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Article

Kefir and Lactobacillus plantarum Cultures in the Production of Fermented Blueberry Juices

by
Anca Dumuta
*,
Zorica Vosgan
,
Liviu Giurgiulescu
*,
Cristina Mihali
and
Lucia Mihalescu
Faculty of Science, Technical University of Cluj-Napoca, 76 Victoriei Str., 430122 Baia Mare, Romania
*
Authors to whom correspondence should be addressed.
Sustainability 2025, 17(23), 10488; https://doi.org/10.3390/su172310488 (registering DOI)
Submission received: 2 September 2025 / Revised: 15 November 2025 / Accepted: 20 November 2025 / Published: 23 November 2025
(This article belongs to the Special Issue Sustainable Research on Food Science and Food Technology)
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Abstract

Developing the nutritional profile of food through the fermentation produced by lactic acid bacteria is part of sustainable development, but it also helps to improve public health. Thus, the objective of the study was to obtain synbiotic juices of fruit origin, which can successfully substitute well-known probiotic dairy products and also be introduced into the diet of vegetarians or people with certain intolerances to animal products. In this regard, two fermented blueberry juices were obtained: NC, using kefir cultures, and NP with Lactobacillus plantarum inoculation. Two more simple blueberry juices were used as controls: NCM, thermostatted at 24 °C and NPM, at 37 °C. The four types of juices were subjected to physicochemical and microbiological analyses. The results show that NP juice is the best substitute from a physicochemical point of view, presenting the highest polyphenol content and the highest DPPH radical scavenging activity even after refrigeration for 5 days. Additionally, the microbiological results of the analyzed juices can recommend these products for industrial production.

1. Introduction

Sustainable food production encompasses three main requirements: it is environmentally and climate-friendly, economically feasible, and socially respectable [1]. Blueberries, due to their unique nutritional composition, are fruits which are extremely suitable for obtaining value-added products of sustainable production [2]. Berry fruits are highly consumed throughout the world because they have attractive colors and their chemical composition highlights their sweet taste, fruity aroma, and beneficial health properties [3]. Due to the exceptional properties, blueberries have been certified by the Food and Agriculture Organization (FAO) as one of the five healthier groups of foods [4]. The blueberry shrub is a member of the Vaccinium genus of the Ericaceae family, with small fruits known for their important content of organic and phenolic acids, flavonoids, anthocyanins and other bioactive substances [5].
The secondary metabolites category of plant tissues includes bioactive compounds, which comprise a large group of polyphenols. Polyphenols can be divided into flavonoids and non-flavonoids. Flavonoids comprise anthocyanins, flavonols, flavanols, flavones and flavanones, while non-flavonoids comprise tannins, stilbenes and phenolic acids like hydroxybenzoic and hydroxycinnamic acids [6]. The flavonols in the highest concentration in blueberries are quercetin, myricetin, and kaempferol, and the flavanols are catechin, epicatechin, and gallocatechin [7,8,9]. Anthocyanins, the most potent antioxidants in blueberries, are natural pigments composed of two benzene rings linked by three carbon atoms, which can react with glucose, rhamnose, galactose and arabinose. There are five kinds of typical anthocyanins: delphinidin, malvidin, peonidin, cyanidin, and pelunidin [10]. After human ingestion, the anthocyanins are deglycosylated in the small intestine and degraded into anthocyanidins for further absorption [11].
The regular consumption of fruits and vegetables is recommended for a healthy lifestyle due to their content of fibers, vitamins, minerals, and phytochemicals [3]. Blueberries can be considered as a functional food due to their high anthocyanin content, which provides health benefits due to their antioxidant capacity [12]. But all the bioactive compounds of the blueberry’s composition lead to health improvements through their multiple roles as antioxidant, anti-inflammatory, antiseptic, antiproliferative, antiaging, and astringent agents, as well as neuro-, cardio-, vision-, and kidney-protective agents [10].
The highest disadvantage of this nutrient-dense fruit exploitation is the fact that it is very perishable because it can be easily damaged under the influence of mechanical or microbiological factors. Thus, blueberries present a strong seasonal availability, a reduced shelf life, and subsequently, economic losses [13]. Therefore, it is necessary to enhance blueberry consumption mainly by functionally derived products in order to make their active compounds available to a more diversified market. Among these derived products, blueberry juice is the most popular because it is highly palatable and healthy [14].
The food industry currently faces a number of sustainability challenges due to adverse conditions, agricultural practices, environmental pollution, and food waste. These can be addressed encouragingly by harnessing the power of microorganisms in ways that reduce their environmental footprint [15]. Also, Saud et al. (2024) [16] consider the fruit and vegetable juices obtained by lactic acid fermentation as a novel nutritional approach for improving health. This is important, especially given that fermentation is a simple and sustainable technology that involves low costs, but also extends the shelf life of fruits and vegetables [17]. A series of new compounds are generated following the fermentation of fruits that improve the sensory characteristics of the finished product and bring great benefits to the consumers health; in addition, this process can maintain a series of essential nutrients and degrade toxic components [18]. Thus, the content of phenolic compounds, exopolysaccharides, vitamins, and minerals increases, leading to a series of functional properties in the finished product, such as antioxidant, hypoglycemic, and antihypertensive activity [19]. Fermented juices also help maintain a healthy gut microbiota, which prevents or improves the health of people suffering from chronic metabolic diseases such as obesity and diabetes, as well as cardiovascular and cerebrovascular diseases [20].
Lactic bacteria play a crucial role in sustainable food production, encompassing environmental, economic, and social aspects [21]. Lactobacillus plantarum-fermented jujube generated more aroma, volatile organic components, and exhibited lower bitterness, astringency, and aftertaste [22]. The combination of Lactobacillus plantarum and Pichia pastoris may supply a new mixed fermentation agent towards fermented jujube products and provides reference values for flavor regulation in the co-fermentation of jujube juice [23]. It can also be mentioned that the stronger actions of fermented products from blueberries, lychees, and apples, compared to unfermented control samples in terms of preventing obesity and hyperglycemia, improved immunomodulatory function and intestinal protection, as well as their biological activities [24,25,26]. The quality of the fermented fruit juices is influenced by factors such as the microorganisms used for fermentation, the fermentation conditions, and the type and quality of the fruits used [20].
In this study, the use of kefir and Lactobacillus plantarum cultures for the production of fermented blueberry juices was explored. The aim of the study was to evaluate the impact of these cultures on the physicochemical and microbiological properties of the fermented blueberry juices, as well as their stability during five days of refrigeration.

2. Materials and Methods

2.1. Preparation of the Blueberry Juices

Blueberries were provided by the Bluettes Saveur blueberry plantation from Satu Mare, Romania. The blueberries juice was obtained by crushing and pressing fresh blueberries, followed by filtering the resulting juice. After pasteurization (71.7 °C/15 s), the obtained juice was divided into 4 equal parts. A total of 2 parts were used to produce the 2 functional juices: one with 0.1% w/v Lactobacillus plantarum (L. plantarum Probiotic by Swanson, Fargo, ND, USA) and one with 0.1% w/v kefir cultures (Probiotic ferment for kefir, eco-bio, by My. Yo, Frankfurt, Germany), and 2 parts represented the simple control juices.
Before inoculation, the microbial purity of the two cultures used was verified. The Lactobacillus plantarum strain was tested on MRS agar (from Sigma-Aldrich, Darmstadt, Germany) to confirm purity and viable cell count. The kefir culture, being a complex microbial consortium, was analyzed by plating on selective media to evaluate total viable counts and to check for the presence of potential contaminants, such as coliforms and molds. These quality control measures were taken to ensure the reproducibility and microbiological safety of the fermentation process.
The study has been performed using juice bottles with a capacity of 250 mL. All juices were subjected to thermostatation at different temperatures for 24 h: the ones for Lactobacillus plantarum study were kept at 37 °C, and those for kefir cultures at a temperature of 25 °C. The 4 types of juice experimentally analyzed in the laboratory were abbrieviated as follows: NPM for the control blueberry juices for the product fermented with Lactobacillus plantarum; NP for the blueberry juices fermented with Lactobacillus plantarum; NCM for the control blueberry juices for the product fermented with kefir cultures; NC for the blueberry juices fermented with kefir cultures.
The four types of juice were analyzed at the following time points:
  • T0—before fermentation (day of inoculation).
  • T1—after fermentation (24 h of incubation).
  • T2—after 3 days of refrigerated storage.
  • T3—after 5 days of refrigerated storage.
The fermentation was carried out under static, microaerophilic conditions, which are generally suitable for L. plantarum, a facultative anaerobe that can tolerate limited oxygen and may benefit from moderate CO2 levels due to its capnophilic tendencies.

2.2. pH Determination by the Potentiometric Method

The determination was carried out using a pH meter (Voltcraft PH-100 ATC, Conrad Electronic, Hirschau, Germany), which measures the potential difference between a reference electrode and a measuring electrode introduced into the juice sample. The calibration of the device was achieved using two buffer solutions with a pH close to the possible pH of the sample. The electrodes were introduced into the sample to be analyzed, and after 1–2 min, the pH was read. The pH measurement was performed three times for each sample, the pH value was thus given by the arithmetic mean of the 3 measurements as long as the difference between the extreme values did not exceed 0.15 pH units.

2.3. Determination of the Refractive Index

The refractive index (RI) determination was performed using the refractometer (PAL-RI digital ATAGO, Atago, Tokyo, Japan). For fruit juices, the refractive index depends on the sugar concentration as well as on the concentration of other soluble materials (organic acids, minerals, amino acids, etc.).
The juices must be thoroughly mixed before determination and brought to a temperature of approx. 20 °C (±0.5 °C). A small portion of the sample was placed on the lower prism of the refractometer; then, the measurement was carried out according to the instructions of the device. Three determinations were made on the same sample according to the International Federation of Fruit Juice Producers (IFU) methods [27].

2.4. Determination of Vitamin C by the Iodometric Method

According to Varga et al. 2004 [28], the iodometric method for determining the vitamin C content is based on the oxidation of this vitamin (ascorbic acid) to dehydroascorbic acid in an acidic medium, using iodine. Starch is used as an indicator, which turns blue when all the vitamin C has been oxidized. The iodine required for the reaction came from a prior reaction between potassium iodate and potassium iodide in an acidic medium. For the vitamin C extraction, 5 mL of the sample was mixed with 20 mL of 2% hydrochloric acid solution. The mixture was left to stand for 15–20 min and then filtered. Meanwhile, the sample was prepared for titration: in a 50 mL Erlenmeyer flask, 1 mL of the vitamin C solution (or the obtained filtrate) was added together with 3 mL of distilled water, 0.5 mL of potassium iodide (Merck, Darmstadt, Germany) solution and 2 mL of starch (from Sigma-Aldrich, Darmstadt, Germany) solution. The mixture was immediately titrated with a potassium iodate (Merck, Darmstadt, Germany) solution until a persistent blue color appeared. The blank sample was processed under identical conditions, replacing the vitamin C solution with 1 mL of 2% hydrochloric acid solution (prepared by appropriate dilution of concentrated hydrochloric acid of PA degree, Warchem, Zakręt, Poland).
The vitamin C concentration was expressed in mg% (mg of vitamin C per 100 mL of sample) according to the relationships:
N = Pd − Pm
C = N × 0.088 × 100
where
  • Pd—the number of mL of potassium iodate solution used for the analyzed sample,
  • Pm—the number of mL of potassium iodate solution used for the blank sample,
  • C—vitamin C concentration, in mg%.

2.5. DPPH Radical Scavenging Activity

The DPPH radical scavenging activity was determined spectrophotometrically using the spectrometer (Perkin Elmer Lambda 35, PerkinElmer, Shelton, CT, USA). The method was proposed by Tongnuanchan et al. (2012) [29]. 0.5 mL of the sample was treated with 10 mL methanol (from Sigma-Aldrich, Darmstadt, Germany) and homogenized for 3 h. Then, it was filtered and a volume of 3 mL methanolic extract was treated with 3 mL of 0.15 mM DPPH (from Merck, Darmstadt, Germany) ethanolic solution (DPPH in 95% ethanol) and kept in the dark for 30 min at room temperature. The mixture was stirred. For the blank sample, 3 mL of methanol was mixed with 3 mL DPPH solution and kept in the dark for 30 min. The absorbance was read at 517 nm and the antioxidant activity was determined with the formula:
DPPH radical scavenging activity (%) = [1 − (A517 nm sample/A517 nm blank)] × 100

2.6. Determination of the Total Polyphenol Content

For the analysis of the polyphenol content, the method based on their oxidation reaction under the action of the Folin–Ciocalteu reagent was used, which is a mixture of sodium phosphotungstate, phosphomolybdic acid, and phosphoric acid according to an adapted procedure based on the method proposed by Singleton et al. (1999) and used in other studies [30,31,32]. The analysis method comprises three important steps, such as drawing the calibration curve with gallic acid (standard polyphenol), polyphenol extraction from the samples to be analyzed using an extractant solution consisting of 1% HCl in 40% methanol or 1% HCl in 40% ethanol, and the spectrophotometric (spectrometer Perkin Elmer Lambda 35, PerkinElmer, Shelton, CT, USA) determination of polyphenols from the extract with the Folin–Ciocalteu reagent.
To plot the calibration curve, a series of gallic acid standard solutions were prepared with concentrations ranging from 10 to 500 mg/L. Then, 1 mL of each standard solution was mixed with 60 mL of distilled water and 1 mL of Folin–Ciocalteu reagent, left to stand for 1 min, and finally, 15 mL of 7.5% Na2CO3 solution was added and the mixture was made up to 100 mL in a volumetric flask. The solution was left to stand for 30 min for color development, then the absorbance was read at 750 nm against distilled water. A blank containing the same reagents was also prepared, but instead of 1 mL of the gallic acid standard solution, 1 mL of 1% HCl solution in 40% methanol was added (the solution used to extract polyphenols from fruits or plants).
The spectrophotometric determination of the polyphenols for the liquid samples using Folin–Ciocalteu reagent was performed by mixing 1 mL of the liquid sample with 60 mL of distilled water in a 100 mL volumetric flask. Then, 1 mL of Folin–Ciocalteu reagent was added and left to stand for 1 min. Afterwards, 15 mL of 7.5% Na2CO3 solution was mixed with the solution and made up to 100 mL in a volumetric flask. The absorbance of the resulting solution was measured after 30 min at a wavelength of 750 nm against distilled water, and the results were expressed in mg/L gallic acid equivalents (GAE). All the reagents used in the method are from Sigma-Aldrich and Merck (Darmstadt, Germany).

2.7. Microbiological Determinations

The microbiological profiles of the blueberry juices samples were determined by ISO standardized analyses: aerobic mesophilic bacteria [33] and yeasts and molds [34]. For each analysis, the samples were mixed with sterile saline solution (8.5% NaCl, from Sigma-Aldrich, Darmstadt, Germany) in a ratio of 1:9, and subsequently, a series of dilutions were performed according to the mentioned standards.
For the total number of aerobic mesophilic bacteria, the PCA (Plate Count Agar) culture medium (from Sigma-Aldrich, Darmstadt, Germany) was used. It was sterilized and cooled to 45 °C, then poured onto the inoculated plates. The inoculum was mixed with the culture medium, left for solidification, and then the Petri dishes were incubated at 30 °C for 72 h. To calculate the number of aerobic mesophilic bacteria/mL samples, plates containing less than 300 colonies were taken into account.
For the enumeration of yeasts and molds, the Sabouraud Chloramphenicol medium (from Sigma-Aldrich, Darmstadt, Germany) was used. It was distributed in the Petri dishes over the inoculum, and after solidification, incubation at 25 °C for 3–5 days followed. The selected plates were the ones where the number of colonies was up to 200, taking into account both yeast colonies that are smooth, moist, raised, or superficial, and mold colonies that show abundant hyphal growth and may appear in different colors.
For the detection of Staphylococcus sp. in the juice samples, the standard ISO 6888-1:2021 method [35] was applied. The inoculum was dispersed in Petri dishes containing Baird–Parker agar medium (from Sigma-Aldrich, Darmstadt, Germany) and incubated for 24–48 h at 37 °C. After 48 h of incubation, specific colonies of staphylococci, which are shiny black with a narrow white border and surrounded by a clear halo, may be observed.
Isolation and identification of Escherichia coli was performed according to ISO 16649-2:2007 [36], with slight modifications. 1 mL of each dilution of the juice sample was transferred to two sterile Petri dishes and then tryptone bile X-glucuronide (TBX) agar (from Sigma-Aldrich, Darmstadt, Germany) (~15 mL selective medium) precooled to 44–47 °C was poured. The solidified mixture was incubated at 44 °C for 24 h, and the development of blue-green colonies was monitored.
After all incubation steps of bacteria, yeasts, and molds, the obtained results were presented as log CFU/mL. All measurements were performed in triplicate.

3. Results

The four types of juices obtained after fermentation were monitored over 5 days of storage under refrigerated conditions.

3.1. pH Values Variation for the Four Types of Blueberry Juices

Following the application of the pH determination method for the four types of juice analyzed in the study, the obtained results are presented in Table 1.
According to Table 1, it can be seen that the control blueberry juice for L. plantarum, NPM, is characterized by a pH value of 3.66 on the day before thermostatation at 37 °C, and then the pH registers an increase in value to 3.67 at T1 and to 3.86 at T2. Five days after opening and storage under refrigeration conditions, the pH increases slightly to a value of 3.93. NP blueberry juice also registers an increase in pH from the day of L. plantarum seeding until post-fermentation time T1, because this bacterium ferments plant substrates at low pH values, contributing to the increase in the environment pH from approximately 3.5 to values around 4.0. After opening, with storage under refrigeration conditions, the pH remains almost constant from 3.94 at 3 days to 3.95 at 5 days.
In the case of the juices prepared for the kefir culture study, a decrease in pH value can be observed, both in the case of the control, and in the juice inoculated with kefir-specific strains. Thus, NCM records a decrease in pH value from 3.86 to 3.67 following the thermostatation operation at 24 °C, reaching 3.65 after 5 days of opening and storage under refrigerated conditions. NC is characterized by the most pronounced decrease in pH value, from 3.93 on the day of inoculation to 3.65 after thermostatation and to 3.66 after 5 days of opening. The decrease in pH value is due to the dissociation of organic acids and the use of sugars by the lactic bacteria of the kefir culture for the production of lactic acid, but also to the formation of carbon dioxide [37].

3.2. Refractive Indices Variation for the Four Types of Blueberry Juices

The results of the refractive index determination for the blueberry juices used in the study are shown in Table 2.
The refractive index value increases on the day of thermostatation in the case of the juices treated at 37 °C, NPM and NP, and then remains almost constant during the 5 days of post-opening storage under refrigerated conditions. Regarding the values recorded in the case of NCM and NC juices, the refractive index decreases almost constantly in the case of NC juice inoculated with kefir culture from moment T0 corresponding to thermostatation at 24 °C until the fifth day after opening due to the fermentation phenomenon, but remains almost constant in the case of NCM juice from T0 until T3.

3.3. The Variation in the Ascorbic Acid (AA) Concentration for the Four Types of Blueberry Juices

After determining the concentration of the ascorbic acid for the four types of juices subject the analysis, the results are presented in Table 3.
According to the previous table, the vitamin C content at T0, corresponding to the thermostatation day, is equal for all four types of juices, with a value of 1.76 mg%. After thermostatation, it decreases only in the case of NCM treated at 24 °C. The vitamin C concentration decreases in the same proportion for NPM, NP and NC 3 days after opening, reaching the same concentration with NCM, 1.32 mg%, which has remained constant since T1 moment.
At five days after opening—T3 moment, the highest concentration of vitamin C is recorded by the samples inoculated with the two cultures, kefir and L. plantarum, with a value of 1.32 mg%. This may be due to the reaction between pectin and functional components of the juices through hydrophobic or hydrogen bonds [6]. The control juices suffer a significant decrease in vitamin C concentration, reaching 0.88 mg% in the case of NPM or even 0.44 mg% in the case of NCM.

3.4. DPPH Radical Scavenging Activity for the Four Types of Blueberry Juices

The results obtained in the case of the DPPH radical scavenging activity method are highlighted in Table 4.
DPPH radical scavenging activity values increase from T0 to T1 in the case of NP, NCM, and NC juices. The highest DPPH radical scavenging activity at T1 is recorded by the juice inoculated with kefir culture, 56.77%, which starts from an activity of 40.88% at the moment before the thermostatation operation at 24 °C. For 3 days after opening, the NC juice remains with the highest DPPH radical scavenging activity value, 51.2%, which decreases to 40.66% five days after opening. For the juice inoculated with L. plantarum, the DPPH radical scavenging activity remains quite high even after 5 days after opening, recording a value of 47.74%. The control juices present a DPPH radical scavenging activity whose value is reduced quite drastically after 5-day storage in refrigeration.

3.5. The Variation in the Polyphenol’s Concentration for the Four Types of Blueberry Juices

The results recorded for the four types of blueberry juices analyzed in this study in terms of the polyphenol concentration are shown in Table 5.
In the previous table, a significant increase in the polyphenol content in all types of inoculated juices after the thermostatation operation can be observed. This fact is due to the disintegration of macromolecular polyphenols into smaller phenolic compounds, possibly through deglycosylation [38]. A total of 3 days after opening, the polyphenol content decreases to values close to the T0 moment. At 5 days after opening, at the T3 moment, the values are similar to those at the T2 moment; the highest value of the polyphenols content are recorded by the juice inoculated with L. plantarum NP, of 132.63 mg EAG/100 mL juice, followed by NC of 130.74 mg EAG/100 mL juice.

3.6. The Microbiological Determinations Result of the Four Types of Blueberry Juices

3.6.1. Determination of the Total Number of Aerobic Mesophilic Bacteria

In order to highlight the microbiological evolution of the blueberry juices during the five days of refrigerated storage, the total number of aerobic mesophilic bacteria was determined. Thus, in Figure 1, the average values obtained, expressed as log CFU/mL, are presented.
Juices inoculated with L. plantarum and kefir cultures present a high number of aerobic mesophilic bacteria, with values ranging between 7.24 and 7.65 log CFU/mL on moment T0. Then, these values decrease as storage progresses, due to their sensitivity to refrigeration conditions, reaching values of 6.27–6.55 log CFU/mL on day 5. The control juices do not show any viable microorganisms due to the pasteurization operation within their production flow during the first days. The slight growth observed after 3 and 5 days shows weak contamination, possibly from the storage environment. The observed microbial growth does not indicate a deficiency in the pasteurization process, which proved to be initially effective, but rather suggests possible post-process contamination, most likely during handling or storage.

3.6.2. The Total Yeast and Mold Counts

The presence of yeasts and molds in blueberry juices was assessed in order to evaluate the microbiological quality, consumer safety, and product stability during refrigerated storage. As a result of the juice pasteurization, good efficiency of the process was found, with yeasts and molds being detected only on the fifth day of refrigeration storage and in a reduced number, both in the control samples and in the one inoculated with L. plantarum. In the case of the blueberry juice inoculated with kefir cultures, the analysis evaluated the microbiological behavior of the product during refrigeration, as well as the monitorization of the controlled fermentative activity of the introduced cultures.
The recorded values are expressed in log CFU/mL and are graphically represented in Figure 2.
The presence of yeasts in kefir-based fermentation is indeed to be expected, as they are part of the natural microbial consortium of kefir cultures. However, the decrease in yeast and mold counts during refrigerated storage may affect the functional properties of the product, particularly its probiotic potential. Microbial density decreased over a 5-day period from 5.97 to 4.06 log CFU/mL, most likely due to the sensitivity of the yeasts to refrigeration conditions and to the phenolic composition of the juice. This reduction may limit the probiotic potential of the final product, indicating the need to adjust the fermentation conditions (e.g., higher temperature or addition of nutrients) to maintain high viability of the probiotic strains.

3.6.3. Detection of Staphylococcus sp. and Escherichia coli

During the microbiological analysis of the blueberry juices, specific tests were performed to detect the presence of pathogenic bacteria Staphylococcus sp. and Escherichia coli.
Thus, it was found that during the storage period of 5 days at 4 °C, these bacteria were not detected, being a short interval for a possible significant proliferation of these microorganisms, especially in pasteurized acidic juices.

4. Discussion

The control juice and the one inoculated with L. plantarum subjected to the thermostatation operation at 37 °C recorded a slight increase in pH, due to the fermentation conditions of this microorganism, but also the maintenance of almost constant pH values during the storage period. Instead, the control juice and the one inoculated with the kefir culture thermostatted at 24 °C showed a decrease in the pH values due to the resulting acids and carbon dioxide [37].
This acidification process is typical of kefir fermentation, which involves a symbiotic consortium of lactic acid bacteria (LAB) and yeasts. Slight pH stabilization after storage may be due to reduced microbial activity under refrigeration and the intrinsic buffering capacity of the juice.
The slight increase in pH observed in the samples inoculated with Lactobacillus plantarum can be explained by the strain’s ability to metabolize the organic acids naturally present in blueberry juice (such as malic or citric acid), which leads to a reduction in acidity. Additionally, the decarboxylation and deamination of phenolic compounds or amino acids may result in the formation of alkaline metabolites (e.g., ammonia), contributing to the pH increase. These mechanisms, combined with the buffering capacity of the natural compounds in the juice, may account for the deviation from the typical acidifying behavior of lactic fermentation.
The refractive index values increased in the case of the juices intended for inoculation with L. Plantarum, NPM and NP, from T0 moment to T1 moment, due to the fermentation operation at 37 °C. This process is accompanied by exopolysaccharides generation [39], an increase in the content of B vitamins [40,41], and mineral salts through the decomposition of mineral–polyphenol compounds [42], but also by hydrolysis processes with water consumption, which lead to the concentration of the products, and thus, to higher values of the refractive index [16]. Then, during the refrigeration storage, the refractive index values of these types of juice remained almost constant. In the case of the juices studied for the kefir cultures, the refractive index decreased slightly due to the fermentation process and the binding of sugars to anthocyanidins (malvidin) with the generation of anthocyanins [43], and then remained almost constant.
After the fermentation process, the polyphenol content increased remarkably: in the case of NP juice, it recorded the highest increase of 2.22 times at T1 compared to T0, and for NC, 2.13 times at T1 compared to T0. The significant increase in the polyphenol content is also correlated with other studies of the changes, due to the phenolic compounds being generated by the glucosidase produced by lactic bacteria, which led to phenolic metabolism and their release [4,44,45,46,47,48,49].
Also, the fermentation process produced by lactic bacteria led to a significant increase in the DPPH radical scavenging activity in the case of the juices inoculated with the bacterial strain (L. plantarum) and the kefir microbial consortium. Thus, in the case of NP juice, there is a 33.33% increase in the DPPH radical scavenging activity of T1 moment compared to T0, and in the case of NC juice, this increase is even about 38.86% at the moment T1 compared to T0. This increase in the DPPH radical scavenging activity is also found in other studies [50,51], according to which the radical scavenging abilities of the blueberry juices were significantly improved by the inoculation of lactic bacteria due to the structure and compositions of these bacteria or their metabolites; also, antioxidant activities of phenolic compounds and anthocyanin have been specified, because these compounds could break the chain reaction of free radicals by transporting hydrogen atoms or chelating metal ions through passing a single electron. Also, the DPPH radical scavenging activity values remained the highest for the two juices inoculated with lactic acid strains during the refrigerated storage period, recording quite high values after 5 days of 47.74% for NP and 40.66% for NC, which also presented the highest values of vitamin C concentration, of 1.32 mg%.
The microbiological characteristics of the analyzed juices are favorable, this being due to both the effect of pasteurization and the natural properties of blueberry juice. The research of Nualkaekul et al. (2011) [52] shows that L. plantarum inoculated into fruit juices survives very well at 4 °C, with only a modest decrease in viability over a 6-week storage period. Thus, in the case of blueberry juice with L. plantarum addition, a decrease in cell viability of ~0.5–1 log over the first 5 days is to be expected [53]. Although longer storage periods have demonstrated very good survival of L. plantarum, the aim of this study was to focus on early viability dynamics in order to optimize fermentation conditions and product quality during the critical initial phase of refrigerated storage. Future studies could extend the monitoring period to evaluate long-term viability and shelf life.
The initial value of 7.65 log CFU/mL and the decline to 6.55 log CFU/mL on moment T3 indicated the survival of L. plantarum under cold storage. Also, in the case of the blueberry juices inoculated with kefir, values ranged between 7.24 and 6.27 log CFU/mL over the same period. This aspect is beneficial for the analyzed juices, because other studies [54,55,56] also mentioned that maintaining a concentration above 6 log CFU/mL is recommended to ensure the probiotic effect, as well as to confer a biopreservation effect through mechanisms such as acidification and competition against undesirable microflora.
In the case of pasteurized juices, yeasts/molds are usually below the detection limit immediately after the treatment [57]. If there is a minimal contamination during storage after opening the containers, yeasts remain undetectable until around day 3 and can slowly reach ~0.5–1 log CFU/mL by day 5 [58]. This fact was also observed in the samples analyzed as controls with the addition of L. plantarum. For the juice with kefir inoculation, the yeasts gradually decreased at 4 °C, with a modest decline of ~0.5–1 log in the first five days, a fact also indicated by the research of Fan et al., 2018 [59], whose study monitored microbial dynamics over a 24-week refrigerated storage period. Usually, in an acidic environment and under refrigeration temperatures, molds are not significantly evident in the first 5 days.
Regarding the presence of pathogenic bacteria, various fruit juices that were heat-treated and stored at 4 °C showed undetectable levels of E. coli and S. aureus immediately after pasteurization and maintained this level for up to 7 days [60]. Blueberry juices have a naturally acidic pH which, together with the heat treatment, leads to the complete inactivation of vegetative bacteria, including S. aureus and E. coli, immediately after their processing [61]; this is also observed in the analyzed samples. An analysis of the effect of the phenolic fractions from blueberries demonstrated that residual extracts actively inhibit E. coli O157:H7 and S. aureus in liquid media [62], suggesting that the growth of these pathogens remains strongly inhibited.

5. Conclusions

This approach contributes to sustainability by promoting plant-based alternatives to fermented dairy products, valorizing local natural resources (such as blueberries), applying clean biotechnological processes (controlled fermentation without chemical additives), and potentially extending the shelf life of fresh plant-based products, thereby reducing food waste.
These juices contained also the prebiotics represented by the carbohydrates of the blueberry juice, in addition to the added probiotics, which maintained a high viability during refrigeration (from 7.24 to 7.65 to 6.27–6.55 log CFU/mL). The behavior of these functional products was also evaluated during a refrigeration storage of 5 days when the pH of the synbiotic juices maintained in stable ranges (e.g., 3.66 → 3.93 for L. plantarum and 3.93 → 3.66 for kefir juice). Both the synbiotic juices present optimal physicochemical and microbiological characteristics, including a higher vitamin C content (1.32 mg%) and increased levels of polyphenols after fermentation (up to 132.63 mg EAG/100 mL) compared to the control samples; thus, these products can be considered for further industrial production. Through innovative technologies and sustainable practices, the blueberry industry can develop a wide range of value-added products appealing to health-conscious and environmentally aware consumers.

Author Contributions

Conceptualization, A.D. and L.G.; methodology, A.D., Z.V. and C.M.; software, C.M.; validation, A.D., C.M. and L.G.; formal analysis, L.M.; investigation, A.D., Z.V. and C.M.; resources, L.M.; data curation, A.D., C.M. and Z.V.; writing—original draft preparation, A.D. and L.M.; writing—review and editing, A.D., Z.V. and L.G.; visualization, C.M. and L.M.; supervision, A.D. and L.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Acknowledgments

The authors acknowledge the Technical University of Cluj-Napoca, North University Center of Baia Mare, and the Bluettes Saveur blueberries plantation from Satu Mare, Romania for the material base provided.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Evolution of the total number of mesophilic aerobic bacteria (log CFU/mL) in the blueberry juices during the 5 days of storage.
Figure 1. Evolution of the total number of mesophilic aerobic bacteria (log CFU/mL) in the blueberry juices during the 5 days of storage.
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Figure 2. Yeasts and molds evolution (log CFU/mL) in the blueberry juices during the 5 days of storage.
Figure 2. Yeasts and molds evolution (log CFU/mL) in the blueberry juices during the 5 days of storage.
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Table 1. pH values of the blueberry juices at T0, T1, T2 and T3.
Table 1. pH values of the blueberry juices at T0, T1, T2 and T3.
Blueberry Juices CodeT0T1T2T3
pH
NPM3.66 ± 0.01 * a **, i ***3.67 ± 0.02 c, i3.86 ± 0.02 c, j3.93 ± 0.03 b, j
NP3.67 ± 0.01 a, i3.94 ± 0.02 b, j3.94 ± 0.03 b, k3.95 ± 0.02 b, j
NCM3.86 ± 0.02 b, j3.67 ± 0.03 a, i3.64 ± 0.03 a, i3.65 ± 0.02 a, i
NC3.93 ±0.02 b, k3.65 ± 0.02 a, i3.65 ± 0.02 a, i3.66 ± 0.03 a, i
* the data are expressed as the mean ± standard deviation of triplicate samples. ** Different letters a, b, c, in the same line indicate statistically significant differences at 95% confidence level according to Fisher’s least significant difference (LSD) procedure for the pH indicator at different moments. *** The letters i, j, k indicate the statistically significant differences at 95% confidence level between the data in the respective columns between the variant (type) of blueberry juices.
Table 2. Refractive indices values of the blueberry juices at moments T0, T1, T2 and T3.
Table 2. Refractive indices values of the blueberry juices at moments T0, T1, T2 and T3.
Blueberry Juices Code T0T1T2T3
Refractive Index
NPM1.3531 ± 0.0002 * a **, i ***1.3554 ± 0.0002 b, j1.3553 ± 0.0002 b, i1.3555 ± 0.0002 b, j
NP1.3542 ± 0.0003 a, j1.3553 ± 0.0002 b, j1.3552 ± 0.0002 b, i1.3551 ± 0.0003 b, ij
NCM1.3550 ± 0.0002 a, k1.3550 ± 0.0002 a, i1.3549 ± 0.0003 a, i1.3549 ± 0.0004 a, i
NC1.3554 ± 0.0001 b, l1.3552 ± 0.0001 a, ij1.3550 ± 0.0002 b, i1.3546 ± 0.0003 a, i
* The data are expressed as mean ± standard deviation of triplicate samples. ** Different letters a, b, in the same line indicate statistically significant differences at 95% confidence level according to Fisher’s LSD procedure for the refractive index at different moments. *** The letters i, j, k, l indicate the statistically significant differences at 95% confidence level between the data in the respective columns between the type of blueberry juices.
Table 3. Ascorbic acid (AA) concentration values of the blueberry juices at T0, T1, T2 and T3.
Table 3. Ascorbic acid (AA) concentration values of the blueberry juices at T0, T1, T2 and T3.
Blueberry Juices CodeT0T1T2T3
AA Concentration, mg%
NPM1.76 ± 0.02 * c **, i ***1.76 ± 0.02 c, i1.32 ± 0.02 b, i0.88 ± 0.03 a, j
NP1.76 ± 0.02 b, i1.76 ± 0.02 b, i1.32 ± 0.02 a, i1.32 ± 0.02 a. k
NCM1.76 ± 0.02 c, i1.32 ± 0.03 b, j1.32 ± 0.03 b, i0.44 ± 0.02 a, i
NC1.76 ± 0.02 c, i1.76 ± 0.02 c, i1.32 ± 0.03 b, i1.32 ± 0.01 b, k
* The data are expressed as mean ± standard deviation of triplicate samples. ** Different letters a, b, c, in the same line indicate statistically significant differences at 95% confidence level according to Fisher’s LSD procedure for AA concentrations at different moments. *** The letters i, j, k indicate the statistically significant differences at 95% confidence level between the data in the respective columns between the type of blueberry juices.
Table 4. DPPH radical scavenging activity of the blueberry juices T0, T1, T2 and T3.
Table 4. DPPH radical scavenging activity of the blueberry juices T0, T1, T2 and T3.
Blueberry Juices Code %T0T1T2T3
DPPH Radical Scavenging Activity
NPM38.55 ± 0.87 * c **, i ***34.33 ± 1.10 b, i46.23 ± 0.96 d, i18.37 ± 0.48 a, j
NP38.40 ± 0.92 a, i51.20 ± 0.49 d, k48.64 ± 0.69 c, j47.74 ± 0.41 b, l
NCM41.41 ± 0.56 b, j48.64 ± 0.87 c, j49.39 ± 0.98 c, j 16.71 ± 0.62 a, i
NC40.88 ± 0.90 a, j56.77 ± 0.68 c, l51.20 ± 0.63 b, k40.66 ± 0.58 a, k
* The data are expressed as mean ± standard deviation of triplicate samples. ** Different letters a, b, c, d, in the same line indicate statistically significant differences at 95% confidence level according to Fisher’s LSD procedure for DPPH radical scavenging activity at different moments. *** The letters i, j, k, l indicate the statistically significant differences at 95% confidence level between the data in the respective columns between the type of blueberry juice.
Table 5. Polyphenol concentration of the blueberry juices at moments T0, T1, T2 and T3, in mg-equivalent gallic acid (EAG)/100 mL.
Table 5. Polyphenol concentration of the blueberry juices at moments T0, T1, T2 and T3, in mg-equivalent gallic acid (EAG)/100 mL.
Blueberry Juices CodeT0T1T2T3
Polyphenols in mg EAG/100 mL
NPM125.61 ± 1.02 * b **, j ***127.41 ± 1.18 b, i120.91 ± 1.15 a, j126.85 ± 1.25 b, j
NP123.83 ± 1.10 a, ij274.84 ± 1.41 c, j122.72 ± 1.25 a, j132.63 ± 1.14 b, k
NCM123.74 ± 1.31 c, i126.07 ± 0.74 d, i114.34 ± 1.35 a, i120.32 ± 1.51 b, i
NC129.22 ± 0.95 a, k275.83 ± 1.48 c, j135.62 ± 1.23 b, k130.74 ± 1.08 a, k
* The data are expressed as mean ± standard deviation of triplicate samples. ** Different letters a, b, c, d, in the same line indicate statistically significant differences at 95% confidence level according to Fisher’s LSD procedure for the polyphenol’s concentration at different moments. *** The letters i, j, k, indicate the statistically significant differences at 95% confidence level between the data in the respective columns between the type of blueberry juice.
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Dumuta, A.; Vosgan, Z.; Giurgiulescu, L.; Mihali, C.; Mihalescu, L. Kefir and Lactobacillus plantarum Cultures in the Production of Fermented Blueberry Juices. Sustainability 2025, 17, 10488. https://doi.org/10.3390/su172310488

AMA Style

Dumuta A, Vosgan Z, Giurgiulescu L, Mihali C, Mihalescu L. Kefir and Lactobacillus plantarum Cultures in the Production of Fermented Blueberry Juices. Sustainability. 2025; 17(23):10488. https://doi.org/10.3390/su172310488

Chicago/Turabian Style

Dumuta, Anca, Zorica Vosgan, Liviu Giurgiulescu, Cristina Mihali, and Lucia Mihalescu. 2025. "Kefir and Lactobacillus plantarum Cultures in the Production of Fermented Blueberry Juices" Sustainability 17, no. 23: 10488. https://doi.org/10.3390/su172310488

APA Style

Dumuta, A., Vosgan, Z., Giurgiulescu, L., Mihali, C., & Mihalescu, L. (2025). Kefir and Lactobacillus plantarum Cultures in the Production of Fermented Blueberry Juices. Sustainability, 17(23), 10488. https://doi.org/10.3390/su172310488

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