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Article

Production of Bio-Improved Butter with Lactic Acid Bacteria Isolated from Traditional Cheese Matrix and Eye Fluid

by
Gokce Keser
and
Tulay Ozcan
*
Department of Food Engineering, Bursa Uludag University, Gorukle, Bursa 16059, Türkiye
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(11), 620; https://doi.org/10.3390/fermentation11110620 (registering DOI)
Submission received: 29 August 2025 / Revised: 17 October 2025 / Accepted: 29 October 2025 / Published: 31 October 2025
(This article belongs to the Special Issue Fermented Dairy Products: From Artisanal Production to Functional Products and Beyond)
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Abstract

This study aimed to investigate the effects of Levilactobacillus brevis, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus strains isolated from Mihalic cheese, also known as “weeping cheese”, on fermentation kinetics, microbial viability, and textural and aromatic properties of the butter matrix. The effects of the isolates were determined on acidification kinetics (Vmax, Tvmax, pHvmax), viability proportion index (VPI), textural parameters (firmness, work of shear, stickiness, work of adhesion), and volatile aroma compounds (GC-MS) formation. This study found that the BLR sample containing Lacticaseibacillus rhamnosus maintained its limited viability under acidic stress conditions despite its high fermentation rate and low pHvmax values. The BLP sample containing Lacticaseibacillus paracasei exhibited high viability due to its low acidification rate and limited pH change. Determining the chemical classes to which the aroma compounds in the BLP sample belonged revealed a composition rich in fatty acids. The BLB sample containing Levilactobacillus brevis produced a high ΔpH value and an aroma profile rich in aldehyde compounds. Examination of the macro-structural properties of the butter samples revealed that the sample containing Lacticaseibacillus rhamnosus, similar to the control sample (BMC), was more compact and rigid during storage. In contrast, samples containing Lacticaseibacillus paracasei and Levilactobacillus brevis had a softer/spreadable texture. These findings demonstrate the potential of lactic acid bacteria isolates from the traditional Mihalic cheese microbiota as biological catalysts for the development/improvement of texture, aroma, and sensory quality in high-fat dairy products and for the industrial production of products modified to meet consumer preferences.

1. Introduction

Mihalic cheese, one of the cheeses produced in the Marmara region of Türkiye, is also known among the public as “Weeping Cheese.” It is a semi-hard, high-salty, light yellow or straw-colored cheese traditionally made from raw sheep’s milk in the provinces of Bursa and Balikesir. Mihalic cheese is also known by different names, such as “Maglic,” “Mahlic,” “Manyas,” and “Kelle,” depending on the region [1,2]. Mihalic cheese is characterized by round eyes, 3–4 mm in diameter. These eyes are uniformly distributed throughout the cheese, decreasing in size toward the edges. While liquid inside these porous structures is considered necessary in consumer preferences, sparse and large eyes are undesirable. During fermentation, eyes are formed by the cheese’s mixed microbiota; during ripening, these eyes are filled with brine and aromatic fluids [1]. Manyas Kelle Cheese, unique to the Manyas district of Balikesir province, was granted a geographical indication in 2020 [3].
In traditional Mihalic cheese production, raw or pasteurized sheep’s milk and/or cow’s milk is mixed with rennet in wooden barrels called “polim.” The curd is cut into rice-sized pieces and boiled at 45 °C. Tools called “needles” are used to separate the whey, and the characteristic eye structure of the cheese is developed at this stage. Once the whey is separated, the curd is cut into 3–5 kg blocks and stored in 18% NaCl for 3 days, 20% NaCl for the next 2 days, and finally in a 22% NaCl brine for 15–30 days. The cheese blocks are placed in wooden barrels containing salt. The cheese is then covered with a brine containing 20–22% NaCl, and the barrels are sealed. The cheese is consumed after being matured in cold barrels (4 ± 1 °C) for approximately 3 months [4,5]. The microbiological, physicochemical, and sensory properties of traditional Mihalic cheese have been investigated in studies. Tirpanci-Sivri and Oksuz [6] identified propionic acid bacteria in 25 samples of traditionally produced Mihalic cheese using MALDI-TOF/MS. The identified isolates were Propionibacterium freudenreichii ssp. freudenreichii (57%), Propionibacterium freudenreichii ssp. shermanii (33%), and Propionibacterium thoenii (10%). In cheeses made from raw milk, such as Mihalic, intense lipolysis is inevitable due to the natural lipase. However, lipolytic agents also originate from rennet paste, starter, co-starter, non-starter bacteria, and exogenous lipase [7]. In a study conducted by Ozcan and Kurdal [1], a combination of lipase from Mucor miehei, protease from Bacillus subtilis, and Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. cremoris, and Lactococcus lactis subsp. diacetylactis cultures were used to accelerate ripening in Mihalic cheeses. The addition of both protease and lipase resulted in improved sensory properties. It was also stated that mesophilic aromatic starter cultures could be used in addition to heat treatment and lipase and protease enzymes to contribute to cheese ripening. Keser et al. [5] reported that using different starter cultures in traditionally produced Mihalic cheeses resulted in changes in proteolytic activity, leading to differences in the cheese’s firmness parameters. In Mihalic cheeses produced from raw and pasteurized milk, the total mesophilic lactic acid bacteria count during ripening was reported as 9.3 × 106–4.6 × 107 cfu/g, the total thermophilic lactic acid bacteria count as 1.0 × 109–1.2 × 109 cfu/g, and the propionic acid bacteria count as 1.2 × 108–6.6 × 108 cfu/g [8].
Fermentation is one of the oldest food protection and preservation methods, a processing method that enhances the characteristic flavor and texture of dairy products. In recent years, with the adoption of sustainable and traceable production approaches, indigenous/autochthonous culture studies have become an important area of fermentation [9,10,11,12]. LAB metabolizes proteins, lipids, and other organic substances during fermentation, forming aroma-active compounds such as aldehydes, alcohols, ketones, and esters. Consumers’ inclination to experience different aromatic flavors and experiences also drives research in the food field. Therefore, studies investigating the volatile and non-volatile aroma compounds formed by indigenous cultures in innovative, “green label (Earth-Friendly; food characterized as “Green” is produced sustainably, climate-protecting, and environmentally conscious)” dairy products have increased in recent years. Research is being conducted on the characterization of volatile components and the metabolic footprint of these untargeted volatile components in the fermented milk system [13,14]. This study investigated the metabolic effects of LAB, which form a unique ecosystem with traditional eye fluids and a cheese matrix, on butter, a milk fat-rich product, through fermentation. Furthermore, innovative and improvable approaches were presented for functional product design studies. The effects of fermentation with isolated strains of Levilactobacillus brevis (Lv. brevis), Lacticaseibacillus paracasei (Ls. paracasei), and Lacticaseibacillus rhamnosus (Ls. rhamnosus) on the microbiological, physicochemical, textural, and sensory properties of butter were evaluated. The impact of symbiotic effects in co-fermentation kinetics on aroma compounds was investigated using instrumental (un-targeted aroma compounds) and sensory (QDA) analyses. The results obtained from this study provide a new theoretical basis to produce high-quality, sustainable, and aromatic butter.

2. Materials and Methods

2.1. Materials

Strains

Lv. brevis, Ls. paracasei, and Ls. rhamnosus were isolated from Mihalic cheeses (Figure 1) collected from local producers in the cities of the Marmara region of Türkiye. The cultures were isolated according to our previous study [12]. The strains were first sub-cultured three times in the MRS broth (De Man Rogosa and Sharpe, Merck, Darmstadt, Germany) overnight at 35 ± 1 °C before use. The mixed culture (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoc mesenteroides subsp. cremoris) was obtained from Chr. Hansen (Hørsholm, Denmark).

2.2. Methods

2.2.1. Preparation of Butter Samples

Butter production was carried out according to the experimental design specified in Table 1. In production, 3% culture (Except for the control sample, cultures were added to all samples at a 1:1 ratio) was added to pasteurized cream at 25 ± 1 °C, and incubation was terminated when the pH reached 5.20–5.22. The cream was cooled at 10 °C for 3 h, and after cooling, buttermilk was separated by churning. Samples were washed with pasteurized water at 4 ± 1 °C and kneaded. The samples were refrigerated at 4 ± 1 °C for 90 days in vacuum packaging. Three replicates were performed per sample [15].

2.2.2. pH Measurement

The pH was measured using a WTW pH 3110 Set 2 portable pH meter (Xylem Analytics Germany Sales GmbH & Co. KG WTW, Weilheim, Germany). The calibration was performed using two buffer solutions at pH 4, and 7. All measurements were performed at 25 °C. In addition, Vmax (Maximum fermentation rate), Tvmax (Time to reach maximum fermentation rate), pHvmax (pH value at maximum fermentation rate), tend of pH 5.20 (Time for pH value to reach 5.20), and ΔpH (Drop rate) values were calculated during fermentation [16].
ΔpH = End of pH value − Beginning of pH value/Fermentation time

2.2.3. Viability Proportion Index (VPI)

Total LAB counts of butter samples were determined by diluting 1 mL of the sample in 9 mL of sterilized NaCl solution at appropriate ratios. The diluted solutions were then incubated on MRS and M17 agar at 37 °C for 72 h, and LAB was counted and expressed as colony-forming units per gram (log10 cfu g−1) [17]. The VPI of total lactic acid bacteria was calculated using the formula below [18].
VPI = Cell population at the end of storage (90th day)/Cell population at the beginning of storage (1st day) (log10 cfu g−1)

2.2.4. Aroma Compounds Analysis

Aroma compounds in butter samples were determined by modifying the parameters applied by Zianni et al. [19]. Headspace Solid-Phase Microextraction (HS-SPME) technique and Gas Chromatography-Mass Spectrometry (GC-MS) were used in the analysis of aroma compounds. 5 g of the sample was weighed and placed in a 20 mL headspace vial, and 1 g of NaCl was added. The vial was left on an automatic shaker (500 rpm) at 60 °C for 15 min. A 50/30 µm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was used for HS-SPME, and the samples were kept at 40 °C for 30 min. The analysis was conducted in splitless mode for 5 min at the 250 °C injection port. A 30 m × 0.25 mm × 0.25 µm thick HP-5MS column was used for GC. The temperature program consisted of an initial hold at 40 °C for 3 min, followed by an increase to 150 °C at 5 °C/min, and then to 250 °C at 10 °C/min for a 10 min hold. Helium was the carrier gas at a constant 1.0 mL/min flow. Ionization was performed in electron collision (EI, 70 eV) mode. Volatile compounds were identified based on the degree of matching of their mass spectra with the NIST library database. Aroma compounds were applied at the beginning of the storage (1st day).

2.2.5. Textural Analysis

Texture Analyser TA-XT Plus (Stable Micro Systems, Godalming, Surrey, UK) was used for textural properties. It was equipped with a TTC Spreadability Rig (HDP/SR), comprising a 90° cone probe and precisely matched perspex cone-shaped product holders. The measurements were carried out on one surface of the rectangular 5 cm × 3 cm block of butter at after keeping the samples at room temperature (20 °C) for 10 min (~10 °C). Parameters including firmness (g), work of shear (g.s), stickiness (g), and work of adhesion were determined (g.s) [15,20].

2.2.6. Quantitative Descriptive Analysis (QDA)

Academic staff and graduate students were selected as panelists and completed training in the sensory evaluation of foods according to ISO (8586) [21] guidelines. Panelists were trained to identify aroma, taste, odor compounds, and textural properties commonly found in dairy products. Ten selected panelists were retrained to recognize and perceive standard aroma and taste compounds. A 15-point scale was used to measure the intensity of taste, aroma, and textural properties in butter samples (0: The property was not perceived at all, 15: The property was perceived very intensely). Butter samples were presented to the panelists at 8–10 °C, coded with three-digit letters. BMC, BMB, BMP, and BMR samples were presented to the panelists on disposable plates, respectively. They were also served unsalted pieces of bread and water to help them clear their palates during the presentation. The panel was repeated three times under the same conditions but at different times. In our study, 10 trained panelists participated in the sensory evaluation, and mean scores for each parameter were calculated from 10 individual evaluations per sample. Each panelist evaluated all samples independently under controlled conditions. The scores of the panelists were used to calculate the average results. Panelists were asked to record their scores on the prepared sensory forms. The panelists also obtained consent forms [13]. The descriptors used and their definitions are given in Table 2.

2.2.7. Statistical Analysis

The butter samples’ fermentation kinetics and microorganism viability were used to evaluate Single-factor ANOVA, while multifactor ANOVA was used to determine textural and sensory properties. Differences between samples were then determined by applying the Fisher test based on the significance of p values (<0.05 or <0.01). Statistical analyses were performed using Minitab 17. Principal Component Analysis (PCA) was used to determine variations in aromatic compounds based on the chemical classes detected in the samples. PCA was performed using the chemical classes (alcohol, aldehyde, ketone, lactone, fatty acid, and hydrocarbon) to which the volatile aroma compounds identified in each sample (BMC, BLB, BLP, BLR) belonged. The spatial distribution of the samples and the relationships between chemical classes were interpreted to evaluate differences caused by the isolated culture. The results were summarized in PCA and graphics using XLSTAT (2021 version, Addinsoft, New York, NY, USA).

3. Results and Discussion

3.1. Acidification and Microorganism Viability

The results of the fermentation activity parameters of butter samples are presented in Table 3. The values representing the maximum rate of acidification during fermentation (Vmax) were significantly different between the samples (p < 0.01). The BLR sample containing Ls. rhamnosus was found to have the highest fermentation rate, and this activity indicated that the bacteria caused faster acidification in lactic acid production compared to the other isolates. The BLR sample also had the longest to maximum acidification during fermentation (Tvmax), indicating a late onset of acidification during fermentation. This time was shorter in the BLP sample, indicating that fermentation began earlier but acidification developed more slowly. The pH value at maximum fermentation rate (pHVmax) was similar in the BMC and BLP samples and lower in the BLR and BLB samples. It has been determined that Ls. rhamnosus and Lv. brevis are more resistant to acidic environments and maintain their activity at lower pH values. Recent studies have determined that some lactic acid bacteria maintain their metabolic activity under low pH conditions. Nasr and Abd Alhalim [22] determined that the LAB isolates from fermented dairy products resistant to gastrointestinal conditions belonged to Ls. rhamnosus strains. They also suggested that these strains possess probiotic potential and could be easily adapted to the fermented dairy industry due to these characteristics. Similarly, Ls. rhamnosus has been reported to cause rapid pH drops in fermentation media containing different carbon sources, an indicator of the physiological adaptability of this microorganism to low pH values [23]. Han et al. [24] reported that an acid-tolerant Lv. brevis strain can sustain fermentation biometabolism at low pH values. A study evaluating isolated Lv. brevis in the production of ginseng-fortified yogurt reported that the bacteria can survive even in acidic environments [25]. The results of the in vitro and food matrix studies are similar to the data obtained in this study, further supporting the successful use of these strains in developing functional dairy products. When the pH values of the samples were examined, it was determined that BLP reached pH 5.20 the earliest. It was determined that Ls. paracasei easily adapted to the environment and decreased pH in a shorter time with the development of acidity. The highest pH change was defined in the BLB (Lv. brevis) sample. The lowest ΔpH value was determined in the BLP (Ls. paracasei) sample. It was determined that Ls. paracasei caused a more limited pH change during fermentation (Table 3). The differences detected between fermentation times may cause changes depending on the ability of each bacterium to utilize carbon and nitrogen sources [26].
Ls. rhamnosus caused a high fermentation rate and low pHvmax, resulting in remarkably rapid acidification in the BLR sample. The BLB sample had the highest ΔpH value and high acidification capacity. However, the BMC and BLP samples had a lower fermentation rate and a more limited pH decrease, resulting in a controlled fermentation compared to the other samples. The study results reveal the different strain-based effects of Lactobacillus spp. on fermentation kinetics. Similarly, in the various research, Ls. rhamnosus has been reported to have the ability to produce fast acid, while Lv. brevis has the potential to produce high acidity [26,27]. Sezer et al. [28] and de Souza Oliveira et al. [29] reported that Ls. rhamnosus exhibited similar fermentation properties in ice cream and whey media. Fan et al. [27] determined that Lv. brevis could lower the pH to 4.3 in a milk-based fermentation system and had acid tolerance. However, Costa et al. [30] reported that fermentation progressed more slowly in Ls. paracasei and S. thermophilus co-culture systems, and the pH decrease was more controlled. The different acidification profiles detected depending on the strains used paralleled the kinetic properties defined based on the genetic and metabolic characteristics of the bacteria.
VPI is an important parameter indicating microorganism viability during fermentation. The results of the VPI values of butter samples at the end of storage are given in Table 4. It was determined that the total LAB count on M17 agar had higher VPI values than the BMC and BLP samples, and that viability was preserved. Although the lowest viability rate was detected in the BLR sample, it was >1 at the positive growth value. The lactic acid bacteria (on MRS agar) count had the highest VPI value in the BMC sample. The VPI value of the LAB count (on MRS agar) in the BLR sample was <1, and their viability was determined at lower levels. These results have significant variations in terms of preservation of viability depending on the strain (p < 0.01). Sezer et al. [28] determined that bacterial viability was preserved up to 95.9% at the end of 90 days at −25 °C in prebiotic-supported ice cream formulations containing Ls. rhamnosus and S. thermophilus. Yilmaz-Ersan et al. [31] also reported adding L. acidophilus and Ls. rhamnosus to a butter matrix resulted in viability of ≥6 log10 cfu g−1 after 22 days of storage. Hurtado-Romero et al. [32] reported that Ls. rhamnosus maintained its viability for 30 days at −20 °C in fermented milk-based frozen products. Compared with these studies, the present study showed high viability of total LAB counts on MRS and M17 agar, especially in BMC and BLP samples, and was determined to be suitable for probiotic stability. It has also been reported that bacteria exposed to shear stress in mechanically technology-based W/O systems, such as butter, can adhere well in the gastrointestinal tract, suggesting a health-improving effect [33,34]. However, the VPI value was 0.928 in the sample containing Ls. rhamnosus can be explained by developmental interactions between species and the effects of metabolic byproducts on cell stability [30]. Although Ls. rhamnosus can develop rapid acidification, it can be exposed to stress in environments such as butter. Furthermore, Sebastián-Nicolás et al. [26] reported decreasing the pH of Ls. rhamnosus GG disrupted cell membrane stability and reduced cell viability.
Furthermore, Fan et al. [35] determined that Ls. rhamnosus viability decreased in yogurt samples under acidic stress, and cell proliferation ceased when optimal conditions were not provided. It has been reported that the formation of lactic acid and other organic acids during the fermentation process inhibits the proliferation of lactic acid bacteria, thus limiting cell division [36]. As a result, the lower VPI value in the BLR sample may be related to the formation of acidic stress with rapid pH decrease and nutrient deficiency by preventing diffusion in the fat matrix.

3.1.1. Textural Properties

Butter and butter alternative products are physically composed of fat globules, fat crystals, air bubbles, and water droplets. All these structures play an essential role in the physical and chemical properties of the products [37]. The results for the textural properties of the butter samples are presented in Table 5. The firmness of the BMC (control) sample was high throughout the storage period. Still, it did not cause a statistically significant difference (p > 0.05), and the structure was found to maintain stability throughout storage. The firmness values of the BLB sample decreased on the 30th and 60th days of storage, but by the 90th day, they were similar to the beginning of the storage period. The firmness values of the BLP sample decreased significantly on the 60th day of storage. The BLR sample had lower firmness values throughout the 60th day, but firmness increased on the 90th day. Consequently, a more compact structure was formed with storage. This increase at the end of storage can be explained by the effect of Ls. rhamnosus’s proteolytic activity and EPS production ability, which occur in the late storage period [38,39]. While the work of the shear parameter reached its highest value on the 90th day of storage in the BMC sample, a significant increase was observed in storage time in the BLR sample. This is an indication of the rheological change that occurs during maturation. High shear work means it exhibits a cohesive structure requiring more energy. In particular, the increase in the work of shear value of BLR on the 90th day of storage created a more resistant structure due to its firmness. Stickiness values did not change in the control sample (BMC), but decreased in the BLB and BLP samples on day 60 of storage. However, this decrease was not statistically significant (p > 0.05). These values increased in the sample containing Ls. rhamnosus and a more adhesive structure emerged at the end of storage. Work of adhesion values decreased on day 30 of storage in the BLB sample but subsequently increased. Generally, lower values were detected in the BLP sample. The BLR sample’s lowest work of adhesion value was observed on day 60, and the highest on day 90. This increase in the BLR containing Ls. rhamnosus at the end of storage suggests that the adhesion capacity of the oil globules in the structure also increased. Determination of overall textural properties revealed that the BLR sample (Ls. rhamnosus) developed its structure during storage, increasing its hardness, shear strength, and adhesive properties.
In contrast, the BLB and BLP samples developed a more unstable structure. Based on the results of this study, it can be concluded that Ls. rhamnosus is the preferred starter culture for improving textural properties in high-fat products, while Lv. brevis and Ls. paracasei are preferred for developing products with softer and more spreadable properties. These findings suggest that microbial cultures shape the final textural properties of the product not only through their effects at the beginning of fermentation but also through the secondary metabolite activities that emerge during long-term storage. While unsaturated fatty acids in butter reduce the hardness of the fat due to their low melting point, saturated fatty acids increase the hardness of the fat. For this reason, Silva et al. [40] found that the ratios of saturated and unsaturated fats in the fatty acids in the butter structure could cause changes in textural properties. Figueiroa et al. [41] and Ozcan et al. [42] explained that storage temperature causes differences in the textural properties of butter, and that this is due to the change in physical properties (transition from solid to liquid state) caused by the amounts of oleic acid (C18:1; melting point: 13–16 °C) and caprylic acid (C8; melting point: 16–17 °C). Ronholt et al. [43] and Tondhoosh et al. [44] reported that hardness significantly increased in butter obtained from cream ripened by slow cooling (due to the formation of a denser crystal network), while brittleness significantly decreased in butter obtained from cream ripened by rapid cooling. In a study evaluating spreadable milk fat products and butters with different fat content in terms of consumer preferences, it was determined that the shear force of the sample with higher fat content was higher, regardless of temperature.
Furthermore, Chudy et al. [45] reported that the shear force of milk fat samples at 4 °C was higher than that measured at 20 °C. The interactions between the crystals forming the network structure prevent the aggregation of liquid fat and crystal clusters [46]. Consequently, Karakus et al. [47] reported that weak bonds between crystals and the network structure lead to reduced hardness, improved spreadability, and a more cohesive product. The chemical properties of milk fat (lipid profile and fat globule size) significantly influence shear force. Various researchers have reported that the best spreadability (low hardness and shear work) is achieved in samples with low fat and high moisture content [45,48,49,50]. In many studies, the wide range of textural properties of milk fat-based products and butter samples has led to inconsistencies in comparing results.

3.1.2. Aroma Compounds

Dairy products produced by lactic fermentation are therapeutic foods with high nutritional value and improved functional and sensory properties. Starter cultures that produce fermented dairy products metabolize milk components to form the basic flavor compounds that constitute the aroma and flavor profile. These metabolites also provide growth-promoting or -inhibiting properties for other bacteria in the consortium [51,52]. The retronasal aroma compounds produced in these products are a multifaceted phenomenon that develops through the interaction of microbial and biochemical processes. These processes occur through two primary reactions: primary (glycolysis, proteolysis, lipolysis), and secondary (amino acid and fatty acid metabolism) [53].
The results for the main aroma compounds detected in the butter samples are given in Figure 2. High amounts of aldehyde compounds were detected in all samples. The aroma profile of the BLB sample containing Lv. brevis consisted primarily of aldehydes. Aldehyde amounts were lower in the BMC, BLP, and BLR samples. Fatty acid content was determined in higher amounts in the BLP and BMC samples than in the others. Alcohol compounds were found in lower amounts in the BLB and BMC samples. The formation of alcohol compounds is associated with the biometabolism of Ls. paracasei and Ls. rhamnosus. Ketone compounds were detected only in the BMC (control) sample, and hydrocarbon compounds were detected only in the BLR (Ls. rhamnosus) sample. Volatile ketone compounds such as acetaldehyde, diacetyl, butanone, and acetone are formed through the metabolism of lactose, a milk component [54]. Proteolytic bacteria are used as precursors in the formation of free amino acids by degrading milk proteins, and these amino acids are used as precursors in the formation of aroma compounds such as esters, lactones, ketones, and sulfur compounds [13,53]. The hydrolysis of triglycerides by the lipase activity of bacterial cultures produces short-chain free fatty acids, which have low perceptual thresholds and significantly affect aroma [13,53]. The amount of fatty acids in the BLP sample containing Ls. paracasei determined that this bacterium has lipolytic properties. It was stated that isolated Lactiplantibacillus plantarum P9 not only improves the sensory quality of the products by increasing the amount of fatty acids and some bioactive peptides, but also improves their functional properties [55]. It was determined that Ls. casei Zhang strain isolated from Koumiss enhanced the aroma profile by increasing the production of acetaldehyde and 2,3-butanedione during fermentation [56]. In this study, the distribution of volatile compounds, determinants of the aroma profile in high-fat milk samples, varied significantly depending on the microorganism species used. In the BMC sample containing mixed culture, it was determined that aldehydes (55%), ketones (10%), lactones (20%), and fatty acids (15%) formed a balanced distribution. Similarly, it is reported in various research that cultures play an active role in synthesizing lactones and ketones [13]. In the BLB sample containing Lv. brevis, the aroma profile was reduced mainly to aldehydes (over 95%), indicating that microbial metabolism resulted in limited compound diversity. Similar findings have been reported in Lv. brevis, which increases aldehyde production [57]. In the BLP sample, the alcohol (10%) and fatty acid (50%) ratios increased significantly under the influence of Ls. paracasei, which is associated with lipid and carbohydrate metabolism during fermentation. These results are parallel with studies confirming that Ls. paracasei can metabolize lipid compounds and its effect on alcohol production [58]. The BLR sample containing Ls. rhamnosus had high hydrocarbon (25%) and alcohol (20%) contents. It is stated that hydrocarbon compounds are among the aroma compounds formed by the biometabolism of Ls. rhamnosus and that the amount of these compounds increases. It has been reported that Ls. rhamnosus can synthesize hydrocarbon compounds from free fatty acids. The aroma composition varies depending on the isolated LAB species, and this variation is considered very important in terms of the sensory properties of foods [59].
PCA was performed to reveal differences in the classification of aroma compounds detected in butter samples (BMC, BLB, BLP, BLR) based on their chemical properties (Figure 3). F1 and F2 components explained 51.16% and 29.32% of the total variance, respectively, and the total was determined to be 80.48%. The BMC sample was associated with compounds with ketone and lactone structures, which explains the creamy and butter-like aromas of samples containing mixed starter cultures. The BLP sample was associated with fatty acids such as tetradecanoic and pentadecanoic acids, and these compounds are reported to increase mouthfeel and creamy taste in dairy products [60]. The BLB and BLR samples are located close to each other in the PCA plane and have a positive correlation with aldehyde compounds. Aldehydes are formed by lipid oxidation and amino acid catabolism. Compounds with aldehyde and ketone structures stand out as the most prominent chemical aroma groups used to detect differences between samples due to their low sensory thresholds [61]. The PCA plot has been evaluated as an effective multivariate analysis method for visualizing differences in aroma composition in microbial cultures and distinguishing samples based on their chemical properties. Furthermore, increased aldehyde levels and reactions, particularly among aroma compounds, have been reported to be associated with flavor changes, leading to reduced shelf life, off-odor formation, textural defects, and decreased nutritional value. Panseri et al. [62] reported different flavor and aroma compounds formed during storage in butter samples produced using sour and sweet cream.

3.1.3. Sensory Properties

The bacterial strains used in fermentation significantly affect the sensory properties of fermented dairy products. The ability of different microbial isolates to produce aroma compounds diversifies the final product’s taste, odor, and texture. Butter samples fermented using different isolated cultures (BMC, BLB (Lv. brevis), BLP (Ls. paracasei), and BLR (Ls. rhamnosus)) were evaluated based on 27 sensory descriptors, and the obtained data were analyzed using heatmap-based hierarchical cluster analysis. The results are presented in Figure 4. To reveal differences, sensory properties were evaluated based on appearance, taste, aroma, and textural parameters. The cluster analysis showed that the BLB and BLR samples were located in Cluster 0 and had particularly high correlation values in textural parameters such as smoothness, spreadability, springiness, and mouth coating. Similarly, studies have also shown that Lv. brevis and Ls. rhamnosus strains have been reported to promote positive textural properties in dairy products, such as creaminess, spreadability, and low particle size perception by consumers [63,64]. BMC and BLP samples are located in Cluster 1 and stand out in this group with aroma and taste characteristics such as acetaldehyde, cheesy aroma, fermented taste, butyric acid taste, and bitterness. Studies have shown that Ls. paracasei improves fermentation aroma by increasing the formation of Strecker aldehydes and volatile compounds from amino acids through its proteolytic activity [59,65]. In general, it has been shown that the starter cultures used in fermented products have a decisive effect on the structural integrity and mouthfeel of the products, as well as their sensory aromatic profiles. In this context, it has been demonstrated, in line with the literature, that the selected culture combinations enable holistic sensory optimization regarding final product quality [66]. In this study, the BLR sample containing Ls. rhamnosus was the most similar to the control sample regarding sensory properties. This suggests that Ls. rhamnosus could be selected to create a product for consumers who demand similar textural properties due to its mouthfeel and stability, and to provide functional properties. It was also concluded that the samples containing Ls. paracasei and Lv. brevis could be used in producing various food products (such as sauces, cream-filled products, and desserts) due to their softness.
The results of the QDA evaluations performed on the butter samples are presented in Table 6. In all samples, fermented taste, creaminess, and spreadability were found to increase significantly at the end of storage (90th day), while sweet taste, melting, diacetyl, and fluidity decreased. While the pronounced bitterness and butyric acid-derived flavors in the BMC and BLP samples were found to be limiting for shelf life, the BLB and BLR samples achieved a more balanced, creamy, and matured sensory profile. Physically, the products were perceived as smoother, stickier, and yellow color at the end of storage, and their particle size and thickness decreased, resulting in a more homogeneous structure.
Sensory evaluation results and instrumental texture measurements demonstrated a consistent relationship between the perceived and measured textural properties of butter samples throughout storage. Both analysis methods revealed that lactic acid bacteria isolated from traditional cheese and eyewash significantly affected the texture and sensory properties of butter throughout storage. Firmness and spreadability values were negatively correlated in sensory and instrumental analyses. While the control sample (BMC) had relatively higher firmness values throughout storage, fluctuations in firmness were more pronounced in samples containing isolated bacteria, particularly in the BLB and BLR groups, suggesting that microbial activity modulates fat structure. Sensory data also support this; at the end of the storage period, spreadability values increased significantly in all samples containing isolated bacteria, particularly in the BLB and BLR samples. This is consistent with the decrease in instrumentally measured firmness and is associated with improved spreadability and a softer mouthfeel. The increase in smoothness values observed during storage parallels the decrease in instrumental hardness and work of shear values observed in some inoculated samples (especially BLB and BLP). This suggests that microbial or enzymatic activities affect fat globule distribution and interfacial properties, causing partial softening in the butter matrix. Consistent results were also obtained between the two analyses for stickiness. Sensory stickiness values increased during storage, while instrumental stickiness values remained generally stable, but in inoculated samples, they showed less change at the beginning of storage. This increase in panelists’ perception of stickiness is likely attributable to changes in the fat phase or to increased surface stickiness caused by exopolysaccharides produced by lactic acid bacteria.
Regarding work of shear, the control sample (BMC) maintained the highest values throughout storage, consistent with the high hardness values. In contrast, the work of shearing in the BLB and BLP samples was initially lower and increased again at day 90. Sensory springiness and smoothness results also support this finding, suggesting that microbial activity may be due to water-oil-protein interactions over time. Work of adhesion values remained generally similar across samples, with a slight decrease in the inoculated groups. This finding is consistent with the sensory perception of increased spreadability and decreased graininess. In general, butter samples containing lactic acid bacteria from traditional cheese, particularly the BLB and BLP groups, exhibited a balanced and favorable texture profile in terms of firmness, smoothness, and spreadability throughout storage.

4. Conclusions

This study investigated the fermentation kinetics, textural, sensory, and aroma components of Lv. brevis, Ls. paracasei, and Ls. rhamnosus strains isolated from the mixed culture ecosystem of traditional Mihalic cheese. This study concluded that Ls. rhamnosus caused the highest fermentation rate and a rapid increase in acidity in the BLR sample. Furthermore, increasing the firmness and shear parameters of this sample during storage allowed the development of a more cohesive product. In general, butter samples containing lactic acid bacteria from traditional cheese, particularly the BLB and BLP groups, exhibited a balanced and favorable texture profile in terms of firmness, smoothness, and spreadability throughout storage. Butter produced with LAB may contribute to the uptake of beneficial metabolites, but it should be considered as a supplementary carrier and not the primary source of probiotics in the diet. These results confirm that they may contribute to the development of industrial starter culture combinations and guide the production of innovative, sustainable, and value-added functional products. Furthermore, revealing the changes induced by the strains isolated in this study through genomic and transcriptomic genetic studies will significantly contribute to the more traceable characterization of aroma profiles.

Author Contributions

Conceptualization and Methodology, T.O.; Software, G.K.; Validation, T.O.; Formal Analysis, G.K.; Investigation, T.O.; Data Curation, T.O. and G.K.; Writing—Original Draft Preparation, G.K. and T.O.; Writing—Review & Editing, T.O.; Supervision, T.O.; Project Administration, T.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Bursa Uludag University (FOA-2022-1074).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in this article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 11 00620 g001
Figure 1. Image of Mihalic cheese produced with the traditional method.
Figure 1. Image of Mihalic cheese produced with the traditional method.
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Figure 2. Relative content of volatile aroma classifications in samples. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus.
Figure 2. Relative content of volatile aroma classifications in samples. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus.
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Figure 3. PCA analysis of volatile compounds in samples. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus. Lowercase letters indicate differences between samples.
Figure 3. PCA analysis of volatile compounds in samples. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus. Lowercase letters indicate differences between samples.
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Figure 4. HCA of the mean QDA values of samples. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus.
Figure 4. HCA of the mean QDA values of samples. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus.
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Table 1. Experimental design of butter samples.
Table 1. Experimental design of butter samples.
SamplesMixed CultureIsolated Cultures
Lv. brevisLs. paracaseiLs. rhamnosus
BMCX
BLBXX
BLPX X
BLRX X
X: The presence of the component is expressed in the samples.
Table 2. Descriptive attributes and definitions.
Table 2. Descriptive attributes and definitions.
DescriptorsDefinitions
Appearance Properties
OpacityThe degree to which a product prevents light from passing through
Yellow ColorVisual perception of yellowness
Brightness The surface of the sample appears bright to matte
Taste, Aroma, and Flavor Properties
Creamy AromaSweet and fatty cream
Cheesy AromaThe aroma of fresh cheese
Buttery AromaFresh or sweet cream
Fermented FlavorTypical fermented yogurt aroma and taste
Acetaldehyde AromaSharp, green, pungent, or fruity aroma, often described as green apple or unripe fruit.
Sweet TasteThe basic taste sensation of sugars or sweeteners
Sour TasteA basic taste associated with acids such as lactic or citric acid
Bitter TasteA basic taste perceived from substances like caffeine or certain peptides
AstringencyA drying, puckering mouthfeel
Diacetyl AromaA buttery or creamy aroma
Butyric Acid AromaThe fatty acid with a strong, rancid, or cheesy odor
Milk Fat AromaThe perception of natural milk fat
Margarine FlavorA flavor often associated with vegetable oils
Textural Properties
SmoothnessA texture without roughness; smooth to the touch
StickinessSticks to any of the teeth, gums, or palate
SpringinessThe elasticity
GrainyThe presence of small particles
SpreadabilityThe ease with which a product can be spread over a piece of bread
Porous StructureNon-homogeny
MeltingTransition from solid to liquid at mouth temperature
Particle SizeThe perceived or measured size of solid components
ThicknessThe perceived or actual body or viscosity of the product
FluidityThe ease with which a product flows, refers to the ability of a substance, particularly lipids, to flow and change shape without breaking apart.
Mouth CoatingThe extent to which the product leaves a coating on the tongue or palate after swallowing
Table 3. Acidification properties of butter samples.
Table 3. Acidification properties of butter samples.
SamplesVmaxTvmax
(min.)		pHvmaxTend of pH 5.20
(min.)		ΔpH
(Beginning of pH-Vmax pH)
BMC0.00483 c425 d5.33 a515 a0.98 c
BLB0.00700 b435 b5.22 b465 c1.12 a
BLP0.00500 c420 c5.31 a450 d0.92 d
BLR0.00840 a475 a5.20 b475 b1.07 b
p**********
BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus. Vmax: Maximum fermentation rate, Tvmax: Time to reach maximum fermentation rate, pHvmax: pH value at maximum fermentation rate, Tend of pH 5.20: Time for pH value to reach 5.20, and ΔpH: Drop rate. **: p < 0.01, lowercase letters indicate differences between samples.
Table 4. Viability Proportion Index (VPI) of butter samples.
Table 4. Viability Proportion Index (VPI) of butter samples.
SamplesLAB (M17 Agar)LAB (MRS Agar)
BMC1.084 a1.043 a
BLB1.077 b1.020 c
BLP1.081 a1.035 b
BLR1.008 c0.928 d
p****
BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus. ** p < 0.01, lowercase letters indicate differences between samples.
Table 5. Textural properties of butter samples.
Table 5. Textural properties of butter samples.
Texture ParametersSamplesStorage Time (Day)
1306090
FirmnessBMC2925.48 ± 119.710 aA1894.64 ± 273.185 abB3621.18 ± 287.785 aA3687.59 ± 382.608 aA
BLB2887.16 ± 332.743 aA1659.77 ± 182.829 bcB1869.32 ± 148.499 bB2960.71 ± 198.962 aA
BLP2290.16 ± 229.227 aA2198.76 ± 131.972 aA 1589.87 ± 93.134 bB2450.47 ± 267.039 aA
BLR2005.12 ± 252.595 aB1223.31 ± 17.240 cC 1437.63 ± 127.938 bBC3760.27 ± 220.730 aA
Work of ShearBMC5229.89 ± 269.522 aA2665.12 ± 298.425 aB3976.62 ± 236.437 aAB4572.59 ± 295.459 aA
BLB4353.81 ± 271.059 abA2147.27 ± 198.731 abB2619.41 ± 220.866 bB3937.68 ± 145.371 aA
BLP3143.74 ± 236.104 bcA2568.90 ± 140.179 aA2231.35 ± 151.135 bA3137.51 ± 295.475 aA
BLR2916.76 ± 202.475 cB1402.18 ± 120.145 bD1957.24 ± 94.035 bC4189.68 ± 111.147 aA
StickinessBMC−2269.92 ± 49.245 aA−1444.26 ± 122.698 aA−2506.24 ± 146.285 aA−2252.14 ± 196.717 aA
BLB−2312.16 ± 96.589 aA−1251.50 ± 54.667 abA−1290.46 ± 65.189 bA−1831.86 ± 87.644 aA
BLP−1594.58 ± 93.049 bA−1679.03 ± 113.816 aA−1174.73 ± 79.847 bA−1734.93 ± 228.461 aA
BLR−1646.90 ± 129.556 bB −884.13 ± 107.554 bC−1069.15 ± 65.304 bBC−2621.79 ± 126.832 aA
Work of
Adhesion		BMC−424.58 ± 41.302 aA−424.06 ± 25.990 aA−505.96 ± 28.680 aA−652.71 ± 54.730 aA
BLB−494.48 ± 71.132 aA−365.31 ± 33.216 aB−416.96 ± 49.382 abAB−475.45 ± 29.226 abA
BLP−356.00 ± 30.163 aA−405.64 ± 32.139 aA−348.31 ± 37.472 bcA−300.92 ± 15.508 bA
BLR−460.17 ± 46.328 aA−303.11 ± 38.863 aB−258.38 ± 21.886 cB−427.43 ± 38.656 bA
BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus. Lowercase letters indicate differences between samples. Uppercase letters indicate differences between storage times.
Table 6. Sensory properties of butter samples.
Table 6. Sensory properties of butter samples.
BMCBLBBLPBLR
Storage Time (Day)190190190190
Appearance Properties
Opacity6.40 aA7.80 aA5.20 abB7.20 aA5.20 abB6.40 abA5.80 abA6.00 abA
Yellow Color6.20 aB9.20 aA6.60 aB8.60 aA7.20 aA7.40 abA6.80 aA6.80 abA
Brightness9.40 abA9.00 abA10.00 aA10.00 aA10.40 aA10.60 aA9.60 abA10.60 aA
Taste, Aroma, and Flavor Properties
Creamy Aroma7.80 bB8.40 aA8.40 aB9.20 aA7.60 bA7.80 bA7.46 bB8.80 aA
Cheesy Aroma0.00 aA0.00 aA0.00 aA0.00 aA0.00 aA0.20 aA0.00 aA0.00 aA
Buttery Aroma10.60 aA10.00 bA11.20 aA10.80 aA11.00 aA9.60 bB10.76 aA11.00 aA
Fermented Flavor5.80 abA5.80 bA6.40 aA7.00 aA7.10 aA8.40 aA6.10 aB8.40 aA
Acetaldehyde Aroma4.00 bB6.00 bA4.80 aB6.80 aA5.00 aB6.60 aA4.56 aB6.60 aA
Sweet Taste8.40 aA6.00 aB9.60 aA6.20 aB6.80 bA5.40 bB8.80 aA5.30 bB
Sour Taste4.30 bB5.80 bA5.40 aB7.00 aA6.40 aB7.80 aA4.32 bB7.00 aA
Bitter Taste0.00 bB2.80 aA0.00 bA0.00 bA0.00 bB3.20 aA0.00 bA0.00 bA
Astringency Taste0.67 abA0.52 abA0.91 aA0.82 aA1.19 aA1.10 aA1.24 aA1.26 aA
Diacetyl Aroma5.80 abA4.00 aB6.20 aA4.00 aB6.20 aA4.40 aB6.04 aA4.20 aB
Butyric Acid Taste0.20 aB2.00 aA0.40 aB2.00 aA0.60 aB1.60 aA0.20 aB1.80 aA
Milk Fat Aroma7.40 aA7.40 aA8.20 aA5.80 bB8.00 aA5.00 bB7.66 aA5.40 bB
Margarine Flavor0.00 bB1.00 aA1.80 aA0.00 bB0.00 bA0.00 bA0.00 bA0.00 bA
Textural Properties
Smoothness7.40 aB12.60 aA7.80 aB11.60 aA7.80 aB12.40 aA7.80 aB9.20 abA
Porous Structure0.00 bA0.00 bA0.00 bB3.00 aA1.44 aA0.00 bB0.00 bA0.00 bA
Stickiness7.80 aB9.40 aA8.20 aA8.40 aA8.00 aB9.40 aA7.50 aB9.00 aA
Spreadability6.40 aB9.80 aA7.00 aB10.00 aA6.50 aB9.60 aA4.58 bB8.80 abA
Springiness7.60 abA6.60 bA8.40 aA8.20 aA8.40 aA6.00 bAB7.80 abA6.00 bAB
Grainy0.40 bB1.40 aA0.00 bB1.38 aA1.62 aA0.24 bB0.19 bB1.80 aA
Melting7.60 aA3.40 aB7.00 aA3.40 aB6.00 abA3.00 aB5.90 abA2.60 aB
Particle Size4.00 aA3.10 aA4.20 aA3.10 aA4.30 aA3.22 aA4.40 aA3.03 aA
Thickness4.00 aA3.20 aA4.40 aA3.00 aA4.30 aA3.20 aA4.20 aA3.00 aA
Fluidity4.80 aA3.40 aA5.40 aA3.40 aA5.00 aA3.00 aA4.50 aA2.80 aA
Mouth Coating7.80 aA6.40 abA7.40 aA7.20 aA7.30 aA8.25 aA7.10 aA7.20 aA
+ 0 = lowest intensity, 7–8 = medium intensity, 15 = highest intensity. BMC: Mixed culture, BLB: Mixed culture + Lv. brevis, BLP: Mixed culture + Ls. paracasei, BLR: Mixed culture + Ls. rhamnosus. Lowercase letters indicate differences between samples. Uppercase letters indicate differences between storage times.
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Keser, G.; Ozcan, T. Production of Bio-Improved Butter with Lactic Acid Bacteria Isolated from Traditional Cheese Matrix and Eye Fluid. Fermentation 2025, 11, 620. https://doi.org/10.3390/fermentation11110620

AMA Style

Keser G, Ozcan T. Production of Bio-Improved Butter with Lactic Acid Bacteria Isolated from Traditional Cheese Matrix and Eye Fluid. Fermentation. 2025; 11(11):620. https://doi.org/10.3390/fermentation11110620

Chicago/Turabian Style

Keser, Gokce, and Tulay Ozcan. 2025. "Production of Bio-Improved Butter with Lactic Acid Bacteria Isolated from Traditional Cheese Matrix and Eye Fluid" Fermentation 11, no. 11: 620. https://doi.org/10.3390/fermentation11110620

APA Style

Keser, G., & Ozcan, T. (2025). Production of Bio-Improved Butter with Lactic Acid Bacteria Isolated from Traditional Cheese Matrix and Eye Fluid. Fermentation, 11(11), 620. https://doi.org/10.3390/fermentation11110620

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