification rate (10−3 pH/min), the time at which the maximum acidification rate was reached (min), and the time required to reach pH 4.6 (min) were evaluated [28].

2.4.2. Titratable Acidity, pH, and Moisture Content

The titratable acidity of the yogurt samples (10 g) was determined by means of titration with 0.1 N NaOH and phenolphthalein as an indicator and expressed as % w/w lactic acid equivalent [27]. The pH of the yogurts was determined using an FC200B pH electrode (Hanna instruments, Woonsocket, RI, USA). The moisture content of the yogurts was determined by oven drying the samples at 105 °C until constant weight (approximately 4 h) [29] and calculated using the following equation:
Moisture   ( % ) =   W 1 − W 2 W 1   × 100
W1: weight of sample before drying; W2: weight of sample after drying.

2.4.3. Water-Holding Capacity and Syneresis

The water-holding capacity (WHC) of the yogurts was determined using the centrifugation method (10 g of yogurt sample, 5000× g for 10 min at 20 °C) [27] and the following equation W H C   ( % ) = 100   ( Y − W E ) Y , where WE = whey expelled in g and Y = initial yogurt sample in g. Syneresis was determined after filtration of 50 mL of unstirred yogurt using filter paper (No.1 filter paper, Whatman Ltd., Maidstone, UK) at 4 °C for 5 h [27]. Then, the volume of whey collected in the beaker was measured, multiplied by 2, and expressed as syneresis (%).

2.4.4. Total Phenolic Content

The total phenolic content of apple pulp extract and yogurt extract was analyzed following the Folin–Ciocalteu method [27]. Diluted apple pulp extract or yogurt extract (total 1 mL), 3 mL of distilled water, 0.250 mL of Folin–Ciocalteu reagent (2M; Sigma Aldrich, Burlington, MA, USA), and 0.750 mL of 20% w/w sodium carbonate were mixed and allowed to stand at room temperature in darkness for 120 min. Then, the absorbance was recorded at 750 nm in a UV/Vis spectrophotometer (Helios Omega, UV-VIS, Thermo Fisher Scientific, Madison, WI, USA). The results were quantified using gallic acid (99.9%; Sigma Aldrich, Burlington, MA, USA) as a standard (R2 = 0.999).

2.4.5. Antioxidant Activity

The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was carried out according to our previous study [27]. More specifically, apple pulp extract or yogurt extract diluted to a total volume of 1.250 mL, and 3.750 mL of DPPH solution (TCI, Zwijndrecht, Belgium) were mixed. After incubation at room temperature in darkness for 30 min, the absorbance at 517 nm was recorded. The results were quantified using Trolox (>98%; Acros Organics, Antwerpen, Belgium) as a standard (R2 = 0.998).
The FRAP (Ferric reducing antioxidant potential) assay was carried out according to the method described in a previous study [30] with some modifications. From apple pulp or yogurt extract, 0.400 mL was added to 3.600 mL of FRAP solution. After 30 min incubation at 37 °C in the dark, the absorbance was recorded at 593 nm and quantified using Trolox (>98%; Acros Organics, Antwerpen, Belgium) as a standard (R2 = 0.999).

2.4.6. Microbiological Analysis

Yogurt samples (10 g) were homogenized with 90 mL of sterile Ringer solution, and subsequent serial dilutions were made. The enumeration of yogurt starters was carried out using MRS agar (at pH 5.4; anaerobic incubation at 37 °C for 72 h) and M-17 agar (aerobic incubation at 37 °C for 48 h) for L. delbrueckii subsp. bulgaricus and S. thermophilus, respectively [27].

2.5. Statistical Analysis

All experiments were conducted in triplicate, with duplicate samples collected for each analysis. The experimental data were statistically evaluated for significance using analysis of variance (ANOVA), followed by Tukey’s Honest Significant Difference (HSD) test for post hoc comparisons (Statistica software, ver. 12.0; StatSoft Inc., Tulsa, OK, USA).

3. Results and Discussion

3.1. Selection of Apple Cultivar

As with other fruits, the chemical content of apples varies, even within the same cultivar, based on a number of factors, including farming practices, production location, maturity, and even storage conditions. In fact, notable differences in antioxidant activity and total phenolic content could be found between cultivars and even between different fruits within the same cultivar [31]. The same applies to phenolic compounds, where variations occur not only among different cultivars but also within different tissues. The majority of phenolic compounds in apples are in the skins, which contain almost two to four times the phenolic content of the pulp [32,33]. The main phenolic compounds detected in apples include a variety of antioxidants and polyphenols that significantly contribute to their health benefits.
In the present study, apple pulps from five cultivars (Gala, Starking, Jonagold, Golden, and Granny Smith) were analyzed for their antioxidant activity and total phenolic content, and the results are presented in Figure 2. The apple cultivar significantly affected the total phenolic content and antioxidant activity of the apple pulp. Jonagold and Golden presented similar values (p > 0.05) of antioxidant activity and total phenolic content. A good correlation between total phenolic content and FRAP analysis was reported (R2 = 0.9915). The highest antioxidant activity (measured using FRAP) and total phenolic content were reported in the case of apple pulp from the Granny Smith cultivar, followed by the Starking and Gala cultivars. In the case of DPPH, the same three cultivars presented the highest antioxidant activity. The good antioxidant activity and total phenolic content of Granny Smith apples, and especially their fleshes, in comparison with other cultivars has also been observed in other studies [31,34]. The phenolic content in apple flesh is influenced by various factors, including agronomic practices, genetic background, pre- and postharvest conditions, and processing methods [31]. Notably, conventional apple cultivars generally show higher levels of phenolics compared to organic ones, which may be attributed to differences in cultivation techniques and the plants’ responses to stress [34]. Therefore, apple pulp derived from the Granny Smith cultivar was employed in the following yogurt experiments.

3.2. Acidification Kinetics of Yogurt

The initial pH of the milk was significantly (p < 0.05) affected by the apple pulp (Figure 3). The higher the concentration of apple pulp in the milk, the lower the initial pH (6.57 for Y0AP and 5.95 for Y15AP). It is well known that the incorporation of fruits and fruit juices into yogurt significantly impacts its acidification and fermentation kinetics. This addition influences the initial pH of milk and the rate of pH decline during fermentation. Fruits and their juices, particularly those high in organic acids, tend to lower the initial pH of the yogurt mix, enhancing the acidification rate during the early stages of fermentation, probably due to their content in simple fermentable sugars like glucose and fructose. For instance, passion fruit juice, due to its high organic acid content, significantly accelerated the acidification rate in the initial 30 min of fermentation compared to control samples without fruit juice. This rapid acidification can suppress the growth of undesirable bacteria and subsequently improve the microbial quality of the yogurt [28]. The results were similar when juices [27], freeze-dried mulberry [35], and soursop puree [36] were incorporated into yogurts.
Moreover, the inclusion of fruits and their juices can also affect the total fermentation time. In the present study, fermentation times between 210 and 230 min were reported. The addition of apple pulp significantly affected the total fermentation time and reduced it by up to 20 min (Y15AP) compared to the control sample without apple pulp (Y0AP). Studies have shown that incorporating fruits, like apples, can slightly reduce the overall fermentation time, due to the additional sugars and organic acids provided by the fruits, which serve as substrates for the bacteria. For example, yogurt produced with freeze-dried immobilized Lactobacillus casei on apple pieces exhibited a reduction in total acidification time compared to yogurt without fruit addition [37], as was also observed by Popescu et al. [18], who studied the addition of apple pomace in yogurt. Similarly, the addition of fruit juices such as blueberry, aronia, and grapes, as well as soursop puree, has been observed to enhance fermentation kinetics [27,36].
From the beginning of fermentation and up to approximately 100 min of incubation, an initial pH reduction was found in all of the yogurt samples. This may be attributed to the metabolic activity of the starter culture that facilitated the accelerated catabolism of lactose. The highest rate of pH reduction was observed between 100 and 200 min post-inoculation and indicates the peak activity of starter culture bacteria, and this period is similar (120–210 min) to yogurts containing soursop puree [36]. However, the maximum rate of acidification was reported in Y0AP (14.7 × 10−3 pH/min) at 137.5 min and the lowest (10.7 × 10−3 pH/min) was reported at 112.5 min in Y15AP, containing the highest quantity of apple pulp (Table 1). The control sample presented the highest acidification rate, also in a previous study with passion fruit juice, wherein a decrease was observed with the increase in juice addition [28]. In the present study, this may be attributed to the differences in the initial pH values of the yogurt samples. Specifically, yogurts enriched with apple pulp exhibited significantly lower starting pH values (5.96 ± 0.01, in the case of Y15AP) compared to the control (6.52 ± 0.07, Y0AP). A lower initial pH limits the extent of pH decline during fermentation and consequently reduces the acidification rate. This drop in pH is attributed to the acid content of Granny Smith apple pulp (0.32 g/100 g) and mainly malic acid [38].
However, the effects are not exclusively beneficial. Excessive addition of fruit juice or fruit pulp can lead to over-acidification and structural degradation of the yogurt gel. For instance, adding more than 7.5% passion fruit juice can weaken the yogurt’s gel structure due to the high acidity, resulting in a less desirable texture and potential syneresis [28]. Therefore, while the incorporation of fruits and fruit juices into yogurt can enhance fermentation kinetics and acidification rates, optimizing the concentration of these additions is crucial to maintaining the desired yogurt quality and texture.

3.3. Quality Assessment of Yogurt

3.3.1. Total Acidity, pH, and Moisture

The pH values and acidity were measured, considered as important factors that affect the shelf life of the produced yogurts (Table 2).
The initial pH values and acidity were affected by the added apple pulp as well as by the starter culture’s bacteria. Generally, pH values decreased, while titratable acidity correspondingly increased for all yogurts during the storage period. Similar results were recorded with the addition of fruit pulp to yogurt [39]. On day 1, a significantly (p < 0.05) higher drop in pH values and an increase in acidity were observed in yogurts with apple pulp compared to the control. Apple pulp is a source of acids, which lower the pH, and carbohydrates, which enhance the metabolic activity of the starter culture [26].
Post-acidification was observed in all yogurts, which decreased pH levels during storage. This post-acidification is a characteristic of the type of starter culture used. Storage time had a significant (p < 0.01) impact in all cases; however, the pH of yogurts was not affected by the presence of apple pulp, particularly after 7 days. At the end of storage (28 days), all yogurts had similar pH values, ranging from 3.98 to 4.05. These findings are notable as they show that adding apple pulp had no impact on the yogurt starters’ ability to ferment during storage. Similar outcomes to the pH results were also reported for acidity. These findings are in accordance with previous studies using fruit juices in yogurt production [27].
The moisture content was also determined, since it is an important property that affects the quality of the produced yogurts. Moisture content ranged from 83.8 to 85.4% w/w for yogurts with apple pulp and from 84.1 to 85.2% w/w for control yogurt, presenting no significant differences based on apple pulp addition. Yogurts with apple pulp had a slight, but non-significant, increase in moisture content on the first day, compared to the control. In addition, there were no significant (p > 0.05) differences in moisture content for all samples during storage. This may be attributed to the similar total solid content of apple pulp and milk used in the presented study, which also resulted in non-significant differences in the textural characteristics of yogurts with and without apple pulp, like hardness, as was revealed in our recent study [25].

3.3.2. Syneresis and Water-Holding Capacity

Syneresis or serum separation is a defect of fermented milk products, such as yogurt, whereby liquid (yogurt whey or serum) is expelled from the yogurt network and depends on many factors, i.e., processing conditions, additives, etc. [40]. Water-holding capacity is the ability of yogurt to retain water and is considered a factor that represents the stability of yogurt. The inclusion of apple pulp to yogurt significantly (p < 0.01) affected these properties. The syneresis and water-holding capacity of the produced yogurts during storage are presented in Table 3.
During storage, yogurts enriched with apple pulp exhibited lower syneresis values and higher water-holding capacity compared to the control, in a concentration-dependent manner. The control sample showed the highest syneresis (28.0–30.8% v/v) and the lowest water-holding capacity (42.8–47.0% w/w), whereas the addition of apple pulp significantly reduced syneresis and increased water-holding capacity in yogurt. These improvements may result from interactions between milk proteins and apple-derived compounds, such as pectin [41] and polyphenols [42]. The presence of dietary fibers in apple pulp likely contributes to stabilizing the yogurt matrix by entrapping water and enhancing gel structure, highlighting apple pulp’s potential as a natural stabilizer [43]. Additionally, apple pectin is known to strengthen the protein gel network and increase its water-binding capacity, further supporting these findings. Similar effects were reported by Popescu et al. [18], who observed improved water-holding capacity and reduced syneresis in yogurts supplemented with apple pomace.
Another important factor affecting syneresis and water-holding capacity is the protein and fat content of the final product. The control yogurt contains 4.5 g of protein and 3.7 g of fat per 100 g. The addition of apple pulp, which does not contain protein and fat, resulted in yogurts with lower protein (4.3 g Y5AP, 4.1 g Y10AP, and 3.8 g Y15AP) and fat content (3.5 g Y5AP, 3.4 g Y10AP, and 3.2 g Y15AP). Reducing the fat content may alter the stability of yogurt and its sensory characteristics since fat globules act as linking protein agents. This effect is more pronounced if a subsequent reduction in protein content occurs. In such cases, it is very important to maintain or even increase the total solid content to prevent specific textural defects. Indeed, in the present study, the addition of apple pulp proved to balance this decrease in fat and protein, resulting in yogurts with improved water-holding capacity.

3.4. Viability of Starter Culture

During yogurt production and storage, it is very important to maintain high numbers of viable yogurt starters. According to the FAO/WHO, these viable cells should be higher than 107 per gram of yogurt during consumption [44]. In the present study, yogurt starters were able to survive well in yogurts with and without apple pulp, maintaining high viable counts (>8.50 log CFU/g). Moreover, during the storage period, the number of starters in yogurts with apple pulp was found to significantly decrease in a concentration-dependent manner compared to the control but was always higher than 8.50 log CFU/g (Figure 4). This may also explain the highest acidification rate observed in the control (Y0AP) sample.
A recent study investigated the effect of jujube pulp fortification on the microbial profile of goat milk yogurts. The findings revealed a gradual decline in the number of yogurt starters’ colonies with prolonged storage, with the control sample exhibiting higher viable counts than the fortified sample [45]. Similar results were observed in yogurts with the addition of fruit juice [46]. This decrease has been attributed either to the natural acidity of fruit pulp/juice or to the presence of high concentrations of polyphenols [41], and it is more pronounced at high concentrations of fruit (>15%) [47]. Finally, in another relevant study, a negative impact was reported in the counts of S. thermophilus with the addition of apple pomace hot water extract (3.3%) [48]. Other studies have highlighted the potential prebiotic effect of fruit juices (at low concentrations) and apple pomace [27,49]. However, these materials usually selectively stimulate the growth and activity of specific bacteria [26], as also reported for some fructooligosaccharides [50]. A slight, though not significant (p > 0.05), reduction in bacterial numbers was reported with increasing storage time, a finding that aligns with those reported in several other studies [27,46,51]. Interestingly, in a recent study, the beneficial effect of apple fiber on probiotic viability during yogurt and simulated gastrointestinal conditions was reported by our group, revealing the great potential of apple products [21]. The viability of yogurt starters during storage is important due to their numerous health benefits and probiotic potential [52,53,54].

3.5. Total Phenolic Content and Antioxidant Activity of Yogurts

The use of different contents of apple pulp (0–15% w/w) in yogurt formulations significantly (p < 0.05) affected the antioxidant activity and total phenolic content of yogurts, resulting in increased values in these parameters (Figure 5 and Figure 6).
The total phenolic content of the control yogurt without apple pulp (Y0AP, 58–66 mg GAE/kg) was similar and slightly higher than that reported in other studies (40–42 mg GAE/kg or GAE/L) [17,19]. Some authors attributed these values of the control yogurt to limitations of the Folin–Ciocalteu method, which may be affected by the presence of reducing sugars [19]. However, it is well known that milk naturally contains amounts of phenolic compounds that primarily originate from the cow’s diet, particularly when they consume phenolic-rich feeds [55]. During the fermentation process, the phenolic compounds undergo transformations, which can affect their concentration and bioavailability in the final yogurt product. Generally, the fermentation process by lactic acid bacteria, such as the yogurt starters, leads to an increase in the concentration of certain phenolic compounds. The total phenolic content significantly (p < 0.001) increased in an apple pulp concentration-dependent manner. Control and Y5AP presented (p > 0.05) similar values of total phenolic content after 14 days of storage. Similar values of total phenolic content were detected in Y10AP and Y15AP, but significantly higher than in the control and Y5AP, after 14 days of storage. A similar trend of increased total phenolic content with increased concentration of apple derivative was reported in other similar studies with apple pomace [19], apple pomace syrup [20], and apple pomace flour [17]. During storage, a significant increase in the total phenolic content was observed in all yogurt samples, consistent with findings from a previous study involving the incorporation of apple pomace powder into stirred yogurt [19]. This increase may be attributed to the progressive release of phenolic compounds from the apple pulp matrix, potentially facilitated by the changes in yogurt characteristics during storage, such as pH, titratable acidity, and protein structure, or by enzymatic activity associated with starter cultures. In contrast, a study incorporating cranberry into yogurt reported a decrease in total phenolic content, which was ascribed to the metabolic activity of starter cultures and probiotic strains capable of metabolizing phenolic compounds [56].
The antioxidant activity of yogurts presented a similar trend with total phenolic content (Figure 6). More specifically, an increase in the antioxidant activity (FRAP and DPPH method) in an apple pulp concentration-dependent manner was reported in the present study, as in similar studies with yogurts with apple pomace derivatives [17,18,20]. A good correlation was reported between antioxidant activity and total phenolic content (R2 = 0.8239 in FRAP and R2 = 0.8743 in DPPH, after 14 days of storage), indicating that the phenolic compounds released from apple pulp are the main contributors to the antioxidant activity of the extracts obtained from the yogurts, as was also reported in the case of apple pomace flour [16]. A slight but non-significant increase in the antioxidant activity of all yogurt samples was reported during storage. In similar studies with the incorporation of fruits, a decrease in antioxidant activity was reported during storage, and it was correlated, as in the case of phenolic compounds, with the metabolic activity of starters and probiotics [56,57].
The phenolic compounds that have been found in apple pulp and pomace of the Granny Smith cultivar include hydroxycinnamic acids, flavan-3-ols, flavonols, dihydrochalcones, and procyanidin-B2, and a correlation has been observed between the content of these compounds and antioxidant activity [58,59,60]. In general, Y10AP and Y15AP presented the highest values of antioxidant activity and total phenolic content among the yogurt samples and may therefore be considered to possess the most optimal functional characteristics. However, the incorporation of apple pulp at higher concentrations was found to significantly affect the textural and sensory attributes of yogurts, as demonstrated in our recent study [25]. Considering both the enhancement of functional characteristics and the preservation of acceptable sensory and textural quality, Y5AP appears to offer the most balanced formulation. It provides a notable improvement in antioxidant and phenolic content compared to the control while maintaining consumer-relevant qualities. Thus, Y5AP may be regarded as the optimal compromise between nutritional enrichment and product quality.

4. Conclusions

In conclusion, the apple pulp was successfully incorporated into yogurt, representing a promising approach to enhancing the functional characteristics of this widely consumed dairy product, thereby making it more appealing to health-conscious consumers. The results of the present study highlight the variations in the phenolic content and antioxidant activity of apple pulp derived from different cultivars and present an insight into its potential application in functional yogurt production. The incorporation of apple pulp lowered the initial pH of the yogurt mixture, slightly reduced the fermentation time, while improving the water-holding capacity and reducing syneresis of yogurts. It is noteworthy that viable counts of starters remained above 108 CFU/g during 28 days of storage. Alongside the increase in total phenolic content, the antioxidant activity of the yogurt was also enhanced with the addition of apple pulp. These findings underline the potential of apple pulp as a functional ingredient in yogurt, enhancing its health benefits by boosting phenolic content and antioxidant capacity.

Author Contributions

Conceptualization, D.D.; methodology, D.D. and P.K.; validation, D.D.; formal analysis, D.D. and P.K.; investigation, D.D. and P.K.; resources, D.D.; writing—original draft preparation, D.D. and P.K.; writing—review and editing, D.D. and P.K.; supervision, D.D.; project administration, D.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank the undergraduate students Gkekas S., Mersini R., Christodoulakou D., Bako A., Giatra M., and Pappa C. for their assistance during the experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CFUColony-forming unit
DPPH1,1-diphenyl-2-picrylhydrazyl
FRAPFerric reducing antioxidant potential
Y0APYogurt without apple pulp
Y5APYogurt containing 5% w/w apple pulp
Y10APYogurt containing 10% w/w apple pulp
Y15APYogurt containing 15% w/w apple pulp

References

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Fermentation 11 00466 g001
Figure 1. The procedure of yogurt production with apple pulp.
Figure 1. The procedure of yogurt production with apple pulp.
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Figure 2. Antioxidant activity and total phenolic content of pulp from different apple cultivars (GAE: gallic acid equivalents; TE: Trolox equivalents. a–d Different lowercase letters in the columns, in the same analysis, indicate significant differences (p < 0.05)).
Figure 2. Antioxidant activity and total phenolic content of pulp from different apple cultivars (GAE: gallic acid equivalents; TE: Trolox equivalents. a–d Different lowercase letters in the columns, in the same analysis, indicate significant differences (p < 0.05)).
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Figure 3. Acidification kinetics during yogurt production (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively).
Figure 3. Acidification kinetics during yogurt production (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively).
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Figure 4. Viability of yogurt starters during storage (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively. a–c Different lowercase letters in the columns, on the same day of storage, indicate significant differences (p < 0.05)).
Figure 4. Viability of yogurt starters during storage (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively. a–c Different lowercase letters in the columns, on the same day of storage, indicate significant differences (p < 0.05)).
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Figure 5. Total phenolic content of yogurts (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively; GAE: gallic acid equivalents. a–d Different lowercase letters in the columns, on the same day of analysis, indicate significant differences—effect of apple pulp (p < 0.05). A–C Different uppercase letters in the columns, in the same sample, indicate significant differences—effect of storage (p < 0.05).
Figure 5. Total phenolic content of yogurts (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively; GAE: gallic acid equivalents. a–d Different lowercase letters in the columns, on the same day of analysis, indicate significant differences—effect of apple pulp (p < 0.05). A–C Different uppercase letters in the columns, in the same sample, indicate significant differences—effect of storage (p < 0.05).
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Figure 6. Antioxidant activity of yogurts measured with the DPPH (A) and FRAP (B) methods (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively; TE: Trolox equivalents. a–d Different lowercase letters in the columns, on the same day of analysis, indicate significant differences—effect of apple pulp (p < 0.05). A–C Different uppercase letters in the columns, at the same sample, indicate significant differences—effect of storage (p < 0.05).
Figure 6. Antioxidant activity of yogurts measured with the DPPH (A) and FRAP (B) methods (Y0AP: yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: yogurts with apple pulp 5, 10, and 15% w/w, respectively; TE: Trolox equivalents. a–d Different lowercase letters in the columns, on the same day of analysis, indicate significant differences—effect of apple pulp (p < 0.05). A–C Different uppercase letters in the columns, at the same sample, indicate significant differences—effect of storage (p < 0.05).
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Table 1. Effect of apple pulp addition on the acidification kinetics of yogurt.
Table 1. Effect of apple pulp addition on the acidification kinetics of yogurt.
ParametersYogurt SamplesSignificance
Y0APY5APY10APY15AP
Vmax (×10−3 pH/min)14.7 ± 0.5 C12.8 ± 0.0 B11.9 ± 0.6 AB10.7 ± 0.8 A***
TVmax (min)137.5 ± 0.0 B137.5 ± 0.0 B112.5 ± 0.0 A112.5 ± 0.0 A***
TpH4.6 (min)230 ± 4 C220 ± 0 B215 ± 3 AB210 ± 2 A***
A–C: Means within the same row with different uppercase superscripts differ significantly (p < 0.05), indicating the effect of apple pulp addition. Y0AP: Yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: Yogurts containing 5%, 10%, and 15% w/w apple pulp, respectively. ***: p < 0.001. Vmax: maximum rate of acidification; TVmax: time to reach Vmax; TpH 4.6: end time of fermentation.
Table 2. Effect of apple pulp and storage on the pH, acidity, and moisture content of yogurts.
Table 2. Effect of apple pulp and storage on the pH, acidity, and moisture content of yogurts.
ParametersStorage
(Days)		Yogurt SamplesSignificance
Y0APY5APY10APY15AP
pH14.32 ± 0.00 aA4.34 ± 0.01 aA4.24 ± 0.01 aB4.24 ± 0.04 aB**
74.23 ± 0.02 abA4.26 ± 0.04 abA4.21 ± 0.01 aA4.27 ± 0.03 aAns
144.15 ± 0.06 bA4.16 ± 0.05 bcA4.17 ± 0.06 abA4.12 ± 0.06 abAns
214.07 ± 0.05 bA4.11 ± 0.06 bcA4.06 ± 0.05 bcA4.04 ± 0.01 bAns
284.03 ± 0.03 bA4.05 ± 0.04 cA3.98 ± 0.03 cA4.00 ± 0.06 bAns
significance ******** 
Acidity
(lactic acid
% w/w)		10.98 ± 0.03 aA1.00 ± 0.03 aA1.01 ± 0.02 aA1.02 ± 0.02 aAns
71.05 ± 0.04 abA1.10 ± 0.03 abA1.12 ± 0.03 bA1.10 ± 0.03 abAns
141.10 ± 0.01 bcA1.19 ± 0.02 bB1.19 ± 0.04 bcB1.19 ± 0.02 bB*
211.12 ± 0.01 bcA1.15 ± 0.03 bA1.18 ± 0.00 bcA1.12 ± 0.01 bAns
281.19 ± 0.01 cAB1.19 ± 0.01 bAB1.25 ± 0.03 cA1.15 ± 0.03 bB*
significance ******** 
Moisture
(% w/w)		184.1 ± 0.1 aA85.2 ± 0.4 aA84.9 ± 1.0 aA85.4 ± 0.1 aAns
784.4 ± 0.5 aA84.7 ± 0.4 aA84.5 ± 0.4 aA84.0 ± 0.9 aAns
1484.7 ± 0.9 aA84.6 ± 0.7 aB85.0 ± 1.2 aB85.4 ± 0.7 aBns
2184.6 ± 0.4 aA83.8 ± 1.0 aA84.4 ± 1.1 aA84.2 ± 0.3 aAns
2885.2 ± 0.3 aA83.9 ± 0.7 aA85.3 ± 0.3 aA83.6 ± 0.5 aBns
significance nsnsnsns 
A–B: Means within the same row for a given day with different uppercase superscripts differ significantly (p < 0.05), indicating the effect of apple pulp addition. a–c: Means within the same column for a given sample with different lowercase superscripts differ significantly (p < 0.05), indicating the effect of storage. Y0AP: Yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: Yogurts containing 5%, 10%, and 15% w/w apple pulp, respectively. ns: not significant; *: p < 0.05; **: p < 0.01.
Table 3. Effect of apple pulp and storage on the syneresis and water-holding capacity of yogurts.
Table 3. Effect of apple pulp and storage on the syneresis and water-holding capacity of yogurts.
ParametersStorage
(Days)		Yogurt SamplesSignificance
Y0APY5APY10APY15AP
Syneresis
(% v/v)		130.7 ± 1.3 A22.3 ± 1.3 aB23.4 ± 1.1 aB22.8 ± 1.1 abB**
728.7 ± 1.8 A21.6 ± 0.6 aB23.1 ± 1.3 aB21.6 ± 0.6 aB*
1430.8 ± 1.7 A22.6 ± 0.6 aB24.7 ± 1.1 abB25.8 ± 1.1 bcB**
2129.7 ± 0.4 A24.1 ± 1.3 abB26.4 ± 0.8 abB26.3 ± 0.4 bcB*
2828.0 ± 0.7 AB25.5 ± 0.4 bB28.5 ± 0.7 bA26.0 ± 0.8 cAB*
significance ns**** 
Water-holding
capacity
(% w/w)		142.8 ± 0.8 A47.3 ± 1.5 AB50.2 ± 1.4 B52.0 ± 1.3 B**
744.8 ± 0.8 A46.4 ± 1.2 A52.8 ± 0.8 B50.3 ± 0.7 B**
1447.0 ± 0.7 A49.2 ± 0.1 AB54.6 ± 1.9 C53.3 ± 1.1 BC**
2146.2 ± 1.8 A48.6 ± 1.0 A54.0 ± 0.8 B54.2 ± 1.2 B**
2846.5 ± 0.7 A46.8 ± 0.2 AB50.4 ± 0.6 B53.4 ± 0.7 C**
significance nsns*ns 
A–C: Means within the same row for a given day with different uppercase superscripts differ significantly (p < 0.05), indicating the effect of apple pulp addition. a–c: Means within the same column for a given sample with different lowercase superscripts differ significantly (p < 0.05), indicating the effect of storage. Y0AP: Yogurt without apple pulp; Y5AP, Y10AP, and Y15AP: Yogurts containing 5%, 10%, and 15% w/w apple pulp, respectively. ns: not significant; *: p < 0.05; **: p < 0.01.
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Dimitrellou, D.; Kandylis, P. Acidification Kinetics, Culture Viability, Physicochemical and Antioxidant Characteristics of Yogurt Fortified with Apple Pulp. Fermentation 2025, 11, 466. https://doi.org/10.3390/fermentation11080466

AMA Style

Dimitrellou D, Kandylis P. Acidification Kinetics, Culture Viability, Physicochemical and Antioxidant Characteristics of Yogurt Fortified with Apple Pulp. Fermentation. 2025; 11(8):466. https://doi.org/10.3390/fermentation11080466

Chicago/Turabian Style

Dimitrellou, Dimitra, and Panagiotis Kandylis. 2025. "Acidification Kinetics, Culture Viability, Physicochemical and Antioxidant Characteristics of Yogurt Fortified with Apple Pulp" Fermentation 11, no. 8: 466. https://doi.org/10.3390/fermentation11080466

APA Style

Dimitrellou, D., & Kandylis, P. (2025). Acidification Kinetics, Culture Viability, Physicochemical and Antioxidant Characteristics of Yogurt Fortified with Apple Pulp. Fermentation, 11(8), 466. https://doi.org/10.3390/fermentation11080466

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