Effect of Dunaliella salina supplement on growth of probiotic bacteria and nutritional values of Kefir

Zina Alajil Alslibi; Abuzer Çelekli; Hüseyin Bozkurt

doi:10.1007/s42770-025-01800-z

. 2025 Oct 19;56(4):2473–2484. doi: 10.1007/s42770-025-01800-z

Effect of Dunaliella salina supplement on growth of probiotic bacteria and nutritional values of Kefir

Zina Alajil Alslibi ¹, Abuzer Çelekli ^2,^✉, Hüseyin Bozkurt ³

PMCID: PMC12660539 PMID: 41110030

Abstract

Probiotic-rich foods like kefir are increasingly recognized for their benefits to gut health and overall well-being. This study aimed to improve the functionality and nutritional value of kefir by supplementing it with Dunaliella salina, a microalga rich in bioactive compounds and minerals. The effects of D. salina at 0%, 0.25%, 0.5%, and 1.0% concentrations were evaluated on the growth of bacteria (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, Lactobacillus acidophilus, and Bifidobacterium animalis subsp. lactis) before and after fermentation over 22 days of storage. Viscosity decreased over time, and samples with D. salina showed significant reductions in Hunter color parameters (p < 0.05). Principal component analysis effectively differentiated the samples based on their biochemical, nutritional, and physical properties, explaining 99.47% of the total variation. Supplementation with 1% D. salina yielded the highest levels of total solids, proteins, minerals, and essential fatty acids (DHA and EPA). Moreover, 1% supplementation significantly increased the final viable counts of L. delbrueckii subsp. bulgaricus, S. thermophiles, and B. animalis subsp. lactis compared to the control (p < 0.05), while the increase in L. acidophilus was not statistically significant. Sensory evaluation revealed that kefir with 0.25% D. salina maintained consumer acceptability comparable to the control, whereas higher concentrations led to slight but acceptable declines in flavor and appearance. These results confirm that D. salina supplementation can enhance probiotic stability and nutritional quality while maintaining satisfactory sensory attributes, supporting its potential as a functional additive in fermented dairy products.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-025-01800-z.

Keywords: Microalgal, Supplementation functional food probiotic bacteria, Kefir, Lactic acid bacteria, Food supplement

Introduction

Fermented items have a higher nutritional value than non-fermented foods because fermentation improves digestion and naturally boosts important amino acids and vitamin levels. More recently, fermented drinks having live microbes have become a crucial dietary approach for enhancing human health [1]. Probiotic bacteria have a variety of characteristics and effects over storage time [2]. The most prevalent lactic acid bacteria (LAB) found in fermented items are responsible for the majority of microbial changes, and they are extremely important in industry.

Kefir is a traditional, fermented dairy product by kefir grains and valued for its potential probiotic properties. It’s possible health benefits are attributed to its complex microbiota [3, 4]. Kefir grain is a symbiotic community of LAB, acetic acid bacteria, and yeasts encased in a complex structure of proteins, lipids, and an exopolysaccharides called kefiran [5]. Kefiran is primarily generated by Lactobacillus kefiranofaciens, Lentilactobacillus (formerly Lactobacillus) kefiri, Lentilactobacillus (formerly Lactobacillus) parakefiri, and other species present in kefir grains through cooperative biosynthesis. LAB commonly found in kefir have long been consumed in fermented foods and are widely considered safe. Many of these strains have been designated as “Generally Recognized as Safe” by the U.S. Food and Drug Administration, and their inclusion in the European Food Safety Authority’s Qualified Presumption of Safety list [6, 7]. LAB are Gram-positive, acid-tolerant bacteria that include genera such as Lactobacillus, Lacticaseibacillus, Lactococcus, and Streptococcus. They primarily produce lactic acid as the main end-product of carbohydrate fermentation. In contrast, Bifidobacterium species, while also producing lactic acid, are not classified as LAB because they additionally generate acetic acid. Several Bifidobacterium taxa—such as Bifidobacterium animalis subsp. lactis—are recognized as containing strains with documented probiotic properties [8]. Members of this genus are important constituents of the human gut microbiota and have been associated with cholesterol reduction, improved lactose digestion, diarrhea mitigation, cancer prevention, immune modulation, and the establishment of a healthy gut microbiome [8]. The FAO/WHO defines probiotics as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [9]. Health effects must be demonstrated in well-designed clinical trials. While kefir contains numerous bacterial and yeast species with potential probiotic properties, few commercially available kefir products specify or contain clinically validated probiotic strains [8, 9]. Therefore, kefir is more accurately described as a fermented food with potential probiotic properties rather than as a probiotic product per se [3, 4].

Consuming foods produced by the fermentation process with the addition of algae, which have unique nutritional and functional properties, is associated with various health outcomes [10]. The type of algae and the microbes employed in the process both have an impact on the fermentation process. The LAB like L. delbrueckii subsp. bulgaricus, L. acidophilus, and S. salivarius subsp. thermophilus are particularly affected by the extracellular products formed by algae [11]. Research has indicated that the health-promoting properties of bioactive chemicals found in algae—such as antioxidants, polyunsaturated fatty acids, pigments, polysaccharides, and vitamins—are of particular importance due to their potential roles in reducing oxidative stress, modulating immune function, and supporting metabolic health [12]. Furthermore, increased bioavailability of essential nutrients may be the outcome of both fermentation and algae activities [12]. Consequently, it can have a beneficial impact on health.

Dunaliella salina is a halophilic green microalga in saline and marine environments. It is well-known for its antioxidant capacity, primarily due to its ability to produce high concentrations of carotenoids—especially β-carotene—which are widely used in dietary supplements and cosmetics [12, 13]. To survive under intense light and osmotic stress, D. salina synthesizes large amounts of β-carotene (provitamin A) and glycerol, adaptations that also contribute to its resilience and biotechnological potential [12, 13]. Dunaliella salina offers a unique carotenoid profile, particularly with higher β-carotene content, which can enhance the nutritional and functional qualities of fermented foods [12]. Moreover, its halophilic nature and capacity to accumulate bioactive compounds under stress conditions make it a promising candidate for improving both the nutritional value and shelf-life of probiotic products [12].

Although numerous studies have focused on the biosynthesis of β-carotene by D. salina, limited information is available on its potential to promote the growth and viability of probiotic strains. This study aims to explore these functional properties and use of D. salina as a functional additive in fermented dairy products. The main objective is that the addition of D. salina improves the survival of probiotic bacteria in fermented dairy products, both during fermentation and storage, by providing essential nutrients, including various fatty acids, amino acids, and specific vitamins [14]. To our knowledge, this study is the first to comprehensively assess the impact of D. salina supplementation on probiotic bacteria growth, nutritional composition, physicochemical properties, and sensory attributes in kefir.

Materials and methods

Experimental design

The procedure started with pasteurization of cow milk for 15 min at 90 °C. Then it was cooled down to 25 °C and kefir grains having mix culture of LAB [Lactobacillus (10.54 log CFU/mL), Lactococcus (10.62 log CFU/mL)], yeast (2.89 log CFU/mL) and Bidifobacterium (7.78 log CFU/mL) (Sevdanem Kefir Grain, 3 × 0.5 gram, Danem, Türkiye) were inoculated as 5% (w/v) according to instructions by the culture’s manufacturer. Then, different concentrations of D. salina (DS) were added into kefir samples as follows: (a) control without DS, (b) 0.25% DS, (c) 0.5% DS, and (d) 1% DS (D. salina, Greenheart store, Poland). The samples were prepared in three independent batches for each treatment.

Fermented was conducted at 25 °C for 18 to 24 h up to desired pH level was reached [15]. After fermentation, kefir samples were chilled and stored in the refrigerator at 4 °C for 22 days after fermentation. Viable counts of the bacterial strains used in fermentation, as well as biochemical variables, including titratable acidity, pH, and Hunter color values, were measured at multiple time points: during the fermentation, immediately after the fermentation, and through the storage time after the 1 st, 6th, 12th, 18th, and 22nd days. Each microbial and biochemical assay was performed in triplicate (n = 3 × 3 = 9 triplicate batches x triplicate analysis). Viscosity measurements were noted after 1 st, 6th, 12th, 18th, and 22nd days of storage, also in triplicate (n = 9). Protein, total solid were recorded at 1 st, 15th and 22nd, each with three replicates (n = 9). Minerals, as well as DHA and EPA levels were recorded after15th, and 22nd days of storage, with three replicates per assay (n = 9).

Microbiological analysis

Bacterial counts were expressed as colony-forming units per milliliter (CFU/mL). The growth of each bacterium was monitored in kefir samples inoculated into selective media described by Dave and Shah [16] and Tabasco et al. [17]. The effects of DS at 0.25%, 0.5%, and 1% on the growth and survival of the studied bacteria were evaluated on days 1, 6, 12, 18, and 22 of storage. Serial tenfold dilutions were prepared in 0.1% peptone water, and appropriate dilutions were plated on selective agar media using spread or pour plate techniques, followed by incubation under optimal conditions for each microorganism. All determinations were performed in triplicate.

The colonies of Lactobacillus acidophilus were counted using a selective media; MRS agar (Merck, Darmstadt, Germany) containing 1% maltose and inoculating an appropriate dilution [16]. Colonies were counted after three days of incubation at 37 °C [11]. By inoculating an appropriate dilution on M17 agar (Merck, Darmstadt, Germany) containing 1% lactose, the pour plate technique was used to count Streptococcus salivarius subsp. thermophilus. Following incubation of the Petri dishes at 45 °C for 24 h, the bacterial colonies were counted. Using the pour-plate method on MRS agar containing 1% fructose, Lactobacillus delbrueckii subsp. bulgaricus was counted after incubating for 72 h at 45 °C [16]. Bifidobacterium.animalis subsp. lactis were counted by using the pour technique at appropriate dilutions in the MRS agar. The colonies of bacteria were counted after three days of incubation of the petries at 45 °C [17]. The methods of selective enumeration and identification of cultures were done.

Chemicals and sensorial analyses

All samples’ pH levels were determined by use of a pH meter (pH Benchtop Meters, HI2211, Malaysia). The pH value of kefir samples, 10 mL, was measured during preparation and after the storage.

The samples were used to investigate and quantify the titratable acidity [18]. Phenolphthalein was used as an indicator and 0.1 M NaOH was used for determining acidity. All the samples were titrated with 0.1 M NaOH to record the value of acidity. The titratable acidity was calculated using the Soxhlet-Henkel point (SH) [18].

Total solid content of samples was measured by gravimetric method depending on Turkish Standard Institute Method TS1330 [19]. Ten mL of samples were boiled for a few minutes at 35 to 40 °C in a beaker, they were allowed to cool to room temperature. After an hour of holding at 102–103 °C, the dishes were allowed to cool to room temperature in a desiccator, the weight was recorded as M0. The weight before drying was measured after adding 5 mL of the sample (M1). The sample was then placed in an oven tuned to 102–103 °C until a constant weight was obtained. After that, the dishes were permitted to arrive to room temperature in a desiccator, the weight was measured (M2). The total solids content of the samples was measured according to (Eq. 1) [19].

The protein values of kefir samples were measured using the Turkish Standard Institute Method TS1330 [19]. In a conical flask, 10 mL of sample, 0.5 mL of phenolphthalein and 0.4 mL of saturated potassium oxalate solution were mixed and permitted to settle for 2 min. After that, the mixture was neutralized until it turned pink color using a burette containing a 0.1 M NaOH solution. Then, 2 mL of 40% formaldehyde solution were added and the titration was carried out with 0.1 M NaOH solution until the same pink color was obtained. The protein content of kefir samples (Eq. 2) was determined as follows [19];

Where a is the mL of NaOH utilized used for milk and b is the mL of NaOH utilized for blank.

Mineral compositions were evaluated by Atomic Absorption Spectrometer (PerkinElmer AAnalyst 800, USA). For analysis of minerals, 2 g of kefir samples were taken in porcelain bottles and put in an oven at 550 °C for 6 h; after obtaining ash, they were allowed to cool to room temperature in a desiccator. Then, 4 mL of 0.1 M HCl was added to the ash samples and up to 100 mL by adding water. Then, the samples were measured in an Atomic Absorption Spectrometer.

The docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were analyzed in kefir samples by use of high-performance liquid chromatography (HPLC, Shimadzu, LC-20AB prominence, Japan). A 0.5 mL aliquot of the sample was mixed with 25 mL of acetonitrile-methanol (50:50, v/v) and centrifuged at 0.014xg for 30 min, then filtration was applied using filter paper (Isolab, Cat no: 105.12.110, USA), after that, filtrate was put in an oven at 50 °C for one hour. Next, 1.5 mL of the samples were taken and placed in a vial to read the results by HPLC. The systems had a solvent delivery pump (Shimadzu LC-20AB, Japan), a photo-diode array detector monitoring UV spectral between 190 and 370 nm (Shimadzu, SPD M40, Japan), and an autosampler (Shimadzu, SIL-20 A HT, Japan) [20]. An inertsil 5 ODS-3 (250 × 4.6 mm, 5 μm) (GL Science INC. Japan) column was used for analysis with a mobile phase (A phase 0.1% H₃PO₄ in CH₃CN and phase B 0.1% H₃PO₄ in H₂O). The injection volume was 20 µl, column oven temperature was 40–45 °C.

The viscosity level of all kefir samples was measured with a rotating viscometer (Brookfield viscometer, DV3T™ viscometer, USA) at 20 rpm in a 150 mL of samples beaker.

The color analysis of kefir samples was measured using Hunter lab ColorFlex (A60-1010-615 Model colorimeter, Hunter lab, Reston, VA). Before recording the data, 10 mL of each class’s sample was put on the apparatus.

Sensory analysis

Sensory analysis was performed by TS EN ISO 8589 [21] standard method to evaluate the appearance, color, flavor, smell, and texture of kefir samples. Twenty competent panelists, graduate students of M.Sc. and Ph.D. from the Department of Food Engineering at Gaziantep University, were selected to evaluate the kefir samples according to 5 pre-defined (appearance, color, flavor, smell, and mouthfeel) attributes. The panelists gave scores for the sensory attributes utilizing a structured nine-point scales as; (9: like extremely, 8: like very much, 7: like moderately, 6: like slightly, 5: neither like nor dislike, 4: dislike slightly, 3: dislike moderately, 2: dislike very much, 1: dislike extremely). The average of scores from the panelists was calculated for each attribute. All protocols about sensory analysis were approved by Research Ethics Board of Gaziantep University Science and Engineering (date and no: 10.06.2024–506480).

Statistical analysis

Using SPSS version 19.0 (SPSS Inc., Chicago, USA), one-way analysis of variance (ANOVA) was carried out to assess the count of probiotic bacteria, pH level, titratable acidity, color levels, and viscosity values of kefir prepared with different concentrations of DS during the storage period. To compare more than two groups, the multiple comparison test of Duncan was employed.

Principal component analysis (PCA, CANOCO version 4.5) was utilized for multivariate data analysis to reduce the number of dependent variables into a smaller set of underlying factors. Additionally, factor analysis was applied, using the mean biochemical content ratings, to identify how the nutritional content could be distinguished [22].

Results and discussions

Bacteria count

The effect of DS supplementation on probiotic viability in kefir is summarized in Table 1. The addition of DS at 0.25%, 0.5%, and 1% significantly (p < 0.05) enhanced the growth of L. delbrueckii subsp. bulgaricus compared to the control, particularly evident before storage and on Day-18. This is consistent with previous findings using Spirulina platensis in fermented dairy products, which promoted the growth of LAB and B. animalis subsp. lactis, likely due to its rich content of nitrogenous compounds, B vitamins, and minerals [11]. Similar trends were observed for S. salivarius subsp. thermophilus, with the highest cell count achieved with 1% DS after 18 days of storage (Table 1). Bifidobacterium animalis subsp. lactis showed a marked (p < 0.05) increase in viability from fermentation through Day-22 in all DS treatments. Growth notably improved after Days-12 and − 18, suggesting enhanced probiotic stability during storage. These results align with findings from Hyrslova et al. [14] reported that DS biomass boosted the growth of several probiotic strains and increased adhesion of Bifidobacterium to epithelial cells. Lactobacillus acidophilus growth also significantly (p < 0.05) increased with rising DS concentrations, reaching 7.96 log CFU/mL in 1% supplementation by the end of storage. These results meet the minimum potential probiotic requirement (7 log CFU/g) set by the Turkish Food Codex [23], confirming the microbial quality of the kefir samples.

Table 1.

Effect of Dunaliella Salina (DS) additives on the number of colonies (log CFU/mL) of Lactobacillus delbrueckii subsp. Bulgaricus (LB), Bifidobacterium animalis subsp. Lactis (BL), and Lactobacillus acidophilus (LA) and S. salivarius subsp. Thermophilus (ST) before the fermentation (B.F.) and after the 1 st, 6th, 12th, 18th, and 22nd days of storage

Bacteria	DS (%)	Time (day)
Bacteria	DS (%)	B.F.	1st	6th	12th	18th	22nd
LB	0	2.00^Aa ± 0.15	4.74^Aa ± 0.32	6.00^Acd ± 0.13	5.92^Ab ± 0.18	5.81^Aa ± 0.12	5.46^Aa ± 0.09
	0.25	2.00^Aab ± 0.15	6.30^Bcd ± 0.09	6.44^B ± 0.18	6.46^Ab ± 0.12	6.48^Bde ± 0.04	6.27^Bd ± 0.08
	0.5	1.12^Aa ± 0.12	6.65^Bc ± 0.09	6.80^BCd ± 0.1	6.74^Abc ± 0.01	6.67^BCd ± 0.09	6.62^Cd ± 0.06
	1	3.40^Aa ± 0.15	6.71^Bc ± 0.16	6.80^Cf ± 0.15	6.82^Bcd ± 0.03	6.84^Cc ± 0.10	6.65^Cd ± 0.11
BL	0	5.00^Aa ± 0.01	6.49^Ab ± 0.20	5.84^Ab ± 0.04	5.95^Aab ± 0.06	6.06^Aa ± 0.06	6.12^Aab ± 0.06
	0.25	4.89^Bb ± 0.06	6.57^Aab ± 0.11	6.60^Bbc ± 0.04	6.73^Acd ± 0.15	6.75^Aab ± 0.08	6.63^Ad ± 0.08
	0.5	5.00^Bb ± 0.00	6.76^Ac ± 0.14	6.69^BCc ± 0.08	6.77^Bde ± 0.12	6.80^Ab ± 0.13	6.68^ABe ± 0.13
	1	5.28^Bc ± 0.07	6.73^Abc ± 0.07	6.86^Cc ± 0.10	6.86^Bcd ± 0.19	6.96^Abc ± 0.17	6.72^Bc ± 0.17
LA	0	1.08^Aa ± 0.08	6.90^Aa ± 0.06	6.87^Aa ± 0.23	6.87^Aa ± 0.27	7.08^Aa ± 0.23	7.15^Aa ± 0.04
	0.25	0.00^Aa ± 0.00	7.45^Ab ± 0.01	7.31^ABb ± 0.19	7.08^Aa ± 0.11	7.37^ABa ± 0.12	7.71^Aab ± 0.16
	0.5	0.00^A ± 0.00	7.24^Aa ± 0.07	7.27^ABab ± 0.16	7.27^Aa ± 0.16	7.29^ABa ± 0.03	7.89^Abc ± 0.11
	1	0.00^A ± 0.00	7.33^Aab ± 0.1	7.51^Ba ± 0.08	7.61^Aa ± 0.15	7.63^Ba ± 0.1	7.96^Aa ± 0.12
ST	0	4.97^Aa ± 0.18	7.01^Aa ± 0.03	7.00^Aa ± 0.12	7.02^Aa ± 0.19	7.25^Ab ± 0.27	6.76^Aab ± 0.12
	0.25	5.17^Aab ± 0.10	7.31^ABb ± 0.10	7.47^Bb ± 0.04	7.62^Aab ± 0.17	7.44^ABb ± 0.03	7.36^Bb ± 0.00
	0.5	5.12^Aa ± 0.07	7.47^Bb ± 0.10	7.59^Bb ± 0.05	7.65^Aa ± 0.25	7.62^ABb ± 0.06	7.50^Bc ± 0.09
	1	5.66^Bbc ± 0.17	7.43^Ba ± 0.183	7.73^Bb ± 0.12	7.75^Aa ± 0.27	7.80^Bb ± 0.07	7.58^Bf ± 0.09

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All result data are the mean of four data n = 4 (2 batches and duplicates) with their standard errors (SE). Different capital letters as; A, B, C, D, etc… indicate the statistical difference between times at α = 0.05 level among products at each time. Different lower-case letters as; a, b, c, d, etc… indicate a statistical difference between concentrations at α = 0.05 levels among products at each sample

The improved potential probiotic viability can be attributed to DS’s nutritional profile, including amino acids, galactose, xylose, proteins, fatty acids, and antioxidants such as beta-carotene and lutein. These components are known to support microbial metabolism and stability during storage [14, 24]. Overall, the incorporation of DS, particularly at 1%, significantly enhanced the final viable counts of L. delbrueckii subsp. bulgaricus, S. thermophilus, B. animalis subsp. lactis compared to the control (p < 0.05), whereas L. acidophilus exhibited an increase that was not statistically significant, highlighting its potential as a functional additive for improving probiotic viability and product quality.

Protein, total solids, fatty acids, and mineral contents

Protein contents of control and 0.25%, 0.5%, and 1% DS containing kefir samples are given in Table 2. All DS-supplemented samples exhibited significantly higher (p < 0.05) protein levels than the control, with the highest content (3.94 g/L) observed in the 1% DS sample on Day-15. The increase is likely due to the high protein content of DS, which is known to enhance the nutritional value of fermented products. The decline in protein content by Day-22 may be attributed to proteolytic activity by LAB, such as Lactobacillus desidiosus and L. fermentum, which remain metabolically active during storage. These bacteria can hydrolyze proteins into peptides and amino acids via proteases [25]. Although LAB generally exhibit versatile extracellular protease activity, certain strains during storage period can promote protein breakdown. Previous Other studies reported kefir protein contents ranging from 2.30% to 3.48% [26], while the Turkish Fermented Dairy Products Communiqué [23] mandates a minimum of 2.7% milk protein in such products. Additionally, more than 30% of the protein in fermented products may be converted into peptides and amino acids, enhancing bioavailability [27].

Table 2.

Effect of Dunaliella Salina additions on the protein, total solid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) and minerals amount in Kefir samples during the storage period

Name	Time (day)	Dunaliella salina (%)
Name	Time (day)	0 (Control)	0.25	0.5	1.0
Protein (g/L)	1st	2.55^a ± 0.12	2.65^{a, b}±0.13	2.72^{a, b}±0.14	2.87^b ± 0.17
	15th	3.37^a ± 0.01	3.57^a ± 0.04	3.74^a ± 0.01	3.94^a ± 0.01
	22nd	2.81^a ± 0.01	3.02^a ± 0.01	3.44^a ± 0.01	3.75^a ± 0.01
Total solid (g/L)	1st	10.28^a ± 0.17	12.00^a ± 0.13	12.24^a ± 0.15	12.38^a ± 0.15
	15th	10.26^a ± 0.01	12.03^a ± 0.15	12.27^a ± 0.01	12.42^a ± 0.01
	22nd	10.27^a ± 0.01	12.04^a ± 0.12	12.28^a ± 0.15	12.43^a ± 0.01
DHA (ppm)	15th	0.0000^a ± 0.0001	0.0217^a ± 0.0015	0.2211^b ± 0.0156	0.2298^b ± 0.0163
DHA (ppm)	22nd	0.0000^a ± 0.0001	0.0163^a ± 0.0012	0.1679^b ± 0.0119	0.1723^b ± 0.0122
EPA (ppm)	15th	0.1464^a ± 0.0103	0.2097^b ± 0.0148	0.2123^b ± 0.0150	0.2140^b ± 0.0153
EPA (ppm)	22nd	0.1232^a ± 0.0087	0.1310^a ± 0.0093	0.1351^a ± 0.0096	0.1373^a ± 0.0097
Minerals (ppm)	Na 15th	5.52^a ± 0.39	6.32^a ± 0.45	6.43^a ± 0.45	6.48^a ± 0.46
	Na 22nd	5.53^a ± 0.39	6.33^a ± 0.45	6.43^a ± 0.46	6.49^a ± 0.46
	Fe 15th	1282.39^a ± 90.68	1435.80^a ± 101.53	2029.55^b ± 143.51	2665.91^c ± 188.51
	Fe 22nd	1282.40^a ± 89.94	1435.81^a ± 101.51	2029.56^b ± 143.52	2665.91^c ± 188.51
	Mn 15th	3169.32^a ± 224.10	4021.58^{a, b}±284.37	4771.58^{b, c}±337.40	5260.23^c ± 371.95
	Mn 22nd	3169.33^a ± 224.11	4021.59^{a, b}±284.37	4771.59^{b, c}±337.41	5260.24^c ± 371.95
	Zn 15th	71.56^a ± 5.06	84.92^a ± 6.01	123.09^b ± 8.70	201.34^c ± 14.24
	Zn 22nd	71.57^a ± 5.06	84.93^a ± 6.01	123.10^b ± 8.71	201.35^c ± 14.24
	Ca 15th	1678.83^a ± 118.83	1918.62^a ± 135.67	2007.90^a ± 141.98	2043.61^a ± 144.50
	Ca 22nd	1678.84^a ± 118.83	1918.63^a ± 135.68	2007.91^a ± 141.98	2043.62^a ± 144.51

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C; Control All result data are the mean of four data n = 4 (2 batches and duplicates) with their standard errors (SE). Different lower-case letters, such as a, b, c, indicate a statistical difference between concentrations at α = 0.05 levels among products each sample

Total solid contents also increased with DS supplementation (Table 2). This may result from ongoing LAB metabolism, which breaks down carbohydrates into organic acids, thereby raising the soluble solids content [28].

The effects of DS inclusion on DHA (ppm) and EPA (ppm) levels are presented in Table 2. A significant increase (p < 0.05) in DHA and EPA concentrations was observed by Day 15 in DS-treated samples, particularly at 1% concentration. DHA levels were notably higher than EPA, which was undetectable in control samples (0% DS). The rise in polyunsaturated fatty acids is attributed to the fatty acid-rich composition of DS, highlighting its potential as a functional additive for improving the nutritional profile of kefir. Similar nutritional enhancements have been reported in other studies involving microalgal enrichment of dairy and probiotic products [11, 12, 14]. Furthermore, peptides, proteins, and amino acids present in DS may serve as nitrogen sources to support microbial growth. The biomass of DS also contains essential fatty acids, minerals, and antioxidants, which contribute to its functional properties [12, 14].

Dunaliella salina also significantly enhanced (p < 0.05) the mineral content of kefir, particularly iron, manganese, zinc, calcium, and sodium (Table 2). Iron content increased to 2665.91 ppm in the 1% DS sample, compared to the control. Calcium and zinc rose to 2043.62 ppm and 201.34 ppm, respectively, while manganese reached 5260.24 ppm by the end of storage. The increased mineral levels are likely due to diffusion from the fermented matrix during storage and reflect the mineral-rich nature of DS biomass. DS supplementation significantly improved the protein, total solids, fatty acid, and mineral content of kefir, supporting its use as a functional additive in fermented dairy products.

Principal component analysis (PCA)

PCA effectively distinguished the kefir samples based on their biochemical properties in relation to DS supplementation, consistent with previous findings on A. platensis-enriched white chocolate [29]. The analysis explained 99.47% of the total variation in biochemical parameters, including protein, total solids, minerals, DHA, and EPA. Notably, the kefir sample containing 1% DS (D1) was strongly associated with improved nutritional and biochemical attributes (Fig. 1). This indicates a clear positive correlation between the 1% DS addition and enhanced biochemical composition in kefir.

Fig. 1 — Principal components of kefir samples depend on the chemical composition of protein, TS-total solid, minerals (Na, Ca, Mn, Fe, Zn), DHA-docosahexaenoic acid, and EPA-eicosapentaenoic acid. D0.25, D0.5, and D1 represent 0.25%, 0.5%, and 1% DS, respectively

pH value and acidity titratable

The incorporation of DS significantly reduced the pH value of kefir samples (p < 0.05) following fermentation and throughout the storage period (1st, 6th, 12th, 18th, and 22nd days), compared to the control (Fig. 2a). This pH decline is likely related to the metabolic activity and proliferation of the starter culture during fermentation and storage [30]. Specifically, lactic acid production and bacterial growth contribute to post-acidification, resulting in decreased pH values over time [30] (Fig. 2a). Metabolic activity and LAB formation led to a lower pH and can reduce viable bacterial counts, low pH also effectively inhibits spoilage microorganisms, thereby extending the product’s shelf life. However, excessively low pH values may impart a sour taste, potentially affecting consumer acceptability. Certain Lactobacillus species ferment lactose into lactic acid, which increases acidity, reduces pH, and causes milk coagulation upon heating [31]. Commercial kefir beverages available in the Turkish market typically exhibit titratable acidity levels between 0.71% and 0.93% and pH values ranging from 3.86 to 4.06 [26]. These pH changes primarily reflect the fermentation of lactose into lactic acid by LAB, with enzymatic activity continuing, albeit at a reduced rate, during refrigerated storage.

Dunaliella salina significantly affected the titratable acidity (p < 0.05), with acidity decreasing as DS concentrations increased (Fig. 2b). Samples supplemented with 0.25%, 0.5% or 1% DS showed a significant reduction in the titratable acidity level compared to the control after the first day until the end of storage (p < 0.05). These effects were observed before and after fermentation as well as during storage (Fig. 2b). According to The Turkısh Food Codex [23], kefir products must have a minimum initial titratable acidity of 0.6%. Similar patterns have been observed in yogurt, where acidity increases during storage alongside pH decline, indicative of ongoing fermentation [32]. The titratable acidity values measured in the present study remained within the national regulatory limits, confirming product compliance.

The observed reduction in acidity with DS addition may have dual effects on product quality. Lower titratable acidity can lead to milder flavor profiles, potentially improving consumer acceptance by reducing sourness associated with over-acidification. On the other hand, reduced acidity could slightly compromise microbial stability, potentially shortening shelf life or altering textural properties if spoilage organisms are less inhibited. Thus, balancing acidity is crucial to optimizing both the sensory and preservation characteristics of kefir enriched with DS.

The viscosity

Statistical studies revealed that the addition of 0.25%, 0.5%, and 1% of DS remarkably (p < 0.05) decreased the viscosity levels in contrast to control samples of kefir after fermentation, on the 1 st, 6th, 12th, 18th and 22nd days of the storage. There was a 0.25%, 0.5%, and 1% improvement in the viscosity assessments accordingly from fermentation through the result of storage; DS was contrasted with the control. So, the incorporation of DS led to a decrease in the viscosity levels in kefir samples compared to the control. Similarly, the viscosity of various kefir samples prepared by various kefir grains between 7.5 and 15.4 mPa.s [33].

Hunter color parameters

The addition of DS at concentrations of 0.25%, 0.5%, and 1% significantly (p < 0.05) influenced the color attributes of kefir samples during and after fermentation, as well as across storage Days-1, −6, −12, −18, and − 22 (Table 3). Specifically, DS supplementation led to a notable decrease in L* values (lightness) compared to the control, indicating a darker appearance. Simultaneously, a* values (redness) significantly increased (p < 0.05) in all DS-treated samples throughout storage, reflecting the influence of red-orange pigments, primarily β-carotene, present in DS. The b* values (yellowness) also showed an increase, particularly in samples with 0.25% DS, although a slight decline was observed over the storage period at higher DS concentrations. These changes in color parameters are primarily attributed to the presence of natural pigments in DS, including chlorophylls and carotenoids, which impart characteristic green, yellow, and orange hues. The orange coloration of DS, visually similar to carrot pigment, contributed significantly (p < 0.05) to increase the a* values of kefir samples, thereby contributing to a more intense visual appearance compared to the control. Comparable alterations in Hunter color values were reported in studies incorporating A. platensis into ayran [11]. Color is a critical sensory attribute that influences both consumer acceptance and perceived freshness of fermented products. Therefore, monitoring color changes offers valuable insights into the visual quality and potential shelf appeal of functional dairy beverages.

Table 3.

Effect of Dunaliella Salina (DS) additions on Hunter color parameters analysis with a*, b*, and L* in the fermentation and the storage period

Hunter Color Paramater	DS (%)	Before fermentation	Storage period (day)
Hunter Color Paramater	DS (%)	Before fermentation	1st	6th	12th	18th	22nd
a*	0.00	0.81^Ac ± 0.01	0.83^Ab ± 0.00	0.15^Ac ± 0.01	0.73^Ab ± 0.01	0.83^Ac ± 0.01	0.64^Ab ± 0.01
	0.25	7.36^Bd ± 0.00	7.48^Bf ± 0.00	7.34^Bd ± 0.00	7.64^Bd ± 0.00	7.44^Bd ± 0.00	7.74^Bg ± 0.00
	0.50	10.01^Cf ± 0.00	10.58^Cf ± 0.00	10.37^Cg ± 0.0	10.45^Cf ± 0.00	10.67^Cg ± 0.00	10.73^Ce ± 0.00
	1.00	13.12^Df ± 0.00	14.2^De ± 0.10	14.2^Df ± 0.0	14.22^Dd ± 0.00	14.43^Dg ± 0.00	14.65^Df ± 0.00
b*	0.00	8.34^Aa ± 0.00	6.82^Aa ± 0.04	7.74^Aa ± 0.00	6.81^Ac ± 0.05	6.82^Aa ± 0.01	8.12^Aa ± 0.01
	0.25	10.44^Bb ± 0.00	12.49^Bc ± 0.01	12.81^Bc ± 0.00	12.84^Bd ± 0.00	12.81^Bb ± 0.00	12.81^Bc ± 0.00
	0.50	11.97^Cb ± 0.00	14.64^Cc ± 0.01	15.06^Cd ± 0.00	15.16^Cd ± 0.00	15.06^Cc ± 0.00	15.06^Cc ± 0.00
	1.00	13.72^Db ± 0.00	17.66^Dc ± 0.00	18.02^Dd ± 0.01	18.24^Da ± 0.01	18.43^Dd ± 0.00	18.93^Dc ± 0.00
L*	0.00	93.13^Dg ± 0.03	39.82^Aab ± 0.08	92.64^Dh ± 0.03	92.94^Dg ± 0.03	94.23^Df ± 0.01	94.54^Df ± 0.02
	0.25	79.91^Cb ± 0.00	84.36^Dab ± 0.00	84.73^Ce ± 0.01	84.75^Ce ± 0.01	84.98^Cd ± 0.00	85.12^Ce ± 0.00
	0.50	74.60^Ba ± 0.15	79.95^Cab ± 0.01	80.28^Bc ± 0.01	80.57^Bd ± 0.02	80.87^Bc ± 0.00	80.97^Bc ± 0.01
	1.00	68.13^Da ± 0.01	74.43^Aa ± 0.03	74.52^Bb ± 0.00	74.56^Aa ± 0.02	74.62^Aa ± 0.00	74.76^Aa ± 0.016

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Capital letters (A, B, C, D, etc…) indicate a statistical difference at α = 0.05 level between items at each time. Lowercase letters (a, b, c, d, e, f, etc…) indicate a statistical difference (α = 0.05) in concentrations between products

Sensory study

Sensory attributes of kefir samples—including appearance, color, flavor, aroma, and mouthfeel—were assessed using a nine-point hedonic scale (Fig. 3). The kefir sample enriched with 0.25% DS exhibited a reddish hue but received sensory scores comparable to the control across all parameters, indicating good consumer acceptability at this concentration. However, increasing the DS concentration to 0.5% and 1% led to progressive declines in sensory scores, particularly in flavor and appearance. The intensified color and possible off-flavors associated with higher DS levels likely contributed to this reduction, suggesting that excessive DS incorporation may adversely impact consumer perception. Despite these decreases, most scores for the higher DS concentrations remained within the acceptable range, indicating that moderate DS incorporation is feasible without compromising sensory quality. The reduction in sensory appeal over storage time may be partially attributed to microbial interactions, particularly the inhibition of yeast and mold growth by probiotic bacteria present in kefir [34], which can alter organoleptic properties during storage. Similar findings have been reported in other fermented dairy products where probiotic strains such as L. acidophilus and B. animalis subsp. lactis improved sensory acceptance through metabolite production [28].

Fig. 3 — Effect of *Dunaliella salina* additions 0% (blue), 0.25% (red), 0.5% (green), and 1% (purple) on the sensory analysis of kefir samples

To improve acceptability at higher DS levels, potential strategies include blending DS with complementary flavors or masking agents to counteract off-flavors, optimizing fermentation conditions to modulate sensory changes, or using microencapsulation techniques to limit direct taste impact. Adjusting the fermentation time or incorporating flavor enhancers may also help balance the color intensity and taste profile, making higher DS enrichments more palatable.

Incorporation of DS biomass into kefir significantly enhanced its nutritional profile, highlighting its potential as a functional food supplement. The observed improvements in protein, fatty acids, minerals, and total solids underscore the nutritional benefits of DS-enriched kefir. Additionally, LAB contribute to the overall quality by producing various metabolites that enhance both organoleptic and nutritional characteristics. The metabolic versatility of probiotic bacteria supports the development of fermented products with improved sensory properties, nutritional value, and safety, underscoring their suitable for functional food applications.

Conclusions

Kefir is a well-known fermented beverage recognized for its health-promoting properties and unique sensory profile. In this study, the incorporation of D. salina (DS) at concentrations of 0%, 0.25%, 0.5%, and 1% enhanced the growth of key probiotic strains including L. delbrueckii subsp. bulgaricus, L. acidophilus, S. salivarius subsp. thermophilus, and B. animalis subsp. lactis. DS addition significantly (p < 0.05) improved the physicochemical properties of kefir—such as pH, titratable acidity, total solids, viscosity, and Hunter color values (L*, a*, and b*)—as well as its nutritional profile, including protein, mineral content, and levels of DHA and EPA during fermentation and throughout 22 days of storage. PCA confirmed that 1% DS supplementation had the most pronounced positive impact on the kefir’s biochemical and nutritional characteristics. Overall, this study demonstrates that DS not only supports probiotic viability but also enhances kefir’s functional and nutritional value. Notably, this research represents one of the first comprehensive assessments of kefir enriched with microalgal biomass, considering both probiotic dynamics and nutritional enrichment. Future studies in food biotechnology may build upon these findings to develop innovative, nutrient-rich kefir-based products with tailored sensory and functional properties.

Electronic supplementary material

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Supplementary Material 1^{(1.8MB, png)}

Supplementary Material 2^{(33KB, doc)}

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Author contributions

Z.A.A. methodology, validation, formal analysis, data curation, writing—original draft preparation. A.Ç. methodology, formal analysis, investigation, data curation, writing—original draft preparation, writing—review and editing. H.B. methodology, formal analysis, investigation, writing—review and editing, visualization.

Code Availability

Not applicable.

Data availability

The data presented in this study are available on request from the corresponding author.

Declarations

Ethics approval

This study was approved by Research Ethics Board of Gaziantep University Science and Engineering (the certificate date and number: 14.06.2024–506480).

Consent for publication

Not applicable.

Consent to participate

Not applicable.

Statement of informed consent, human/animal rights

No conflicts, informed consent, human or animal rights applicable to this study.

Clinical trial number

not applicable.

Conflict of interest

The authors have declared no conflicts of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(1.8MB, png)}

Supplementary Material 2^{(33KB, doc)}

Supplementary Material 3^{(71.5KB, doc)}

Data Availability Statement

Not applicable.

The data presented in this study are available on request from the corresponding author.

[CR1] 1.Marco LM, Heeney D, Binda S, Cifelli CJ, Cotter PD, Foligné B, Gänzle M, Kort R, Pasin G, Pihlanto A, Smid EJ, Hutkins R (2017) Health benefits of fermented foods: microbiota and beyond. Curr Opin Biotechnol 44:94–102. 10.1016/j.copbio.2016.11.010 [DOI] [PubMed] [Google Scholar]

[CR2] 2.Babich O, Ivanova S, Michaud P, Sukhikh S, Prosekov A, Larichev A, Popov A, Noskova S, Pungin A, Ulrikh E, Astakhova L, Rudoy D (2023) Fermentation of micro- and microalgae as a way to produce value-added products. 10.1016/j.btre.2023.e00827. Biotechnology Reports e00827 [DOI] [PMC free article] [PubMed]

[CR3] 3.Shah AB, Aizhamal B, Muhammad Z, Ali SI, Yang J, Chen Y, Shah FUH, Shah FU (2024) Probiotic significance of Lactobacillus strains: a comprehensive review on health impacts, research gaps, and future prospects. Gut Microbes 16(1):2431643. 10.1080/19490976.2024.2431643 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Guzel-Seydim ZB, Kok-Tas T, Greene AK, Seydim AC (2011) Review: functional properties of kefir. Crit Rev Food Sci Nutr 51(3):261–268. 10.1080/10408390903579029 [DOI] [PubMed] [Google Scholar]

[CR5] 5.Hernández-Martínez A, Jiménez-Pérez C, Cruz-Guerrero A, García-Garibay M, Gómez-Ruiz L, Rodríguez-Serrano GM, Domínguez-González KN (2024) Production of exopolysaccharides through fermentation of secondary whey with kefir grains. Biol Life Sci Forum 40:1–10. 10.3390/blsf2024040001 [Google Scholar]

[CR6] 6.Food US, Administration D (2017) Microorganisms & Microbial-Derived Ingredients Used in Food. U.S. Food and Drug Administration. https://www.fda.gov/food/generally-recognized-safe-gras/microorganisms-microbial-derived-ingredients-used-food

[CR7] 7.Obis D, Ouwehand AC (2024) The Safety of Lactic acid bacteria for Use in Foods. Lactic acid bacteria, edited by Vinderola G, Ouwehand A, Salminen S, Wright A. CRC, Boca Raton, FL, pp 707–724 [Google Scholar]

[CR8] 8.Cheng J, Laitila A, Ouwehand AC (2021) Bifidobacterium animalis subsp. Lactis HN019 effects on gut health: a review. Front Nutr 8:790561. 10.3389/fnut.2021.790561 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR9] 9.FAO/WHO (2001) Report of a joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Córdoba Argentina

[CR10] 10.Garofalo C, Norici A, Mollo L, Osimani A, Aquilanti L (2022) Fermentation of microalgal biomass for innovative food production. Microorganisms 10(10):2069. 10.3390/microorganisms10102069 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Çelekli A, Alslibi ZA, Bozkurt H (2019) Influence of incorporated Spirulina platensis on the growth of microflora and physicochemical properties of Ayran as a functional food. Algal Res 44:101710. 10.1016/j.algal.2019.101710 [Google Scholar]

[CR12] 12.Çelekli A, Özbal B, Bozkurt H (2024) Challenges in functional food products with the incorporation of some microalgae. Foods 13:1–15. 10.3390/foods13010001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.da Silva MROB, Moura YAS, Converti A, Almeida JMA, Costa AM, Silva JA, Magalhães FEC, Maciel MIS (2021) Assessment of the potential of Dunaliella microalgae for different biotechnological applications: a systematic review. Algal Res 58:102396. 10.1016/j.algal.2021.102396 [Google Scholar]

[CR14] 14.Hyrslova I, Krausova G, Mrvikova I, Curdova M, Cabicarova T, Stankova B, Doskocil I, Hyrsl P, Roubalova R, Rada V (2022) Functional properties of Dunaliella Salina and its positive effect on probiotics. Mar Drugs 20(12):1–13. 10.3390/md20120781 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Güzel-Seydim ZB, Seydim AC, Greene AK, Bodine AB (2000) Determination of organic acids and volatile flavor substances in kefir during fermentation. J Food Compos Anal 13(1):35–43. 10.1006/jfca.1999.0842 [Google Scholar]

[CR16] 16.Dave RI, Shah NP (1998) Ingredient supplementation effects on viability of probiotic bacteria in yogurt. J Dairy Sci 81(11):2804–2816. 10.3168/jds.S0022-0302(98)75839-4 [DOI] [PubMed] [Google Scholar]

[CR17] 17.Tabasco R, Paarup T, Janer C, Peláez C, Requena T (2007) Selective enumeration and identification of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. Paracasei subsp. Paracasei and bifidobacterium lactis in fermented milk. Int Dairy J 17(9):1107–1114. 10.1016/j.idairyj.2007.01.010 [Google Scholar]

[CR18] 18.Çelekli A, Alslibi ZA, Bozkurt H (2020) Boosting effects of Spirulina platensis, whey protein, and probiotics on the growth of microflora and the nutritional value of Ayran. Eng Rep (Hoboken) 2:e12235. 10.1002/eng2.12235 [Google Scholar]

[CR19] 19.TSI (2006) Turkish Standards Institution. (A Turkish Stand Method TS1330) Ankara, Turkey

[CR20] 20.Shaish A, Ben-Amotz A, Avron M (1992) Biosynthesis of β-carotene in Dunaliella. Methods in enzymology, vol 213. Elsevier, pp 439–444. 10.1016/0076-6879(92)13146-8

[CR21] 21.TS EN ISO (2010) EN ISO 8589, sensory analysis; general guidance for the design of test rooms. Geneva, Switzerland [Google Scholar]

[CR22] 22.ter Braak CJF, Smilauer P (2002) CANOCO reference manual and CanoDraw for Windows user’s guide: software for canonical community ordination (version 4.5). www.canoco.com

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[CR24] 24.Hadebe N (2016) Isolation and characterization of prebiotic oligosaccharides from algal extracts and their effect on gut microflora. Durban University of Technology, Durban, South Africa. 10.51415/10321/1754 [Google Scholar]

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PERMALINK

Effect of Dunaliella salina supplement on growth of probiotic bacteria and nutritional values of Kefir

Zina Alajil Alslibi

Abuzer Çelekli

Hüseyin Bozkurt

Abstract

Graphical abstract

Supplementary Information

Introduction

Materials and methods

Experimental design

Microbiological analysis

Chemicals and sensorial analyses

Sensory analysis

Statistical analysis

Results and discussions

Bacteria count

Table 1.

Protein, total solids, fatty acids, and mineral contents

Table 2.

Principal component analysis (PCA)

Fig. 1.

pH value and acidity titratable

Fig. 2.

The viscosity

Hunter color parameters

Table 3.

Sensory study

Fig. 3.

Conclusions

Electronic supplementary material

Author contributions

Code Availability

Data availability

Declarations

Ethics approval

Consent for publication

Consent to participate

Statement of informed consent, human/animal rights

Clinical trial number

Conflict of interest

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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