Enhancing Kefir with Raspberry Pomace: Storage-Dependent Changes in Quality and Stability

Abstract

This study evaluated the effects of raspberry pomace addition on kefir’s chemical, microbiological, and sensory properties during a 14-day refrigerated storage. Kefir samples were prepared using 10% and 20% raspberry pomace, either retaining or straining out pomace after fermentation. Raspberry pomace notably enhanced antioxidant activity, peaking at 95.91% DPPH radical reduction on day 10, and increased total polyphenol content to 78.24 mg gallic acid/L. Pomace addition also improved microbiological stability, maintaining higher lactic acid bacteria (7.48 log CFU/mL) and stable yeast counts. Repeated measures ANOVA revealed a significant effect of storage duration on the concentrations of all analyzed parameters. Results showed that pomace-enriched samples, particularly those retaining pomace during storage, exhibited significantly higher levels of lactic, acetic, and citric acids, as well as ethanol and residual sugars. Sensory evaluations revealed kefir samples with strained raspberry pomace had the highest consumer acceptability, scoring 7.8 out of 9 for overall acceptance due to balanced flavor and improved texture. These results highlight raspberry pomace’s potential as a valuable ingredient for improvement in kefir, offering a promising approach to functional dairy innovation.

1. Introduction

Kefir is a traditional fermented milk drink with a mildly tangy taste, known as one of the oldest fermented dairy products. It is produced through the fermentation of milk by kefir grains—a unique polysaccharide matrix that contains a symbiotic culture of yeast and bacteria. The combined activity of kefir’s microbiota results in a beverage with distinctive sensory qualities [1,2]. It possesses both probiotic and prebiotic properties, owing to its rich composition of beneficial microorganisms, enzymes, polysaccharides, polyphenols, vitamins, and minerals. The chemical composition of kefir is variable and not clearly defined. It depends on the type of milk, milk fat content, the microbial composition of the kefir grains, and the technological process of production [3]. The chemical composition is primarily determined by the maturation of kefir, as certain biochemical changes occur during fermentation and storage, influenced by lactic acid and alcoholic fermentation [4].

Kefir has a creamy texture and a mildly sour, slightly effervescent taste, which many find refreshing and enjoyable. However, some consumers may find its natural tartness or acidity too strong, creating a need for improvement through flavor balancing, sweetness adjustment, or the addition of fruit and natural flavorings to enhance its taste [2]. The sensory properties of kefir can be enhanced by the addition of fruits, such as raspberries, cherries, peaches, or strawberries, as well as their extracts or by-products. Previous studies have demonstrated that the addition of berry aromas [5], fruit juices [6], or encapsulated juices [7], as well as other ingredients such as plant or agro-food waste extracts [8] and coffee [9], can enhance sensory, rheological, and functional properties of kefir, significantly improving consumers’ acceptance, technological properties, and overall health benefits.

Fruit pomace, the solid residue remaining after juice extraction is of particular interest for incorporation in food and dairy products, due to its valuable nutritional content [10]. During juice processing, approximately 20–30% of the original berry mass remains as a by-product, commonly referred to as pomace [11].

Raspberry pomace, a by-product of juice production consisting of pulp and seeds, is especially valuable due to its high content of phenols, flavonoids, dietary fiber, and essential fatty acids, particularly linoleic and α-linolenic acids [11]. Additionally, it is rich in micronutrients, with potassium, magnesium, and calcium being the most abundant [12,13,14]. Despite its nutritional potential, substantial quantities of raspberry pomace are discarded during juice processing, leading to environmental concerns due to waste accumulation. To mitigate this, various valorization strategies have been explored, including incorporating dried and ground raspberry pomace into gluten-free cookies [15], breads [13], or fruit bars [16], which enhanced their nutritional profile and antioxidant activity. However, despite the growing interest in functional foods enriched with fruit by-products, there is a scarcity of research examining the effects of fruit pomace incorporation on the chemical, microbiological, and sensory attributes of kefir and kefir-like products, especially during storage. Understanding the changes in the chemical composition of kefir during storage and the maturation period is essential for ensuring its quality, stability, and consumer acceptability, particularly when organic non-standard ingredient such as fruit pomace is added, since the fermentation process continues during cold storage, with a potential to alter key chemical and sensory properties.

Hence, this study aimed to investigate the impact of the addition of raspberry pomace and kefir maturation during cold storage on its chemical and microbiological composition, as well as sensory properties. The influence of varying concentrations of raspberry pomace and different technological procedures on selected chemical, microbiological, and sensory parameters was assessed over 14 days of storage at 4 °C.

2. Materials and Methods

2.1. Kefir Production

Kefir samples were prepared using UHT cow’s milk with 2.8% milk fat (Imlek, Belgrade, Serbia) and raspberry pomace (Fruvita, Belgrade, Serbia) at different concentrations. Raspberry pomace (dry matter content 51.67%, pH 3.3) was used without pretreatment and applied in its original form, as received from the juice producer immediately after pressing. Samples were divided into two groups: samples K1 and K3 contained 10% raspberry pomace, while samples K2 and K4 contained 20%. Home-made kefir grains were previously propagated under sterile conditions using UHT cow’s milk at 25 °C for 24 h. After each fermentation cycle, the grains were gently washed with sterile distilled water and re-inoculated into fresh milk. This procedure was repeated daily for one month prior to the experiment to maintain grain activity and increase biomass. Kefir starter was prepared by inoculating 5% (w/v) kefir grains into milk and incubating at 25 °C for 24 h. Afterwards, kefir grains were removed, and the resulting kefir starter was used for the second step of fermentation. Fermentation was carried out in sterile 700 mL glass jars to simulate typical home-scale fermentation conditions. Each jar was filled with 200 mL of UHT cow’s milk and inoculated with 10% (v/v) of kefir starter. The second step of the fermentation process was conducted under static conditions at 23 °C for 14 h in a temperature-controlled incubator. All jars and equipment were sterilized prior to use by autoclaving at 121 °C for 15 min to prevent external microbial contamination. Fermentation was carried out in sterile 700 mL glass jar to simulate typical home-scale fermentation conditions. Each jar was filled with 200 mL of UHT cow’s milk and inoculated with 10% (v/v) of kefir starter. The second step of the fermentation process was conducted under static conditions at 23 °C for 14 h in a temperature-controlled incubator. All jars and equipment were sterilized prior to the use by autoclaving at 121 °C for 15 min to prevent external microbial contamination. Following fermentation, samples K3 and K4 were strained into new sterile containers using a sterile sieve to remove the pomace whereas, in samples K1 and K2, the pomace remained throughout maturation. Maturation was carried out at 4 °C ± 1 °C for 14 days. The following sample labels were used throughout the study: K1 (10% raspberry pomace, retained during maturation), K2 (20% pomace, retained during maturation), K3 (10% pomace, removed after fermentation), and K4 (20% pomace, removed after fermentation). A kefir sample prepared without the addition of raspberry pomace was used as the control. Each experiment was performed in triplicate.

2.2. Physicochemical Characterization

Kefir samples were analyzed for pH and titratable acidity. The pH of kefir samples was measured using a HANNA HI 9318 pH meter (Leighton Buzzard, UK). The titratable acidity of kefir samples was determined using an AOAC titration method [17] and expressed in °SH degrees.

2.3. Microbiological Analysis

The enumeration of microorganisms was performed by the method of serial dilutions, aseptically transferring 1 mL kefir sample into 9 mL of sterile physiological saline solution (0.9 g NaCl/L) and then plating the samples on De Man, Rogosa, and Sharpe (MRS) (Torlak, Belgrade, Serbia) medium for enumeration of lactic acid bacteria (LAB), and on Sabouraud Maltose agar (SMA) (Torlak, Belgrade, Serbia) for yeast counting. The enumeration of LAB and yeast was performed after 2 days of incubation at 37 °C and 3 days of incubation at 27 °C, respectively. Viable count results were expressed as log averages of colony-forming units per mL of kefir (log CFU/mL).

2.4. Determination of Antioxidant Capacity and Polyphenol Content of Kefir Samples

2.4.1. Extract Preparation

Kefir extracts were prepared according to the method described by Yilmaz-Ersan, et al., 2018 [18]. Two grams of kefir samples were mixed with 20 mL of an extraction solution (methanol/water, 70:30 v/v) and stirred on a magnetic stirrer (SCILOGEX SCI280-Pro, Rocky Hill, CT, USA) at 20 °C in a dark place for 4 h. The mixture was then centrifuged for 10 min at 7000 rpm, and the supernatant was filtered through filter paper. The filtrate was used to determine the antioxidant activity and total polyphenol content.

2.4.2. Antioxidant Activity

The antioxidant activity of the extracts was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, based on the method described by Yilmaz-Ersan et al. [18], with slight modifications. Two milliliters of the extract were mixed with 8 mL of 0.1 mM DPPH reagent (Sigma-Aldrich, Sternheim, Germany) (0.1 mM), incubated for 30 min in the dark, and the absorbance was measured at 517 nm (AS) using a 2100 UV spectrophotometer (Cole-Parmer, Vernon Hills, IL, USA). The control sample (AC) contained 8 mL of DPPH and 2 mL of methanol, while methanol was used as a blank. The degree of reduction of free radicals was calculated according to Equation (1):

DPPH radical reducing ability (%) = (AC − AS)/AC × 100

(1)

The experiment was performed in triplicate for each sample individually, and the results are presented as the average value ± standard deviation.

2.4.3. Total Phenolic Content

The total phenolic content (TPC) was measured by applying the Folin–Ciocalteu method described previously by Malićanin and coworkers [19]. Briefly, 0.5 mL of extract was mixed with 4.5 mL distilled water and 0.5 mL Folin–Ciocalteu reagent and then left in the dark for 5 min at room temperature. After adding 5 mL of 7.5% sodium carbonate, the mixture was incubated for 90 min under the same conditions. Absorbance was measured at 765 nm using a blank made of distilled water, Folin–Ciocalteu reagent, and sodium carbonate. A calibration curve was created using gallic acid (30–300 μg/mL), with results expressed as mg gallic acid equivalents per mL of kefir.

2.5. HPLC Analysis

Separation and quantification of sugars and organic acids were carried out using high-performance liquid chromatography (HPLC) with an Agilent 1100 Series system (Waldbron, Germany) according to a previously described method [20] with slight modifications. Briefly, five grams of each sample were transferred to labeled centrifuge tubes, and 25 mL of 0.01 N H₂SO₄ was added. The mixture was vortexed for 1 min and then stirred at 240 rpm for 30 min. The tubes were cooled and centrifuged at 7000× g for 7 min. The supernatant was filtered through 0.45 µm filters into HPLC vials for analysis. Detection of the compounds was performed on an Aminex HPX-87H column (7.8 × 300 mm, Bio rad Laboratories, Hercules, CA, USA) with 5 mM H₂SO₄ used as isocratic eluent with the following operating conditions: injection volume 20 μL, temperature 50 °C, and eluent flow of 0.6 mL/min. The concentration of each compound was calculated according to the external standard and expressed as g/L [21]. The limit of detection (LOD) and limit of quantification (LOQ) for quantified compounds were in the range of 0.002–0.25 g/L and 0.008–0.75 g/L, respectively.

2.6. Sensory Analysis

The sensory acceptability of kefir samples was assessed by 35 untrained panelists during the product’s maturation. All participants were volunteers, and the evaluation was conducted in accordance with ISO standard guidelines [22]. They evaluated the samples in duplicate on descriptors that were developed before evaluation, during a 1 h long training. Fifty milliliters of each sample were served in opaque cups in random order, and panelists recorded their observations using a 9-point hedonic scale (1 = dislike extremely, 5 = neither like nor dislike, and 9 = like extremely) [22].

2.7. Statistical Analysis

All the experiments were carried out in triplicate and presented as the average value ± standard deviation. One-way analysis of variance (ANOVA), followed by Tukey’s test, was used to determine whether there were statistically significant differences between the analyzed parameters across different samples at a significance level of 5% (p < 0.05). Additionally, the effect of storage duration was assessed using one-way repeated measures ANOVA. All analyses were performed using PASW Statistics 18 (Version 18.0, Chicago: SPSS Inc., Chicago, IL, USA; IBM Corp., Armont, NY, USA).

3. Results

3.1. Physicochemical Parameters

Titratable acidity and pH are fundamental indicators of kefir’s fermentation dynamics and maturation, as they directly influence its microbial stability, texture, and sensory profile. The pH values of all kefir samples decreased, while the titratable acidity (°SH) of all kefir samples increased over the 14-day storage period, in line with the observed pH decline (

Table 1. pH and titratable acidity in kefir samples during storage.

			Storage Duration, Days
Analysis	Sample	Inoculation	0	3	6	10	14
pH	Control	6.09 d ± 0.01	4.92 c ± 0.02	4.86 e ± 0.01	4.79 d ± 0.01	4.66 d ± 0.01	4.54 e ± 0.01
	K1	5.72 c ± 0.00	4.46 b ± 0.02	4.30 b ± 0.01	4.21 b ± 0.01	4.19 c ± 0.01	4.09 b ± 0.01
	K2	5.48 b ± 0.00	4.42 a ± 0.01	4.21 a ± 0.02	4.16 a ± 0.01	4.05 a ± 0.01	3.99 a ± 0.01
	K3	5.72 c ± 0.02	4.43 a ± 0.01	4.36 d ± 0.01	4.24 c ± 0.01	4.21 c ± 0.02	4.13 d ± 0.01
	K4	5.44 a ± 0.02	4.44 ab ± 0.01	4.33 c ± 0.02	4.23 c ± 0.01	4.16 b ± 0.01	4.11 c ± 0.01
Titratable acidity °SH	Control	6.0 a ± 0.030	20.0 c ± 0.045	17.0 b ± 0.035	15.8 a ± 0.020	16.0 a ± 0.040	15.0 a ± 0.025
	K1	9.2 b ± 0.030	13.2 a ± 0.025	20.0 d ± 0.050	20.8 d ± 0.035	21.0 c ± 0.020	21.8 c ± 0.045
	K2	12.0 d ± 0.050	15.2 b ± 0.030	16.0 a ± 0.024	24.6 e ± 0.045	25.0 d ± 0.035	27.8 e ± 0.050
	K3	9.4 c ± 0.028	13.2 a ± 0.020	19.4 c ± 0.040	17.0 b ± 0.030	19.6 b ± 0.045	20.6 b ± 0.025
	K4	12.0 d ± 0.020	20.4 d ± 0.035	24.0 e ± 0.050	20.6 c ± 0.030	26.0 e ± 0.045	25.4 d ± 0.025

Different letters (a–e) indicate statistically significant differences in the same column between results for the same parameter (p < 0.05). K1—kefir with 10% raspberry pomace, K2—kefir with 20% raspberry pomace, K3—kefir with 10% raspberry pomace strained after fermentation, K4—kefir with 20% raspberry pomace strained after fermentation.

). At inoculation, the pH ranged from 5.05 to 6.08, with a significant drop observed in all samples immediately after fermentation. The lowest pH values were recorded in samples containing 20% raspberry pomace. Throughout the storage period, a gradual decrease in pH was observed, with the most pronounced reduction in K2, reaching 3.99 by the last day of storage. In contrast, the control sample exhibited the highest final pH, indicating the least acidification during storage. Among the pomace-containing samples, those in which the pomace remained in the beverage throughout storage showed a more significant pH decline compared to those where the pomace was removed before storage. Repeated measures ANOVA revealed a significant effect of storage duration [F (5, 95) = 2410.10; p < 0.001 and F (5, 95) = 61.85] on pH and titratable acidity, respectively.

3.2. Antioxidative Activity and Polyphenol Content in Kefir Samples

Fermented dairy products, including kefir, are known for their significant antioxidant activity, which is influenced not only by the type of milk and microbial culture used but also by the maturation period and the presence of added ingredients. Therefore, this study aimed to evaluate the impact of raspberry pomace addition and maturation time on the antioxidant activity and polyphenol concentration in kefir samples.

The findings of this study demonstrate that the addition of raspberry pomace significantly influenced antioxidant activity (Figure 1). All kefir samples exhibited a higher degree of DPPH radical reduction compared to the control, ranging from 15.5 ± 1.13% to 95.91 ± 0.78%, depending on the phase of the maturation period. The control sample consistently showed the lowest DPPH reduction at all time points, indicating that raspberry pomace enhances antioxidant activity, with higher concentrations leading to greater effects.

An increase in antioxidant activity was observed throughout the storage period following fermentation. The most significant increase occurred between day 0 and day 10, followed by a slight decline. The highest DPPH radical reduction was recorded in samples K2 and K4, peaking at day 10 (95.91 ± 0.78%, % and 88.69 ± 0.10%, respectively). The decline in DPPH reduction on day 14 suggests a possible degradation of antioxidant compounds over extended storage, indicating that the optimal storage period for maintaining antioxidant activity is 10 days.

Similarly, the addition of raspberry pomace significantly increased the polyphenol concentration in kefir samples (Figure 2). A rise in polyphenol levels was observed in all kefir samples except for the control. In line with the antioxidant activity results, the highest polyphenol concentration was recorded on the tenth day of storage. Sample K2 reached a maximum polyphenol concentration of 78.24 ± 3.29 mg/L of gallic acid, which is nine times higher than that of the control.

The results further indicate that the retention of raspberry pomace in kefir samples during maturation had a significant impact on both antioxidant activity and polyphenol content. Samples K1 and K2, where the pomace remained throughout the maturation period consistently exhibited higher DPPH reduction values and polyphenol content compared to K3 and K4, where the pomace was removed after fermentation. This effect was particularly pronounced in the case of polyphenols. Storage duration had a statistically significant effect on antioxidant activity and total polyphenol content [F(4, 76) = 137.08; F(4, 76) = 32.84; all p < 0.001].

3.3. Enumeration of Viable Bacteria

Figure 3 shows the changes in the microorganism populations during cold storage of the kefir. The LAB counts in all kefir samples increased significantly after fermentation (8.28 ± 0.01–8.59 ± 0.01 log CFU/mL), peaking between days 3 and 6, followed by a gradual decline throughout further storage. In particular, LAB levels remained high by the sixth day of storage for all samples except for the control, which showed a more pronounced decline compared to pomace-containing kefirs. The reduction in LAB counts became more evident after day 10, with values ranging from 7.34 ± 0.03 to 8.35 ± 0.08 log CFU/mL. By the fourteenth day of storage, further decreases were recorded, with K2 retaining the highest count (7.48 ± 0.03 log CFU/mL) and the control showing the lowest LAB levels (6.95 ± 0.06 log CFU/mL).

The yeast population followed a similar pattern. At inoculation, yeast counts were similar across all samples, ranging from 4.24 to 4.26 log CFU/mL, indicating no initial differences in yeast population. After fermentation, a significant increase in yeast counts was observed in all samples, reaching values between 6.29 ± 0.07 and 6.55 ± 0.01 log CFU/mL. During storage, yeast populations exhibited fluctuations across the samples. By the sixth day of storage, samples with retained raspberry pomace (K2 and K1) exhibited greater stability compared to the other samples that showed minor reductions in yeast counts. A general decline was observed from day 10 onward, with the lowest final yeast populations recorded in the control (5.67 ± 0.06 log CFU/mL) and K4 (5.64 ± 0.01 log CFU/mL), while K1 and K2 retained the highest yeast counts.

3.4. HPLC Analysis

The sugar and organic acid composition of kefir is a key indicator of microbial activity and fermentation efficiency, directly influencing sensory properties, nutritional value, and consumer acceptability. This study aimed to investigate how the addition of raspberry pomace and its presence during cold storage influences the sugar and organic acids composition of kefir, addressing a gap in the current knowledge on functional ingredient interactions in fermented dairy products. The results of the HPLC analysis are given in

Table 2. HPLC analysis of sugar and organic acid content (g/L) in kefir samples enriched with raspberry pomace during cold storage.

Compound	Day	Control	K1	K2	K3	K4
Lactose	Inoculation	50.24 a ± 2.04	49.9 a ± 1.64	48.19 a ± 1.88	49.79 a ± 1.71	49.23 a ± 1.96
	0	41.33 a ± 1.79	40.87 a ± 1.47	40.19 a ± 1.63	40.79 a ± 1.39	40.23 a ± 1.55
	6	20.55 a ± 1.63	20.63 a ± 1.71	21.03 a ± 1.55	19.33 a ± 1.95	20.13 a ± 1.80
	10	13.88 a ± 1.28	12.92 a ± 1.04	14.11 a ± 2.12	11.99 a ± 1.63	14.55 a ± 1.29
	14	10.11 a ± 1.55	9.15 a ± 1.30	9.88 a ± 1.79	10.35 a ± 1.46	11.23 a ± 1.63
Glucose	Inoculation	10.22 a ± 0.73	15.36 bc ± 0.69	16.52 c ± 0.74	14.01 b ± 0.65	15.49 bc ± 0.69
	0	15.16 a ± 0.65	17.27 b ± 0.73	18.59 b ± 0.66	16.33 ab ± 0.69	15.55 a ± 0.65
	6	10.18 a ± 0.77	12.40 b ± 0.65	15.64 c ± 0.77	12.31 b ± 0.73	12.30 b ± 0.73
	10	10.25 a ± 0.69	12.38 bc ± 0.74	13.61 c ± 0.69	11.36 ab ± 0.65	12.27 bc ± 0.69
	14	11.22 ab ± 0.65	13.46 c ± 0.79	12.60 bc ± 0.73	10.33 a ± 0.69	11.37 ab ± 0.65
Fructose	Inoculation	0.09 a ± 0.014	0.36 b ± 0.032	0.50 c ± 0.041	0.38 b ± 0.031	0.47 c ± 0.028
	0	0.13 a ± 0.009	0.58 c ± 0.021	0.63 d ± 0.033	0.47 b ± 0.026	0.53 bc ± 0.037
	6	0.14 a ± 0.008	0.74 c ± 0.040	0.77 c ± 0.038	0.51 b ± 0.019	0.56 b ± 0.009
	10	0.16 a ± 0.009	0.85 d ± 0.033	0.94 e ± 0.051	0.52 b ± 0.017	0.58 c ± 0.023
	14	0.17 a ± 0.006	0.95 c ± 0.028	1.02 d ± 0.042	0.55 b ± 0.025	0.60 b ± 0.025
Etanol	Inoculation	nd	nd	nd	nd	nd
	0	1.27 b ± 0.02	1.67 d ± 0.02	1.91 e ± 0.03	1.00 a ± 0.02	1.47 c ± 0.03
	6	1.10 a ± 0.02	1.81 c ± 0.03	2.14 d ± 0.02	1.15 a ± 0.01	1.70 b ± 0.02
	10	1.21 a ± 0.03	1.88 c ± 0.02	2.43 d ± 0.04	1.61 b ± 0.02	1.84 c ± 0.03
	14	1.62 a ± 0.02	1.99 d ± 0.02	2.45 e ± 0.03	1.70 b ± 0.02	1.83 c ± 0.02
Citric acid	Inoculation	nd	nd	nd	nd	nd
	0	0.19 a ± 0.016	0.33 b ± 0.024	0.69 e ± 0.032	0.43 c ± 0.012	0.61 d ± 0.020
	6	0.19 a ± 0.017	0.35 b ± 0.021	0.62 d ± 0.020	0.54 c ± 0.016	0.60 d ± 0.024
	10	0.21 a ± 0.012	1.28 e ± 0.032	1.00 d ± 0.024	0.59 b ± 0.017	0.66 c ± 0.021
	14	0.33 a ± 0.016	1.42 d ± 0.028	1.40 d ± 0.021	0.55 b ± 0.013	0.77 c ± 0.019
Malic acid	Inoculation	nd	nd	nd	nd	nd
	0	0.01 a ± 0.002	0.01 ac ± 0.004	0.03 c ± 0.001	0.02 b ± 0.008	0.03 c ± 0.002
	6	0.02 a ± 0.002	0.03 b ± 0.001	0.08 d ± 0.003	0.03 b ± 0.001	0.04 c ± 0.002
	10	0.01 a ± 0.001	0.04 c ± 0.001	0.07 d ± 0.002	0.03 b ± 0.002	0.03 b ± 0.001
	14	0.01 a ± 0.001	0.05 d ± 0.002	0.06 e ± 0.002	0.03 b ± 0.001	0.04 c ± 0.003
Lactic acid	Inoculation	1.11 c ± 0.05	0.92 b ± 0.02	1.24 d ± 0.04	0.84 b ± 0.03	0.74 a ± 0.02
	0	7.18 a ± 0.12	7.09 a ± 0.09	8.71 b ± 0.16	7.00 a ± 0.08	7.06 a ± 0.08
	6	7.39 a ± 0.14	7.46 a ± 0.13	8.94 c ± 0.17	7.78 b ± 0.11	7.66 ab ± 0.11
	10	7.38 a ± 0.13	7.68 b ± 0.12	8.85 c ± 0.17	7.55 ab ± 0.09	7.53 ab ± 0.08
	14	7.15 a ± 0.13	8.07 c ± 0.15	8.82 d ± 0.18	7.99 bc ± 0.14	7.70 b ± 0.14
Acetic acid	Inoculation	nd	nd	nd	nd	nd
	0	0.77 a ± 0.012	0.81 b ± 0.016	0.82 bc ± 0.014	0.85 c ± 0.012	0.88 c ± 0.020
	6	0.76 a ± 0.011	0.90 b ± 0.014	0.94 c ± 0.016	0.89 b ± 0.016	0.91 bc ± 0.016
	10	0.80 a ± 0.011	1.14 e ± 0.024	1.10 d ± 0.017	0.95 b ± 0.014	0.99 c ± 0.016
	14	0.99 a ± 0.016	1.15 c ± 0.018	1.27 d ± 0.014	1.04 b ± 0.016	1.01 ab ± 0.012

Different letters indicate statistically significant differences in the same row between results for the same parameter (p < 0.05). K1—kefir with 10% raspberry pomace, K2—kefir with 20% raspberry pomace, K3—kefir with 10% raspberry pomace strained after fermentation, K4—kefir with 20% raspberry pomace strained after fermentation.

.

Repeated measures ANOVA revealed a significant effect of storage duration on the concentrations of all analyzed compounds. For carbohydrates, the effect was significant for lactose, glucose, and fructose [F(4, 76) = 10675.11; F(4, 76) = 99.82; F(4, 76) = 32.65; all p < 0.001]. Similarly, storage time significantly influenced the levels of ethanol, citric, malic, and acetic acid [F(3, 57) = 56.61; F(3, 57) = 18.77; F(3, 57) = 18.76; F(4, 76) = 2461.99; F(3, 57) = 70.44; all p < 0.001].

In particular, the lactose concentration in all kefir samples decreased significantly during fermentation and cold storage. Initial lactose content before fermentation ranged from 48.19 to 50.24 g/L, with no statistically significant differences between treatments. By the end of the storage period, lactose content had decreased in all samples, reaching the lowest levels in K1 (9.15 ± 1.30 g/L) and K2 (9.88 ± 1.79 g/L). However, there were no statistically significant differences among samples throughout the storage period (p > 0.05), indicating that the addition and removal of raspberry pomace, regardless of concentration, did not affect the rate of lactose degradation.

On the other hand, glucose content showed notable variations between samples and over time. Samples with added raspberry pomace generally maintained higher glucose concentrations than the control at all time points, particularly K2, which consistently showed the highest levels. Over the cold storage, glucose levels declined in all samples, reflecting microbial utilization during the maturation period. Although the presence of raspberry pomace contributed to initially higher glucose levels, samples in which the pomace was removed after fermentation (K3 and K4) showed slightly reduced glucose concentrations compared to K1 and K2 during storage.

As expected, fructose concentrations were initially low in the control sample, while raspberry pomace-enriched samples exhibited significantly higher levels at inoculation. Following fermentation, the fructose content continued to rise in all treated samples, especially in K2 and K1 (0.63 ± 0.033 g/L and 0.58 ± 0.021 g/L, respectively), indicating a pomace-related contribution. This trend continued throughout the storage period, with an observed steady increase in all samples. These findings suggest that raspberry pomace serves as a source of fermentable and slowly hydrolyzing fructose-containing compounds [23].

Throughout storage, ethanol levels increased, reaching a maximum of 2.45 ± 0.03 g/L in K2. Samples where pomace remained in the beverage consistently showed significantly higher ethanol concentrations than the control and treatments where pomace was removed, suggesting that raspberry pomace supports ongoing fermentative activity, which is also confirmed by microbial analysis. The control sample exhibited a slower increase, reaching 1.62 ± 0.02 g/L till the end of the storage.

Lactic acid levels increased significantly across all treatments, reaching 8.71 ± 0.16 g/L in K2 and 7.18 ± 0.12 g/L in the control after fermentation. During storage, lactic acid levels remained elevated, with K2 consistently showing significantly higher concentrations than the other samples, peaking at 8.94 ± 0.17 g/L on day 6 and ending at 8.82 ± 0.18 g/L on day 14 (p < 0.05). The consistently higher lactic acid levels in K1 and K2 are a consequence of enhanced LAB activity in the presence of raspberry pomace.

Citric acid followed a similar pattern. It was undetectable at inoculation but present after fermentation, with concentrations ranging from 0.19 ± 0.016 g/L in the control to 0.69 ± 0.032 g/L in K2. By the end of the storage period, levels rose significantly, reaching 1.42 ± 0.028 g/L in K1 and 1.40 ± 0.021 g/L in K2, which were significantly higher than those in K3 (0.55 ± 0.013 g/L) and the control (0.33 ± 0.016 g/L) (p < 0.05). Malic acid also increased over time, although at much lower concentrations. Similarly, acetic acid appeared in all samples post-fermentation and increased throughout storage. Immediately after fermentation, the lowest value was recorded in the control (0.77 ± 0.012 g/L) and the highest in K4 (0.88 ± 0.020 g/L). By the end of the storage period, K2 exhibited the highest acetic acid concentration at 1.27 ± 0.014 g/L, which was significantly higher than the control (0.99 ± 0.016 g/L) (p < 0.05). These findings suggest that raspberry pomace contributes as a source of citric, malic, and acetic acid.

3.5. Sensory Analysis

From the consumer’s perspective, kefir and similarly fermented dairy products should have a homogeneous texture, a mildly acidic taste, and a pleasant, well-balanced flavor [24]. Since many consumers are not accustomed to the distinct sensory characteristics of kefir, incorporating fruit or fruit-based ingredients presents a promising approach to enhancing its sensory appeal and making it more acceptable to the average consumer. Hence, the sensory properties of kefir samples were evaluated over 14 days of refrigerated storage to assess changes in smell, color, texture, taste, acidity, and overall acceptability (Figure 4). Sensory analysis demonstrated that kefir samples with raspberry pomace generally exhibited higher scores for smell and color compared to the control, while texture and taste varied depending on the formulation. Initially, all samples were well-accepted, with K4 receiving the highest overall rating due to its balanced acidity and favorable texture. However, a notable decline in sensory scores across all samples occurred after 10 days of storage, particularly in acidity and overall acceptability. Over time, acidity and taste scores declined, with the most pronounced reduction observed in K1 and K2, where acidity scores decreased significantly. Texture scores remained stable in K3 and K4 but decreased in K1 and K2, likely due to microbial activity. Smell and color scores remained relatively high in pomace-enriched samples, suggesting that the added fruit component contributed to aromatic compounds influencing those attributes.

Despite having the highest antioxidative and polyphenol content, K1 and K2 did not achieve the best sensory acceptance, particularly in terms of acidity and texture. This is likely due to the presence of unstrained raspberry pomace particles, which influenced mouthfeel and overall perception of the product. Overall acceptability remained high during the first 10 days but declined in later stages, particularly in samples where acidity fluctuations were more pronounced. These findings indicate that, while raspberry pomace positively influenced certain sensory characteristics in the early storage period, it contributed to acidity variations and texture changes over time, ultimately affecting long-term consumer acceptance. Compared to the control, K3 and K4 maintained better texture and color scores throughout storage, likely due to their more balanced formulation. K4, in particular, exhibited the highest overall acceptability, suggesting that its composition provided the best balance between sensory attributes and stability. While the control sample showed stable texture, it had lower scores for color and smell than K3 and K4, highlighting the potential of raspberry pomace to enhance sensory characteristics when properly integrated into kefir formulations.

4. Discussion

This study investigated the influence of raspberry pomace, added in varying amounts and treated differently during processing, on the chemical composition, microbial viability, and sensory attributes of kefir during cold storage.

Among the parameters evaluated, acidity and pH emerged as key indicators of fermentation progress and product stability. Acidity is a critical parameter influencing the quality and organoleptic properties of fermented dairy products, while pH serves as an indicator of microbial activity and determines the fermentation phase. A low pH can inhibit the growth of microorganisms in kefir grains and alter the sensory characteristics of the final product [25]. It is expected for the acidity to increase during fermentation due to the production of organic acids, reaching a certain threshold that primarily depends on the acidification capacity of the microbial culture involved [26]. The optimal pH range for kefir is typically between 4.2 and 4.6 [27,28], which is consistent with our findings. The reduction in pH and a corresponding increase in titratable acidity during fermentation is attributed to the accumulation of metabolites such as lactic, acetic, malic, and propionic acids produced by LAB [29]. Aroua et al. (2023) also reported increased acidity values in donkey’s and cow’s milk kefir during 28 days of storage [30]. In this study, the lowest pH value and highest titratable acidity were observed in the sample containing 20% raspberry pomace that remained during cold storage (sample K2), suggesting an additional acidifying effect from the organic acids present in the pomace. A key finding is the difference in acidification trends between samples where pomace was retained and those where it was removed before storage. Samples where the pomace was retained during cold storage exhibited consistently lower pH values and higher acidity, suggesting that the prolonged presence of pomace facilitated further microbial activity, potentially through the sustained release of fermentable substrates and bioactive compounds. From a technological perspective, the findings suggest that incorporating raspberry pomace in kefir formulations can enhance acidification, potentially extending shelf life by suppressing the growth of spoilage microorganisms [31]. However, the increased acidity may also impact sensory characteristics, implying the need for optimization of pomace concentration and maturation duration to balance functional benefits with consumer acceptance.

The results of this study confirm that the addition of raspberry pomace significantly enhances the antioxidant activity and polyphenol content of kefir. This effect is attributed to both the continuous extraction of bioactive compounds from the pomace and the metabolic activity of fermentative microorganisms [29,32]. The sustained presence of raspberry pomace during storage led to a more pronounced increase in antioxidant activity, suggesting a gradual release of polyphenolic compounds over time. These findings are consistent with previous research indicating that the incorporation of various fruit-based ingredients, such as raspberries [12], strawberries [12,33], chokeberries [34], pomegranates [6], black mulberries [6], or coffee [9] enhances the antioxidant properties of fermented dairy products. Moreover, earlier studies have shown that product storage and maturation positively influence antioxidant activity [30,35], further supporting the trends observed in this study.

The observed increase in antioxidant activity over the storage period can be attributed to two primary mechanisms: the metabolic activity of kefir microbiota and the gradual diffusion of bioactive compounds from the raspberry pomace. During fermentation and maturation, lactic acid bacteria contribute to proteolysis, leading to the release of bioactive peptides with antioxidant properties. Additionally, the production of organic acids enhances the extraction of polyphenols from the pomace, further improving antioxidant capacity [35]. The highest DPPH radical reduction was recorded on day 10, after which a slight decline was observed, likely due to the degradation of certain antioxidant compounds over prolonged storage.

The increase in polyphenol concentration over time is likely influenced by the acidic environment of kefir. The low pH values may disrupt the bonds between polyphenols and proteins, facilitating the release of free polyphenolic compounds into the beverage [26]. This phenomenon has been previously reported in fermented dairy products, where enzymatic activity, particularly from β-glucosidase, enhances the hydrolysis of complex phenolic structures into simpler, more bioavailable forms [36,37]. Baniasadi and coworkers reported similar values of polyphenol concentrations in kefir from cow and goat milk, which ranged from 65 to 73.15 mg GAE/100 mL and 158.31 to 73.5 mg GAE/100 mL, respectively [35].

The microbial dynamics observed in this study are consistent with previous findings on kefir fermentation and storage, reporting initial LAB counts between 7.30 and 8.96 log CFU/mL [4,9,30,38,39,40]. In our study, LAB and yeast populations increased during fermentation and storage, peaking between days 3 and 6, followed by a gradual decline during further storage. A previous study also reported the highest LAB count of 10⁸ CFU/mL followed by a gradual decrease of approximately 1.5 log units between days 7 and 14 due to environmental stressors, such as acidification, nutrient depletion, and storage temperature effects [4]. Despite the decline in microbial counts over time, all kefir formulations maintained viable LAB and yeast populations above the probiotic threshold (7 log CFU/mL for LAB, 4 log CFU/mL for yeasts) throughout the 14-day storage period [9]. Notably, the addition of raspberry pomace appeared to enhance microbial retention, particularly in samples K1 and K2, which exhibited higher LAB and yeast counts compared to the control. Similar protective effects of fruit-based substrates on kefir microbiota have been observed in kefir fortified with anthocyanin-rich juices [6]. When it comes to yeast population, previous studies have reported a wide range of yeast counts in kefir (4.04 and 7.61 log CFU/mL) highlighting the influence of formulation and storage conditions on microbial dynamics [4,6], which also aligns with our findings. The decline in yeast viability during storage may be attributed to acid accumulation and cell proteolysis due to low pH, competition for nutrients, and reduced fermentative activity under cold storage conditions [30,38]. However, the higher yeast retention in K1 and K2 suggests a potential protective effect of pomace.

Results of this study have shown how the addition of raspberry pomace not only contributes to bioactive compounds but also influences carbohydrate metabolism during fermentation and cold storage. The observed reduction in lactose concentration aligns with previous studies reporting the rapid degradation of lactose during fermentation by LAB and yeasts [20,41]. The enzymatic hydrolysis of lactose into glucose and galactose by β-galactosidase initiates carbohydrate catabolism, resulting in the production of lactic acid via glycolysis. Our findings are consistent with those of Da Costa et al. who reported a time-dependent decrease in lactose during fermentation, especially in samples supplemented with fermentable fiber sources [42]. In contrast to Leite and coworkers, who observed no significant changes in lactose content during short-term storage, our study detected continued lactose degradation up to day 14, although at a slower rate, which could be attributed to residual enzymatic activity or prolonged microbial viability during cold storage.

The glucose content in raspberry pomace-enriched kefir samples was higher than in traditional and goat milk kefir reported in previous studies, aligning more closely with values found in lactose-free or fruit-fortified fermented dairy products [20,42,43]. Glucose levels were significantly higher in raspberry pomace-enriched samples compared to the control at all time points. This agrees with a previous study, which noted that glucose levels may rise during early fermentation due to lactose hydrolysis before being consumed by microorganisms [41]. Raspberry pomace likely contributes additional glucose through the hydrolysis of fruit-derived oligosaccharides, and its retention in the product (samples K1 and K2) enhances this effect.

Fructose levels also increased over time, with the highest values recorded in K2 and K1. These results differ from previous findings, where fructose levels remained relatively constant during storage [6]. The consistent accumulation of fructose in raspberry pomace-containing samples suggests that this by-product may serve as a source of fermentable sugars. Similar conclusions were drawn in recent work by Zielińska and coworkers, who found that micronized raspberry pomace releases simple sugars during storage, including fructose [44]. This fructose may originate from the enzymatic breakdown of complex carbohydrates.

The results of our study confirm that lactic acid is the predominant organic acid in kefir, with concentrations increasing sharply after fermentation and continuing to rise moderately during storage, which is in agreement with a previous study on Brazilian kefir, which observed a rapid rise in lactic acid to 7.38 g/L after 24 h of fermentation and a further increase to 9.54 g/L after 28 days of storage [20]. In our study, the highest lactic acid levels were recorded in K2 (8.94 ± 0.17 g/L on day 6), which remained significantly higher than in other treatments throughout storage (p < 0.05). Similar concentrations were reported by Gul et al. in kefirs made from cow and buffalo milk, with final values around 9.2 g/L [38]. The elevated levels in K2 and K1 suggest that raspberry pomace may enhance microbial activity, particularly LAB, likely due to its complex content of fermentable fibers, phenolics, and micronutrients, as supported by Naibaho and coworkers in a study on yogurt fortified with brewers’ spent grain [45].

Acetic acid, the second most abundant acid detected, showed a gradual increase in all samples during storage, with the highest concentration again observed in K2, in levels that are comparable to those reported by Leite and coworkers [20], who found acetic acid concentrations of up to 1.16 g/L during storage and slightly above values in Norwegian milk kefir (~0.8 g/kg) [46]. The production of acetic acid in kefir is primarily linked to heterofermentative LAB and yeast metabolism, particularly under conditions favoring mixed-acid fermentation [25,42].

Citric and malic acids, though present at lower concentrations, contributed to the organic acid profile of kefir and were clearly influenced by the presence of raspberry pomace. Citric acid is a key intermediate in microbial metabolism and plays an important role in flavor development, acting as a precursor for compounds like acetoin and diacetyl [42]. The literature shows that its concentration in kefir can vary depending on milk type and processing method [38] and that it tends to remain relatively stable during storage [46], which is in accordance with our study. The elevated citric acid levels in pomace-retained samples suggest that raspberry pomace supplies additional citric acid by supporting microbial pathways that enhance its accumulation. Malic acid, while detected in much smaller amounts, also appeared more prominently in pomace-enriched samples. Although limited research has focused on malic acid in kefir, the concentrations obtained in our study are much lower than those published on goat and cow milk, which reported malic acid concentrations of 0.145 g/L in cow milk kefir and a significantly higher level of 3.082 g/L in goat milk kefir, indicating that both the milk type and microbial composition can have a profound effect on organic acid metabolism [47].

Ethanol production increased significantly in all treatments after fermentation, with the highest levels consistently observed in K2 (2.45 ± 0.03 g/L at day 14), suggesting enhanced yeast activity in the presence of pomace. This aligns with previous findings that emphasized the role of yeast species, such as Kluyveromyces spp. and Saccharomyces spp., in ethanol production. Our ethanol values fall within the range reported for Brazilian kefir (0.14–1.36 g/L) [20], which also noted significant ethanol increases during storage. Ethanol, though present in low concentrations, contributes to kefir’s characteristic aroma and refreshing sensory properties and may also enhance its antimicrobial potential during storage [46].

The results of the sensory evaluation indicate that the addition of raspberry pomace influenced the sensory attributes of kefir samples, particularly in terms of smell, color, texture, and acidity. The observed decline in sensory acceptability over time is consistent with reports by Irigoyen and coworkers, who found that acceptability levels peaked during the early storage period and declined as flavor intensity and astringency increased [4]. Similarly, Aroua et al. demonstrated that sensory attributes of cow and donkey kefirs deteriorated over extended storage [30]. In this study, the acidity of kefir samples varied depending on the concentration of raspberry pomace. Samples K1 and K2, which contained the highest concentrations of pomace, exhibited greater acidity fluctuations over time, likely due to the accumulation of organic acids, including malic and acetic acid [26]. The increasing acidity, combined with microbial activity, contributed to a reduction in overall acceptability after 10 days of storage. The texture of kefir samples was also affected by the addition of raspberry pomace. While the presence of pomace improved color and smell scores, it negatively impacted the texture in K1 and K2 compared to other samples. This finding supports the results of Alirezalu and coworkers, who observed a decline in brightness, smoothness, and mouthfeel attributes in yogurts fortified with natural additives over time [48]. Compared to the control, K3 and K4 maintained better sensory stability throughout storage, particularly in terms of texture and overall acceptability. These samples exhibited more balanced acidity and texture, suggesting that a moderate concentration of raspberry pomace may enhance sensory appeal without introducing excessive acidity or undesirable textural changes. This is in contrast to findings by Kabakci and coworkers, who reported no significant changes in general acceptability for fruit juice-fortified kefirs up to 8 weeks of storage, suggesting that the impact of fruits addition may depend on their composition, particle size, and texture [6].

The findings of this study underscore the importance of optimizing both the concentration and processing methods of raspberry pomace to balance functional enhancement with sensory acceptability. While higher pomace concentrations (20%) and retention of pomace during storage significantly improved antioxidant activity, polyphenol content, and microbial viability, these samples also exhibited intensified acidity and textural changes that negatively affected sensory acceptance after 10 days. In contrast, strained samples with moderate pomace content (particularly K4) maintained favorable sensory scores while still offering notable functional benefits. These observations suggest that a 10% pomace concentration with removal post-fermentation may represent an optimal formulation for maintaining a desirable balance.

5. Conclusions

The incorporation of raspberry pomace into kefir significantly enhanced its functional, chemical, and microbiological properties during refrigerated storage. Pomace-enriched samples, particularly those retaining pomace during storage, exhibited higher levels of antioxidant activity (up to 95.91% DPPH reduction), polyphenols (up to 78.24 mg GAE/L), lactic acid (up to 8.94 g/L), and ethanol (up to 2.45 g/L), as well as improved microbial viability. Even though all samples recorded a reduction in the microbial count over time, all kefir formulations maintained viable LAB and yeast populations above the probiotic threshold. However, high pomace content and prolonged storage led to increased acidity and changes in texture, which negatively affected sensory acceptance and antioxidant activity after 10 days, indicating this period of time as an optimal storage duration. Among all formulations, strained samples with moderate pomace content achieved the best sensory scores, demonstrating that optimizing pomace concentration and processing methods are essential for balancing functional benefits and consumer preference. Overall, raspberry pomace shows strong potential for improving kefir’s nutritional value, offering a sustainable valorization for fruit-processing by-products. Future research should focus on optimizing pomace particle size and concentration to improve texture and minimize sensory drawbacks.